Restricted mobility of side chains on concave surfaces of solenoid proteins may impart heightened potential for intermolecular interactions.

PubMed

Ramya, L; Gautham, N; Chaloin, Laurent; Kajava, Andrey V

2015-09-01

Significant progress has been made in the determination of the protein structures with their number today passing over a hundred thousand structures. The next challenge is the understanding and prediction of protein-protein and protein-ligand interactions. In this work we address this problem by analyzing curved solenoid proteins. Many of these proteins are considered as "hub molecules" for their high potential to interact with many different molecules and to be a scaffold for multisubunit protein machineries. Our analysis of these structures through molecular dynamics simulations reveals that the mobility of the side-chains on the concave surfaces of the solenoids is lower than on the convex ones. This result provides an explanation to the observed preferential binding of the ligands, including small and flexible ligands, to the concave surface of the curved solenoid proteins. The relationship between the landscapes and dynamic properties of the protein surfaces can be further generalized to the other types of protein structures and eventually used in the computer algorithms, allowing prediction of protein-ligand interactions by analysis of protein surfaces. © 2015 Wiley Periodicals, Inc.

Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus

PubMed Central

Foster, Timothy J.; Geoghegan, Joan A.; Ganesh, Vannakambadi K.; Höök, Magnus

2014-01-01

Staphylococcus aureus is an important opportunistic pathogen and persistently colonizes about 20% of the human population. Its surface is ‘decorated’ with proteins that are covalently anchored to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections. PMID:24336184

Hydrophilic crosslinked-polymeric surface capable of effective suppression of protein adsorption

NASA Astrophysics Data System (ADS)

Kamon, Yuri; Inoue, Naoko; Mihara, Erika; Kitayama, Yukiya; Ooya, Tooru; Takeuchi, Toshifumi

2016-08-01

We investigated the nonspecific adsorption of proteins towards three hydrophilic crosslinked-polymeric thin layers prepared by surface-initiated atom transfer radical polymerization using N,N‧-methylenebisacrylamide, 2-(methacryloyloxy)ethyl-[N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), or 6,6‧-diacryloyl-trehalose crosslinkers. Protein binding experiments were performed by surface plasmon resonance with six proteins of different pI values including α-lactalbumin, bovine serum albumin (BSA), myoglobin, ribonuclease A, cytochrome C, and lysozyme in buffer solution at pH 7.4. All of the obtained crosslinked-polymeric thin layers showed low nonspecific adsorption of negatively charged proteins at pH 7.4 such as α-lactalbumin, BSA, and myoglobin. Nonspecific adsorption of positively charged proteins including ribonuclease A, cytochrome C, and lysozyme was the lowest for poly(MMPC). These results suggest poly(MMPC) can effectively reduce nonspecific adsorption of a wide range of proteins that are negatively or positively charged at pH 7.4. MMPC is a promising crosslinker for a wide range of polymeric materials requiring low nonspecific protein binding.

Surface Proteins of Gram-Positive Pathogens: Using Crystallography to Uncover Novel Features in Drug and Vaccine Candidates

NASA Astrophysics Data System (ADS)

Baker, Edward N.; Proft, Thomas; Kang, Haejoo

Proteins displayed on the cell surfaces of pathogenic organisms are the front-line troops of bacterial attack, playing critical roles in colonization, infection and virulence. Although such proteins can often be recognized from genome sequence data, through characteristic sequence motifs, their functions are often unknown. One such group of surface proteins is attached to the cell surface of Gram-positive pathogens through the action of sortase enzymes. Some of these proteins are now known to form pili: long filamentous structures that mediate attachment to human cells. Crystallographic analyses of these and other cell surface proteins have uncovered novel features in their structure, assembly and stability, including the presence of inter- and intramolecular isopeptide crosslinks. This improved understanding of structures on the bacterial cell surface offers opportunities for the development of some new drug targets and for novel approaches to vaccine design.

Structure and Modification of Electrode Materials for Protein Electrochemistry.

PubMed

Jeuken, Lars J C

The interactions between proteins and electrode surfaces are of fundamental importance in bioelectrochemistry, including photobioelectrochemistry. In order to optimise the interaction between electrode and redox protein, either the electrode or the protein can be engineered, with the former being the most adopted approach. This tutorial review provides a basic description of the most commonly used electrode materials in bioelectrochemistry and discusses approaches to modify these surfaces. Carbon, gold and transparent electrodes (e.g. indium tin oxide) are covered, while approaches to form meso- and macroporous structured electrodes are also described. Electrode modifications include the chemical modification with (self-assembled) monolayers and the use of conducting polymers in which the protein is imbedded. The proteins themselves can either be in solution, electrostatically adsorbed on the surface or covalently bound to the electrode. Drawbacks and benefits of each material and its modifications are discussed. Where examples exist of applications in photobioelectrochemistry, these are highlighted.

A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel ITIM protein

PubMed Central

Senis, Yotis A.; Tomlinson, Michael G.; García, Ángel; Dumon, Stephanie; Heath, Victoria L.; Herbert, John; Cobbold, Stephen P.; Spalton, Jennifer C.; Ayman, Sinem; Antrobus, Robin; Zitzmann, Nicole; Bicknell, Roy; Frampton, Jon; Authi, Kalwant; Martin, Ashley; Wakelam, Michael J.O.; Watson, Stephen P.

2007-01-01

Summary The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we have identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomic and genomic approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography; biotin/NeutrAvidin affinity chromatography; and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68 and 22 surface membrane, intracellular membrane and membrane proteins of unknown sub-cellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomic studies, we analysed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multi-transmembrane proteins. Strikingly, 17 of the 25 most megakaryocyte-specific genes (relative to 30 other SAGE libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2-containing phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation. PMID:17186946

PL-PatchSurfer: a novel molecular local surface-based method for exploring protein-ligand interactions.

PubMed

Hu, Bingjie; Zhu, Xiaolei; Monroe, Lyman; Bures, Mark G; Kihara, Daisuke

2014-08-27

Structure-based computational methods have been widely used in exploring protein-ligand interactions, including predicting the binding ligands of a given protein based on their structural complementarity. Compared to other protein and ligand representations, the advantages of a surface representation include reduced sensitivity to subtle changes in the pocket and ligand conformation and fast search speed. Here we developed a novel method named PL-PatchSurfer (Protein-Ligand PatchSurfer). PL-PatchSurfer represents the protein binding pocket and the ligand molecular surface as a combination of segmented surface patches. Each patch is characterized by its geometrical shape and the electrostatic potential, which are represented using the 3D Zernike descriptor (3DZD). We first tested PL-PatchSurfer on binding ligand prediction and found it outperformed the pocket-similarity based ligand prediction program. We then optimized the search algorithm of PL-PatchSurfer using the PDBbind dataset. Finally, we explored the utility of applying PL-PatchSurfer to a larger and more diverse dataset and showed that PL-PatchSurfer was able to provide a high early enrichment for most of the targets. To the best of our knowledge, PL-PatchSurfer is the first surface patch-based method that treats ligand complementarity at protein binding sites. We believe that using a surface patch approach to better understand protein-ligand interactions has the potential to significantly enhance the design of new ligands for a wide array of drug-targets.

PL-PatchSurfer: A Novel Molecular Local Surface-Based Method for Exploring Protein-Ligand Interactions

PubMed Central

Hu, Bingjie; Zhu, Xiaolei; Monroe, Lyman; Bures, Mark G.; Kihara, Daisuke

2014-01-01

Structure-based computational methods have been widely used in exploring protein-ligand interactions, including predicting the binding ligands of a given protein based on their structural complementarity. Compared to other protein and ligand representations, the advantages of a surface representation include reduced sensitivity to subtle changes in the pocket and ligand conformation and fast search speed. Here we developed a novel method named PL-PatchSurfer (Protein-Ligand PatchSurfer). PL-PatchSurfer represents the protein binding pocket and the ligand molecular surface as a combination of segmented surface patches. Each patch is characterized by its geometrical shape and the electrostatic potential, which are represented using the 3D Zernike descriptor (3DZD). We first tested PL-PatchSurfer on binding ligand prediction and found it outperformed the pocket-similarity based ligand prediction program. We then optimized the search algorithm of PL-PatchSurfer using the PDBbind dataset. Finally, we explored the utility of applying PL-PatchSurfer to a larger and more diverse dataset and showed that PL-PatchSurfer was able to provide a high early enrichment for most of the targets. To the best of our knowledge, PL-PatchSurfer is the first surface patch-based method that treats ligand complementarity at protein binding sites. We believe that using a surface patch approach to better understand protein-ligand interactions has the potential to significantly enhance the design of new ligands for a wide array of drug-targets. PMID:25167137

Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment.

PubMed Central

Mayor, S; Maxfield, F R

1995-01-01

A diverse set of cell surface eukaryotic proteins including receptors, enzymes, and adhesion molecules have a glycosylphosphoinositol-lipid (GPI) modification at the carboxy-terminal end that serves as their sole means of membrane anchoring. These GPI-anchored proteins are poorly solubilized in nonionic detergent such as Triton X-100. In addition these detergent-insoluble complexes from plasma membranes are significantly enriched in several cytoplasmic proteins including nonreceptor-type tyrosine kinases and caveolin/VIP-21, a component of the striated coat of caveolae. These observations have suggested that the detergent-insoluble complexes represent purified caveolar membrane preparations. However, we have recently shown by immunofluorescence and electron microscopy that GPI-anchored proteins are diffusely distributed at the cell surface but may be enriched in caveolae only after cross-linking. Although caveolae occupy only a small fraction of the cell surface (< 4%), almost all of the GPI-anchored protein at the cell surface becomes incorporated into detergent-insoluble low-density complexes. In this paper we show that upon detergent treatment the GPI-anchored proteins are redistributed into a significantly more clustered distribution in the remaining membranous structures. These results show that GPI-anchored proteins are intrinsically detergent-insoluble in the milieu of the plasma membrane, and their co-purification with caveolin is not reflective of their native distribution. These results also indicate that the association of caveolae, GPI-anchored proteins, and signalling proteins must be critically re-examined. Images PMID:7579703

Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

PubMed Central

Volkmann, Gerrit; Liu, Xiang-Qin

2009-01-01

Background Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. Methodology A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. Conclusions We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups. PMID:20027230

Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

PubMed

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

2010-07-26

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

PubMed Central

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L.; Conrad, Daniel H.; Xu, Ping

2010-01-01

Background Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. Methods and Findings We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. Conclusions The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases. PMID:20668678

Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d.

PubMed

Doxey, Andrew C; Cheng, Zhenyu; Moffatt, Barbara A; McConkey, Brendan J

2010-08-03

Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins. The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

Molecular Characteristics and Biological Functions of Surface-Active and Surfactant Proteins.

PubMed

Sunde, Margaret; Pham, Chi L L; Kwan, Ann H

2017-06-20

Many critical biological processes take place at hydrophobic:hydrophilic interfaces, and a wide range of organisms produce surface-active proteins and peptides that reduce surface and interfacial tension and mediate growth and development at these boundaries. Microorganisms produce both small lipid-associated peptides and amphipathic proteins that allow growth across water:air boundaries, attachment to surfaces, predation, and improved bioavailability of hydrophobic substrates. Higher-order organisms produce surface-active proteins with a wide variety of functions, including the provision of protective foam environments for vulnerable reproductive stages, evaporative cooling, and gas exchange across airway membranes. In general, the biological functions supported by these diverse polypeptides require them to have an amphipathic nature, and this is achieved by a diverse range of molecular structures, with some proteins undergoing significant conformational change or intermolecular association to generate the structures that are surface active.

Non-interacting surface solvation and dynamics in protein-protein interactions.

PubMed

Visscher, Koen M; Kastritis, Panagiotis L; Bonvin, Alexandre M J J

2015-03-01

Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein-protein binding, even for simple lock-and-key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein-protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein-protein surfaces. We compare properties of the interface, rim, and non-interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non-interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non-interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein-protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein-protein complexes from unintended protein-protein interactions. © 2014 Wiley Periodicals, Inc.

Surface Passivation for Single-molecule Protein Studies

PubMed Central

Chandradoss, Stanley D.; Haagsma, Anna C.; Lee, Young Kwang; Hwang, Jae-Ho; Nam, Jwa-Min; Joo, Chirlmin

2014-01-01

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation. PMID:24797261

Rapid Covalent Immobilization of Proteins by Phenol-Based Photochemical Cross-Linking.

PubMed

Ren, Jun; Tian, Kaikai; Jia, Lingyun; Han, Xiuyou; Zhao, Mingshan

2016-10-19

A strategy for photoinduced covalent immobilization of proteins on phenol-functionalized surfaces is described. Under visible light irradiation, the reaction can be completed within seconds at ambient temperature, with high yields in aqueous solution of physiological conditions. Protein immobilization is based on a ruthenium-catalyzed radical cross-linking reaction between proteins and phenol-modified surfaces, and the process has proven mild enough for lipase, Staphylococcus aureus protein A, and streptavidin to preserve their bioactivity. This strategy was successfully applied to antibody immobilization on different material platforms, including agarose beads, cellulose membranes, and glass wafers, thus providing a generic procedure for rapid biomodification of surfaces.

Polyethylene Glycol Propionaldehydes

NASA Technical Reports Server (NTRS)

Harris, Joe M.; Sedaghat-Herati, Mohammad R.; Karr, Laurel J.

1992-01-01

New class of compounds derived from polyethylene glycol (PEG's) namely, PEG-propionaldehydes, offers two important advantages over other classes of PEG aldehyde derivatives: compounds exhibit selective chemical reactivity toward amino groups and are stable in aqueous environment. PEG's and derivatives used to couple variety of other molecules, such as, to tether protein molecules to surfaces. Biotechnical and biomedical applications include partitioning of two phases in aqueous media; immobilization of such proteins as enzymes, antibodies, and antigens; modification of drugs; and preparation of protein-rejecting surfaces. In addition, surfaces coated with PEG's and derivatives used to control wetting and electroosmosis. Another potential application, coupling to aminated surfaces.

Surface enhanced Raman spectroscopy detection of biomolecules using EBL fabricated nanostructured substrates.

PubMed

Peters, Robert F; Gutierrez-Rivera, Luis; Dew, Steven K; Stepanova, Maria

2015-03-20

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

Patch Finder Plus (PFplus): a web server for extracting and displaying positive electrostatic patches on protein surfaces.

PubMed

Shazman, Shula; Celniker, Gershon; Haber, Omer; Glaser, Fabian; Mandel-Gutfreund, Yael

2007-07-01

Positively charged electrostatic patches on protein surfaces are usually indicative of nucleic acid binding interfaces. Interestingly, many proteins which are not involved in nucleic acid binding possess large positive patches on their surface as well. In some cases, the positive patches on the protein are related to other functional properties of the protein family. PatchFinderPlus (PFplus) http://pfp.technion.ac.il is a web-based tool for extracting and displaying continuous electrostatic positive patches on protein surfaces. The input required for PFplus is either a four letter PDB code or a protein coordinate file in PDB format, provided by the user. PFplus computes the continuum electrostatics potential and extracts the largest positive patch for each protein chain in the PDB file. The server provides an output file in PDB format including a list of the patch residues. In addition, the largest positive patch is displayed on the server by a graphical viewer (Jmol), using a simple color coding.

Patch Finder Plus (PFplus): A web server for extracting and displaying positive electrostatic patches on protein surfaces

PubMed Central

Shazman, Shula; Celniker, Gershon; Haber, Omer; Glaser, Fabian; Mandel-Gutfreund, Yael

2007-01-01

Positively charged electrostatic patches on protein surfaces are usually indicative of nucleic acid binding interfaces. Interestingly, many proteins which are not involved in nucleic acid binding possess large positive patches on their surface as well. In some cases, the positive patches on the protein are related to other functional properties of the protein family. PatchFinderPlus (PFplus) http://pfp.technion.ac.il is a web-based tool for extracting and displaying continuous electrostatic positive patches on protein surfaces. The input required for PFplus is either a four letter PDB code or a protein coordinate file in PDB format, provided by the user. PFplus computes the continuum electrostatics potential and extracts the largest positive patch for each protein chain in the PDB file. The server provides an output file in PDB format including a list of the patch residues. In addition, the largest positive patch is displayed on the server by a graphical viewer (Jmol), using a simple color coding. PMID:17537808

Influence of surface features of hydroxyapatite on the adsorption of proteins relevant to bone regeneration.

PubMed

Fernández-Montes Moraleda, Belén; San Román, Julio; Rodríguez-Lorenzo, Luís M

2013-08-01

Protein-surface interaction may determine the success or failure of an implanted device. Not much attention have been paid to the specific surface parametes of hydroxyapatite (OHAp) that modulates and determines the formation and potential activity of the layer of proteins that is first formed when the material get in contact with the host tissue. the influence of specific surface area (SSA), crystallite size (CS) and particle size (PS) of OHAp on the adsorption of proteins relevant for bone regeneration is evaluated in this article. OHAp have been prepared by a wet chemical reaction of Ca(OH)2 with H3PO4. One set of reactions included poly acrylic acid in the reactant solution to modify the properties of the powder. Fibrinogen (Fg) Fraction I, type I: from Human plasma, (67% Protein), and Fibronectin (Fn) from Human plasma were selected to perform the adsorption experiments. The analysis of protein adsorption was carried out by UV/Vis spectrometry. A lower SSA and a different aspect ratio are obtained when the acrylic acid is included in the reaction badge. The deconvolution of the amide I band on the Raman spectra of free and adsorbed proteins reveals that the interaction apatite-protein happens through the carboxylate groups of the proteins. The combined analysis of CS, SSA and PS should be considered on the design of OHAp materials intended to interact with proteins. Copyright © 2013 Wiley Periodicals, Inc.

Single-molecule resolution of protein dynamics on polymeric membrane surfaces: the roles of spatial and population heterogeneity.

PubMed

Langdon, Blake B; Mirhossaini, Roya B; Mabry, Joshua N; Sriram, Indira; Lajmi, Ajay; Zhang, Yanxia; Rojas, Orlando J; Schwartz, Daniel K

2015-02-18

Although polymeric membranes are widely used in the purification of protein pharmaceuticals, interactions between biomolecules and membrane surfaces can lead to reduced membrane performance and damage to the product. In this study, single-molecule fluorescence microscopy provided direct observation of bovine serum albumin (BSA) and human monoclonal antibody (IgG) dynamics at the interface between aqueous buffer and polymeric membrane materials including regenerated cellulose and unmodified poly(ether sulfone) (PES) blended with either polyvinylpyrrolidone (PVP), polyvinyl acetate-co-polyvinylpyrrolidone (PVAc-PVP), or polyethylene glycol methacrylate (PEGM) before casting. These polymer surfaces were compared with model surfaces composed of hydrophilic bare fused silica and hydrophobic trimethylsilane-coated fused silica. At extremely dilute protein concentrations (10(-3)-10(-7) mg/mL), protein surface exchange was highly dynamic with protein monomers desorbing from the surface within ∼1 s after adsorption. Protein oligomers (e.g., nonspecific dimers, trimers, or larger aggregates), although less common, remained on the surface for 5 times longer than monomers. Using newly developed super-resolution methods, we could localize adsorption sites with ∼50 nm resolution and quantify the spatial heterogeneity of the various surfaces. On a small anomalous subset of the adsorption sites, proteins adsorbed preferentially and tended to reside for significantly longer times (i.e., on "strong" sites). Proteins resided for shorter times overall on surfaces that were more homogeneous and exhibited fewer strong sites (e.g., PVAc-PVP/PES). We propose that strong surface sites may nucleate protein aggregation, initiated preferentially by protein oligomers, and accelerate ultrafiltration membrane fouling. At high protein concentrations (0.3-1.0 mg/mL), fewer strong adsorption sites were observed, and surface residence times were reduced. This suggests that at high concentrations, adsorbed proteins block strong sites from further protein adsorption. Importantly, this demonstrates that strong binding sites can be modified by changing solution conditions. Membrane surfaces are intrinsically heterogeneous; by employing single-molecule techniques, we have provided a new framework for understanding protein interactions with such surfaces.

Listeria monocytogenes biofilm-associated protein (BapL) may contribute to surface attachment of L. monocytogenes but is absent from many field isolates.

PubMed

Jordan, Suzanne J; Perni, Stefano; Glenn, Sarah; Fernandes, Isabel; Barbosa, Manuela; Sol, Manuela; Tenreiro, Rogerio P; Chambel, Lelia; Barata, Belarmino; Zilhao, Isabel; Aldsworth, Timothy G; Adriao, Andreia; Faleiro, M Leonor; Shama, Gilbert; Andrew, Peter W

2008-09-01

Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.

NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip-translocon protein-protein interactions.

PubMed

McShan, Andrew C; Kaur, Kawaljit; Chatterjee, Srirupa; Knight, Kevin M; De Guzman, Roberto N

2016-08-01

The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein-protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097-1107. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

PubMed Central

Tacchi, Jessica L.; Raymond, Benjamin B. A.; Haynes, Paul A.; Berry, Iain J.; Widjaja, Michael; Bogema, Daniel R.; Woolley, Lauren K.; Jenkins, Cheryl; Minion, F. Chris; Padula, Matthew P.; Djordjevic, Steven P.

2016-01-01

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC–MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

Novel surface attachment mechanism of the Streptococcus pneumoniae protein PspA.

PubMed Central

Yother, J; White, J M

1994-01-01

Pneumococcal surface protein A (PspA) of Streptococcus pneumoniae has been found to utilize a novel mechanism for anchoring to the bacterial cell surface. In contrast to that of surface proteins from other gram-positive bacteria, PspA anchoring required choline-mediated interactions between the membrane-associated lipoteichoic acid and the C-terminal repeat region of PspA. Release of PspA from the cell surface could be effected by deletion of 5 of the 10 C-terminal repeat units, by high concentrations of choline, or by growth in choline-deficient medium. Other pneumococcal proteins, including autolysin, which has a similar C-terminal repeat region, were not released by these treatments. The attachment mechanism utilized by PspA thus appears to be uniquely adapted to exploit the unusual structure of the pneumococcal cell surface. Further, it has provided the means for rapid and simple isolation of immunogenic PspA from S. pneumoniae. Images PMID:7910604

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763

Biopharmaceutical production: Applications of surface plasmon resonance biosensors.

PubMed

Thillaivinayagalingam, Pranavan; Gommeaux, Julien; McLoughlin, Michael; Collins, David; Newcombe, Anthony R

2010-01-15

Surface plasmon resonance (SPR) permits the quantitative analysis of therapeutic antibody concentrations and impurities including bacteria, Protein A, Protein G and small molecule ligands leached from chromatography media. The use of surface plasmon resonance has gained popularity within the biopharmaceutical industry due to the automated, label free, real time interaction that may be exploited when using this method. The application areas to assess protein interactions and develop analytical methods for biopharmaceutical downstream process development, quality control, and in-process monitoring are reviewed. 2009 Elsevier B.V. All rights reserved.

Surface plasmon resonance biosensing of the monomer and the linked dimer of the variants of protein G under mass transport limitation.

PubMed

Imamura, Hiroshi; Honda, Shinya

2016-12-01

This article presented the data related to the research article entitled "Calibration-free concentration analysis for an analyte prone to self-association" (H. Imamura, S. Honda, 2017) [1]. The data included surface plasmon resonance (SPR) responses of the variants of protein G with different masses under mass transport limitation. The friction factors of the proteins analyzed by an ultracentrifugation were recorded. Calculation of the SPR response of the proteins was also described.

Bacterial Ice Crystal Controlling Proteins

PubMed Central

Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

2014-01-01

Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

Bacterial ice crystal controlling proteins.

PubMed

Lorv, Janet S H; Rose, David R; Glick, Bernard R

2014-01-01

Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

Biofilm Matrix Proteins.

PubMed

Fong, Jiunn N C; Yildiz, Fitnat H

2015-04-01

Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins, and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this article, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation.

Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL

PubMed Central

Ko, Hyeok-Jin; Park, Eunhye; Song, Joseph; Yang, Taek Ho; Lee, Hee Jong; Kim, Kyoung Heon

2012-01-01

Autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of Escherichia coli for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from Saccharophagus degradans 2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 104 molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing E. coli. PMID:22344647

A script to highlight hydrophobicity and charge on protein surfaces

PubMed Central

Hagemans, Dominique; van Belzen, Ianthe A. E. M.; Morán Luengo, Tania; Rüdiger, Stefan G. D.

2015-01-01

The composition of protein surfaces determines both affinity and specificity of protein-protein interactions. Matching of hydrophobic contacts and charged groups on both sites of the interface are crucial to ensure specificity. Here, we propose a highlighting scheme, YRB, which highlights both hydrophobicity and charge in protein structures. YRB highlighting visualizes hydrophobicity by highlighting all carbon atoms that are not bound to nitrogen and oxygen atoms. The charged oxygens of glutamate and aspartate are highlighted red and the charged nitrogens of arginine and lysine are highlighted blue. For a set of representative examples, we demonstrate that YRB highlighting intuitively visualizes segments on protein surfaces that contribute to specificity in protein-protein interfaces, including Hsp90/co-chaperone complexes, the SNARE complex and a transmembrane domain. We provide YRB highlighting in form of a script that runs using the software PyMOL. PMID:26528483

Applications of Functional Amyloids from Fungi: Surface Modification by Class I Hydrophobins.

PubMed

Piscitelli, Alessandra; Cicatiello, Paola; Gravagnuolo, Alfredo Maria; Sorrentino, Ilaria; Pezzella, Cinzia; Giardina, Paola

2017-06-26

Class I hydrophobins produced from fungi are amongst the first proteins recognized as functional amyloids. They are amphiphilic proteins involved in the formation of aerial structures such as spores or fruiting bodies. They form chemically robust layers which can only be dissolved in strong acids. These layers adhere to different surfaces, changing their wettability, and allow the binding of other proteins. Herein, the modification of diverse types of surfaces with Class I hydrophobins is reported, highlighting the applications of the coated surfaces. Indeed, these coatings can be exploited in several fields, spanning from biomedical to industrial applications, which include biosensing and textile manufacturing.

The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini

PubMed Central

Mulvenna, Jason; Sripa, Banchob; Brindley, Paul J.; Gorman, Jeffrey; Jones, Malcolm K.; Colgrave, Michelle L.; Jones, Alun; Nawaratna, Sujeevi; Laha, Thewarach; Suttiprapa, Sutas; Smout, Michael J.; Loukas, Alex

2011-01-01

Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and Multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products (ES) and, utilizing selective labeling and sequential solubilization, from the host-exposed tegument. The ES included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti-apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. Proteases in ES included cathepsins, calpain and a protein with homology to autoimmune prostatitis antigen 2. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno-modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin-2. Novel roles in pathogenesis were suggested for the tegument-host interface since more than ten biotinylated surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke. PMID:20049860

Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.

PubMed

Shirvan, Ali Nazari; Aitken, Robert

2016-01-01

Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. Copyright © 2016. Published by Elsevier Editora Ltda.

Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression

PubMed Central

2015-01-01

The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells. PMID:26301573

Understanding the nanoparticle-protein corona complexes using computational and experimental methods.

PubMed

Kharazian, B; Hadipour, N L; Ejtehadi, M R

2016-06-01

Nanoparticles (NP) have capability to adsorb proteins from biological fluids and form protein layer, which is called protein corona. As the cell sees corona coated NPs, the protein corona can dictate biological response to NPs. The composition of protein corona is varied by physicochemical properties of NPs including size, shape, surface chemistry. Processing of protein adsorption is dynamic phenomena; to that end, a protein may desorb or leave a surface vacancy that is rapidly filled by another protein and cause changes in the corona composition mainly by the Vroman effect. In this review, we discuss the interaction between NP and proteins and the available techniques for identification of NP-bound proteins. Also we review current developed computational methods for understanding the NP-protein complex interactions. Copyright © 2016. Published by Elsevier Ltd.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, John J.; Barbour, Alan G.

1996-11-05

A method is provided herein for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.

New approaches for solving old problems in neuronal protein trafficking.

PubMed

Bourke, Ashley M; Bowen, Aaron B; Kennedy, Matthew J

2018-04-10

Fundamental cellular properties are determined by the repertoire and abundance of proteins displayed on the cell surface. As such, the trafficking mechanisms for establishing and maintaining the surface proteome must be tightly regulated for cells to respond appropriately to extracellular cues, yet plastic enough to adapt to ever-changing environments. Not only are the identity and abundance of surface proteins critical, but in many cases, their regulated spatial positioning within surface nanodomains can greatly impact their function. In the context of neuronal cell biology, surface levels and positioning of ion channels and neurotransmitter receptors play essential roles in establishing important properties, including cellular excitability and synaptic strength. Here we review our current understanding of the trafficking pathways that control the abundance and localization of proteins important for synaptic function and plasticity, as well as recent technological advances that are allowing the field to investigate protein trafficking with increasing spatiotemporal precision. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of Host Proteins Associated with Retroviral Vector Particles by Proteomic Analysis of Highly Purified Vector Preparations▿

PubMed Central

Segura, María Mercedes; Garnier, Alain; Di Falco, Marcos Rafael; Whissell, Gavin; Meneses-Acosta, Angélica; Arcand, Normand; Kamen, Amine

2008-01-01

The Moloney murine leukemia virus (MMLV) belongs to the Retroviridae family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate-zonal ultracentrifugation. Viral proteins were fractionated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin β1 (fibronectin receptor subunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and late endosomal markers CD63 and Lamp-2. Identification of membrane proteins on the retroviral surface is particularly attractive, since they can serve as anchoring sites for the insertion of tags for targeting or purification purposes. The implications of our findings for retrovirus-mediated gene therapy are discussed. PMID:18032515

Electrostatic Interactions between OmpG Nanopore and Analyte Protein Surface Can Distinguish between Glycosylated Isoforms.

PubMed

Fahie, Monifa A; Chen, Min

2015-08-13

The flexible loops decorating the entrance of OmpG nanopore move dynamically during ionic current recording. The gating caused by these flexible loops changes when a target protein is bound. The gating is characterized by parameters including frequency, duration, and open-pore current, and these features combine to reveal the identity of a specific analyte protein. Here, we show that OmpG nanopore equipped with a biotin ligand can distinguish glycosylated and deglycosylated isoforms of avidin by their differences in surface charge. Our studies demonstrate that the direct interaction between the nanopore and analyte surface, induced by the electrostatic attraction between the two molecules, is essential for protein isoform detection. Our technique is remarkably sensitive to the analyte surface, which may provide a useful tool for glycoprotein profiling.

Personalized protein corona on nanoparticles and its clinical implications.

PubMed

Corbo, Claudia; Molinaro, Roberto; Tabatabaei, Mateen; Farokhzad, Omid C; Mahmoudi, Morteza

2017-02-28

It is now well understood that once in contact with biological fluids, nanoscale objects lose their original identity and acquire a new biological character, referred to as a protein corona. The protein corona changes many of the physicochemical properties of nanoparticles, including size, surface charge, and aggregation state. These changes, in turn, affect the biological fate of nanoparticles, including their pharmacokinetics, biodistribution, and therapeutic efficacy. It is progressively being accepted that even slight variations in the composition of a protein source (e.g., plasma and serum) can substantially change the composition of the corona formed on the surface of the exact same nanoparticles. Recently it has been shown that the protein corona is strongly affected by the patient's specific disease. Therefore, the same nanomaterial incubated with plasma proteins of patients with different pathologies adsorb protein coronas with different compositions, giving rise to the concept of personalized protein corona. Herein, we review this concept along with recent advances on the topic, with a particular focus on clinical relevance.

Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface

PubMed Central

Marín, Elvira; Parra-Giraldo, Claudia M.; Hernández-Haro, Carolina; Hernáez, María L.; Nombela, César; Monteoliva, Lucía; Gil, Concha

2015-01-01

Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in NS than in HIS, as validated by immunofluorescence. PMID:26696967

Boosting protein stability with the computational design of β-sheet surfaces.

PubMed

Kim, Doo Nam; Jacobs, Timothy M; Kuhlman, Brian

2016-03-01

β-sheets often have one face packed against the core of the protein and the other facing solvent. Mutational studies have indicated that the solvent-facing residues can contribute significantly to protein stability, and that the preferred amino acid at each sequence position is dependent on the precise structure of the protein backbone and the identity of the neighboring amino acids. This suggests that the most advantageous methods for designing β-sheet surfaces will be approaches that take into account the multiple energetic factors at play including side chain rotamer preferences, van der Waals forces, electrostatics, and desolvation effects. Here, we show that the protein design software Rosetta, which models these energetic factors, can be used to dramatically increase protein stability by optimizing interactions on the surfaces of small β-sheet proteins. Two design variants of the β-sandwich protein from tenascin were made with 7 and 14 mutations respectively on its β-sheet surfaces. These changes raised the thermal midpoint for unfolding from 45°C to 64°C and 74°C. Additionally, we tested an empirical approach based on increasing the number of potential salt bridges on the surfaces of the β-sheets. This was not a robust strategy for increasing stability, as three of the four variants tested were unfolded. © 2016 The Protein Society.

Interaction of gold and silver nanoparticles with human plasma: Analysis of protein corona reveals specific binding patterns.

PubMed

Lai, Wenjia; Wang, Qingsong; Li, Lumeng; Hu, Zhiyuan; Chen, Jiankui; Fang, Qiaojun

2017-04-01

Determining how nanomaterials interact with plasma will assist in understanding their effects on the biological system. This work presents a systematic study of the protein corona formed from human plasma on 20nm silver and gold nanoparticles with three different surface modifications, including positive and negative surface charges. The results show that all nanoparticles, even those with positive surface modifications, acquire negative charges after interacting with plasma. Approximately 300 proteins are identified on the coronas, while 99 are commonly found on each nanomaterial. The 20 most abundant proteins account for over 80% of the total proteins abundance. Remarkably, the surface charge and core of the nanoparticles, as well as the isoelectric point of the plasma proteins, are found to play significant roles in determining the nanoparticle coronas. Albumin and globulins are present at levels of less than 2% on these nanoparticle coronas. Fibrinogen, which presents in the plasma but not in the serum, preferably binds to negatively charged gold nanoparticles. These observations demonstrate the specific plasma protein binding pattern of silver and gold nanoparticles, as well as the importance of the surface charge and core in determining the protein corona compositions. The potential downstream biological impacts of the corona proteins were also investigated. Copyright © 2017 Elsevier B.V. All rights reserved.

Silica surface characterization as a function of formation and surface treatment using traditional methods and proteins as surface probes

NASA Astrophysics Data System (ADS)

Korwin-Edson, Michelle Lynn

Previous works have shown that cells proliferate differently depending on the chemistry of the glass on which they are growing. Since proteins form the bonds between cells and glass, the hypothesis of this study is that proteins can distinguish between surface chemical variations of glass. This theory was examined through the use of various silica forms, a few select proteins, four surface treatment procedures, and a variety of characterization techniques. The silica forms include amorphous slides, cane, fiber, microspheres, fumed silica and quartz crystal terminals. The proteins selected were human serum albumin, mouse Immunoglobulin G, streptavidin, antimouse IgG, and biotin. The surface treatments utilized to bring about chemical variation on the silica surface were HF acid etching, ethanol cleaning, water plasma treatments, and 1000°C heat treatments. The characterization techniques encompassed both traditional material techniques and biological methods. The techniques studied were atomic force microscopy (AFM), chemical force microscopy (CFM), glancing incidence X-ray analysis (GIXA), fluorescence spectrometry, polyacrylamide gel electrophoresis (SDS-PAGE), and bicinchoninic acid (BCA) assay. It was the main goal of this project to determine the feasibility of these techniques in utilizing proteins as glass surface probes. Proteins were adsorbed to all of the various forms and the binding ability was studied by either stripping off the protein and quantifying them, or by deductive reasoning through the use of "depleted" protein solutions. Fluorimetry and BCA assay both utilized the depleted solutions, but the high error associated with this protocol was prohibitive. SDS-PAGE with streptavidin was very difficult due to staining problems, however the IgG proteins were able to be quantified with some success. GIXA showed that the protein layer thickness is monolayer in nature, which agrees well with the AFM fluid tapping data on protein height, but in addition showed features on the order of ten protein agglomerations. CFM is by far the most promising technique for utilizing proteins as surface probes. Functionalized tips of -COOH, streptavidin and -CH3 are able to discern between surface treatments, but not forms. A general conclusion is that adhesion forces are greatest for -COOH, then streptavidin, and least for -CH3.

The scope of phage display for membrane proteins.

PubMed

Vithayathil, Rosemarie; Hooy, Richard M; Cocco, Melanie J; Weiss, Gregory A

2011-12-09

Numerous examples of phage display applied to soluble proteins demonstrate the power of the technique for protein engineering, affinity reagent discovery and structure-function studies. Recent reports have expanded phage display to include membrane proteins (MPs). The scope and limitations of MP display remain undefined. Therefore, we report data from the phage display of representative types of membrane-associated proteins including plasma, nuclear, peripheral, single and multipass. The peripheral MP neuromodulin displays robustly with packaging by conventional M13-KO7 helper phage. The monotopic MP Nogo-66 can also display on the phage surface, if packaged by the modified M13-KO7(+) helper phage. The modified phage coat of KO7(+) can better mimic the zwitterionic character of the plasma membrane. Four examples of putatively α-helical, integral MPs failed to express as fusions to an anchoring phage coat protein and therefore did not display on the phage surface. However, the β-barrel MPs ShuA (Shigella heme uptake A) and MOMP (major outer membrane protein), which pass through the membrane 22 and 16 times, respectively, can display surprisingly well on the surfaces of both conventional and KO7(+) phages. The results provide a guide for protein engineering and large-scale mutagenesis enabled by the phage display of MPs. Copyright © 2011 Elsevier Ltd. All rights reserved.

iview: an interactive WebGL visualizer for protein-ligand complex.

PubMed

Li, Hongjian; Leung, Kwong-Sak; Nakane, Takanori; Wong, Man-Hon

2014-02-25

Visualization of protein-ligand complex plays an important role in elaborating protein-ligand interactions and aiding novel drug design. Most existing web visualizers either rely on slow software rendering, or lack virtual reality support. The vital feature of macromolecular surface construction is also unavailable. We have developed iview, an easy-to-use interactive WebGL visualizer of protein-ligand complex. It exploits hardware acceleration rather than software rendering. It features three special effects in virtual reality settings, namely anaglyph, parallax barrier and oculus rift, resulting in visually appealing identification of intermolecular interactions. It supports four surface representations including Van der Waals surface, solvent excluded surface, solvent accessible surface and molecular surface. Moreover, based on the feature-rich version of iview, we have also developed a neat and tailor-made version specifically for our istar web platform for protein-ligand docking purpose. This demonstrates the excellent portability of iview. Using innovative 3D techniques, we provide a user friendly visualizer that is not intended to compete with professional visualizers, but to enable easy accessibility and platform independence.

Micromechanical sensors based on conformational change of proteins

NASA Astrophysics Data System (ADS)

Yang, Xin; Buchapudi, Koutilya R.; Gao, Hongyan; Xu, Xiaohe; Ji, Hai-Feng

2008-04-01

Microcantilevers (MCLs) hold a position as a cost-effective and highly sensitive sensor platform for medical diagnostics, environmental, and fast throughput analysis. One of recently focus in this technology is the development of biosensors based on the conformational change of proteins on MCL surfaces. The surface stress changes due to conformational change of the proteins upon interaction with specific analytes are promising as transducers of chemical information. We will discuss our recent results on several biosensors due to conformational change of proteins. The proteins include glucose oxidase (GOx), organophosphorus hydrolyses (OPH), Calmodulin (CaM), and Horseradish peroxidase (HRP).

Electrostatic and dispersion interactions during protein adsorption on topographic nanostructures.

PubMed

Elter, Patrick; Lange, Regina; Beck, Ulrich

2011-07-19

Recently, biomaterials research has focused on developing functional implant surfaces with well-defined topographic nanostructures in order to influence protein adsorption and cellular behavior. To enhance our understanding of how proteins interact with such surfaces, we analyze the adsorption of lysozyme on an oppositely charged nanostructure using a computer simulation. We present an algorithm that combines simulated Brownian dynamics with numerical field calculation methods to predict the preferred adsorption sites for arbitrarily shaped substrates. Either proteins can be immobilized at their initial adsorption sites or surface diffusion can be considered. Interactions are analyzed on the basis of Derjaguin-Landau-Verway-Overbeek (DLVO) theory, including electrostatic and London dispersion forces, and numerical solutions are derived using the Poisson-Boltzmann and Hamaker equations. Our calculations show that for a grooved nanostructure (i.e., groove and plateau width 8 nm, height 4 nm), proteins first contact the substrate primarily near convex edges because of better geometric accessibility and increased electric field strengths. Subsequently, molecules migrate by surface diffusion into grooves and concave corners, where short-range dispersion interactions are maximized. In equilibrium, this mechanism leads to an increased surface protein concentration in the grooves, demonstrating that the total amount of protein per surface area can be increased if substrates have concave nanostructures.

Prediction of Protein-Protein Interaction Sites Using Electrostatic Desolvation Profiles

PubMed Central

Fiorucci, Sébastien; Zacharias, Martin

2010-01-01

Abstract Protein-protein complex formation involves removal of water from the interface region. Surface regions with a small free energy penalty for water removal or desolvation may correspond to preferred interaction sites. A method to calculate the electrostatic free energy of placing a neutral low-dielectric probe at various protein surface positions has been designed and applied to characterize putative interaction sites. Based on solutions of the finite-difference Poisson equation, this method also includes long-range electrostatic contributions and the protein solvent boundary shape in contrast to accessible-surface-area-based solvation energies. Calculations on a large set of proteins indicate that in many cases (>90%), the known binding site overlaps with one of the six regions of lowest electrostatic desolvation penalty (overlap with the lowest desolvation region for 48% of proteins). Since the onset of electrostatic desolvation occurs even before direct protein-protein contact formation, it may help guide proteins toward the binding region in the final stage of complex formation. It is interesting that the probe desolvation properties associated with residue types were found to depend to some degree on whether the residue was outside of or part of a binding site. The probe desolvation penalty was on average smaller if the residue was part of a binding site compared to other surface locations. Applications to several antigen-antibody complexes demonstrated that the approach might be useful not only to predict protein interaction sites in general but to map potential antigenic epitopes on protein surfaces. PMID:20441756

Geometric Detection Algorithms for Cavities on Protein Surfaces in Molecular Graphics: A Survey

PubMed Central

Simões, Tiago; Lopes, Daniel; Dias, Sérgio; Fernandes, Francisco; Pereira, João; Jorge, Joaquim; Bajaj, Chandrajit; Gomes, Abel

2017-01-01

Detecting and analyzing protein cavities provides significant information about active sites for biological processes (e.g., protein-protein or protein-ligand binding) in molecular graphics and modeling. Using the three-dimensional structure of a given protein (i.e., atom types and their locations in 3D) as retrieved from a PDB (Protein Data Bank) file, it is now computationally viable to determine a description of these cavities. Such cavities correspond to pockets, clefts, invaginations, voids, tunnels, channels, and grooves on the surface of a given protein. In this work, we survey the literature on protein cavity computation and classify algorithmic approaches into three categories: evolution-based, energy-based, and geometry-based. Our survey focuses on geometric algorithms, whose taxonomy is extended to include not only sphere-, grid-, and tessellation-based methods, but also surface-based, hybrid geometric, consensus, and time-varying methods. Finally, we detail those techniques that have been customized for GPU (Graphics Processing Unit) computing. PMID:29520122

Suppression of protein adsorption on a charged phospholipid polymer interface.

PubMed

Xu, Yan; Takai, Madoka; Ishihara, Kazuhiko

2009-02-09

High capability of a charged interface to suppress adsorption of both anionic and cationic proteins was reported. The interface was covalently constructed on quartz by modifying with an anionic phospholipid copolymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-potassium 3-methacryloyloxypropyl sulfonate (PMPS)-co-3-methacryloxypropyl trimethoxysilane (MPTMSi)) (PMBSSi). The PMBSSi interfaces were very hydrophilic and homogeneous and could function effectively for a long time even under long-term fluidic working conditions. The PMBSSi density on the interface, which was controllable by adjusting the PMBSSi concentration of the modification solution, affected the surface properties, including the surface contact angle, the surface roughness, and the surface zeta-potential. When a PMBSSi modification was applied, the adsorption of various proteins (isoelectric point varying from 1.0 to 11.0) on quartz was reduced to at least 87% in amount, despite the various electrical natures these proteins have. The protein adsorption behavior on the PMBSSi interface depended more on the PMBSSi density than on the surface charge. The PMBSSi modification had a stable impact on the surface, not only at the physiologic ionic strength, but also over a range of the ionic strength, suggesting that electrostatic interactions do not dominate the behavior of protein adsorption to the PMBSSi surface.

Protein adsorption at charged surfaces: the role of electrostatic interactions and interfacial charge regulation.

PubMed

Hartvig, Rune A; van de Weert, Marco; Østergaard, Jesper; Jorgensen, Lene; Jensen, Henrik

2011-03-15

The understanding of protein adsorption at charged surfaces is important for a wide range of scientific disciplines including surface engineering, separation sciences and pharmaceutical sciences. Compared to chemical entities having a permanent charge, the adsorption of small ampholytes and proteins is more complicated as the pH near a charged surface can be significantly different from the value in bulk solution. In this work, we have developed a phenomenological adsorption model which takes into account the combined role of interfacial ion distribution, interfacial charge regulation of amino acids in the proximity of the surface, electroneutrality, and mass balance. The model is straightforward to apply to a given set of experimental conditions as most model parameters are obtained from bulk properties and therefore easy to estimate or are directly measurable. The model provides a detailed understanding of the importance of surface charge on adsorption and in particular of how changes in surface charge, concentration, and surface area may affect adsorption behavior. The model is successfully used to explain the experimental adsorption behavior of the two model proteins lysozyme and α-lactalbumin. It is demonstrated that it is possible to predict the pH and surface charge dependent adsorption behavior from experimental or theoretical estimates of a preferred orientation of a protein at a solid charged interface.

Yeast cell surface display for lipase whole cell catalyst and its applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yun; Zhang, Rui; Lian, Zhongshuai

The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chainmore » length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.« less

Distinct Adsorption Configurations and Self-Assembly Characteristics of Fibrinogen on Chemically Uniform and Alternating Surfaces including Block Copolymer Nanodomains

PubMed Central

2015-01-01

Understanding protein–surface interactions is crucial to solid-state biomedical applications whose functionality is directly correlated with the precise control of the adsorption configuration, surface packing, loading density, and bioactivity of protein molecules. Because of the small dimensions and highly amphiphilic nature of proteins, investigation of protein adsorption performed on nanoscale topology can shed light on subprotein-level interaction preferences. In this study, we examine the adsorption and assembly behavior of a highly elongated protein, fibrinogen, on both chemically uniform (as-is and buffered HF-treated SiO2/Si, and homopolymers of polystyrene and poly(methyl methacrylate)) and varying (polystyrene-block-poly(methyl methacrylate)) surfaces. By focusing on high-resolution imaging of individual protein molecules whose configurations are influenced by protein–surface rather than protein–protein interactions, fibrinogen conformations characteristic to each surface are identified and statistically analyzed for structural similarities/differences in key protein domains. By exploiting block copolymer nanodomains whose repeat distance is commensurate with the length of the individual protein, we determine that fibrinogen exhibits a more neutral tendency for interaction with both polystyrene and poly(methyl methacrylate) blocks relative to the case of common globular proteins. Factors affecting fibrinogen–polymer interactions are discussed in terms of hydrophobic and electrostatic interactions. In addition, assembly and packing attributes of fibrinogen are determined at different loading conditions. Primary orientations of fibrinogen and its rearrangements with respect to the underlying diblock nanodomains associated with different surface coverage are explained by pertinent protein interaction mechanisms. On the basis of two-dimensional stacking behavior, a protein assembly model is proposed for the formation of an extended fibrinogen network on the diblock copolymer. PMID:24708538

Interplay between grain structure and protein adsorption on functional response of osteoblasts: ultrafine-grained versus coarse-grained substrates.

PubMed

Misra, R D K; Nune, C; Pesacreta, T C; Somani, M C; Karjalainen, L P

2013-01-01

The rapid adsorption of proteins is the starting and primary biological response that occurs when a biomedical device is implanted in the physiological system. The biological response, however, depends on the surface characteristics of the device. Considering the significant interest in nano-/ultrafine surfaces and nanostructured coatings, we describe here, the interplay between grain structure and protein adsorption (bovine serum albumin: BSA) on osteoblasts functions by comparing nanograined/ultrafine-grained (NG/UFG) and coarse-grained (CG: grain size in the micrometer range) substrates by investigating cell-substrate interactions. The protein adsorption on NG/UFG surface was beneficial in favorably modulating biological functions including cell attachment, proliferation, and viability, whereas the effect was less pronounced on protein adsorbed CG surface. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on protein adsorbed NG/UFG surface. The functional response followed the sequence: NG/UFG(BSA) > NG/UFG > CG(BSA) > CG. The differences in the cellular response on bare and protein adsorbed NG/UFG and CG surfaces are attributed to cumulative contribution of grain structure and degree of hydrophilicity. The study underscores the potential advantages of protein adsorption on artificial biomedical devices to enhance the bioactivity and regulate biological functions. Copyright © 2012 Wiley Periodicals, Inc.

Protein adsorption on tailored substrates: long-range forces and conformational changes

NASA Astrophysics Data System (ADS)

Bellion, M.; Santen, L.; Mantz, H.; Hähl, H.; Quinn, A.; Nagel, A.; Gilow, C.; Weitenberg, C.; Schmitt, Y.; Jacobs, K.

2008-10-01

Adsorption of proteins onto solid surfaces is an everyday phenomenon that is not yet fully understood. To further the current understanding, we have performed in situ ellipsometry studies to reveal the adsorption kinetics of three different proteins, lysozyme, α-amylase and bovine serum albumin. As substrates we offer Si wafers with a controlled Si oxide layer thickness and a hydrophilic or hydrophobic surface functionalization, allowing the tailoring of the influence of short- and long-range interactions. Our studies show that not only the surface chemistry determines the properties of an adsorbed protein layer but also the van der Waals contributions of a composite substrate. We compare the experimental findings to results of a colloidal Monte Carlo approach that includes conformational changes of the adsorbed proteins induced by density fluctuations.

Plasma immersion ion implantation of polyurethane shape memory polymer: Surface properties and protein immobilization

NASA Astrophysics Data System (ADS)

Cheng, Xinying; Kondyurin, Alexey; Bao, Shisan; Bilek, Marcela M. M.; Ye, Lin

2017-09-01

Polyurethane-type shape memory polymers (SMPU) are promising biomedical implant materials due to their ability to recover to a predetermined shape from a temporary shape induced by thermal activation close to human body temperature and their advantageous mechanical properties including large recovery strains and low recovery stresses. Plasma Immersion Ion Implantation (PIII) is a surface modification process using energetic ions that generates radicals in polymer surfaces leading to carbonisation and oxidation and the ability to covalently immobilise proteins without the need for wet chemistry. Here we show that PIII treatment of SMPU significantly enhances its bioactivity making SMPU suitable for applications in permanent implantable biomedical devices. Scanning Electron Microscopy (SEM), contact angle measurements, surface energy measurements, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to characterise the PIII modified surface, including its after treatment aging kinetics and its capability to covalently immobilise protein directly from solution. The results show a substantial improvement in wettability and dramatic changes of surface chemical composition dependent on treatment duration, due to the generation of radicals and subsequent oxidation. The SMPU surface, PIII treated for 200s, achieved a saturated level of covalently immobilized protein indicating that a full monolayer coverage was achieved. We conclude that PIII is a promising and efficient surface modification method to enhance the biocompatibility of SMPU for use in medical applications that demand bioactivity for tissue integration and stability in vivo.

Probing the surface of a sweet protein: NMR study of MNEI with a paramagnetic probe

PubMed Central

Niccolai, Neri; Spadaccini, Roberta; Scarselli, Maria; Bernini, Andrea; Crescenzi, Orlando; Spiga, Ottavia; Ciutti, Arianna; Di Maro, Daniela; Bracci, Luisa; Dalvit, Claudio; Temussi, Piero A.

2001-01-01

The design of safe sweeteners is very important for people who are affected by diabetes, hyperlipemia, and caries and other diseases that are linked to the consumption of sugars. Sweet proteins, which are found in several tropical plants, are many times sweeter than sucrose on a molar basis. A good understanding of their structure–function relationship can complement traditional SAR studies on small molecular weight sweeteners and thus help in the design of safe sweeteners. However, there is virtually no sequence homology and very little structural similarity among known sweet proteins. Studies on mutants of monellin, the best characterized of sweet proteins, proved not decisive in the localization of the main interaction points of monellin with its receptor. Accordingly, we resorted to an unbiased approach to restrict the search of likely areas of interaction on the surface of a typical sweet protein. It has been recently shown that an accurate survey of the surface of proteins by appropriate paramagnetic probes may locate interaction points on protein surface. Here we report the survey of the surface of MNEI, a single chain monellin, by means of a paramagnetic probe, and a direct assessment of bound water based on an application of ePHOGSY, an NMR experiment that is ideally suited to detect interactions of small ligands to a protein. Detailed surface mapping reveals the presence, on the surface of MNEI, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis. PMID:11468346

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface proteinmore » A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.« less

High throughput atmospheric pressure plasma-induced graft polymerization for identifying protein-resistant surfaces.

PubMed

Gu, Minghao; Kilduff, James E; Belfort, Georges

2012-02-01

Three critical aspects of searching for and understanding how to find highly resistant surfaces to protein adhesion are addressed here with specific application to synthetic membrane filtration. They include the (i) discovery of a series of previously unreported monomers from a large library of monomers with high protein resistance and subsequent low fouling characteristics for membrane ultrafiltration of protein-containing fluids, (ii) development of a new approach to investigate protein-resistant mechanisms from structure-property relationships, and (iii) adaptation of a new surface modification method, called atmospheric pressure plasma-induced graft polymerization (APP), together with a high throughput platform (HTP), for low cost vacuum-free synthesis of anti-fouling membranes. Several new high-performing chemistries comprising two polyethylene glycol (PEG), two amines and one zwitterionic monomers were identified from a library (44 commercial monomers) of five different classes of monomers as strong protein-resistant monomers. Combining our analysis here, using the Hansen solubility parameters (HSP) approach, and data from the literature, we conclude that strong interactions with water (hydrogen bonding) and surface flexibility are necessary for producing the highest protein resistance. Superior protein-resistant surfaces and subsequent anti-fouling performance was obtained with the HTP-APP as compared with our earlier HTP-photo graft-induced polymerization (PGP). Copyright © 2011 Elsevier Ltd. All rights reserved.

Protein Adsorption in Three Dimensions

PubMed Central

Vogler, Erwin A.

2011-01-01

Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly-formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and initially-adsorbed protein. Interphase protein concentration CI increases as VI decreases, resulting in slow reduction in interfacial energetics. Steady-state is governed by a net partition coefficient P=(/CBCI). In the process of occupying space within the interphase, adsorbing protein molecules must displace an equivalent volume of interphase water. Interphase water is itself associated with surface-bound water through a network of transient hydrogen bonds. Displacement of interphase water thus requires an amount of energy that depends on the adsorbent surface chemistry/energy. This “adsorption-dehydration” step is the significant free-energy cost of adsorption that controls the maximum amount of protein that can be adsorbed at steady state to a unit adsorbent-surface area (the adsorbent capacity). As adsorbent hydrophilicity increases, protein adsorption monotonically decreases because the energetic cost of surface dehydration increases, ultimately leading to no protein adsorption near an adsorbent water wettability (surface energy) characterized by a water contact angle θ → 65°. Consequently, protein does not adsorb (accumulate at interphase concentrations greater than bulk solution) to more hydrophilic adsorbents exhibiting θ < 65° . For adsorbents bearing strong Lewis acid/base chemistry such as ion-exchange resins, protein/surface interactions can be highly favorable, causing protein to adsorb in multilayers in a relatively thick interphase. A straightforward, three-component free energy relationship captures salient features of protein adsorption to all surfaces predicting that the overall free energy of protein adsorption ΔGadso is a relatively small multiple of thermal energy for any surface chemistry (except perhaps for bioengineered surfaces bearing specific ligands for adsorbing protein) because a surface chemistry that interacts chemically with proteins must also interact with water through hydrogen bonding. In this way, water moderates protein adsorption to any surface by competing with adsorbing protein molecules. This Leading Opinion ends by proposing several changes to the protein-adsorption paradigm that might advance answers to the three core questions that frame the “protein-adsorption problem” that is so fundamental to biomaterials surface science. PMID:22088888

Mechanisms of protein stabilization and prevention of protein aggregation by glycerol.

PubMed

Vagenende, Vincent; Yap, Miranda G S; Trout, Bernhardt L

2009-11-24

The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Glycerol is known to shift the native protein ensemble to more compact states. Glycerol also inhibits protein aggregation during the refolding of many proteins. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. In this study, we derive mechanisms of glycerol-induced protein stabilization by combining the thermodynamic framework of preferential interactions with molecular-level insight into solvent-protein interactions gained from molecular simulations. Contrary to the common conception that preferential hydration of proteins in polyol/water mixtures is determined by the molecular size of the polyol and the surface area of the protein, we present evidence that preferential hydration of proteins in glycerol/water mixtures mainly originates from electrostatic interactions that induce orientations of glycerol molecules at the protein surface such that glycerol is further excluded. These interactions shift the native protein toward more compact conformations. Moreover, glycerol preferentially interacts with large patches of contiguous hydrophobicity where glycerol acts as an amphiphilic interface between the hydrophobic surface and the polar solvent. Accordingly, we propose that glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol. These mechanisms agree well with experimental data available in the literature, and we discuss the extent to which these mechanisms apply to other cosolvents, including polyols, arginine, and urea.

Rational modification of protein stability by targeting surface sites leads to complicated results

PubMed Central

Xiao, Shifeng; Patsalo, Vadim; Shan, Bing; Bi, Yuan; Green, David F.; Raleigh, Daniel P.

2013-01-01

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions. PMID:23798426

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

PubMed

Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

2018-01-23

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Generation and Surface Localization of Intact M Protein in Streptococcus pyogenes Are Dependent on sagA

PubMed Central

Biswas, Indranil; Germon, Pierre; McDade, Kathleen; Scott, June R.

2001-01-01

The M protein is an important surface-located virulence factor of Streptococcus pyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that the sag locus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription of emm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019–6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation in sagA, the first gene in the sag locus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of the emm gene was unaffected by the sagA mutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in the sagA mutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made the sagA mutant strain susceptible to phagocytosis. Thus, although sagA does not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor. PMID:11598078

Prediction of protein-protein interaction sites using electrostatic desolvation profiles.

PubMed

Fiorucci, Sébastien; Zacharias, Martin

2010-05-19

Protein-protein complex formation involves removal of water from the interface region. Surface regions with a small free energy penalty for water removal or desolvation may correspond to preferred interaction sites. A method to calculate the electrostatic free energy of placing a neutral low-dielectric probe at various protein surface positions has been designed and applied to characterize putative interaction sites. Based on solutions of the finite-difference Poisson equation, this method also includes long-range electrostatic contributions and the protein solvent boundary shape in contrast to accessible-surface-area-based solvation energies. Calculations on a large set of proteins indicate that in many cases (>90%), the known binding site overlaps with one of the six regions of lowest electrostatic desolvation penalty (overlap with the lowest desolvation region for 48% of proteins). Since the onset of electrostatic desolvation occurs even before direct protein-protein contact formation, it may help guide proteins toward the binding region in the final stage of complex formation. It is interesting that the probe desolvation properties associated with residue types were found to depend to some degree on whether the residue was outside of or part of a binding site. The probe desolvation penalty was on average smaller if the residue was part of a binding site compared to other surface locations. Applications to several antigen-antibody complexes demonstrated that the approach might be useful not only to predict protein interaction sites in general but to map potential antigenic epitopes on protein surfaces. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Protein Crystal Growth Apparatus for Microgravity

NASA Technical Reports Server (NTRS)

Carter, Daniel C. (Inventor); Dowling, Timothy E. (Inventor)

1997-01-01

Apparatus for growing protein crystals under microgravity environment includes a plurality of protein growth assemblies stacked one above the other within a canister. Each of the protein growth assemblies includes a tray having a number of spaced apart growth chambers recessed below an upper surface. the growth chambers each having an upstanding pedestal and an annular reservoir about the pedestal for receiving a wick and precipitating agents. A well is recessed below the top of each pedestal to define a protein crystal growth receptacle. A flexible membrane is positioned on the upper surface of each tray and a sealing plate is positioned above each membrane, each sealing plate having a number of bumpers corresponding in number and alignment to the pedestals for forcing the membrane selectively against the upper end of the respective pedestal to seal the reservoir and the receptacle when the sealing plate is forced down.

Nanoscale Dewetting Transition in Protein Complex Folding

PubMed Central

Hua, Lan; Huang, Xuhui; Liu, Pu; Zhou, Ruhong; Berne, Bruce J.

2011-01-01

In a previous study, a surprising drying transition was observed to take place inside the nanoscale hydrophobic channel in the tetramer of the protein melittin. The goal of this paper is to determine if there are other protein complexes capable of displaying a dewetting transition during their final stage of folding. We searched the entire protein data bank (PDB) for all possible candidates, including protein tetramers, dimers, and two-domain proteins, and then performed the molecular dynamics (MD) simulations on the top candidates identified by a simple hydrophobic scoring function based on aligned hydrophobic surface areas. Our large scale MD simulations found several more proteins, including three tetramers, six dimers, and two two-domain proteins, which display a nanoscale dewetting transition in their final stage of folding. Even though the scoring function alone is not sufficient (i.e., a high score is necessary but not sufficient) in identifying the dewetting candidates, it does provide useful insights into the features of complex interfaces needed for dewetting. All top candidates have two features in common: (1) large aligned (matched) hydrophobic areas between two corresponding surfaces, and (2) large connected hydrophobic areas on the same surface. We have also studied the effect on dewetting of different water models and different treatments of the long-range electrostatic interactions (cutoff vs PME), and found the dewetting phenomena is fairly robust. This work presents a few proteins other than melittin tetramer for further experimental studies of the role of dewetting in the end stages of protein folding. PMID:17608515

IMMUNOBLOT ANALYSIS OF PROTEINS ASSOCIATED WITH SELF-ASSEMBLED MONOLAYER SURFACES OF DEFINED CHEMISTRIES

PubMed Central

Cornelius, Rena M.; Shankar, Sucharita P.; Brash, John L.; Babensee, Julia E.

2011-01-01

Intact and fragmented proteins, eluted from self assembled monolayer (SAM) surfaces of alkanethiols of different chemistries (-CH3, -OH, -COOH, -NH2 ), following exposure to human plasma (HP) or human serum (HS), were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques. The SAM surfaces were incubated for 1 hour with 10% (v/v) sterile-filtered heat-inactivated (h.i.) HS or 1% (v/v) sterile-filtered h.i. HP preparations [both in phosphate buffered saline (PBS)]. Adsorbed proteins were eluted using 10% SDS/2.3% dithioerythritol for characterization of protein profiles. The type of incubating medium may be an important determinant of adsorbed protein profiles, since some variations were observed in eluates from filtered versus control unfiltered h.i. 10% HS or 1% HP. Albumin and apolipoprotein A1 were consistently detected in both filtered h.i 10% HS and 1% HP eluates from all SAM surfaces and from control tissue culture-treated polystyrene (TCPS). Interestingly, Factor H and Factor I, antithrombin, prothrombin, high molecular weight kininogen (HMWK) and IgG were present in eluates from OH, COOH and NH2 SAM surfaces and in eluates from TCPS, but not in eluates from CH3 SAM surfaces, following exposure to filtered h.i. 10% HS. These results suggest that CH3 SAM surfaces were the least pro-inflammatory of all SAM surfaces. Overall, similar trends were observed in the profiles of proteins eluted from surfaces exposed to filtered 10% HS or 1% HP. However the unique profiles of adsorbed proteins on different SAM surface chemistries may be related to their differential interactions with cells, including immune/inflammatory cells. PMID:21509932

Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface*

PubMed Central

Trevino, R. Sean; Lauckner, Jane E.; Sourigues, Yannick; Pearce, Margaret M.; Bousset, Luc; Melki, Ronald; Kopito, Ron R.

2012-01-01

The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces. PMID:22753412

Proteins feel more than they see: fine-tuning of binding affinity by properties of the non-interacting surface.

PubMed

Kastritis, Panagiotis L; Rodrigues, João P G L M; Folkers, Gert E; Boelens, Rolf; Bonvin, Alexandre M J J

2014-07-15

Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct contributions from the interface, the non-interacting surface (NIS) also plays an important role in binding affinity, especially polar and charged residues. Their percentage on the NIS is conserved over orthologous complexes indicating an evolutionary selection pressure. Their effect on binding affinity can be explained by long-range electrostatic contributions and surface-solvent interactions that are known to determine the local frustration of the protein complex surface. Including these in a simple model significantly improves the affinity prediction of protein complexes from structural models. The impact of mutations outside the interacting surface on binding affinity is supported by experimental alanine scanning mutagenesis data. These results enable the development of more sophisticated and integrated biophysical models of binding affinity and open new directions in experimental control and modulation of biomolecular interactions. Copyright © 2014. Published by Elsevier Ltd.

'2TM proteins': an antigenically diverse superfamily with variable functions and export pathways.

PubMed

Kaur, Jasweer; Hora, Rachna

2018-01-01

Malaria is a disease that affects millions of people annually. An intracellular habitat and lack of protein synthesizing machinery in erythrocytes pose numerous difficulties for survival of the human pathogen Plasmodium falciparum . The parasite refurbishes the infected red blood cell (iRBC) by synthesis and export of several proteins in an attempt to suffice its metabolic needs and evade the host immune response. Immune evasion is largely mediated by surface display of highly polymorphic protein families known as variable surface antigens. These include the two trans-membrane (2TM) superfamily constituted by multicopy repetitive interspersed family (RIFINs), subtelomeric variable open reading frame (STEVORs) and Plasmodium falciparum Maurer's cleft two trans-membrane proteins present only in P. falciparum and some simian infecting Plasmodium species. Their hypervariable region flanked by 2TM domains exposed on the iRBC surface is believed to generate antigenic diversity. Though historically named "2TM superfamily," several A-type RIFINs and some STEVORs assume one trans-membrane topology. RIFINs and STEVORs share varied functions in different parasite life cycle stages like rosetting, alteration of iRBC rigidity and immune evasion. Additionally, a member of the STEVOR family has been implicated in merozoite invasion. Differential expression of these families in laboratory strains and clinical isolates propose them to be important for host cell survival and defense. The role of RIFINs in modulation of host immune response and presence of protective antibodies against these surface exposed molecules in patient sera highlights them as attractive targets of antimalarial therapies and vaccines. 2TM proteins are Plasmodium export elements positive, and several of these are exported to the infected erythrocyte surface after exiting through the classical secretory pathway within parasites. Cleaved and modified proteins are trafficked after packaging in vesicles to reach Maurer's clefts, while information regarding delivery to the iRBC surface is sparse. Expression and export timing of the RIFIN and Plasmodium falciparum erythrocyte membrane protein1 families correspond to each other. Here, we have compiled and comprehended detailed information regarding orthologues, domain architecture, surface topology, functions and trafficking of members of the "2TM superfamily." Considering the large repertoire of proteins included in the 2TM superfamily and recent advances defining their function in malaria biology, a surge in research carried out on this important protein superfamily is likely.

Expansion of divergent SEA domains in cell surface proteins and nucleoporin 54.

PubMed

Pei, Jimin; Grishin, Nick V

2017-03-01

SEA (sea urchin sperm protein, enterokinase, agrin) domains, many of which possess autoproteolysis activity, have been found in a number of cell surface and secreted proteins. Despite high sequence divergence, SEA domains were also proposed to be present in dystroglycan based on a conserved autoproteolysis motif and receptor-type protein phosphatase IA-2 based on structural similarity. The presence of a SEA domain adjacent to the transmembrane segment appears to be a recurring theme in quite a number of type I transmembrane proteins on the cell surface, such as MUC1, dystroglycan, IA-2, and Notch receptors. By comparative sequence and structural analyses, we identified dystroglycan-like proteins with SEA domains in Capsaspora owczarzaki of the Filasterea group, one of the closest single-cell relatives of metazoans. We also detected novel and divergent SEA domains in a variety of cell surface proteins such as EpCAM, α/ε-sarcoglycan, PTPRR, collectrin/Tmem27, amnionless, CD34, KIAA0319, fibrocystin-like protein, and a number of cadherins. While these proteins are mostly from metazoans or their single cell relatives such as choanoflagellates and Filasterea, fibrocystin-like proteins with SEA domains were found in several other eukaryotic lineages including green algae, Alveolata, Euglenozoa, and Haptophyta, suggesting an ancient evolutionary origin. In addition, the intracellular protein Nucleoporin 54 (Nup54) acquired a divergent SEA domain in choanoflagellates and metazoans. © 2016 The Protein Society.

Glycobiology of the ocular surface: Mucins and lectins

PubMed Central

Argüeso, Pablo

2013-01-01

Glycosylation is an important and common form of posttranscriptional modification of proteins in cells. A vast array of biological functions has been ascribed to glycans during the last decade thanks to a rapid evolution in glycomic technologies. Glycogenes highly expressed at the human ocular surface include families of glycosyltransferases, proteoglycans, glycan degradation proteins, as well as mucins and carbohydrate-binding proteins such as the galectins. On the apical glycocalyx, mucin O-glycans promote boundary lubrication, prevent bacterial adhesion and endocytic activity, and maintain epithelial barrier function through interactions with galectins. The emerging roles attributed to glycans are contributing to the appreciation of their biological capabilities at the ocular surface. PMID:23325272

Protein immobilization on the surface of polydimethylsiloxane and polymethyl methacrylate microfluidic devices.

PubMed

Khnouf, Ruba; Karasneh, Dina; Albiss, Borhan Aldeen

2016-02-01

PDMS and PMMA are two of the most used polymers in the fabrication of lab-on-chip or microfluidic devices. In order to use these polymers in biological applications, it is sometimes essential to be able to bind biomolecules such as proteins and DNA to the surface of these materials. In this work, we have evaluated a number of processes that have been developed to bind protein to PDMS surfaces which include passive adsorption, passive adsorption with glutaraldehyde cross-linking, (3-aminopropyl) triethoxysilane functionalization followed by glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride cross-linkers. It has been shown that the latter technique--using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride--results in more than twice the bonding of protein to the surface of PDMS microchannels than proteins binding passively. We have also evaluated a few techniques that have been tested for the functionalization of PMMA microchannels where we have found that the use of polyethyleneimine (PEI) has led to the strongest protein-PMMA microchannel bond. We finally demonstrated the effect of PDMS curing methodology on protein adsorption to its surface, and showed that increased curing time is the factor that reduces passive adsorption the most. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Unique surface adsorption behaviors of serum proteins on chemically uniform and alternating surfaces

NASA Astrophysics Data System (ADS)

Song, Sheng

With increasing interests of studying proteins adsorption on the surfaces with nanoscale features in biomedical field, it is crucial to have fundamental understandings on how the proteins are adsorbed on such a surface and what factors contribute to the driving forces of adsorption. Besides, exploring more available nanoscale templates would greatly offer more possibilities one could design surface bio-detection methods with favorable protein-surface interactions. Thus, to fulfill the purpose, the work in this dissertation has been made into three major sections. First, to probe the intermediate states which possibly exist between stable and unstable phases described in mean-field theory diagram, a solvent vapor annealing method is chosen to slowly induce the copolymer polystyrene-block-polyvinylpyridine (PS-b-PVP)'s both blocks undergoing micro-phase separations from initial spherical nanodomains into terminal cylindrical nanodomains. During this process, real time atomic force microscopy (AFM) has been conducted to capture other six intermediate states with different morphologies on the polymeric film surfaces. Secondly, upon recognizing each intermediate state, the solution of immunoglobulin gamma (IgG) proteins has been deposited on the surface and been rinsed off with buffer solution before the protein-bounded surface is imaged by AFM. It has been found IgG showing a strong adsorption preference on PS over P4VP block. Among all the six intermediate states, the proteins are almost exclusively adsorbed on PS nanodomains regardless the concentration and deposition time. Thirdly, a trinodular shape protein fibrinogen (Fg) is selected for investigating how geometry and surface charge of proteins would interplay with cylindrical nanodomains on a surface developed from Polystyrene -block-Poly-(methyl methacrylate) PS-b-PMMA. Also, Fg adsorptions on chemically homogeneous surfaces are included here to have a better contrast of showing how much difference it can make by using it on a nanoscale surface. Interestingly, higher concentration of protein solution promotes the occurrences of single phase packed Fg on the PS domain. The densely packed network has formed where each Fg keeps its main body in PS domain and leaves its two alpha C chains on nearby PMMA domain. We believe this conformation and orientation would maximize both the hydrophobic and electrostatic interactions between Fg and the underlying surface.

Mechanism for the antibacterial action of epigallocatechin gallate (EGCg) on Bacillus subtilis.

PubMed

Nakayama, Motokazu; Shimatani, Kanami; Ozawa, Tadahiro; Shigemune, Naofumi; Tomiyama, Daisuke; Yui, Koji; Katsuki, Mao; Ikeda, Keisuke; Nonaka, Ai; Miyamoto, Takahisa

2015-01-01

Catechins are a class of polyphenols and have high anti-bacterial activity against various microorganisms. Here, we report the mechanism for antibacterial activity of epigallocatechin gallate (EGCg) against Gram-positive bacteria Bacillus subtilis, which is highly sensitive to EGCg. Transmission electron microscope analysis revealed that deposits containing EGCg were found throughout the cell envelope from the outermost surface to the outer surface of cytoplasmic membrane. Aggregating forms of proteins and EGCg were identified as spots that disappeared or showed markedly decreased intensity after the treatment with EGCg compared to the control by two-dimensional electrophoresis. Among the identified proteins included 4 cell surface proteins, such as oligopeptide ABC transporter binding lipoprotein, glucose phosphotransferase system transporter protein, phosphate ABC transporter substrate-binding protein, and penicillin-binding protein 5. Observations of glucose uptake of cells and cell shape B. subtilis after the treatment with EGCg suggested that EGCg inhibits the major functions of these proteins, leading to growth inhibition of B. subtilis.

Prion protein induced signaling cascades in monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krebs, Bjarne; Dorner-Ciossek, Cornelia; Schmalzbauer, Ruediger

2006-02-03

Prion proteins play a central role in transmission and pathogenesis of transmissible spongiform encephalopathies. The cellular prion protein (PrP{sup C}), whose physiological function remains elusive, is anchored to the surface of a variety of cell types including neurons and cells of the lymphoreticular system. In this study, we investigated the response of a mouse monocyte/macrophage cell line to exposure with PrP{sup C} fusion proteins synthesized with a human Fc-tag. PrP{sup C} fusion proteins showed an attachment to the surface of monocyte/macrophages in nanomolar concentrations. This was accompanied by an increase of cellular tyrosine phosphorylation as a result of activated signalingmore » pathways. Detailed investigations exhibited activation of downstream pathways through a stimulation with PrP fusion proteins, which include phosphorylation of ERK{sub 1,2} and Akt kinase. Macrophages opsonize and present antigenic structures, contact lymphocytes, and deliver cytokines. The findings reported here may become the basis of understanding the molecular function of PrP{sup C} in monocytes and macrophages.« less

Functional cell-surface display of a lipase-specific chaperone.

PubMed

Wilhelm, Susanne; Rosenau, Frank; Becker, Stefan; Buest, Sebastian; Hausmann, Sascha; Kolmar, Harald; Jaeger, Karl-Erich

2007-01-02

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

Involvement of neutral endopeptidase in neoplastic progression.

PubMed

Sumitomo, Makoto; Shen, Ruoqian; Nanus, David M

2005-08-01

Neutral endopeptidase 24.11 (NEP) is a 90-110 kDa cell surface cell surface peptidase that is normally expressed by numerous tissues, including prostate, kidney, intestine, endometrium, adrenal glands and lung. This enzyme cleaves peptide bonds on the amino side of hydrophobic amino acids and inactivates a variety of physiologically active peptides, including atrial natriuretic factor, substance P, bradykinin, oxytocin, Leu- and Met-enkephalins, neurotensin, bombesin, endothelin-1, and bombesin-like peptides. NEP reduces the local concentration of peptide available for receptor binding and signal transduction. Loss or decreases in NEP expression have been reported in a variety of malignancies. Reduced NEP may promote peptide-mediated proliferation by allowing accumulation of higher peptide concentrations at the cell surface, and facilitate the development or progression of neoplasia. We have used prostate cancer as model in which to study the involvement of NEP in malignancy. Using a variety of experimental approaches, including recombinant NEP, cell lines expressing wild-type and mutant NEP protein, and cell lines expressing NEP protein with a mutated cytoplasmic domain, we have examined the effects of NEP on cell migration and cell survival. We have shown that the effects of NEP are mediated by its ability to catalytically inactivate substrates such as bombesin and endothelin-1, but also through direct protein-protein interaction with other protein such as Lyn kinase [which associates with the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) resulting in NEP-Lyn-PI3-K protein complex], ezrin/radixin/moesin (ERM) proteins, and the PTEN tumor suppressor protein. We review the mechanisms of NEP's tumor suppressive action and how NEP loss contributes to tumor progression.

Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA

NASA Astrophysics Data System (ADS)

Ziach, Krzysztof; Chollet, Céline; Parissi, Vincent; Prabhakaran, Panchami; Marchivie, Mathieu; Corvaglia, Valentina; Bose, Partha Pratim; Laxmi-Reddy, Katta; Godde, Frédéric; Schmitter, Jean-Marie; Chaignepain, Stéphane; Pourquier, Philippe; Huc, Ivan

2018-05-01

Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein-DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer-protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein-DNA interactions.

Protein sequences bound to mineral surfaces persist into deep time

PubMed Central

Demarchi, Beatrice; Hall, Shaun; Roncal-Herrero, Teresa; Freeman, Colin L; Woolley, Jos; Crisp, Molly K; Wilson, Julie; Fotakis, Anna; Fischer, Roman; Kessler, Benedikt M; Rakownikow Jersie-Christensen, Rosa; Olsen, Jesper V; Haile, James; Thomas, Jessica; Marean, Curtis W; Parkington, John; Presslee, Samantha; Lee-Thorp, Julia; Ditchfield, Peter; Hamilton, Jacqueline F; Ward, Martyn W; Wang, Chunting Michelle; Shaw, Marvin D; Harrison, Terry; Domínguez-Rodrigo, Manuel; MacPhee, Ross DE; Kwekason, Amandus; Ecker, Michaela; Kolska Horwitz, Liora; Chazan, Michael; Kröger, Roland; Thomas-Oates, Jane; Harding, John H; Cappellini, Enrico; Penkman, Kirsty; Collins, Matthew J

2016-01-01

Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C). DOI: http://dx.doi.org/10.7554/eLife.17092.001 PMID:27668515

Implications of protein- and Peptide-based nanoparticles as potential vehicles for anticancer drugs.

PubMed

Elzoghby, Ahmed O; Elgohary, Mayada M; Kamel, Nayra M

2015-01-01

Protein-based nanocarriers have gained considerable attention as colloidal carrier systems for the delivery of anticancer drugs. Protein nanocarriers possess various advantages including their low cytotoxicity, abundant renewable sources, high drug-binding capacity, and significant uptake into the targeted tumor cells. Moreover, the unique protein structure offers the possibility of site-specific drug conjugation and tumor targeting using various ligands modifying the surface of protein nanocarriers. In this chapter, we highlight the most important applications of protein nanoparticles (NPs) for the delivery of anticancer drugs. We examine the various techniques that have been utilized for the preparation of anticancer drug-loaded protein NPs. Finally, the current chapter also reviews the major outcomes of the in vitro and in vivo investigations of surface-modified tumor-targeted protein NPs. © 2015 Elsevier Inc. All rights reserved.

Surface-independent antibacterial coating using silver nanoparticle-generating engineered mussel glue.

PubMed

Jo, Yun Kee; Seo, Jeong Hyun; Choi, Bong-Hyuk; Kim, Bum Jin; Shin, Hwa Hui; Hwang, Byeong Hee; Cha, Hyung Joon

2014-11-26

During implant surgeries, antibacterial agents are needed to prevent bacterial infections, which can cause the formation of biofilms between implanted materials and tissue. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on various surfaces even in wet environment. Here, we proposed a novel surface-independent antibacterial coating strategy based on the fusion of MAP to a silver-binding peptide, which can synthesize silver nanoparticles having broad antibacterial activity. This sticky recombinant fusion protein enabled the efficient coating on target surface and the easy generation of silver nanoparticles on the coated-surface under mild condition. The biosynthesized silver nanoparticles showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria and also revealed good cytocompatibility with mammalian cells. In this coating strategy, MAP-silver binding peptide fusion proteins provide hybrid environment incorporating inorganic silver nanoparticle and simultaneously mediate the interaction of silver nanoparticle with surroundings. Moreover, the silver nanoparticles were fully synthesized on various surfaces including metal, plastic, and glass by a simple, surface-independent coating manner, and they were also successfully synthesized on a nanofiber surface fabricated by electrospinning of the fusion protein. Thus, this facile surface-independent silver nanoparticle-generating antibacterial coating has great potential to be used for the prevention of bacterial infection in diverse biomedical fields.

Quantitative protein expression and cell surface characteristics of Escherichia coli MG1655 biofilms.

PubMed

Mukherjee, Joy; Ow, Saw Yen; Noirel, Josselin; Biggs, Catherine A

2011-02-01

Cell surface physicochemical characterization techniques were combined with quantitative changes in protein expression, to investigate the biological and biophysical changes of Escherichia coli MG1655 cells when grown as a biofilm (BIO). The overall surface charge of BIO cells was found to be less negative, highlighting the need for a lower electrophoretic mobility for attachment to occur. Comparison of the chemical functional groups on the cell surface showed similar profiles, with the absorbance intensity higher for proteins and carbohydrates in the BIO cells. Quantitative proteomic analysis demonstrated that 3 proteins were significantly increased, and 9 proteins significantly decreased in abundance, in cells grown as a BIO compared to their planktonic counterparts, with 7 of these total 12 proteins unique to this study. Proteins showing significant increased or decreased abundance include proteins involved in acid resistance, DNA protection and binding and ABC transporters. Further predictive analysis of the metabolic pathways showed an increased abundance of the amino acid metabolism and tricarboxylic acid (TCA) cycle, with a decrease in expression within the pentose phosphate and glycolysis pathways. It is therefore hypothesized that cells grown as a BIO are still energetically viable potentially using amino acids as an indirect carbon backbone source into the TCA cycle. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Neutrophil cell surface receptors and their intracellular signal transduction pathways☆

PubMed Central

Futosi, Krisztina; Fodor, Szabina; Mócsai, Attila

2013-01-01

Neutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surface receptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surface receptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2 + signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases. PMID:23994464

Proteogenomic analysis of the total and surface-exposed proteomes of Plasmodium vivax salivary gland sporozoites

PubMed Central

Swearingen, Kristian E.; Lindner, Scott E.; Flannery, Erika L.; Vaughan, Ashley M.; Morrison, Robert D.; Patrapuvich, Rapatbhorn; Koepfli, Cristian; Muller, Ivo; Jex, Aaron; Moritz, Robert L.; Kappe, Stefan H. I.; Sattabongkot, Jetsumon

2017-01-01

Plasmodium falciparum and Plasmodium vivax cause the majority of human malaria cases. Research efforts predominantly focus on P. falciparum because of the clinical severity of infection and associated mortality rates. However, P. vivax malaria affects more people in a wider global range. Furthermore, unlike P. falciparum, P. vivax can persist in the liver as dormant hypnozoites that can be activated weeks to years after primary infection, causing relapse of symptomatic blood stages. This feature makes P. vivax unique and difficult to eliminate with the standard tools of vector control and treatment of symptomatic blood stage infection with antimalarial drugs. Infection by Plasmodium is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver. The most advanced malaria vaccine for P. falciparum (RTS,S, a subunit vaccine containing of a portion of the major sporozoite surface protein) conferred limited protection in Phase III trials, falling short of WHO-established vaccine efficacy goals. However, blocking the sporozoite stage of infection in P. vivax, before the establishment of the chronic liver infection, might be an effective malaria vaccine strategy to reduce the occurrence of relapsing blood stages. It is also thought that a multivalent vaccine comprising multiple sporozoite surface antigens will provide better protection, but a comprehensive analysis of proteins in P. vivax sporozoites is not available. To inform sporozoite-based vaccine development, we employed mass spectrometry-based proteomics to identify nearly 2,000 proteins present in P. vivax salivary gland sporozoites. Analysis of protein post-translational modifications revealed extensive phosphorylation of glideosome proteins as well as regulators of transcription and translation. Additionally, the sporozoite surface proteins CSP and TRAP, which were recently discovered to be glycosylated in P. falciparum salivary gland sporozoites, were also observed to be similarly modified in P. vivax sporozoites. Quantitative comparison of the P. vivax and P. falciparum salivary gland sporozoite proteomes revealed a high degree of similarity in protein expression levels, including among invasion-related proteins. Nevertheless, orthologs with significantly different expression levels between the two species could be identified, as well as highly abundant, species-specific proteins with no known orthologs. Finally, we employed chemical labeling of live sporozoites to isolate and identify 36 proteins that are putatively surface-exposed on P. vivax salivary gland sporozoites. In addition to identifying conserved sporozoite surface proteins identified by similar analyses of other Plasmodium species, our analysis identified several as-yet uncharacterized proteins, including a putative 6-Cys protein with no known ortholog in P. falciparum. PMID:28759593

Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics

PubMed Central

Autelitano, François; Loyaux, Denis; Roudières, Sébastien; Déon, Catherine; Guette, Frédérique; Fabre, Philippe; Ping, Qinggong; Wang, Su; Auvergne, Romane; Badarinarayana, Vasudeo; Smith, Michael; Guillemot, Jean-Claude; Goldman, Steven A.; Natesan, Sridaran; Ferrara, Pascual; August, Paul

2014-01-01

Glioblastoma multiform (GBM) remains clinical indication with significant “unmet medical need”. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells. PMID:25360666

Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

PubMed

Autelitano, François; Loyaux, Denis; Roudières, Sébastien; Déon, Catherine; Guette, Frédérique; Fabre, Philippe; Ping, Qinggong; Wang, Su; Auvergne, Romane; Badarinarayana, Vasudeo; Smith, Michael; Guillemot, Jean-Claude; Goldman, Steven A; Natesan, Sridaran; Ferrara, Pascual; August, Paul

2014-01-01

Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Mechanisms for Covalent Immobilization of Horseradish Peroxidase on Ion-Beam-Treated Polyethylene

PubMed Central

Kondyurin, Alexey V.; Naseri, Pourandokht; Tilley, Jennifer M. R.; Nosworthy, Neil J.; Bilek, Marcela M. M.; McKenzie, David R.

2012-01-01

The surface of polyethylene was modified by plasma immersion ion implantation. Structure changes including carbonization and oxidation were observed. High surface energy of the modified polyethylene was attributed to the presence of free radicals on the surface. The surface energy decay with storage time after treatment was explained by a decay of the free radical concentration while the concentration of oxygen-containing groups increased with storage time. Horseradish peroxidase was covalently attached onto the modified surface by the reaction with free radicals. Appropriate blocking agents can block this reaction. All aminoacid residues can take part in the covalent attachment process, providing a universal mechanism of attachment for all proteins. The native conformation of attached protein is retained due to hydrophilic interactions in the interface region. The enzymatic activity of covalently attached protein remained high. The long-term activity of the modified layer to attach protein is explained by stabilisation of unpaired electrons in sp2 carbon structures. A high concentration of free radicals can give multiple covalent bonds to the protein molecule and destroy the native conformation and with it the catalytic activity. The universal mechanism of protein attachment to free radicals could be extended to various methods of radiation damage of polymers. PMID:24278665

Detecting Local Ligand-Binding Site Similarity in Non-Homologous Proteins by Surface Patch Comparison

PubMed Central

Sael, Lee; Kihara, Daisuke

2012-01-01

Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. PMID:22275074

Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison.

PubMed

Sael, Lee; Kihara, Daisuke

2012-04-01

Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. Copyright © 2011 Wiley Periodicals, Inc.

Fibrinogen adsorption on blocked surface of albumin.

PubMed

Holmberg, Maria; Hou, Xiaolin

2011-05-01

We have investigated the adsorption of albumin and fibrinogen onto PET (polyethylene terephthalate) and glass surfaces and how pre-adsorption of albumin onto these surfaces can affect the adsorption of later added fibrinogen. For materials and devices being exposed to blood, adsorption of fibrinogen is often a non-wanted event, since fibrinogen is part of the clotting cascade and unspecific adsorption of fibrinogen can have an influence on the activation of platelets. Albumin is often used as blocking agent for avoiding unspecific protein adsorption onto surfaces in devices designed to handle biological samples, including protein solutions. It is based on the assumption that proteins adsorbs as a monolayer on surfaces and that proteins do not adsorb on top of each other. By labelling albumin and fibrinogen with two different radioactive iodine isotopes that emit gamma radiation with different energies, the adsorption of both albumin and fibrinogen has been monitored simultaneously on the same sample. Information about topography and coverage of adsorbed protein layers has been obtained using AFM (Atomic Force Microscopy) analysis in liquid. Our studies show that albumin adsorbs in a multilayer fashion on PET and that fibrinogen adsorbs on top of albumin when albumin is pre-adsorbed on the surfaces. Copyright © 2010 Elsevier B.V. All rights reserved.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

PubMed

Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display

PubMed Central

Gagic, Dragana; Wen, Wesley; Collett, Michael A; Rakonjac, Jasna

2013-01-01

Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-“docking” protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli. PMID:23233310

Characterization of protein hydration by solution NMR spectroscopy

NASA Astrophysics Data System (ADS)

Wand, Joshua

A comprehensive understanding of the interactions between protein molecules and hydration water remains elusive. Solution nuclear magnetic resonance (NMR) spectroscopy has been proposed as a means to characterize these interactions but is plagued with artifacts when employed in bulk aqueous solution. Encapsulation of proteins in reverse micelles prepared in short chain alkane solvents can overcome these technical limitations. Application of this approach has revealed that the interaction of water with the surface of protein molecules is quite heterogeneous with some regions of the protein having long-lived interactions while other regions show relatively transient hydration. Results from several proteins will be presented including ubiquitin, staphylococcal nuclease, interleukin 1beta, hen egg white lysozyme (HEWL) and T4 lysozyme. Ubiquitin and interleukin 1beta are signaling proteins and interact with other proteins through formation of dry protein-protein interfaces. Interestingly, the protein surfaces of the free proteins show relatively slowed (restricted) motion at the surface, which is indicative of low residual entropy. Other regions of the protein surface have relatively high mobility water. These results are consistent with the idea that proteins have evolved to maximize the hydrophobic effect in optimization of binding with protein partners. As predicted by simulation and theory, we find that hydration of internal hydrophobic cavities of interleukin 1beta and T4 lysozyme is highly disfavored. In contrast, the hydrophilic polar cavity of HEWL is occupied by water. Initial structural correlations suggest that hydration of alpha helical structure is characterized by relatively mobile water while those of beta strands and loops are more ordered and slowed. These and other results from this set of proteins reveals that the dynamical and structural character of hydration of proteins is heterogeneous and complex. Supported by the National Science Foundation.

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

PubMed

Hovingh, Elise S; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion

PubMed Central

Hovingh, Elise S.; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed. PMID:28066340

The Human Cell Surfaceome of Breast Tumors

PubMed Central

da Cunha, Júlia Pinheiro Chagas; Galante, Pedro Alexandre Favoretto; de Souza, Jorge Estefano Santana; Pieprzyk, Martin; Carraro, Dirce Maria; Old, Lloyd J.; Camargo, Anamaria Aranha; de Souza, Sandro José

2013-01-01

Introduction. Cell surface proteins are ideal targets for cancer therapy and diagnosis. We have identified a set of more than 3700 genes that code for transmembrane proteins believed to be at human cell surface. Methods. We used a high-throuput qPCR system for the analysis of 573 cell surface protein-coding genes in 12 primary breast tumors, 8 breast cell lines, and 21 normal human tissues including breast. To better understand the role of these genes in breast tumors, we used a series of bioinformatics strategies to integrates different type, of the datasets, such as KEGG, protein-protein interaction databases, ONCOMINE, and data from, literature. Results. We found that at least 77 genes are overexpressed in breast primary tumors while at least 2 of them have also a restricted expression pattern in normal tissues. We found common signaling pathways that may be regulated in breast tumors through the overexpression of these cell surface protein-coding genes. Furthermore, a comparison was made between the genes found in this report and other genes associated with features clinically relevant for breast tumorigenesis. Conclusions. The expression profiling generated in this study, together with an integrative bioinformatics analysis, allowed us to identify putative targets for breast tumors. PMID:24195083

Plasmonic Thermal Decomposition/Digestion of Proteins: A Rapid On-Surface Protein Digestion Technique for Mass Spectrometry Imaging.

PubMed

Zhou, Rong; Basile, Franco

2017-09-05

A method based on plasmon surface resonance absorption and heating was developed to perform a rapid on-surface protein thermal decomposition and digestion suitable for imaging mass spectrometry (MS) and/or profiling. This photothermal process or plasmonic thermal decomposition/digestion (plasmonic-TDD) method incorporates a continuous wave (CW) laser excitation and gold nanoparticles (Au-NPs) to induce known thermal decomposition reactions that cleave peptides and proteins specifically at the C-terminus of aspartic acid and at the N-terminus of cysteine. These thermal decomposition reactions are induced by heating a solid protein sample to temperatures between 200 and 270 °C for a short period of time (10-50 s per 200 μm segment) and are reagentless and solventless, and thus are devoid of sample product delocalization. In the plasmonic-TDD setup the sample is coated with Au-NPs and irradiated with 532 nm laser radiation to induce thermoplasmonic heating and bring about site-specific thermal decomposition on solid peptide/protein samples. In this manner the Au-NPs act as nanoheaters that result in a highly localized thermal decomposition and digestion of the protein sample that is independent of the absorption properties of the protein, making the method universally applicable to all types of proteinaceous samples (e.g., tissues or protein arrays). Several experimental variables were optimized to maximize product yield, and they include heating time, laser intensity, size of Au-NPs, and surface coverage of Au-NPs. Using optimized parameters, proof-of-principle experiments confirmed the ability of the plasmonic-TDD method to induce both C-cleavage and D-cleavage on several peptide standards and the protein lysozyme by detecting their thermal decomposition products with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The high spatial specificity of the plasmonic-TDD method was demonstrated by using a mask to digest designated sections of the sample surface with the heating laser and MALDI-MS imaging to map the resulting products. The solventless nature of the plasmonic-TDD method enabled the nonenzymatic on-surface digestion of proteins to proceed with undetectable delocalization of the resulting products from their precursor protein location. The advantages of this novel plasmonic-TDD method include short reaction times (<30 s/200 μm), compatibility with MALDI, universal sample compatibility, high spatial specificity, and localization of the digestion products. These advantages point to potential applications of this method for on-tissue protein digestion and MS-imaging/profiling for the identification of proteins, high-fidelity MS imaging of high molecular weight (>30 kDa) proteins, and the rapid analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples.

Deciphering Cryptic Binding Sites on Proteins by Mixed-Solvent Molecular Dynamics.

PubMed

Kimura, S Roy; Hu, Hai Peng; Ruvinsky, Anatoly M; Sherman, Woody; Favia, Angelo D

2017-06-26

In recent years, molecular dynamics simulations of proteins in explicit mixed solvents have been applied to various problems in protein biophysics and drug discovery, including protein folding, protein surface characterization, fragment screening, allostery, and druggability assessment. In this study, we perform a systematic study on how mixtures of organic solvent probes in water can reveal cryptic ligand binding pockets that are not evident in crystal structures of apo proteins. We examine a diverse set of eight PDB proteins that show pocket opening induced by ligand binding and investigate whether solvent MD simulations on the apo structures can induce the binding site observed in the holo structures. The cosolvent simulations were found to induce conformational changes on the protein surface, which were characterized and compared with the holo structures. Analyses of the biological systems, choice of probes and concentrations, druggability of the resulting induced pockets, and application to drug discovery are discussed here.

Outer Membrane Targeting of Passenger Proteins by the Vacuolating Cytotoxin Autotransporter of Helicobacter pylori

PubMed Central

Fischer, Wolfgang; Buhrdorf, Renate; Gerland, Elke; Haas, Rainer

2001-01-01

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. These proteins are supposed to integrate into the outer membrane by β-barrel structures, characteristic of the family of autotransporter proteins. By using the SOMPES (shuttle vector-based outer membrane protein expression) system for outer membrane protein production, we were able to functionally express in H. pylori the cholera toxin B subunit genetically fused to the C-terminal VacA domain. We demonstrate that the fusion protein is translocated to the H. pylori outer membrane and that the CtxB domain is exposed on the H. pylori surface. Thus, we provide the first experimental evidence that the C-terminal β-domain of VacA can transport a foreign passenger protein to the H. pylori surface and hence acts as a functional autotransporter. PMID:11598049

Biomimetic surface coatings from modular amphiphilic proteins

NASA Astrophysics Data System (ADS)

Harden, James; Wan, Fan; Fischer, Stephen; Dick, Scott

2010-03-01

Recombinant DNA methods have been used to develop a library of diblock protein polymers for creating designer biofunctional interfaces. These proteins are composed of a surface-active, amphiphilic block joined to a disordered, water soluble block with an end terminal bioactive domain. The amphiphilic block has a strong affinity for many synthetic polymer surfaces, providing a facile means of imparting biological functionality to otherwise bio-neutral materials through physical self-assembly. We have incorporated a series of bioactive end domains into this diblock motif, including sequences that encode specific cell binding and signaling functions of extracellular matrix constituents (e.g. RGD and YIGSR). In this talk, we show that these diblock constructs self-assemble into biofunctional surface coatings on several model synthetic polymer materials. We demonstrate that surface adsorption of the proteins has minimal impacts on the presentation of the bioactive domains in the soluble block, and through the use of microscopic and cell proliferation assays, we show that the resulting biofunctional interfaces are capable of inducing appropriate cellular responses in a variety of human cell types.

Deciphering the mechanisms for targeting and interaction of Arabidopsis Lipid Droplet Associated Protein (LDAP) to the lipid droplet surface

USDA-ARS?s Scientific Manuscript database

We recently identified a new class of lipid-droplet associated proteins (LDAPs) in plants that share extensive sequence similarity with abundant structural proteins that coat rubber particles in rubber-producing plants. A majority of higher plants, however, including those that do not produce rubber...

A unique cell-surface protein phenotype distinguishes human small-cell from non-small-cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baylin, S.B.; Gazdar, A.F.; Minna, J.D.

1982-08-01

Radioiodination (/sup 125/I) and two-dimensional polyacrylamide gel electrophoresis was used to determine that small-(oat) cell lung carcinoma (SCC)-a tumor with neuroedocrine features-possesses a surface protein pattern distinct from the other types of lung cancer cells (squamous, adeno-, and large-cell undifferentiated carcinoma). Twelve distinguishing proteins, 40 to 70 kilodaltons (kDal), characterized four separate lines of SCC; three of these, designated E (60 kDal; pI = 7.3), S (30 kDal; pI = 6.0), and U 57 kDal; pI = 5.6), may be unique SCC gene products and were identified only in (/sup 35/S)methionine labeling of SCC and not in non-SCC or humanmore » fibroblasts. Two lines of adeno-, one of squamous, and one of undifferentiated large-cell lung carcinoma exhibited similar surface protein patterns to one another. Nine distinguishing proteins (40 to 100 kDal) and at least five large proteins (>100 kDal) were unique to these lines. The surface protein phenotypes for SCC and non-SCC were distinct from those for human lymphoblastoid cells and fibroblasts. However, the neuroendocrine features of SCC were further substantiated because 6 of the 12 distinguishing SCC surface proteins, including E and U, were identified on human neuroblastoma cells. The proteins identified should (i) help define differentiation steps for normal and neoplastic bronchial epithelial cells, (ii) prove useful in better classifying lung cancers, and (iii) be instrumental in tracing formation of neuroendocrine cells.« less

Electrosynthesized MIPs for transferrin: Plastibodies or nano-filters?

PubMed

Zhang, Xiaorong; Yarman, Aysu; Erdossy, Júlia; Katz, Sagie; Zebger, Ingo; Jetzschmann, Katharina J; Altintas, Zeynep; Wollenberger, Ulla; Gyurcsányi, Róbert E; Scheller, Frieder W

2018-05-15

Molecularly imprinted polymer (MIP) nanofilms for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of ~5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered. Copyright © 2018 Elsevier B.V. All rights reserved.

Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

PubMed

MacLean, Lorna; Price, Helen; O'Toole, Peter

2016-01-01

Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

Role of Flippases in Protein Glycosylation in the Endoplasmic Reticulum

PubMed Central

Rush, Jeffrey S.

2015-01-01

Glycosylation is essential to the synthesis, folding, and function of glycoproteins in eukaryotes. Proteins are co- and posttranslationally modified by a variety of glycans in the endoplasmic reticulum (ER); modifications include C- and O-mannosylation, N-glycosylation, and the addition of glycosylphosphatidylinositol membrane anchors. Protein glycosylation in the ER of eukaryotes involves enzymatic steps on both the cytosolic and lumenal surfaces of the ER membrane. The glycans are first assembled as precursor glycolipids, on the cytosolic surface of the ER, which are tethered to the membrane by attachment to a long-chain polyisoprenyl phosphate (dolichol) containing a reduced α-isoprene. The lipid-anchored building blocks then migrate transversely (flip) across the ER membrane to the lumenal surface, where final assembly of the glycan is completed. This strategy allows the cell to export high-energy biosynthetic intermediates as lipid-bound glycans, while constraining the glycosyl donors to the site of assembly on the membrane surface. This review focuses on the flippases that participate in protein glycosylation in the ER. PMID:26917968

A modified Poisson-Boltzmann equation applied to protein adsorption.

PubMed

Gama, Marlon de Souza; Santos, Mirella Simões; Lima, Eduardo Rocha de Almeida; Tavares, Frederico Wanderley; Barreto, Amaro Gomes Barreto

2018-01-05

Ion-exchange chromatography has been widely used as a standard process in purification and analysis of protein, based on the electrostatic interaction between the protein and the stationary phase. Through the years, several approaches are used to improve the thermodynamic description of colloidal particle-surface interaction systems, however there are still a lot of gaps specifically when describing the behavior of protein adsorption. Here, we present an improved methodology for predicting the adsorption equilibrium constant by solving the modified Poisson-Boltzmann (PB) equation in bispherical coordinates. By including dispersion interactions between ions and protein, and between ions and surface, the modified PB equation used can describe the Hofmeister effects. We solve the modified Poisson-Boltzmann equation to calculate the protein-surface potential of mean force, treated as spherical colloid-plate system, as a function of process variables. From the potential of mean force, the Henry constants of adsorption, for different proteins and surfaces, are calculated as a function of pH, salt concentration, salt type, and temperature. The obtained Henry constants are compared with experimental data for several isotherms showing excellent agreement. We have also performed a sensitivity analysis to verify the behavior of different kind of salts and the Hofmeister effects. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein immobilization techniques for microfluidic assays

PubMed Central

Kim, Dohyun; Herr, Amy E.

2013-01-01

Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

Using a biomimetic membrane surface experiment to investigate the activity of the magnetite biomineralisation protein Mms6† †Electronic supplementary information (ESI) available: Including Mms6 protein and peptide sequences, additional QCM-D and SEM data and protein modelling. See DOI: 10.1039/c5ra16469a Click here for additional data file.

PubMed Central

Bird, Scott M.; Rawlings, Andrea E.; Galloway, Johanna M.

2016-01-01

Magnetotactic bacteria are able to synthesise precise nanoparticles of the iron oxide magnetite within their cells. These particles are formed in dedicated organelles termed magnetosomes. These lipid membrane compartments use a range of biomineralisation proteins to nucleate and regulate the magnetite crystallisation process. A key component is the membrane protein Mms6, which binds to iron ions and helps to control the formation of the inorganic core. We have previously used Mms6 on gold surfaces patterned with a self-assembled monolayer to successfully produce arrays of magnetic nanoparticles. Here we use this surface system as a mimic of the interior face of the magnetosome membrane to study differences between intact Mms6 and the acid-rich C-terminal peptide subregion of the Mms6 protein. When immobilised on surfaces, the peptide is unable to reproduce the particle size or homogeneity control exhibited by the full Mms6 protein in our experimental setup. Moreover, the peptide is unable to support anchoring of a dense array of nanoparticles to the surface. This system also allows us to deconvolute particle binding from particle nucleation, and shows that Mms6 particle binding is less efficient when supplied with preformed magnetite nanoparticles when compared to particles precipitated from solution in the presence of the surface immobilised Mms6. This suggests that Mms6 binds to iron ions rather than to magnetite surfaces in our system, and is perhaps a nucleating agent rather than a controller of magnetite crystal growth. The comparison between the peptide and the protein under identical experimental conditions indicates that the full length sequence is required to support the full function of Mms6 on surfaces. PMID:27019707

Versatile multi-functionalization of protein nanofibrils for biosensor applications

NASA Astrophysics Data System (ADS)

Sasso, L.; Suei, S.; Domigan, L.; Healy, J.; Nock, V.; Williams, M. A. K.; Gerrard, J. A.

2014-01-01

Protein nanofibrils offer advantages over other nanostructures due to the ease in their self-assembly and the versatility of surface chemistry available. Yet, an efficient and general methodology for their post-assembly functionalization remains a significant challenge. We introduce a generic approach, based on biotinylation and thiolation, for the multi-functionalization of protein nanofibrils self-assembled from whey proteins. Biochemical characterization shows the effects of the functionalization onto the nanofibrils' surface, giving insights into the changes in surface chemistry of the nanostructures. We show how these methods can be used to decorate whey protein nanofibrils with several components such as fluorescent quantum dots, enzymes, and metal nanoparticles. A multi-functionalization approach is used, as a proof of principle, for the development of a glucose biosensor platform, where the protein nanofibrils act as nanoscaffolds for glucose oxidase. Biotinylation is used for enzyme attachment and thiolation for nanoscaffold anchoring onto a gold electrode surface. Characterization via cyclic voltammetry shows an increase in glucose-oxidase mediated current response due to thiol-metal interactions with the gold electrode. The presented approach for protein nanofibril multi-functionalization is novel and has the potential of being applied to other protein nanostructures with similar surface chemistry.Protein nanofibrils offer advantages over other nanostructures due to the ease in their self-assembly and the versatility of surface chemistry available. Yet, an efficient and general methodology for their post-assembly functionalization remains a significant challenge. We introduce a generic approach, based on biotinylation and thiolation, for the multi-functionalization of protein nanofibrils self-assembled from whey proteins. Biochemical characterization shows the effects of the functionalization onto the nanofibrils' surface, giving insights into the changes in surface chemistry of the nanostructures. We show how these methods can be used to decorate whey protein nanofibrils with several components such as fluorescent quantum dots, enzymes, and metal nanoparticles. A multi-functionalization approach is used, as a proof of principle, for the development of a glucose biosensor platform, where the protein nanofibrils act as nanoscaffolds for glucose oxidase. Biotinylation is used for enzyme attachment and thiolation for nanoscaffold anchoring onto a gold electrode surface. Characterization via cyclic voltammetry shows an increase in glucose-oxidase mediated current response due to thiol-metal interactions with the gold electrode. The presented approach for protein nanofibril multi-functionalization is novel and has the potential of being applied to other protein nanostructures with similar surface chemistry. Electronic supplementary information (ESI) available: Cyclic voltammetry characterization of biosensor platforms including bare Au electrodes (Fig. S1), biosensor response to various glucose concentrations (Fig. S2), and AFM roughness measurements due to WPNF modifications (Fig. S3). See DOI: 10.1039/c3nr05752f

Jet blown PTFE for control of biocompatibility

NASA Astrophysics Data System (ADS)

Leibner, Evan Scott

The development of fully hemocompatible cardiovascular biomaterials will have a major impact on the practice of modern medicine. Current artificial surfaces, unlike native vascular surfaces, are not able to control clot and thrombus formation. Protein interactions are an important component in hemocompatibility and can result in decreased patency due to thrombus formation or surface passivation which can improve endothelization. It is believed that controlling these properties, specifically the nanometer sizes of the fibers on the material's surface, will allow for better control of biological responses. The biocompatibility of Teflon, a widely used polymer for vascular grafts, would be improved with nanostructured control of surface features. Due to the difficultly in processing polytetrafluoroethylene (PTFE), it has not been possible to create nanofibrous PTFE surfaces. The novel technique of Jet Blowing allows for the formation of nanostructured PTFE (nPTFE). A systematic investigation into controlling polymer properties by varying the processing conditions of temperature, pressure, and gas used in the Jet Blowing allows for an increased understanding of the effects of plasticization on the material's properties. This fundamental understanding of the material science behind the Jet Blowing process has enabled control of the micro and nanoscale structure of nPTFE. While protein adsorption, a key component of biocompatibility, has been widely studied, it is not fully understood. Major problems in the field of biomaterials include a lack of standard protocols to measure biocompatibility, and inconstant literature on protein adsorption. A reproducible protocol for measuring protein adsorption onto superhydrophobic surfaces (ePTFE and nPTFE) has been developed. Both degassing of PBS buffer solutions and evacuation of the air around the expanded PTFE (ePTFE) prior to contact with protein solutions are essential. Protein adsorption experiments show a four-fold difference in the measure of proteins adsorbed using radiometry (I-125 labeled human serum albumin (HSA)) and electrophoresis (unlabeled HSA). This provides evidence that the standard method of radiolabeled protein for measuring adsorption does not fully account for changes to the HSA molecules due to labeling. The differences between measured protein values can be attributed to the radiolabel affecting the HSA hydrophobicity resulting in a change in the protein's interactions with the hydrophobic surface. Additionally, our work has provided repeatable results showing that the amount of protein adsorbed onto the polymer surface, after washing, accounted for only 65% of the amount of protein that was removed from solution based on depletion analysis. This implies that measurement of the amount of strongly bound protein on the material significantly underestimates the actual amount of protein adsorbing into the surface region of the material interface. HSA adsorption isotherms demonstrate an increase in protein adsorption capacity on the nPTFE surface compared to adsorption on the same surface area of ePTFE. Preliminary cell work shows that the nPTFE surfaces had a larger number of cells growing on the surface of the material when compared to ePTFE surfaces. The research also shows that while most endothelial cells were not viable on the ePTFE surface after 96 hours, they remained alive on the nPTFE surface during that same time period. Surface functionalization using ammonia plasma has been performed. X-ray photoelectron spectroscopy (XPS) analysis revealed the presence of amine groups on the nPTFE surface. The amine groups can be used to couple polypeptides onto the PTFE surface in the future. The selection of different peptides will allow for selective control of cell adhesion. This research shows that nPTFE has potential for improved biocompatibility over standard ePTFE, based on increased protein adsorption capacity, increased viability of endothelial cells, and the ability to plasma modify the PTFE surface.

Arming Technology in Yeast-Novel Strategy for Whole-cell Biocatalyst and Protein Engineering.

PubMed

Kuroda, Kouichi; Ueda, Mitsuyoshi

2013-09-09

Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called "arming technology", can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach.

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

PubMed

Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

2002-12-01

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

PubMed Central

Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

2002-01-01

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit. PMID:12515387

Hierarchical zwitterionic modification of a SERS substrate enables real-time drug monitoring in blood plasma

NASA Astrophysics Data System (ADS)

Sun, Fang; Hung, Hsiang-Chieh; Sinclair, Andrew; Zhang, Peng; Bai, Tao; Galvan, Daniel David; Jain, Priyesh; Li, Bowen; Jiang, Shaoyi; Yu, Qiuming

2016-11-01

Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive analytical technique with molecular specificity, making it an ideal candidate for therapeutic drug monitoring (TDM). However, in critical diagnostic media including blood, nonspecific protein adsorption coupled with weak surface affinities and small Raman activities of many analytes hinder the TDM application of SERS. Here we report a hierarchical surface modification strategy, first by coating a gold surface with a self-assembled monolayer (SAM) designed to attract or probe for analytes and then by grafting a non-fouling zwitterionic polymer brush layer to effectively repel protein fouling. We demonstrate how this modification can enable TDM applications by quantitatively and dynamically measuring the concentrations of several analytes--including an anticancer drug (doxorubicin), several TDM-requiring antidepressant and anti-seizure drugs, fructose and blood pH--in undiluted plasma. This hierarchical surface chemistry is widely applicable to many analytes and provides a generalized platform for SERS-based biosensing in complex real-world media.

Hierarchical zwitterionic modification of a SERS substrate enables real-time drug monitoring in blood plasma

PubMed Central

Sun, Fang; Hung, Hsiang-Chieh; Sinclair, Andrew; Zhang, Peng; Bai, Tao; Galvan, Daniel David; Jain, Priyesh; Li, Bowen; Jiang, Shaoyi; Yu, Qiuming

2016-01-01

Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive analytical technique with molecular specificity, making it an ideal candidate for therapeutic drug monitoring (TDM). However, in critical diagnostic media including blood, nonspecific protein adsorption coupled with weak surface affinities and small Raman activities of many analytes hinder the TDM application of SERS. Here we report a hierarchical surface modification strategy, first by coating a gold surface with a self-assembled monolayer (SAM) designed to attract or probe for analytes and then by grafting a non-fouling zwitterionic polymer brush layer to effectively repel protein fouling. We demonstrate how this modification can enable TDM applications by quantitatively and dynamically measuring the concentrations of several analytes—including an anticancer drug (doxorubicin), several TDM-requiring antidepressant and anti-seizure drugs, fructose and blood pH—in undiluted plasma. This hierarchical surface chemistry is widely applicable to many analytes and provides a generalized platform for SERS-based biosensing in complex real-world media. PMID:27834380

Searching for protein binding sites from Molecular Dynamics simulations and paramagnetic fragment-based NMR studies.

PubMed

Bernini, Andrea; Henrici De Angelis, Lucia; Morandi, Edoardo; Spiga, Ottavia; Santucci, Annalisa; Assfalg, Michael; Molinari, Henriette; Pillozzi, Serena; Arcangeli, Annarosa; Niccolai, Neri

2014-03-01

Hotspot delineation on protein surfaces represents a fundamental step for targeting protein-protein interfaces. Disruptors of protein-protein interactions can be designed provided that the sterical features of binding pockets, including the transient ones, can be defined. Molecular Dynamics, MD, simulations have been used as a reliable framework for identifying transient pocket openings on the protein surface. Accessible surface area and intramolecular H-bond involvement of protein backbone amides are proposed as descriptors for characterizing binding pocket occurrence and evolution along MD trajectories. TEMPOL induced paramagnetic perturbations on (1)H-(15)N HSQC signals of protein backbone amides have been analyzed as a fragment-based search for surface hotspots, in order to validate MD predicted pockets. This procedure has been applied to CXCL12, a small chemokine responsible for tumor progression and proliferation. From combined analysis of MD data and paramagnetic profiles, two CXCL12 sites suitable for the binding of small molecules were identified. One of these sites is the already well characterized CXCL12 region involved in the binding to CXCR4 receptor. The other one is a transient pocket predicted by Molecular Dynamics simulations, which could not be observed from static analysis of CXCL12 PDB structures. The present results indicate how TEMPOL, instrumental in identifying this transient pocket, can be a powerful tool to delineate minor conformations which can be highly relevant in dynamic discovery of antitumoral drugs. Copyright © 2013 Elsevier B.V. All rights reserved.

Models of globular proteins in aqueous solutions

NASA Astrophysics Data System (ADS)

Wentzel, Nathaniel James

Protein crystallization is a continuing area of research. Currently, there is no universal theory for the conditions required to crystallize proteins. A better understanding of protein crystallization will be helpful in determining protein structure and preventing and treating certain diseases. In this thesis, we will extend the understanding of globular proteins in aqueous solutions by analyzing various models for protein interactions. Experiments have shown that the liquid-liquid phase separation curves for lysozyme in solution with salt depend on salt type and salt concentration. We analyze a simple square well model for this system whose well depth depends on salt type and salt concentration, to determine the phase coexistence surfaces from experimental data. The surfaces, calculated from a single Monte Carlo simulation and a simple scaling argument, are shown as a function of temperature, salt concentration and protein concentration for two typical salts. Urate Oxidase from Asperigillus flavus is a protein used for studying the effects of polymers on the crystallization of large proteins. Experiments have determined some aspects of the phase diagram. We use Monte Carlo techniques and perturbation theory to predict the phase diagram for a model of urate oxidase in solution with PEG. The model used includes an electrostatic interaction, van der Waals attraction, and a polymerinduced depletion interaction. The results agree quantitatively with experiments. Anisotropy plays a role in globular protein interactions, including the formation of hemoglobin fibers in sickle cell disease. Also, the solvent conditions have been shown to play a strong role in the phase behavior of some aqueous protein solutions. Each has previously been treated separately in theoretical studies. Here we propose and analyze a simple, combined model that treats both anisotropy and solvent effects. We find that this model qualitatively explains some phase behavior, including the existence of a lower critical point under certain conditions.

Comparative analysis of Staphylococcus epidermidis strains utilizing quantitative and cell surface shaving proteomics.

PubMed

Solis, Nestor; Cain, Joel A; Cordwell, Stuart J

2016-01-01

Staphylococcus epidermidis is an opportunistic pathogen that is an emerging risk factor in hospitals worldwide and is often difficult to eradicate as virulent strains produce a protective biofilm matrix. We utilized cell shaving proteomics to profile surface-exposed proteins from two fully genome sequenced S. epidermidis strains: the avirulent, non-biofilm forming ATCC12228 and the virulent, strongly adherent biofilm forming ATCC35984 (RP62A). A false positive control strategy was employed to calculate the probabilities of proteins being truly surface-exposed. A total of 78 surface-exposed proteins were identified, of which only 19 proteins were common to ATCC12228 and RP62A, and which thus represents the core surfaceome. S. epidermidis RP62A displayed additional proteins involved in biofilm formation (cell wall-associated Bhp and intercellular adhesion protein IcaB), surface antigenicity, peptidoglycan biosynthesis and antibiotic resistance. We concurrently profiled whole cell proteomes of the two strains using iTRAQ quantitation and LC-MS/MS. A total of 1610 proteins were confidently identified (representing 64% of the theoretical S. epidermidis proteome). One hundred and ninety one proteins were differentially abundant between strains. Proteins associated with RP62A were clustered into functions including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated defense, sulfate assimilation, antibiotic resistance and biofilm formation. Validation of the sulfate assimilation and cysteine/methionine biosynthesis pathways showed RP62A contained elevated levels (~25% increase) of methionine that are likely linked to biofilm formation. Cell shaving and quantitative proteomics identified proteins associated with a biofilm-forming, virulent strain of S. epidermidis (RP62A). These proteins show RP62A maintains an active CRISPR-mediated defense, as well as heightened antibiotic resistance in comparison to a non-virulent, non-biofilm forming strain. Increased abundances of sulfate assimilation proteins lead to elevated intracellular methionine. Proteins and their exposed peptides identified on the surface of S. epidermidis RP62A may be useful vaccine antigens in clinical settings if administered in at-risk patients prior to surgical implantations. Copyright © 2015 Elsevier B.V. All rights reserved.

Streptococcus agalactiae Non-Pilus, Cell Wall-Anchored Proteins: Involvement in Colonization and Pathogenesis and Potential as Vaccine Candidates

PubMed Central

Pietrocola, Giampiero; Arciola, Carla Renata; Rindi, Simonetta; Montanaro, Lucio; Speziale, Pietro

2018-01-01

Group B Streptococcus (GBS) remains an important etiological agent of several infectious diseases including neonatal septicemia, pneumonia, meningitis, and orthopedic device infections. This pathogenicity is due to a variety of virulence factors expressed by Streptococcus agalactiae. Single virulence factors are not sufficient to provoke a streptococcal infection, which is instead promoted by the coordinated activity of several pathogenicity factors. Such determinants, mostly cell wall-associated and secreted proteins, include adhesins that mediate binding of the pathogen to host extracellular matrix/plasma ligands and cell surfaces, proteins that cooperate in the invasion of and survival within host cells and factors that neutralize phagocytosis and/or modulate the immune response. The genome-based approaches and bioinformatics tools and the extensive use of biophysical and biochemical methods and animal model studies have provided a great wealth of information on the molecular structure and function of these virulence factors. In fact, a number of new GBS surface-exposed or secreted proteins have been identified (GBS immunogenic bacterial adhesion protein, leucine-rich repeat of GBS, serine-rich repeat proteins), the three-dimensional structures of known streptococcal proteins (αC protein, C5a peptidase) have been solved and an understanding of the pathogenetic role of “old” and new determinants has been better defined in recent years. Herein, we provide an update of our current understanding of the major surface cell wall-anchored proteins from GBS, with emphasis on their biochemical and structural properties and the pathogenetic roles they may have in the onset and progression of host infection. We also focus on the antigenic profile of these compounds and discuss them as targets for therapeutic intervention. PMID:29686667

A Proteomic Approach to Characterize Protein Shedding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahram, Mamoun; Adkins, Joshua N.; Auberry, Deanna L.

2005-01-01

Shedding (i.e., proteolysis of ectodomains of membrane proteins) plays an important pathophysiological role. In order to study the feasibility of identifying shed proteins, we analyzed serum-free media of human mammary epithelial cells by mass spectrometry following induction of shedding by the phorbol ester, 4β-phorbol 12-myristate 13-acetate (PMA). Different means of sample preparation, including biotinylation of cell surface proteins, isolation of glycosylated proteins, and preparation of crude protein fraction, were carried out to develop the optimal method of sample processing. The collected proteins were digested with trypsin and analyzed by reversed-phase capillary liquid chromatography interfaced to an ion-trap mass spectrometer. Themore » resulting peptide spectra were interpreted using the program SEQUEST. Analyzing the sample containing the crude protein mixture without chemical modification or separation resulted in the greatest number of identifications, including putatively shed proteins. Overall, 93 membrane-associated proteins were identified including 57 that contain at least one transmembrane domain and 36 that indirectly associate with the extracellular surface of the plasma membrane. Of the 57 transmembrane proteins, 43 were identified by extracellular peptides providing strong evidence for them originating from regulated proteolysis or shedding processes. We combined results from the different experiments and used a peptide count method to estimate changes in protein abundance. Using this approach, we identified 2 proteins, syndecan-4 and hepatoma-derived growth factor, whose abundances increased in media of cells treated with PMA. We also detected proteins whose abundances decreased after PMA treatment such as 78-kDa glucose-regulated protein and calreticulin. Further analysis using immunoblotting validated the abundance changes for syndecan-4 and 78-kDa glucose-regulated protein as a result of PMA treatment. These results demonstrate that mass spectrometry can be used to identify low-abundance shed proteins and to estimate changes in protein abundances.« less

The Effect of Simulated Microgravity Environment of RWV Bioreactors on Surface Reactions and Adsorption of Serum Proteins on Bone-bioactive Microcarriers

NASA Technical Reports Server (NTRS)

Radin, Shula; Ducheyne, P.; Ayyaswamy, P. S.

2003-01-01

Biomimetically modified bioactive materials with bone-like surface properties are attractive candidates for use as microcarriers for 3-D bone-like tissue engineering under simulated microgravity conditions of NASA designed rotating wall vessel (RWV) bioreactors. The simulated microgravity environment is attainable under suitable parametric conditions of the RWV bioreactors. Ca-P containing bioactive glass (BG), whose stimulatory effect on bone cell function had been previously demonstrated, was used in the present study. BG surface modification via reactions in solution, resulting formation of bone-like minerals at the surface and adsorption of serum proteins is critical for obtaining the stimulatory effect. In this paper, we report on the major effects of simulated microgravity conditions of the RWV on the BG reactions surface reactions and protein adsorption in physiological solutions. Control tests at normal gravity were conducted at static and dynamic conditions. The study revealed that simulated microgravity remarkably enhanced reactions involved in the BG surface modification, including BG dissolution, formation of bone-like minerals at the surface and adsorption of serum proteins. Simultaneously, numerical models were developed to simulate the mass transport of chemical species to and from the BG surface under normal gravity and simulated microgravity conditions. The numerical results showed an excellent agreement with the experimental data at both testing conditions.

Surface characterization and protein interaction of a series of model poly[acrylonitrile-co-(N-vinyl pyrrolidone)] nanocarriers for drug targeting.

PubMed

Staufenbiel, Sven; Merino, Marian; Li, Wenzhong; Huang, Mao-Dong; Baudis, Stefan; Lendlein, Andreas; Müller, Rainer H; Wischke, Christian

2015-05-15

The surface properties of intravenously injected nanoparticles determine the acquired blood protein adsorption pattern and subsequently the organ distribution and cellular recognition. A series of poly[acrylonitrile-co-(N-vinyl pyrrolidone)] (PANcoNVP) model nanoparticles (133-181 nm) was synthesized, in which the surface properties were altered by changing the molar content of NVP (0-33.8 mol%) as the more hydrophilic repeating unit. The extent of achieved surface property variation was comprehensively characterized. The residual sodium dodecyl sulfate (SDS) content from the synthesis was in the range 0.3-1.6 μgml(-1), potentially contributing to the surface properties. Surface hydrophobicity was determined by Rose Bengal dye adsorption, hydrophobic interaction chromatography (HIC) and aqueous two-phase partitioning (TPP). Particle charge was quantified by zeta potential (ZP) measurements including ZP-pH profiles. The interaction with proteins was analyzed by ZP measurements in serum and by adsorption studies with single proteins. Compared to hydrophobic polystyrene model nanoparticles, all PANcoNVP particles were very hydrophilic. Differences in surface hydrophobicity could be detected, which did not linearly correlate with the systematically altered bulk composition of the PANcoNVP nanoparticles. This proves the high importance of a thorough surface characterization applying a full spectrum of methods, complementing predictions solely based on bulk polymer composition. Copyright © 2015. Published by Elsevier B.V.

The Role of Airborne Proteins in Atopic Dermatitis

PubMed Central

Hostetler, Sarah Grim; Kaffenberger, Benjamin; Hostetler, Todd

2010-01-01

Atopic dermatitis is a common, chronic skin condition. A subpopulation of patients may have cutaneous exposure to common airborne proteins exacerbating their disease through direct proteolytic activity, direct activation of proteinase-activated receptor-2 itch receptors, and immunoglobulin E binding. The most common airborne proteins significant in atopic dermatitis include house dust mites, cockroach, pet dander, and multiple pollens. The literature on atopy patch testing, skin-prick testing, and specific IgE is mixed, with greater support for the use of atopy patch test. Patients with airborne proteins contributing to their disease typically have lesions predominately on air-exposed skin surfaces including the face, neck, and arms; a history of exacerbations after exposure to airborne proteins; severe disease resistant to conventional therapies; and concurrent asthma. Treatment strategies include airborne protein avoidance, removal of airborne proteins from the skin, and barrier repair. Further research is needed to establish the benefit of allergen-specific immunotherapy. PMID:20725535

Handheld Chem/Biosensor Using Extreme Conformational Changes in Designed Binding Proteins to Enhance Surface Plasmon Resonance (SPR)

DTIC Science & Technology

2016-04-01

AFCEC-CX-TY-TR-2016-0007 HANDHELD CHEM/ BIOSENSOR USING EXTREME CONFORMATIONAL CHANGES IN DESIGNED BINDING PROTEINS TO ENHANCE SURFACE PLASMON...Include area code) 03/24/2016 Abstract 08/14/2015--03/31/2016 Handheld chem/ biosensor using extreme conformational changes in designed binding...Baltimore, Maryland on 17-21 April 2016. We propose the development of a highly sensitive handheld chem/ biosensor device using a novel class of engineered

Comparison of the structural basis for thermal stability between archaeal and bacterial proteins.

PubMed

Ding, Yanrui; Cai, Yujie; Han, Yonggang; Zhao, Bingqiang

2012-01-01

In this study, the structural basis for thermal stability in archaeal and bacterial proteins was investigated. There were many common factors that confer resistance to high temperature in both archaeal and bacterial proteins. These factors include increases in the Lys content, the bends and blanks of secondary structure, the Glu content of salt bridge; decreases in the number of main-side chain hydrogen bond and exposed surface area, and changes in the bends and blanks of amino acids. Certainly, the utilization of charged amino acids to form salt bridges is a primary factor. In both heat-resistant archaeal and bacterial proteins, most Glu and Asp participate in the formation of salt bridges. Other factors may influence either archaeal or bacterial protein thermostability, which includes the more frequent occurrence of shorter 3(10)-helices and increased hydrophobicity in heat-resistant archaeal proteins. However, there were increases in average helix length, the Glu content in salt bridges, temperature factors and decreases in the number of main-side chain hydrogen bonds, uncharged-uncharged hydrogen bonds, hydrophobicity, and buried and exposed polar surface area in heat-resistant bacterial proteins. Evidently, there are few similarities and many disparities between the heat-resistant mechanisms of archaeal and bacterial proteins.

Predicting Cell Association of Surface-Modified Nanoparticles Using Protein Corona Structure - Activity Relationships (PCSAR).

PubMed

Kamath, Padmaja; Fernandez, Alberto; Giralt, Francesc; Rallo, Robert

2015-01-01

Nanoparticles are likely to interact in real-case application scenarios with mixtures of proteins and biomolecules that will absorb onto their surface forming the so-called protein corona. Information related to the composition of the protein corona and net cell association was collected from literature for a library of surface-modified gold and silver nanoparticles. For each protein in the corona, sequence information was extracted and used to calculate physicochemical properties and statistical descriptors. Data cleaning and preprocessing techniques including statistical analysis and feature selection methods were applied to remove highly correlated, redundant and non-significant features. A weighting technique was applied to construct specific signatures that represent the corona composition for each nanoparticle. Using this basic set of protein descriptors, a new Protein Corona Structure-Activity Relationship (PCSAR) that relates net cell association with the physicochemical descriptors of the proteins that form the corona was developed and validated. The features that resulted from the feature selection were in line with already published literature, and the computational model constructed on these features had a good accuracy (R(2)LOO=0.76 and R(2)LMO(25%)=0.72) and stability, with the advantage that the fingerprints based on physicochemical descriptors were independent of the specific proteins that form the corona.

Taking Orders from Light: Photo-Switchable Working/Inactive Smart Surfaces for Protein and Cell Adhesion.

PubMed

Zhang, Junji; Ma, Wenjing; He, Xiao-Peng; Tian, He

2017-03-15

Photoresponsive smart surfaces are promising candidates for a variety of applications in optoelectronics and sensing devices. The use of light as an order signal provides advantages of remote and noninvasive control with high temporal and spatial resolutions. Modification of the photoswitches with target biomacromolecules, such as peptides, DNA, and small molecules including folic acid derivatives and sugars, has recently become a popular strategy to empower the smart surfaces with an improved detection efficiency and specificity. Herein, we report the construction of photoswitchable self-assembled monolayers (SAMs) based on sugar (galactose/mannose)-decorated azobenzene derivatives and determine their photoswitchable, selective protein/cell adhesion performances via electrochemistry. Under alternate UV/vis irradiation, interconvertible high/low recognition and binding affinity toward selective lectins (proteins that recognize sugars) and cells that highly express sugar receptors are achieved. Furthermore, the cis-SAMs with a low binding affinity toward selective proteins and cells also exhibit minimal response toward unselective protein and cell samples, which offers the possibility in avoiding unwanted contamination and consumption of probes prior to functioning for practical applications. Besides, the electrochemical technique used facilitates the development of portable devices based on the smart surfaces for on-demand disease diagnosis.

New Method for Quantitation of Lipid Droplet Volume From Light Microscopic Images With an Application to Determination of PAT Protein Density on the Droplet Surface.

PubMed

Dejgaard, Selma Y; Presley, John F

2018-06-01

Determination of lipid droplet (LD) volume has depended on direct measurement of the diameter of individual LDs, which is not possible when LDs are small or closely apposed. To overcome this problem, we describe a new method in which a volume-fluorescence relationship is determined from automated analysis of calibration samples containing well-resolved LDs. This relationship is then used to estimate total cellular droplet volume in experimental samples, where the LDs need not be individually resolved, or to determine the volumes of individual LDs. We describe quantitatively the effects of various factors, including image noise, LD crowding, and variation in LD composition on the accuracy of this method. We then demonstrate this method by utilizing it to address a scientifically interesting question, to determine the density of green fluorescent protein (GFP)-tagged Perilipin-Adipocyte-Tail (PAT) proteins on the LD surface. We find that PAT proteins cover only a minority of the LD surface, consistent with models in which they primarily serve as scaffolds for binding of regulatory proteins and enzymes, but inconsistent with models in which their major function is to sterically block access to the droplet surface.

Stress Proteins and Initiation of Immune Response: Chaperokine activity of Hsp72

PubMed Central

Asea, Alexzander

2006-01-01

From its original description as solely an intracellular molecular chaperone, heat shock proteins have now been shown to function as initiators of the host's immune response. Although the exact mechanism by which intracellular heat shock proteins leave cells is still incompletely understood, recent work from several labs suggest that heat shock proteins are released by both passive (necrotic) and active (physiological) mechanisms. Binding to specific surface receptors is a prerequisite for the initiation of an immune response. To date, several cell surface proteins have been described as the receptor for seventy kilo-Dalton heat shock protein (Hsp70) including Toll-like receptors 2 and 4 with their cofactor CD14, the scavenger receptor CD36, the low-density lipoprotein receptor-related protein CD91, the C-type lectin receptor LOX-1, and another member of the scavenger super-family SR-A plus the co-stimulatory molecule, CD40. Binding of Hsp70 to these surface receptors specifically activates intracellular signaling cascades, which in turn exert immunoregulatory effector functions; a process known as the chaperokine activity of Hsp70. This review will highlight recent advances in understanding the mechanism by which Hsp70 initiates the host's immune response. PMID:16385842

Stress proteins and initiation of immune response: chaperokine activity of hsp72.

PubMed

Asea, Alexzander

2005-01-01

From its original description as solely an intracellular molecular chaperone, heat shock proteins have now been shown to function as initiators of the host's immune response. Although the exact mechanism by which intracellular heat shock proteins leave cells is still incompletely understood, recent work from several labs suggest that heat shock proteins are released by both passive (necrotic) and active (physiological) mechanisms. Binding to specific surface receptors is a prerequisite for the initiation of an immune response. To date, several cell surface proteins have been described as the receptor for seventy kilo-Dalton heat shock protein (Hsp70) including Toll-like receptors 2 and 4 with their cofactor CD14, the scavenger receptor CD36, the low-density lipoprotein receptor-related protein CD91, the C-type lectin receptor LOX-1, and another member of the scavenger super-family SR-A plus the co-stimulatory molecule, CD40. Binding of Hsp70 to these surface receptors specifically activates intracellular signaling cascades, which in turn exert immunoregulatory effector functions; a process known as the chaperokine activity of Hsp70. This review will highlight recent advances in understanding the mechanism by which Hsp70 initiates the host's immune response.

A molecular modeling based method to predict elution behavior and binding patches of proteins in multimodal chromatography.

PubMed

Banerjee, Suvrajit; Parimal, Siddharth; Cramer, Steven M

2017-08-18

Multimodal (MM) chromatography provides a powerful means to enhance the selectivity of protein separations by taking advantage of multiple weak interactions that include electrostatic, hydrophobic and van der Waals interactions. In order to increase our understanding of such phenomena, a computationally efficient approach was developed that combines short molecular dynamics simulations and continuum solvent based coarse-grained free energy calculations in order to study the binding of proteins to Self Assembled Monolayers (SAM) presenting MM ligands. Using this method, the free energies of protein-MM SAM binding over a range of incident orientations of the protein can be determined. The resulting free energies were then examined to identify the more "strongly bound" orientations of different proteins with two multimodal surfaces. The overall free energy of protein-MM surface binding was then determined and correlated to retention factors from isocratic chromatography. This correlation, combined with analytical expressions from the literature, was then employed to predict protein gradient elution salt concentrations as well as selectivity reversals with different MM resin systems. Patches on protein surfaces that interacted strongly with MM surfaces were also identified by determining the frequency of heavy atom contacts with the atoms of the MM SAMs. A comparison of these patches to Electrostatic Potential and hydrophobicity maps indicated that while all of these patches contained significant positive charge, only the highest frequency sites also possessed hydrophobicity. The ability to identify key binding patches on proteins may have significant impact on process development for the separation of bioproduct related impurities. Copyright © 2017 Elsevier B.V. All rights reserved.

Human Common Salivary Protein 1 (CSP-1) Promotes Binding of Streptococcus mutans to Experimental Salivary Pellicle and Glucans Formed on Hydroxyapatite Surface

PubMed Central

Ambatipudi, Kiran S.; Hagen, Fred K.; Delahunty, Claire M.; Han, Xuemei; Shafi, Rubina; Hryhorenko, Jennifer; Gregoire, Stacy; Marquis, Robert E.; Melvin, James E.; Koo, Hyun; Yates, John R.

2010-01-01

Summary The saliva proteome includes host defense factors and specific bacterial-binding proteins that modulate microbial growth and colonization of tooth surface in the oral cavity. A multidimensional mass spectrometry approach identified the major host-derived salivary proteins which interacted with Streptococcus mutans (strain UA159), the primary microorganism associated with the pathogenesis of dental caries. Two abundant host proteins were found to tightly bind to S. mutans cells, common salivary protein-1 (CSP-1) and deleted in malignant brain tumor 1 (DMBT1, also known as salivary agglutinin or gp340). In contrast to gp340, limited functional information is available on CSP-1. The sequence of CSP-1 shares 38.1% similarity with rat CSP-1. Recombinant CSP-1 (rCSP-1) protein did not cause aggregation of S. mutans cells and was devoid of any significant biocidal activity (2.5 to 10 μg/ml). However, S. mutans cells exposed to rCSP-1 (10 μg/ml) in saliva displayed enhanced adherence to experimental salivary pellicle and to glucans in the pellicle formed on hydroxyapatite surfaces. Thus, our data demonstrate that the host salivary protein CSP-1 binds to S. mutans cells and may influence the initial colonization of this pathogenic bacterium onto tooth surface. PMID:20858015

Fluorescent proteins as efficient tools for evaluating the surface PEGylation of silica nanoparticles

NASA Astrophysics Data System (ADS)

Zhang, Wei; Ma, Minyan; Zhang, Xiao-ai; Zhang, Ze-yu; Saleh, Sayed M.; Wang, Xu-dong

2017-06-01

Surface PEGylation is essential for preventing non-specific binding of biomolecules when silica nanoparticles are utilized for in vivo applications. Methods for installing poly(ethylene glycol) on a silica surface have been widely explored but varies from study to study. Because there is a lack of a satisfactory method for evaluating the properties of silica surface after PEGylation, the prepared nanoparticles are not fully characterized before use. In some cases, even non-PEGylated silica nanoparticles were produced, which is unfortunately not recognized by the end-user. In this work, a fluorescent protein was employed, which acts as a sensitive material for evaluating the surface protein adsorption properties of silica nanoparticles. Eleven different methods were systematically investigated for their reaction efficiency towards surface PEGylation. Results showed that both reaction conditions (including pH, catalyst) and surface functional groups of parent silica nanoparticles play critical roles in producing fully PEGylated silica nanoparticles. Great care needs to be taken in choosing the proper coupling chemistry for surface PEGylation. The data and method shown here will guarantee high-quality PEGylated silica nanoparticles to be produced and guide their applications in biology, chemistry, industry and medicine.

MUC1, the renaissance molecule.

PubMed

Gendler, S J

2001-07-01

MUC1 is a large, heavily glycosylated mucin expressed on the apical surfaces of most simple, secretory epithelia including the mammary gland, gastrointestinal, respiratory, urinary and reproductive tracts. Although MUC1 was thought to be an epithelial-specific protein, it is now known to be expressed on a variety of hematopoietic cells as well. Mucins function in protection and lubrication of epithelial surfaces. Transmembrane mucins, which contain cytoplasmic tail domains, appear to have additional functions through their abilities to interact with many proteins involved in signal transduction and cell adhesion. The goal of this review is to highlight recent discoveries that suggest that MUC1 may be a multifunctional protein, located on the surfaces of cells as a sensor of the environment, poised to signal to the interior when things go awry.

MECHANISTIC PATHWAYS AND BIOLOGICAL ROLES FOR RECEPTOR-INDEPENDENT ACTIVATORS OF G-PROTEIN SIGNALING

PubMed Central

Blumer, Joe B.; Smrcka, Alan V.; Lanier, S.M.

2007-01-01

Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents, plays an important role in adaptive processes of organs, and aberrant processing of signals through these transducing systems is a component of various disease states. In addition to GPCR-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Gαβγ heterotrimer or Gα and Gαβγ subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Gα and Gαβγ) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Gαβγ. Such regulatory accessory proteins include the family of RGS proteins that accelerate the GTPase activity of Gα and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor independent activators of G-protein signaling or AGS proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways and provide a platform for diverse functions of both the heterotrimeric Gαβγ and the individual Gα and Gαβγ subunits. PMID:17240454

The effect of geometrical presentation of multimodal cation-exchange ligands on selective recognition of hydrophobic regions on protein surfaces.

PubMed

Woo, James; Parimal, Siddharth; Brown, Matthew R; Heden, Ryan; Cramer, Steven M

2015-09-18

The effects of spatial organization of hydrophobic and charged moieties on multimodal (MM) cation-exchange ligands were examined by studying protein retention behavior on two commercial chromatographic media, Capto™ MMC and Nuvia™ cPrime™. Proteins with extended regions of surface-exposed aliphatic residues were found to have enhanced retention on the Capto MMC system as compared to the Nuvia cPrime resin. The results further indicated that while the Nuvia cPrime ligand had a strong preference for interactions with aromatic groups, the Capto MMC ligand appeared to interact with both aliphatic and aromatic clusters on the protein surfaces. These observations were formalized into a new set of protein surface property descriptors, which quantified the local distribution of electrostatic and hydrophobic potentials as well as distinguishing between aromatic and aliphatic properties. Using these descriptors, high-performing quantitative structure-activity relationship (QSAR) models (R(2)>0.88) were generated for both the Capto MMC and Nuvia cPrime datasets at pH 5 and pH 6. Descriptors of electrostatic properties were generally common across the four models; however both Capto MMC models included descriptors that quantified regions of aliphatic-based hydrophobicity in addition to aromatic descriptors. Retention was generally reduced by lowering the ligand densities on both MM resins. Notably, elution order was largely unaffected by the change in surface density, but smaller and more aliphatic proteins tended to be more affected by this drop in ligand density. This suggests that modulating the exposure, shape and density of the hydrophobic moieties in multimodal chromatographic systems can alter the preference for surface exposed aliphatic or aromatic residues, thus providing an additional dimension for modulating the selectivity of MM protein separation systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen

PubMed Central

Gründel, Anne; Pfeiffer, Melanie; Jacobs, Enno

2015-01-01

In different bacteria, primarily cytosolic and metabolic proteins are characterized as surface localized and interacting with different host factors. These moonlighting proteins include glycolytic enzymes, and it has been hypothesized that they influence the virulence of pathogenic species. The presence of surface-displayed glycolytic enzymes and their interaction with human plasminogen as an important host factor were investigated in the genome-reduced and cell wall-less microorganism Mycoplasma pneumoniae, a common agent of respiratory tract infections of humans. After successful expression of 19 glycolytic enzymes and production of polyclonal antisera, the localization of proteins in the mycoplasma cell was characterized using fractionation of total proteins, colony blot, mild proteolysis and immunofluorescence of M. pneumoniae cells. Eight glycolytic enzymes, pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate dehydrogenase (Ldh), phosphoglycerate mutase (Pgm), pyruvate kinase (Pyk), and transketolase (Tkt), were confirmed as surface expressed and all are able to interact with plasminogen. Plasminogen bound to recombinant proteins PdhB, GapA, and Pyk was converted to plasmin in the presence of urokinase plasminogen activator and plasmin-specific substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Furthermore, human fibrinogen was degraded by the complex of plasminogen and recombinant protein PdhB or Pgm. In addition, surface-displayed proteins (except PdhC) bind to human lung epithelial cells, and the interaction was reduced significantly by preincubation of cells with antiplasminogen. Our results suggest that plasminogen binding and activation by different surface-localized glycolytic enzymes of M. pneumoniae may play a role in successful and long-term colonization of the human respiratory tract. PMID:26667841

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

PubMed Central

Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

2016-01-01

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants. PMID:27602024

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover.

PubMed

Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

2016-01-01

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants.

Multiplex detection of IgG and IgM to Rift Valley fever virus nucleoprotein, nonstructural proteins, and glycoprotein in ovine and bovine

USDA-ARS?s Scientific Manuscript database

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were...

Context-specific target definition in influenza a virus hemagglutinin-glycan receptor interactions.

PubMed

Shriver, Zachary; Raman, Rahul; Viswanathan, Karthik; Sasisekharan, Ram

2009-08-28

Protein-glycan interactions are important regulators of a variety of biological processes, ranging from immune recognition to anticoagulation. An important area of active research is directed toward understanding the role of host cell surface glycans as recognition sites for pathogen protein receptors. Recognition of cell surface glycans is a widely employed strategy for a variety of pathogens, including bacteria, parasites, and viruses. We present here a representative example of such an interaction: the binding of influenza A hemagglutinin (HA) to specific sialylated glycans on the cell surface of human upper airway epithelial cells, which initiates the infection cycle. We detail a generalizable strategy to understand the nature of protein-glycan interactions both structurally and biochemically, using HA as a model system. This strategy combines a top-down approach using available structural information to define important contacts between glycans and HA, with a bottom-up approach using data-mining and informatics approaches to identify the common motifs that distinguish glycan binders from nonbinders. By probing protein-glycan interactions simultaneously through top-down and bottom-up approaches, we can scientifically validate a series of observations. This in turn provides additional confidence and surmounts known challenges in the study of protein-glycan interactions, such as accounting for multivalency, and thus truly defines concepts such as specificity, affinity, and avidity. With the advent of new technologies for glycomics-including glycan arrays, data-mining solutions, and robust algorithms to model protein-glycan interactions-we anticipate that such combination approaches will become tractable for a wide variety of protein-glycan interactions.

Functionalized gold nanostars for label-free detection of PKA phosphorylation using surface-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

He, Shuai; Kah, James C. Y.

2017-04-01

Protein phosphorylation controls fundamental biological processes. Dysregulation of protein kinase is associated with a series of human diseases including cancer. Protein kinase A (PKA) activity has been reported to serve as a potential prognostic marker for cancer. To this end, we developed a non-radioactive, rapid, cheap and robust scheme based on surface-enhanced Raman spectroscopy (SERS) for label-free detection of PKA phosphorylation using gold nanostars (AuNS) functionalized with BSA-kemptide. While bovine serum albumin (BSA) proteins stabilized the AuNS, kemptide, which is a high affinity substrate peptide specific for PKA, were phosphorylated in vitro to generate Raman signals that were identified by performing principal component analysis (PCA) on the acquired SERS spectra.

Simplified method for preparation of concentrated exoproteins produced by Staphylococcus aureus grown on surface of cellophane bag containing liquid medium.

PubMed

Ikigai, H; Seki, K; Nishihara, S; Masuda, S

1988-01-01

A simplified method for preparation of concentrated exoproteins including protein A and alpha-toxin produced by Staphylococcus aureus was successfully devised. The concentrated proteins were obtained by cultivating S. aureus organisms on the surface of a liquid medium-containing cellophane bag enclosed in a sterilized glass flask. With the same amount of medium, the total amount of proteins obtained by the method presented here was identical with that obtained by conventional liquid culture. The concentration of proteins obtained by the method, however, was high enough to observe their distinct bands stained on polyacrylamide gel electrophoresis. This method was considered quite useful not only for large-scale cultivation for the purification of staphylococcal proteins but also for small-scale study using the proteins. The precise description of the method was presented and its possible usefulness was discussed.

Effect of enzymatic treatment of extracted sunflower proteins on solubility, amino acid composition, and surface activity.

PubMed

Conde, José Miñones; Escobar, María del Mar Yust; Pedroche Jiménez, Justo J; Rodríguez, Francisco Millán; Rodríguez Patino, Juan M

2005-10-05

Industrial proteins from agriculture of either animal or vegetable origin, including their peptide derivatives, are of great importance, from the qualitative and quantitative point of view, in food formulations (emulsions and foams). A fundamental understanding of the physical, chemical, and functional properties of these proteins is essential if the performance of proteins in foods is to be improved and if underutilized proteins, such as plant proteins (and their hydrolysates and peptides derivatives), are to be increasingly used in traditional and new processed food products (safe, high-quality, health foods with good nutritional value). In this contribution we have determined the main physicochemical characteristics (solubility, composition, and analysis of amino acids) of a sunflower protein isolate (SPI) and its hydrolysates with low (5.62%), medium (23.5%), and high (46.3%) degrees of hydrolysis. The hydrolysates were obtained by enzymatic treatment with Alcalase 2.4 L for DH 5.62 and 23.5% and with Alcalase 2.4 L and Flavorzyme 1000 MG sequentially for DH 46.3%. The protein concentration dependence on surface pressure (surface pressure isotherm), a measure of the surface activity of the products (SPI and its hydrolysates), was obtained by tensiometry. We have observed that the degree of hydrolysis has an effect on solubility, composition, and content of the amino acids of the SPI and its hydrolysates. The superficial activity and the adsorption efficiency were also affected by the degree of hydrolysis.

Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane

PubMed Central

Salanenka, Yuliya; Verstraeten, Inge; Löfke, Christian; Tabata, Kaori; Naramoto, Satoshi; Glanc, Matouš; Friml, Jiří

2018-01-01

The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses. PMID:29463731

Changes in the Expression of Biofilm-Associated Surface Proteins in Staphylococcus aureus Food-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants

PubMed Central

Polansky, Ondrej; Babak, Vladimir; Kulich, Pavel

2016-01-01

Sublethal concentrations (sub-MICs) of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on three Staphylococcus aureus biofilm formers (strong B+++, moderate B++, and weak B+) that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25–2.5% ethanol and 2500 μg/mL chloramine T significantly enhanced S. aureus biofilm formation. To visualize differences in biofilm compactness between S. aureus biofilms in control medium, 1.25% ethanol, or 2500 μg/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors. PMID:27868063

Protein-Nanoparticle Interactions: Improving Immobilized Lytic Enzyme Activity and Surface Energy Effects

NASA Astrophysics Data System (ADS)

Downs, Emily Elizabeth

Protein-nanostructure conjugates, particularly particles, are a subject of significant interest due to changes in their fundamental behavior compared to bulk surfaces. As the size scale of nano-structured materials and proteins are on the same order of magnitude, nanomaterial properties can heavily influence how proteins adsorb and conform to the surface. Previous work has demonstrated the ability of nanoscale surfaces to modulate protein activity, conformation, and retention by modifying the particle surface curvature, morphology, and surface charge. This work has improved our understanding of the protein material interactions, but a complete understanding is still lacking. The goal of this thesis is to investigate two missing areas of understanding using two distinct systems. The first system utilizes a particle with controlled surface energy to observe the impact of surface energy on protein-particle interactions, while the second system uses a modified Listeria-specific protein to determine how protein structure and flexibility affects protein adsorption and activity on particles. Spherical, amorphous, and uniformly doped Zn-silica particles with tailored surface energies were synthesized to understand the impact of surface energy on protein adsorption behavior. Particle surface energy increased with a decrease in particle size and greater dopant concentrations. Protein adsorption and structural loss increased with both particle size and particle surface energy. Higher surface energies promoted protein-particle association and increased protein unfolding. Particle curvature and protein steric hindrance effects limited adsorption and structural loss on smaller particles. Protein surface charge heterogeneity was also found to be linked to both protein adsorption and unfolding behavior on larger particles. Greater surface charge heterogeneity led to higher adsorption concentrations and multilayer formation. These multilayers transitioned from protein-particle interactions to protein-protein interactions and were thicker with greater surface energy, which resulted in the recovery of secondary structure in the outermost layer. To help understand the impact of protein structure on nano-bio conjugate interactions, a listeria specific protein was used. This system was chosen as it has applications in the food industry in preventing bacterial contamination. The insertion of an amino acid linker between the enzymatic and binding domain of the protein improved the flexibility between domains, leading to increased adsorption, and improved activity in both cell-wall and plating assays. Additionally, linker modified protein incorporated into the silica-polymer nanocomposite showed significant activity in a real-world example of contaminated lettuce. This thesis study has isolated the impact of surface energy and protein flexibility on protein adsorption and structure. Particle surface energy affects adsorbed protein concentration and conformation. Coupled with protein surface charge, surface energy was also found to dictate multilayer thickness. The conformational flexibility of the protein was shown to help in controlling not only protein adsorption concentration but also in retaining protein activity after immobilization. Also, a controllable synthesis method for particles with adjustable surface energy, an ideal platform for studying protein-particle interactions, has been established.

Angiotensin II reduces the surface abundance of KV 1.5 channels in arterial myocytes to stimulate vasoconstriction.

PubMed

Kidd, Michael W; Bulley, Simon; Jaggar, Jonathan H

2017-03-01

Several different voltage-dependent K + (K V ) channel isoforms are expressed in arterial smooth muscle cells (myocytes). Vasoconstrictors inhibit K V currents, but the isoform selectivity and mechanisms involved are unclear. We show that angiotensin II (Ang II), a vasoconstrictor, stimulates degradation of K V 1.5, but not K V 2.1, channels through a protein kinase C- and lysosome-dependent mechanism, reducing abundance at the surface of mesenteric artery myocytes. The Ang II-induced decrease in cell surface K V 1.5 channels reduces whole-cell K V 1.5 currents and attenuates K V 1.5 function in pressurized arteries. We describe a mechanism by which Ang II stimulates protein kinase C-dependent K V 1.5 channel degradation, reducing the abundance of functional channels at the myocyte surface. Smooth muscle cells (myocytes) of resistance-size arteries express several different voltage-dependent K + (K V ) channels, including K V 1.5 and K V 2.1, which regulate contractility. Myocyte K V currents are inhibited by vasoconstrictors, including angiotensin II (Ang II), but the mechanisms involved are unclear. Here, we tested the hypothesis that Ang II inhibits K V currents by reducing the plasma membrane abundance of K V channels in myocytes. Angiotensin II (applied for 2 h) reduced surface and total K V 1.5 protein in rat mesenteric arteries. In contrast, Ang II did not alter total or surface K V 2.1, or K V 1.5 or K V 2.1 cellular distribution, measured as the percentage of total protein at the surface. Bisindolylmaleimide (BIM; a protein kinase C blocker), a protein kinase C inhibitory peptide or bafilomycin A (a lysosomal degradation inhibitor) each blocked the Ang II-induced decrease in total and surface K V 1.5. Immunofluorescence also suggested that Ang II reduced surface K V 1.5 protein in isolated myocytes; an effect inhibited by BIM. Arteries were exposed to Ang II or Ang II plus BIM (for 2 h), after which these agents were removed and contractility measurements performed or myocytes isolated for patch-clamp electrophysiology. Angiotensin II reduced both whole-cell K V currents and currents inhibited by Psora-4, a K V 1.5 channel blocker. Angiotensin II also reduced vasoconstriction stimulated by Psora-4 or 4-aminopyridine, another K V channel inhibitor. These data indicate that Ang II activates protein kinase C, which stimulates K V 1.5 channel degradation, leading to a decrease in surface K V 1.5, a reduction in whole-cell K V 1.5 currents and a loss of functional K V 1.5 channels in myocytes of pressurized arteries. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

PRince: a web server for structural and physicochemical analysis of protein-RNA interface.

PubMed

Barik, Amita; Mishra, Abhishek; Bahadur, Ranjit Prasad

2012-07-01

We have developed a web server, PRince, which analyzes the structural features and physicochemical properties of the protein-RNA interface. Users need to submit a PDB file containing the atomic coordinates of both the protein and the RNA molecules in complex form (in '.pdb' format). They should also mention the chain identifiers of interacting protein and RNA molecules. The size of the protein-RNA interface is estimated by measuring the solvent accessible surface area buried in contact. For a given protein-RNA complex, PRince calculates structural, physicochemical and hydration properties of the interacting surfaces. All these parameters generated by the server are presented in a tabular format. The interacting surfaces can also be visualized with software plug-in like Jmol. In addition, the output files containing the list of the atomic coordinates of the interacting protein, RNA and interface water molecules can be downloaded. The parameters generated by PRince are novel, and users can correlate them with the experimentally determined biophysical and biochemical parameters for better understanding the specificity of the protein-RNA recognition process. This server will be continuously upgraded to include more parameters. PRince is publicly accessible and free for use. Available at http://www.facweb.iitkgp.ernet.in/~rbahadur/prince/home.html.

Silver nanoparticle protein corona composition in cell culture media.

PubMed

Shannahan, Jonathan H; Lai, Xianyin; Ke, Pu Chun; Podila, Ramakrishna; Brown, Jared M; Witzmann, Frank A

2013-01-01

The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC) on their surface. This PC influences the nanomaterial's surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs) including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP) colloidal silver (20 or 110 nm diameter). To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively) compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively), suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index), the PC on 20 nm AgNPs (PVP and citrate) consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of electrostatic and hydrophobic interactions in the formation of the PC which may have broad biological and toxicological implications.

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

PubMed Central

Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

2016-01-01

Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm1

PubMed Central

Mu-Forster, Chen; Wasserman, Bruce P.

1998-01-01

Starch granules from maize (Zea mays) contain a characteristic group of polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol 111: 821–829). Zeins comprise about 50% of the granule-associated proteins, and in this study their spatial distribution within the starch granule was determined. Proteolysis of starch granules at subgelatinization temperatures using the thermophilic protease thermolysin led to selective removal of the zeins, whereas granule-associated proteins of 32 kD or above, including the waxy protein, starch synthase I, and starch-branching enzyme IIb, remained refractory to proteolysis. Granule-associated proteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher. The origin of surface-localized δ-zein was probed by comparing δ-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of δ-zein relative to granules prepared from whole endosperm, thus indicating that δ-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the zeins are deposited on the granule surface as aggregates. In contrast, the granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual granule intrinsic proteins exist as monomers and are not deposited in the form of multimeric clusters within the starch matrix. PMID:9536075

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

PubMed

Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

2015-07-01

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Mina; Moreland, K. Trent; Chen, Jiajun

Here, we investigated the roles of three proteins associated with the formation of otoconia including fetuin A, osteopontin (OPN), and otoconin 90 (OC90). In situ atomic force microscopy (AFM) studies of the effects of these proteins on the growth of atomic steps on calcite surfaces were performed to obtain insight into their effects on the growth kinetics. We also used scanning electron microscopy to examine the effects of these proteins on crystal morphology. All three proteins were found to be potent inhibitors of calcite growth, although fetuin A promoted growth at concentrations below about 40 nM and only became anmore » inhibitor at higher concentrations. We then used in situ optical microscopy to observe calcite nucleation on films of these proteins adsorbed onto mica surfaces. By measuring the calcite nucleation rate as a function of supersaturation, the value of the interfacial energy that controls the free energy barrier to heterogeneous nucleation was determined for each protein. OPN and OC90 films led to significantly reduced interfacial energies as compared to the value for homogeneous calcite nucleation in bulk solution. The value for fetuin A was equal to that for bulk solution within experimental error. Zeta potential measurements showed all of the proteins possessed negative surface charge and varied in magnitude according to sequence fetuin A > OC90 > OPN. In addition, the interfacial energies exhibited an inverse scaling with the zeta potential. In analogy to previous measurements on polysaccharide films, this scaling indicates the differences between the proteins arise from the effect of protein surface charge on the solution–substrate interfacial energy.« less

Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.

PubMed

Bai, Fang; Morcos, Faruck; Cheng, Ryan R; Jiang, Hualiang; Onuchic, José N

2016-12-13

Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

Selective adsorption of bovine hemoglobin on functional TiO2 nano-adsorbents: surface physic-chemical properties determined adsorption activity

NASA Astrophysics Data System (ADS)

Guo, Shiguang; Zhang, Jianghua; Shao, Mingxue; Zhang, Xia; Liu, Yufeng; Xu, Junli; Meng, Hao; Han, Yide

2015-04-01

Surface functionalized nanoparticles are efficient adsorbents which have shown good potential for protein separation. In this work, we chose two different types of organic molecules, oleic acid (OA) and 3-glycidoxypropyltrimethoxy silane (GPTMS), to functionalize the surface of TiO2 nanoparticles, and we studied the effects of this modification on their surface physicochemical properties in correlation with their selective adsorption of proteins. The results showed that the surface zeta potential and the surface water wettability of the modified TiO2 were significantly changed in comparison with the original TiO2 nanoparticles. The adsorption activities of bovine hemoglobin (BHb) and bovine serum albumin (BSA) on these functionalized TiO2 samples were investigated under different conditions, including pH values, contact time, ion strength, and initial protein concentration. In comparison with the non-specific adsorption of original TiO2, however, both the OA-TiO2 and GPTMS-TiO2 exhibited increased BHb adsorption and decreased BSA adsorption at the same time. Using a binary protein mixture as the adsorption object, a higher separation factor (SF) was obtained for OA-TiO2 under optimum conditions. The different adsorption activities of BHb and BSA on the modified TiO2 were correlated with different interactions at the protein/solid interface, and the chemical force as well as the electrostatic force played an important role in the selective adsorption process.

Cytomegalovirus immune evasion by perturbation of endosomal trafficking

PubMed Central

Lučin, Pero; Mahmutefendić, Hana; Blagojević Zagorac, Gordana; Ilić Tomaš, Maja

2015-01-01

Cytomegaloviruses (CMVs), members of the herpesvirus family, have evolved a variety of mechanisms to evade the immune response to survive in infected hosts and to establish latent infection. They effectively hide infected cells from the effector mechanisms of adaptive immunity by eliminating cellular proteins (major histocompatibility Class I and Class II molecules) from the cell surface that display viral antigens to CD8 and CD4 T lymphocytes. CMVs also successfully escape recognition and elimination of infected cells by natural killer (NK) cells, effector cells of innate immunity, either by mimicking NK cell inhibitory ligands or by downregulating NK cell-activating ligands. To accomplish these immunoevasion functions, CMVs encode several proteins that function in the biosynthetic pathway by inhibiting the assembly and trafficking of cellular proteins that participate in immune recognition and thereby, block their appearance at the cell surface. However, elimination of these proteins from the cell surface can also be achieved by perturbation of their endosomal route and subsequent relocation from the cell surface into intracellular compartments. Namely, the physiological route of every cellular protein, including immune recognition molecules, is characterized by specific features that determine its residence time at the cell surface. In this review, we summarize the current understanding of endocytic trafficking of immune recognition molecules and perturbations of the endosomal system during infection with CMVs and other members of the herpesvirus family that contribute to their immune evasion mechanisms. PMID:25263490

Role of Matricellular Proteins in Disorders of the Central Nervous System.

PubMed

Jayakumar, A R; Apeksha, A; Norenberg, M D

2017-03-01

Matricellular proteins (MCPs) are actively expressed non-structural proteins present in the extracellular matrix, which rapidly turnover and possess regulatory roles, as well as mediate cell-cell interactions. MCPs characteristically contain binding sites for other extracellular proteins, cell surface receptors, growth factors, cytokines and proteases, that provide structural support for surrounding cells. MCPs are present in most organs, including brain, and play a major role in cell-cell interactions and tissue repair. Among the MCPs found in brain include thrombospondin-1/2, secreted protein acidic and rich in cysteine family (SPARC), including Hevin/SC1, Tenascin C and CYR61/Connective Tissue Growth Factor/Nov family of proteins, glypicans, galectins, plasminogen activator inhibitor (PAI-1), autotaxin, fibulin and perisostin. This review summarizes the potential role of MCPs in the pathogenesis of major neurological disorders, including Alzheimer's disease, amyotrophic lateral sclerosis, ischemia, trauma, hepatic encephalopathy, Down's syndrome, autism, multiple sclerosis, brain neoplasms, Parkinson's disease and epilepsy. Potential therapeutic opportunities of MCP's for these disorders are also considered in this review.

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein-DNA Complexes That Carry Out DNA Repair.

PubMed

LeBlanc, Sharonda; Wilkins, Hunter; Li, Zimeng; Kaur, Parminder; Wang, Hong; Erie, Dorothy A

2017-01-01

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein-protein and protein-DNA interactions. It can be used to capture snapshots of protein-DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein-protein and protein-DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein-protein and protein-DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein-DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein-protein and protein-nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure-function relationships that govern DNA repair processes. © 2017 Elsevier Inc. All rights reserved.

Modulating protein behaviors on responsive surface by external electric fields: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Xie, Yun; Pan, Yufang; Zhang, Rong; Liang, Ying; Li, Zhanchao

2015-01-01

Molecular dynamics simulations were employed to investigate the modulation of protein behaviors on the electrically responsive zwitterionic phosphorylcholine self-assembled monolayers (PC-SAMs). Results show that PC-SAMs could sensitively respond to the applied electric fields and exhibit three states with different charge distributions, namely both the negatively charged phosphate groups and the positively charged choline groups are exposed to the solution in the absence of electric fields (state 1), phosphate groups exposed in the presence of positive electric fields (state 2), and choline groups exposed in the presence of negative electric fields (state 3). Under state 1, the adsorption of Cyt c on the PC-SAM is reversible and the orientations of Cyt c are randomly distributed. Under state 2, the adsorption of Cyt c is enhanced due to the electrostatic attractions between the exposed phosphate groups and the positively charged protein; when adsorbed on the PC-SAMs, Cyt c tends to adopt the orientation with the heme plane perpendicular to the surface plane, and the percentage of this orientation increases as the field strength rises up. Under state 3, the adsorption of Cyt c is retarded because of the electrostatic repulsions between the exposed choline groups and the protein; however, if the gaps between PC chains are large enough, Cyt c could insert into the PC-SAM and access the phosphate groups after overcoming a slight energy barrier. Under three states, the basic backbone structures of Cyt c are well kept within the simulation time since the conformation of Cyt c is mainly affected by the surface-generated electric fields, whose strengths are modulated by the external electric fields and are not strong enough to deform protein. The results indicate the possibility of regulating protein behaviors, including promoting or retarding protein adsorption and regulating protein orientations, on responsive surfaces by applying electric fields on the surfaces without worrying protein deformation, which may be helpful in the applications of protein separation and controlled drug delivery.

Profiling of acyl-CoA oxidase-deficient and peroxisome proliferator Wy14,643-treated mouse liver protein by surface-enhanced laser desorption/ionization ProteinChip Biology System.

PubMed

Chu, Ruiyin; Zhang, Weihua; Lim, Hanjo; Yeldandi, Anjana V; Herring, Chris; Brumfield, Laura; Reddy, Janardan K; Davison, Matthew

2002-01-01

Peroxisome proliferators induce hepatic peroxisome proliferation and hepatocellular carcinomas in rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolizes lipids, including fatty acids. Mice lacking fatty acyl-CoA oxidase (AOX-/-), the first enzyme of the peroxisomal beta-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation. To investigate proteins involved in peroxisome proliferation, we adopted a novel surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology to compare the protein profiles of control (wild-type), AOX-/-, and wild-type mice treated with peroxisome proliferator, Wy-14,643. The results indicated that the protein profiles of AOX-/- mice were similar to the wild-type mice treated with Wy14,643, but significantly different from the nontreated wild-type mice. Using four different ProteinChip Arrays, a total of 40 protein peaks showed more than twofold changes. Among these differentially expressed peaks, a downregulated peak was identified as the major urinary protein in both AOX-/- and Wyl4,643-treated mice by SELDI. The identification of MUP was further confirmed by two-dimensional electrophoresis and liquid chromatography coupled tandem mass spectrometry (LC-MS-MS). This SELDI method offers several technical advantages for detection of differentially expressed proteins, including ease and speed of screening, no need for chromatographic processing, and small sample size.

Effect of otoconial proteins fetuin A, osteopontin, and otoconin 90 on the nucleation and growth of calcite

DOE PAGES

Hong, Mina; Moreland, K. Trent; Chen, Jiajun; ...

2014-10-30

Here, we investigated the roles of three proteins associated with the formation of otoconia including fetuin A, osteopontin (OPN), and otoconin 90 (OC90). In situ atomic force microscopy (AFM) studies of the effects of these proteins on the growth of atomic steps on calcite surfaces were performed to obtain insight into their effects on the growth kinetics. We also used scanning electron microscopy to examine the effects of these proteins on crystal morphology. All three proteins were found to be potent inhibitors of calcite growth, although fetuin A promoted growth at concentrations below about 40 nM and only became anmore » inhibitor at higher concentrations. We then used in situ optical microscopy to observe calcite nucleation on films of these proteins adsorbed onto mica surfaces. By measuring the calcite nucleation rate as a function of supersaturation, the value of the interfacial energy that controls the free energy barrier to heterogeneous nucleation was determined for each protein. OPN and OC90 films led to significantly reduced interfacial energies as compared to the value for homogeneous calcite nucleation in bulk solution. The value for fetuin A was equal to that for bulk solution within experimental error. Zeta potential measurements showed all of the proteins possessed negative surface charge and varied in magnitude according to sequence fetuin A > OC90 > OPN. In addition, the interfacial energies exhibited an inverse scaling with the zeta potential. In analogy to previous measurements on polysaccharide films, this scaling indicates the differences between the proteins arise from the effect of protein surface charge on the solution–substrate interfacial energy.« less

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device.

PubMed

Battle, Katrina N; Jackson, Joshua M; Witek, Małgorzata A; Hupert, Mateusz L; Hunsucker, Sally A; Armistead, Paul M; Soper, Steven A

2014-03-21

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.

An internal thioester in a pathogen surface protein mediates covalent host binding

PubMed Central

Walden, Miriam; Edwards, John M; Dziewulska, Aleksandra M; Bergmann, Rene; Saalbach, Gerhard; Kan, Su-Yin; Miller, Ona K; Weckener, Miriam; Jackson, Rosemary J; Shirran, Sally L; Botting, Catherine H; Florence, Gordon J; Rohde, Manfred; Banfield, Mark J; Schwarz-Linek, Ulrich

2015-01-01

To cause disease and persist in a host, pathogenic and commensal microbes must adhere to tissues. Colonization and infection depend on specific molecular interactions at the host-microbe interface that involve microbial surface proteins, or adhesins. To date, adhesins are only known to bind to host receptors non-covalently. Here we show that the streptococcal surface protein SfbI mediates covalent interaction with the host protein fibrinogen using an unusual internal thioester bond as a ‘chemical harpoon’. This cross-linking reaction allows bacterial attachment to fibrin and SfbI binding to human cells in a model of inflammation. Thioester-containing domains are unexpectedly prevalent in Gram-positive bacteria, including many clinically relevant pathogens. Our findings support bacterial-encoded covalent binding as a new molecular principle in host-microbe interactions. This represents an as yet unexploited target to treat bacterial infection and may also offer novel opportunities for engineering beneficial interactions. DOI: http://dx.doi.org/10.7554/eLife.06638.001 PMID:26032562

In situ cell surface proteomics reveals differentially expressed membrane proteins in retinal pigment epithelial cells during autoimmune uveitis.

PubMed

Uhl, P B; Szober, C M; Amann, B; Alge-Priglinger, C; Ueffing, M; Hauck, S M; Deeg, C A

2014-09-23

Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from healthy horses and horses with ERU were captured by in situ biotinylation and analysed with high resolution mass spectrometry coupled to liquid chromatography (LC-MS/MS) to identify differentially expressed proteins. With label free differential proteomics, a total of 27 differently expressed cell surface proteins in diseased RPE could be detected. Significant down-regulation of three very interesting proteins, synaptotagmin 1, basigin and collectrin was verified and further characterised. We applied an innovative and successful method to detect changes in the plasma cell surface proteome of RPE cells in a spontaneous inflammatory eye disease, serving as a valuable model for human autoimmune uveitis. We were able to identify 27 differentially expressed plasma cell membrane proteins, including synaptotagmin 1, basigin and collectrin, which play important roles in cell adhesion, transport and cell communication. Copyright © 2014 Elsevier B.V. All rights reserved.

Toward a Molecular Understanding of Protein Solubility: Increased Negative Surface Charge Correlates with Increased Solubility

PubMed Central

Kramer, Ryan M.; Shende, Varad R.; Motl, Nicole; Pace, C. Nick; Scholtz, J. Martin

2012-01-01

Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins. Protein precipitants can be used to obtain comparative solubility measurements and, in some cases, estimations of solubility in buffer alone. Protein precipitants fall into three broad classes: salts, long-chain polymers, and organic solvents. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained for the two different precipitants agree closely with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone. PMID:22768947

Identification of Collagen-Binding Proteins in Lactobacillus spp. with Surface-Enhanced Laser Desorption/Ionization–Time of Flight ProteinChip Technology

PubMed Central

Howard, Jeffrey C.; Heinemann, Christine; Thatcher, Bradley J.; Martin, Brian; Gan, Bing Siang; Reid, Gregor

2000-01-01

Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)–time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions. PMID:11010889

Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors.

PubMed

Nishimura, Tamako; Morone, Nobuhiro; Suetsugu, Shiro

2018-04-17

Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin-Amphiphysin-Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Surface association and the MreB cytoskeleton regulate pilus production, localization and function in Pseudomonas aeruginosa.

PubMed

Cowles, Kimberly N; Gitai, Zemer

2010-06-01

Spatial organization of bacterial proteins influences many cellular processes, including division, chromosome segregation and motility. Virulence-associated proteins also localize to specific destinations within bacterial cells. However, the functions and mechanisms of virulence factor localization remain largely unknown. In this work, we demonstrate that polar assembly of the Pseudomonas aeruginosa PAO1 type IV pilus is regulated by surface association in a manner that affects gene transcription, protein levels and protein localization. We also uncover one mechanism for this regulation that acts through the actin homologue MreB. Inactivation of MreB leads to mislocalization of the pilus retraction ATPase PilT, mislocalization of the pili themselves and a reduction in motility. Furthermore, the role of MreB in polar localization of PilT is modulated by surface association, corroborating our results that environmental factors influence the regulation of pilus production. Specifically, MreB mediates both the initiation and maintenance of PilT localization when cells are grown in suspension but only affects the initiation of localization when cells are grown on a surface. Together, these results suggest that the bacterial cytoskeleton provides a mechanism for the polar localization of P. aeruginosa pili and demonstrate that protein localization may represent an important aspect of virulence factor regulation in bacterial pathogens.

Lacritin and other new proteins of the lacrimal functional unit.

PubMed

McKown, Robert L; Wang, Ningning; Raab, Ronald W; Karnati, Roy; Zhang, Yinghui; Williams, Patricia B; Laurie, Gordon W

2009-05-01

The lacrimal functional unit (LFU) is defined by the 2007 International Dry Eye WorkShop as 'an integrated system comprising the lacrimal glands, ocular surface (cornea, conjunctiva and meibomian glands) and lids, and the sensory and motor nerves that connect them'. The LFU maintains a healthy ocular surface primarily through a properly functioning tear film that provides protection, lubrication, and an environment for corneal epithelial cell renewal. LFU cells express thousands of proteins. Over 200 new LFU proteins have been discovered in the last decade. Lacritin is a new LFU-specific growth factor in human tears that flows through ducts to target corneal epithelial cells on the ocular surface. When applied topically in rabbits, lacritin appears to increase the volume of basal tear secretion. Lacritin is one of only a handful of tear proteins preliminarily reported to be downregulated in blepharitis and in two dry eye syndromes. Computational analysis predicts an ordered C-terminal domain that binds the corneal epithelial cell surface proteoglycan syndecan-1 (SDC1) and is required for lacritin's low nanomolar mitogenic activity. The lacritin-binding site on the N-terminus of SDC1 is exposed by heparanase. Heparanase is constitutively expressed by the corneal epithelium and appears to be a normal constituent of tears. Binding triggers rapid signaling to downstream NFAT and mTOR. A wealth of other new proteins, originally designated as hypothetical when first identified by genomic sequencing, are expressed by the human LFU including: ALS2CL, ARHGEF19, KIAA1109, PLXNA1, POLG, WIPI1 and ZMIZ2. Their demonstrated or implied roles in human genetic disease or basic cellular functions are fuel for new investigation. Addressing topical areas in ocular surface physiology with new LFU proteins may reveal interesting new biological mechanisms and help get to the heart of ocular surface dysfunction.

Theoretical models for electrochemical impedance spectroscopy and local ζ-potential of unfolded proteins in nanopores

NASA Astrophysics Data System (ADS)

Vitarelli, Michael J.; Talaga, David S.

2013-09-01

Single solid-state nanopores find increasing use for electrical detection and/or manipulation of macromolecules. These applications exploit the changes in signals due to the geometry and electrical properties of the molecular species found within the nanopore. The sensitivity and resolution of such measurements are also influenced by the geometric and electrical properties of the nanopore. This paper continues the development of an analytical theory to predict the electrochemical impedance spectra of nanopores by including the influence of the presence of an unfolded protein using the variable topology finite Warburg impedance model previously published by the authors. The local excluded volume of, and charges present on, the segment of protein sampled by the nanopore are shown to influence the shape and peak frequency of the electrochemical impedance spectrum. An analytical theory is used to relate the capacitive response of the electrical double layer at the surface of the protein to both the charge density at the protein surface and the more commonly measured zeta potential. Illustrative examples show how the theory predicts that the varying sequential regions of surface charge density and excluded volume dictated by the protein primary structure may allow for an impedance-based approach to identifying unfolded proteins.

Retriever, a multiprotein complex for retromer-independent endosomal cargo recycling

PubMed Central

McNally, Kerrie E.; Faulkner, Rebecca; Steinberg, Florian; Gallon, Matthew; Ghai, Rajesh; Pim, David; Langton, Paul; Pearson, Neil; Danson, Chris M.; Nägele, Heike; Morris, Lindsey M; Singla, Arnika; Overlee, Brittany L; Heesom, Kate J.; Sessions, Richard; Banks, Lawrence; Collins, Brett M; Berger, Imre; Billadeau, Daniel D.; Burstein, Ezra; Cullen, Peter J.

2018-01-01

Following endocytosis and entry into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are alternatively retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multi-protein complex which orchestrates cargo retrieval and recycling and importantly, is biochemically and functionally distinct to the established retromer pathway. Composed of a heterotrimer of DSCR3, C16orf62 and VPS29, and bearing striking similarity with retromer, we have called this complex ‘retriever’. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to the CCC and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5β1-integrin. Through quantitative proteomic analysis we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, which require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major new endosomal retrieval and recycling pathway. PMID:28892079

Morphology and the Strength of Intermolecular Contact in Protein Crystals

NASA Technical Reports Server (NTRS)

Matsuura, Yoshiki; Chernov, Alexander A.

2002-01-01

The strengths of intermolecular contacts (macrobonds) in four lysozyme crystals were estimated based on the strengths of individual intermolecular interatomic interaction pairs. The periodic bond chain of these macrobonds accounts for the morphology of protein crystals as shown previously. Further in this paper, the surface area of contact, polar coordinate representation of contact site, Coulombic contribution on the macrobond strength, and the surface energy of the crystal have been evaluated. Comparing location of intermolecular contacts in different polymorphic crystal modifications, we show that these contacts can form a wide variety of patches on the molecular surface. The patches are located practically everywhere on this surface except for the concave active site. The contacts frequently include water molecules, with specific intermolecular hydrogen-bonds on the background of non-specific attractive interactions. The strengths of macrobonds are also compared to those of other protein complex systems. Making use of the contact strengths and taking into account bond hydration we also estimated crystal-water interfacial energies for different crystal faces.

Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery.

PubMed

Patching, Simon G

2014-01-01

Surface plasmon resonance (SPR) spectroscopy is a rapidly developing technique for the study of ligand binding interactions with membrane proteins, which are the major molecular targets for validated drugs and for current and foreseeable drug discovery. SPR is label-free and capable of measuring real-time quantitative binding affinities and kinetics for membrane proteins interacting with ligand molecules using relatively small quantities of materials and has potential to be medium-throughput. The conventional SPR technique requires one binding component to be immobilised on a sensor chip whilst the other binding component in solution is flowed over the sensor surface; a binding interaction is detected using an optical method that measures small changes in refractive index at the sensor surface. This review first describes the basic SPR experiment and the challenges that have to be considered for performing SPR experiments that measure membrane protein-ligand binding interactions, most importantly having the membrane protein in a lipid or detergent environment that retains its native structure and activity. It then describes a wide-range of membrane protein systems for which ligand binding interactions have been characterised using SPR, including the major drug targets G protein-coupled receptors, and how challenges have been overcome for achieving this. Finally it describes some recent advances in SPR-based technology and future potential of the technique to screen ligand binding in the discovery of drugs. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding. Copyright © 2013 Elsevier B.V. All rights reserved.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding.

PubMed

Shahi, Payam; Kim, Samuel C; Haliburton, John R; Gartner, Zev J; Abate, Adam R

2017-03-14

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

NASA Astrophysics Data System (ADS)

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-03-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

PubMed Central

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-01-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing. PMID:28290550

Cariogenic potential of foods. II. Relationship of food composition, plaque microbial counts, and salivary parameters to caries in the rat model.

PubMed

Mundorff-Shrestha, S A; Featherstone, J D; Eisenberg, A D; Cowles, E; Curzon, M E; Espeland, M A; Shields, C P

1994-01-01

A series of rat caries experiments was carried out to test the relative cariogenic potential and to identify the major carcinogenic elements of 22 popular snack foods. Parameters that were measured included rat caries, number of cariogenic bacteria in plaque, salivary parameters including flow rate, buffering capacity, total protein, lysozyme and amylase content, and composition of test foods including protein, fat, phosphorus, calcium, fluoride, galactose, glucose, total reducing sugar, sucrose, and starch. Many interesting relationships were observed between food components, numbers of plaque bacteria, salivary components, and specific types of carious lesions. Protein, fat, and phosphorus in foods were all associated with inhibition of both sulcal and buccolingual (smooth-surface) caries. Food fluoride was associated with inhibition of buccolingual caries, whereas calcium was related to inhibition of sulcal caries. Glucose, reducing sugar, and sucrose in foods were all related to promotion of both sulcal and smooth-surface caries. The numbers of Streptococcus sobrinus in plaque were associated with promotion of smooth-surface caries only, whereas lactobacilli, non-mutans bacteria, and total viable flora were related to promotion of both smooth-surface and sulcal caries. The salivary flow rate was associated with inhibition of both buccolingual and sulcal caries. Salivary buffering capacity (at pH 7) and salivary lysozyme delivery were associated with inhibition of number and severity of sulcal caries, while the salivary amylase content was related to the promotion of the number of sulcal lesions.

A New Poly(Methyl Methacrylate) Membrane Dialyzer, NF, with Adsorptive and Antithrombotic Properties.

PubMed

Oshihara, Wataru; Fujieda, Hiroaki; Ueno, Yoshiyuki

2017-01-01

Poly(methyl methacrylate) (PMMA) membranes adsorb several kinds of proteins and can remove high-molecular-weight proteins, including uremic toxins, which are not removed efficiently by hemodialysis or hemodiafiltration. However, the antithrombogenicity of PMMA membranes is insufficient due to their adsorptive properties. Coagulation during hemodialysis occurs because proteins that are adsorbed to the PMMA membrane undergo structural changes and are recognized by platelets, which are then activated by adhesion to the membrane surface. In developing a new PMMA membrane dialyzer, NF, we intended to inhibit platelet adhesion to the membrane surface by suppressing the structural change in the proteins adsorbed on the membrane. In addition, we give examples of clinical trials of the NF in Japan and describe its advantages. Key Message: PMMA membrane dialyzers have been used for 40 years. The PMMA dialyzer NF can suppress the adhesion of platelets to the membrane while maintaining protein adsorption. © 2017 S. Karger AG, Basel.

In Vitro Characterization of the Two-Stage Non-Classical Reassembly Pathway of S-Layers

PubMed Central

Breitwieser, Andreas; Iturri, Jagoba; Toca-Herrera, Jose-Luis; Sleytr, Uwe B.; Pum, Dietmar

2017-01-01

The recombinant bacterial surface layer (S-layer) protein rSbpA of Lysinibacillus sphaericus CCM 2177 is an ideal model system to study non-classical nucleation and growth of protein crystals at surfaces since the recrystallization process may be separated into two distinct steps: (i) adsorption of S-layer protein monomers on silicon surfaces is completed within 5 min and the amount of bound S-layer protein sufficient for the subsequent formation of a closed crystalline monolayer; (ii) the recrystallization process is triggered—after washing away the unbound S-layer protein—by the addition of a CaCl2 containing buffer solution, and completed after approximately 2 h. The entire self-assembly process including the formation of amorphous clusters, the subsequent transformation into crystalline monomolecular arrays, and finally crystal growth into extended lattices was investigated by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Moreover, contact angle measurements showed that the surface properties of S-layers change from hydrophilic to hydrophobic as the crystallization proceeds. This two-step approach is new in basic and application driven S-layer research and, most likely, will have advantages for functionalizing surfaces (e.g., by spray-coating) with tailor-made biological sensing layers. PMID:28216572

Protein disulfide isomerase a multifunctional protein with multiple physiological roles

NASA Astrophysics Data System (ADS)

Ali Khan, Hyder; Mutus, Bulent

2014-08-01

Protein disulfide isomerase (PDI), is a member of the thioredoxin superfamily of redox proteins. PDI has three catalytic activities including, thiol-disulfide oxireductase, disulfide isomerase and redox-dependent chaperone. Originally, PDI was identified in the lumen of the endoplasmic reticulum and subsequently detected at additional locations, such as cell surfaces and the cytosol. This review will provide an overview of the recent advances in relating the structural features of PDI to its multiple catalytic roles as well as its physiological and pathophysiological functions related to redox regulation and protein folding.

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

DOE PAGES

Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.; ...

2014-12-31

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

Analysis of self-assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus.

PubMed

Liu, Jun; Falke, Sven; Drobot, Bjoern; Oberthuer, Dominik; Kikhney, Alexey; Guenther, Tobias; Fahmy, Karim; Svergun, Dmitri; Betzel, Christian; Raff, Johannes

2017-01-01

The formation of stable and functional surface layers (S-layers) via self-assembly of surface-layer proteins on the cell surface is a dynamic and complex process. S-layers facilitate a number of important biological functions, e.g., providing protection and mediating selective exchange of molecules and thereby functioning as molecular sieves. Furthermore, S-layers selectively bind several metal ions including uranium, palladium, gold, and europium, some of them with high affinity. Most current research on surface layers focuses on investigating crystalline arrays of protein subunits in Archaea and bacteria. In this work, several complementary analytical techniques and methods have been applied to examine structure-function relationships and dynamics for assembly of S-layer protein slp-B53 from Lysinibacillus sphaericus: (1) The secondary structure of the S-layer protein was analyzed by circular dichroism spectroscopy; (2) Small-angle X-ray scattering was applied to gain insights into the three-dimensional structure in solution; (3) The interaction with bivalent cations was followed by differential scanning calorimetry; (4) The dynamics and time-dependent assembly of S-layers were followed by applying dynamic light scattering; (5) The two-dimensional structure of the paracrystalline S-layer lattice was examined by atomic force microscopy. The data obtained provide essential structural insights into the mechanism of S-layer self-assembly, particularly with respect to binding of bivalent cations, i.e., Mg 2+ and Ca 2+ . Furthermore, the results obtained highlight potential applications of S-layers in the fields of micromaterials and nanobiotechnology by providing engineered or individual symmetric thin protein layers, e.g., for protective, antimicrobial, or otherwise functionalized surfaces.

Characterization of serum proteins attached to distinct sol-gel hybrid surfaces.

PubMed

Araújo-Gomes, Nuno; Romero-Gavilán, Francisco; Sánchez-Pérez, Ana M; Gurruchaga, Marilo; Azkargorta, Mikel; Elortza, Felix; Martinez-Ibañez, María; Iloro, Ibon; Suay, Julio; Goñi, Isabel

2018-05-01

The success of a dental implant depends on its osseointegration, an important feature of the implant biocompatibility. In this study, two distinct sol-gel hybrid coating formulations [50% methyltrimethoxysilane: 50% 3-glycidoxypropyl-trimethoxysilane (50M50G) and 70% methyltrimethoxysilane with 30% tetraethyl orthosilicate (70M30T)] were applied onto titanium implants. To evaluate their osseointegration, in vitro and in vivo assays were performed. Cell proliferation and differentiation in vitro did not show any differences between the coatings. However, four and eight weeks after in vivo implantation, the fibrous capsule area surrounding 50M50G-implant was 10 and 4 times, respectively, bigger than the area of connective tissue surrounding the 70M30T treated implant. Thus, the in vitro results gave no prediction or explanation for the 50M50G-implant failure in vivo. We hypothesized that the first protein layer adhered to the surface may have direct implication in implant osseointegration, and perhaps correlate with the in vivo outcome. Human serum was used for adsorption analysis on the biomaterials, the first layer of serum proteins adhered to the implant surface was analyzed by proteomic analysis, using mass spectrometry (LC-MS/MS). From the 171 proteins identified; 30 proteins were significantly enriched on the 50M50G implant surface. This group comprised numerous proteins of the immune complement system, including several subcomponents of the C1 complement, complement factor H, C4b-binding protein alpha chain, complement C5 and C-reactive protein. This result suggests that these proteins enriched in 50M50G surface might trigger the cascade leading to the formation of the fibrous capsule observed. The implications of these results could open up future possibilities to predict the biocompatibility problems in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1477-1485, 2018. © 2017 Wiley Periodicals, Inc.

Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

PubMed Central

Martin-DeLeon, Patricia A

2015-01-01

A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca2+-ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca2+ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion–competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca2+-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach. PMID:26112481

Epididymosomes: transfer of fertility-modulating proteins to the sperm surface.

PubMed

Martin-DeLeon, Patricia A

2015-01-01

A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca 2 + -ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca 2 + efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca 2 + -dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.

Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling interfacial chemistry using fluorous-phase surfactants.

PubMed

Roach, L Spencer; Song, Helen; Ismagilov, Rustem F

2005-02-01

Control of surface chemistry and protein adsorption is important for using microfluidic devices for biochemical analysis and high-throughput screening assays. This paper describes the control of protein adsorption at the liquid-liquid interface in a plug-based microfluidic system. The microfluidic system uses multiphase flows of immiscible fluorous and aqueous fluids to form plugs, which are aqueous droplets that are completely surrounded by fluorocarbon oil and do not come into direct contact with the hydrophobic surface of the microchannel. Protein adsorption at the aqueous-fluorous interface was controlled by using surfactants that were soluble in fluorocarbon oil but insoluble in aqueous solutions. Three perfluorinated alkane surfactants capped with different functional groups were used: a carboxylic acid, an alcohol, and a triethylene glycol group that was synthesized from commercially available materials. Using complementary methods of analysis, adsorption was characterized for several proteins (bovine serum albumin (BSA) and fibrinogen), including enzymes (ribonuclease A (RNase A) and alkaline phosphatase). These complementary methods involved characterizing adsorption in microliter-sized droplets by drop tensiometry and in nanoliter plugs by fluorescence microscopy and kinetic measurements of enzyme catalysis. The oligoethylene glycol-capped surfactant prevented protein adsorption in all cases. Adsorption of proteins to the carboxylic acid-capped surfactant in nanoliter plugs could be described by using the Langmuir model and tensiometry results for microliter drops. The microfluidic system was fabricated using rapid prototyping in poly(dimethylsiloxane) (PDMS). Black PDMS microfluidic devices, fabricated by curing a suspension of charcoal in PDMS, were used to measure the changes in fluorescence intensity more sensitively. This system will be useful for microfluidic bioassays, enzymatic kinetics, and protein crystallization, because it does not require surface modification during fabrication to control surface chemistry and protein adsorption.

The human fatty acid-binding protein family: Evolutionary divergences and functions

PubMed Central

2011-01-01

Fatty acid-binding proteins (FABPs) are members of the intracellular lipid-binding protein (iLBP) family and are involved in reversibly binding intracellular hydrophobic ligands and trafficking them throughout cellular compartments, including the peroxisomes, mitochondria, endoplasmic reticulum and nucleus. FABPs are small, structurally conserved cytosolic proteins consisting of a water-filled, interior-binding pocket surrounded by ten anti-parallel beta sheets, forming a beta barrel. At the superior surface, two alpha-helices cap the pocket and are thought to regulate binding. FABPs have broad specificity, including the ability to bind long-chain (C16-C20) fatty acids, eicosanoids, bile salts and peroxisome proliferators. FABPs demonstrate strong evolutionary conservation and are present in a spectrum of species including Drosophila melanogaster, Caenorhabditis elegans, mouse and human. The human genome consists of nine putatively functional protein-coding FABP genes. The most recently identified family member, FABP12, has been less studied. PMID:21504868

The gene for fibroblast activation protein {alpha} (FAP), a putative cell surface-bound serine protease expressed in cancer stroma and wound healing, maps to chromosome band 2q23

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathew, S.; Murty, V.V.V.S.; Chaganti, R.S.K.

The human fibroblast activation protein {alpha} (FAP{alpha}) is an inducible cell surface glycoprotein of M{sub r} 95,000 recognized by a number of monoclonal antibodies (mAbs), including the prototype mAb F19. Immunohistochemical studies have shown that FAP{alpha} expression in vivo is tightly regulated, with transient expression in some fetal mesenchymal tissues but absence of expression in most normal adult tissues. Reexpression of FAP{alpha} is observed in the reactive stromal fibroblasts of several common types of epithelial cancers, including >90% of breast, colorectal, and lung carcinomas and healing wounds. Cloning and sequence analysis of an FAP{alpha}-specific cDNA has revealed that the moleculemore » is encoded by a novel gene, FAP, which shows sequence similarity to members of the serine protease family of integral membrane proteins, namely dipeptidyl peptidase IV (DPPIV, also known as lymphocyte activation antigen, CD26, or adenosine dearoinase binding protein) and DPPX, a DPPIV-related molecule of unknown function. 15 refs., 1 fig.« less

Formation of stable submicron peptide or protein particles by thin film freezing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnston, Keith P.; Engstrom, Joshua; Williams, III, Robert O.

The present invention includes compositions and methods for preparing micron-sized or submicron-sized particles by dissolving a water soluble effective ingredient in one or more solvents; spraying or dripping droplets solvent such that the effective ingredient is exposed to a vapor-liquid interface of less than 50, 100, 150, 200, 250, 200, 400 or 500 cm.sup.-1 area/volume to, e.g., increase protein stability; and contacting the droplet with a freezing surface that has a temperature differential of at least 30.degree. C. between the droplet and the surface, wherein the surface freezes the droplet into a thin film with a thickness of less thanmore » 500 micrometers and a surface area to volume between 25 to 500 cm.sup.-1.« less

Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing.

PubMed

Bidlingmaier, Scott; Ha, Kevin; Lee, Nam-Kyung; Su, Yang; Liu, Bin

2016-04-01

Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D2synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

NASA Astrophysics Data System (ADS)

Martin, Nicholas J.; Bunch, Josephine; Cooper, Helen J.

2013-08-01

Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology

PubMed Central

2011-01-01

Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution. PMID:21338175

Water at protein surfaces studied with femtosecond nonlinear spectroscopy

NASA Astrophysics Data System (ADS)

Bakker, Huib J.

We report on an investigation of the structure and dynamics of water molecules near protein surfaces with femtosecond nonlinear spectroscopic techniques. We measured the reorientation dynamics of water molecules near the surface of several globular protein surfaces, using polarization-resolved femtosecond infrared spectroscopy. We found that water molecules near the protein surface have a much slower reorientation than water molecules in bulk liquid water. The number of slow water molecules scales scales with the size of the hydrophobic surface of the protein. When we denature the proteins by adding an increasing amount of urea to the protein solution, we observe that the water-exposed surface increases by 50% before the secondary structure of the proteins changes. This finding indicates that protein unfolding starts with the protein structure becoming less tight, thereby allowing water to enter. With surface vibrational sum frequency generation (VSFG) spectroscopy, we studied the structure of water at the surface of antifreeze protein III. The measured VSFG spectra showed the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution, at temperatures well above the freezing point. This ordered ice-like hydration layers at the protein surface likely plays an important role in the specific recognition and binding of anti-freeze protein III to nascent ice crystallites, and thus in its anti-freeze mechanism. This research is supported by the ''Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO).

Identification of surface domain structure on enamel crystals using polyamidoamine dendrimer

NASA Astrophysics Data System (ADS)

Chen, Haifeng; Clarkson, Brian H.; Orr, Bradford; Majoros, Istvan; Banaszak Holl, Mark M.

2002-03-01

The control of hydroxyapatite crystal nucleation and crystal growth is central to the mineralization and remineralization of enamel and dentin of teeth. However, the precise biomolecular mechanisms involved remain obscure. The intimate association between the crystal's surface and extracellular protein components implies a modulating role for organic crystal interactions probably mediated via specific crystal surface domains. These include lattice defects and specific stereochemical arrays on associated organic molecules. The nature of protein-crystal interaction depends upon the physical forces of attraction / repulsion between specific biomolecular groups and crystal surface domains. The proposed study is to utilize specific polyamidoamine (PAMAM) dendrimers, also known as “artificial proteins”, acting as nanoprobe. These will be used to probe specific surface domain on the surface of the naturally derived crystals of hydroxyapatite and to determine how control of growth and dissolution may be affected at the biomolecular level. The hydroxyapatite crystals are extracted from the maturation stage enamel of rats. Three types of PAMAM dendrimers, respectively with amine-, carboxylic acid and methyl-capped surface, will be applied in the study. The dendrimer binding on the surface of the hydoxyapatite crystals will be characterized using atomic force microscopy (AFM). The different dendrimer binding on the crystals will disclose the specific surface domain structure on the crystals, which is assumed to be important in binding the extracellular protein.

Modulation of protein stability and aggregation properties by surface charge engineering.

PubMed

Raghunathan, Govindan; Sokalingam, Sriram; Soundrarajan, Nagasundarapandian; Madan, Bharat; Munussami, Ganapathiraman; Lee, Sun-Gu

2013-09-01

An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15-17) and s-GFP(+5-6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.

Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins.

PubMed

Mizumoto, Shuji; Yamada, Shuhei; Sugahara, Kazuyuki

2015-10-01

Recent functional studies on chondroitin sulfate-dermatan sulfate (CS-DS) demonstrated its indispensable roles in various biological events including brain development and cancer. CS-DS proteoglycans exert their physiological activity through interactions with specific proteins including growth factors, cell surface receptors, and matrix proteins. The characterization of these interactions is essential for regulating the biological functions of CS-DS proteoglycans. Although amino acid sequences on the bioactive proteins required for these interactions have already been elucidated, the specific saccharide sequences involved in the binding of CS-DS to target proteins have not yet been sufficiently identified. In this review, recent findings are described on the interaction between CS-DS and some proteins which are especially involved in the central nervous system and cancer development/metastasis. Copyright © 2015. Published by Elsevier Ltd.

Mapping and mutagenesis of the amino-terminal transcriptional repression domain of the Drosophila Krüppel protein.

PubMed Central

Licht, J D; Hanna-Rose, W; Reddy, J C; English, M A; Ro, M; Grossel, M; Shaknovich, R; Hansen, U

1994-01-01

We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region. Images PMID:8196644

Cell-surface prion protein interacts with glycosaminoglycans.

PubMed Central

Pan, Tao; Wong, Boon-Seng; Liu, Tong; Li, Ruliang; Petersen, Robert B; Sy, Man-Sun

2002-01-01

We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23-35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs. PMID:12186633

Proteome analysis of the plant pathogen Xylella fastidiosa reveals major cellular and extracellular proteins and a peculiar codon bias distribution.

PubMed

Smolka, Marcus Bustamante; Martins-de-Souza, Daniel; Martins, Daniel; Winck, Flavia Vischi; Santoro, Carlos Eduardo; Castellari, Rafael Ramos; Ferrari, Fernanda; Brum, Itaraju Junior; Galembeck, Eduardo; Della Coletta Filho, Helvécio; Machado, Marcos Antonio; Marangoni, Sergio; Novello, Jose Camillo

2003-02-01

The bacteria Xylella fastidiosa is the causative agent of a number of economically important crop diseases, including citrus variegated chlorosis. Although its complete genome is already sequenced, X. fastidiosa is very poorly characterized by biochemical approaches at the protein level. In an initial effort to characterize protein expression in X. fastidiosa we used one- and two-dimensional gel electrophoresis and mass spectrometry to identify the products of 142 genes present in a whole cell extract and in an extracellular fraction of the citrus isolated strain 9a5c. Of particular interest for the study of pathogenesis are adhesion and secreted proteins. Homologs to proteins from three different adhesion systems (type IV fimbriae, mrk pili and hsf surface fibrils) were found to be coexpressed, the last two being detected only as multimeric complexes in the high molecular weight region of one-dimensional electrophoresis gels. Using a procedure to extract secreted proteins as well as proteins weakly attached to the cell surface we identified 30 different proteins including toxins, adhesion related proteins, antioxidant enzymes, different types of proteases and 16 hypothetical proteins. These data suggest that the intercellular space of X. fastidiosa colonies is a multifunctional microenvironment containing proteins related to in vivo bacterial survival and pathogenesis. A codon usage analysis of the most expressed proteins from the whole cell extract revealed a low biased distribution, which we propose is related to the slow growing nature of X. fastidiosa. A database of the X. fastidiosa proteome was developed and can be accessed via the internet (URL: www.proteome.ibi.unicamp.br).

Acquired pellicle as a modulator for dental erosion.

PubMed

Vukosavljevic, Dusa; Custodio, William; Buzalaf, Marilia A R; Hara, Anderson T; Siqueira, Walter L

2014-06-01

Dental erosion is a multifactorial condition that can result in the loss of tooth structure and function, potentially increasing tooth sensitivity. The exposure of enamel to acids from non-bacterial sources is responsible for the progression of erosion. These erosive challenges are counteracted by the anti-erosive properties of the acquired pellicle (AP), an integument formed in vivo as a result of selective adsorption of salivary proteins on the tooth surface, containing also lipids and glycoproteins. This review provides an in-depth discussion regarding how the physical structure of the AP, along with its composition, contributes to AP anti-erosive properties. The physical properties that contribute to AP protective nature include pellicle thickness, maturation time, and site of development. The pellicle contains salivary proteins embedded within its structure that demonstrate anti-erosive properties; however, rather than individual proteins, protein-protein interactions play a fundamental role in the protective nature of the AP. In addition, dietary and synthetic proteins can modify the pellicle, enhancing its protective efficiency against dental erosion. The salivary composition of the AP and its corresponding protein-profile may be employed as a diagnostic tool, since it likely contains salivary biomarkers for oral diseases that initiate at the enamel surface, including dental erosion. Finally, by modifying the composition and structure of the AP, this protein integument has the potential to be used as a target-specific treatment option for oral diseases related to tooth demineralization. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Sequence composition and environment effects on residue fluctuations in protein structures

NASA Astrophysics Data System (ADS)

Ruvinsky, Anatoly M.; Vakser, Ilya A.

2010-10-01

Structure fluctuations in proteins affect a broad range of cell phenomena, including stability of proteins and their fragments, allosteric transitions, and energy transfer. This study presents a statistical-thermodynamic analysis of relationship between the sequence composition and the distribution of residue fluctuations in protein-protein complexes. A one-node-per-residue elastic network model accounting for the nonhomogeneous protein mass distribution and the interatomic interactions through the renormalized inter-residue potential is developed. Two factors, a protein mass distribution and a residue environment, were found to determine the scale of residue fluctuations. Surface residues undergo larger fluctuations than core residues in agreement with experimental observations. Ranking residues over the normalized scale of fluctuations yields a distinct classification of amino acids into three groups: (i) highly fluctuating-Gly, Ala, Ser, Pro, and Asp, (ii) moderately fluctuating-Thr, Asn, Gln, Lys, Glu, Arg, Val, and Cys, and (iii) weakly fluctuating-Ile, Leu, Met, Phe, Tyr, Trp, and His. The structural instability in proteins possibly relates to the high content of the highly fluctuating residues and a deficiency of the weakly fluctuating residues in irregular secondary structure elements (loops), chameleon sequences, and disordered proteins. Strong correlation between residue fluctuations and the sequence composition of protein loops supports this hypothesis. Comparing fluctuations of binding site residues (interface residues) with other surface residues shows that, on average, the interface is more rigid than the rest of the protein surface and Gly, Ala, Ser, Cys, Leu, and Trp have a propensity to form more stable docking patches on the interface. The findings have broad implications for understanding mechanisms of protein association and stability of protein structures.

Magnetic capture of polydopamine-encapsulated Hela cells for the analysis of cell surface proteins.

PubMed

Liu, Yiying; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

2018-02-10

A novel method to characterize cell surface proteins and complexes has been developed. Polydopamine (PDA)-encapsulated Hela cells were prepared for plasma membrane proteome research. Since the PDA protection, the encapsulated cells could be maintained for more than two weeks. Amino groups functionalized magnetic nanoparticles were also used for cell capture by the reaction with the PDA coatings. Plasma membrane fragments were isolated and enriched with assistance of an external magnetic field after disruption of the coated cells by ultrasonic treatment. Plasma membrane proteins (PMPs) and complexes were well preserved on the fragments and identified by shot-gun proteomic analytical strategy. 385 PMPs and 1411 non-PMPs were identified using the method. 85.2% of these PMPs were lipid-raft associated proteins. Ingenuity Pathway Analysis was employed for bio-information extraction from the identified proteins. It was found that 653 non-PMPs had interactions with 140 PMPs. Among them, epidermal growth factor receptor and its complexes, and a series of important pathways including STAT3 pathway were observed. All these results demonstrated that the new approach is of great importance in applying to the research of physiological function and mechanism of the plasma membrane proteins. This work developed a novel strategy for the proteomic analysis of cell surface proteins. According to the results, 73.3% of total identified proteins were lipid-raft associated proteins, which imply that the proposed method is of great potential in the identification of lipid-raft associated proteins. In addition, a series of protein-protein interactions and pathways related to Hela cells were pointed out. All these results demonstrated that our proposed approach is of great importance and could well be applied to the physiological function and mechanism research of plasma membrane proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Yeast arming systems: pros and cons of different protein anchors and other elements required for display.

PubMed

Andreu, Cecilia; Del Olmo, Marcel Lí

2018-03-01

Yeast display is a powerful strategy that consists in exposing peptides or proteins of interest on the cell surface of this microorganism. Ever since initial experiments with this methodology were carried out, its scope has extended and many applications have been successfully developed in different science and technology fields. Several yeast display systems have been designed, which all involve introducting into yeast cells the gene fusions that contain the coding regions of a signal peptide, an anchor protein, to properly attach the target to the cell surface, and the protein of interest to be exposed, all of which are controlled by a strong promoter. In this work, we report the description of such elements for the alternative systems introduced by focusing particularly on anchor proteins. The comparisons made between them are included whenever possible, and the main advantages and inconveniences of each one are discussed. Despite the huge number of publications on yeast surface display and the revisions published to date, this topic has not yet been widely considered. Finally, given the growing interest in developing systems for non-Saccharomyces yeasts, the main strategies reported for some are also summarized.

Thermodynamics of coupled protein adsorption and stability using hybrid Monte Carlo simulations.

PubMed

Zhong, Ellen D; Shirts, Michael R

2014-05-06

A better understanding of changes in protein stability upon adsorption can improve the design of protein separation processes. In this study, we examine the coupling of the folding and the adsorption of a model protein, the B1 domain of streptococcal protein G, as a function of surface attraction using a hybrid Monte Carlo (HMC) approach with temperature replica exchange and umbrella sampling. In our HMC implementation, we are able to use a molecular dynamics (MD) time step that is an order of magnitude larger than in a traditional MD simulation protocol and observe a factor of 2 enhancement in the folding and unfolding rate. To demonstrate the convergence of our systems, we measure the travel of our order parameter the fraction of native contacts between folded and unfolded states throughout the length of our simulations. Thermodynamic quantities are extracted with minimum statistical variance using multistate reweighting between simulations at different temperatures and harmonic distance restraints from the surface. The resultant free energies, enthalpies, and entropies of the coupled unfolding and absorption processes are in qualitative agreement with previous experimental and computational observations, including entropic stabilization of the adsorbed, folded state relative to the bulk on surfaces with low attraction.

[Optimization of prokaryotic expression conditions of Leptospira interrogans trigeminy genus-specific protein antigen based on surface response analysis].

PubMed

Wang, Jiang; Luo, Dongjiao; Sun, Aihua; Yan, Jie

2008-07-01

Lipoproteins LipL32 and LipL21 and transmembrane protein OMPL1 have been confirmed as the superficial genus-specific antigens of Leptospira interrogans, which can be used as antigens for developing a universal genetic engineering vaccine. In order to obtain high expression of an artificial fusion gene lipL32/1-lipL21-ompL1/2, we optimized prokaryotic expression conditions. We used surface response analysis based on the central composite design to optimize culture conditions of a new antigen protein by recombinant Escherichia coli DE3.The culture conditions included initial pH, induction start time, post-induction time, Isopropyl beta-D-thiogalactopyranoside (IPTG) concentration, and temperature. The maximal production of antigen protein was 37.78 mg/l. The optimal culture conditions for high recombinant fusion protein was determined: initial pH 7.9, induction start time 2.5 h, a post-induction time of 5.38 h, 0.20 mM IPTG, and a post-induction temperature of 31 degrees C. Surface response analysis based on CCD increased the target production. This statistical method reduced the number of experiments required for optimization and enabled rapid identification and integration of the key culture condition parameters for optimizing recombinant protein expression.

Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence.

PubMed

Sugden, Scott M; Bego, Mariana G; Pham, Tram N Q; Cohen, Éric A

2016-03-03

The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection.

Suspended marine particulate proteins in coastal and oligotrophic waters

NASA Astrophysics Data System (ADS)

Bridoux, Maxime C.; Neibauer, Jaqui; Ingalls, Anitra E.; Nunn, Brook L.; Keil, Richard G.

2015-03-01

Metaproteomic analyses were performed on suspended sediments collected in one coastal environment (Washington margin, Pacific Ocean, n = 5) and two oligotrophic environments (Atlantic Ocean near BATS, n = 5, and Pacific Ocean near HOTS, n = 5). Using a database of 2.3 million marine proteins developed using the NCBI database, 443 unique peptides were detected from which 363 unique proteins were identified. Samples from the euphotic zone contained on average 2-3x more identifiable proteins than deeper waters (150-1500 m) and these proteins were predominately from photosynthetic organisms. Diatom peptides dominate the spectra of the Washington margin while peptides from cyanobacteria, such as Synechococcus sp. dominated the spectra of both oligotrophic sites. Despite differences in the exact proteins identified at each location, there is good agreement for protein function and cellular location. Proteins in surface waters code for a variety of cellular functions including photosynthesis (24% of detected proteins), energy production (10%), membrane production (9%) and genetic coding and reading (9%), and are split 60-40 between membrane proteins and intracellular cytoplasmic proteins. Sargasso Sea surface waters contain a suite of peptides consistent with proteins involved in circadian rhythms that promote both C and N fixation at night. At depth in the Sargasso Sea, both muscle-derived myosin protein and the muscle-hydrolyzing proteases deseasin MCP-01 and metalloprotease Mcp02 from γ-proteobacteria were observed. Deeper waters contain peptides predominately sourced from γ-proteobacteria (37% of detected proteins) and α-proteobacteria (26%), although peptides from membrane and photosynthetic proteins attributable to phytoplankton were still observed (13%). Relative to surface values, detection frequencies for bacterial membrane proteins and extracellular enzymes rose from 9 to 16 and 2 to 4% respectively below the thermocline and the overall balance between membrane proteins and intracellular proteins grows to an approximate 75-25 split. Unlike the phytoplankton membrane proteins, which are detrital in nature, the bacterial protein suite at depth is consistent with living biomass.

Effects of surface wettability and contact time on protein adhesion to biomaterial surfaces

PubMed Central

Xu, Li-Chong; Siedlecki, Christopher A.

2013-01-01

Atomic force microscopy (AFM) was used to directly measure the adhesion forces between three test proteins and low density polyethylene (LDPE) surfaces treated by glow discharge plasma to yield various levels of water wettability. The adhesion of proteins to the LDPE substrates showed a step dependence on the wettability of surfaces as measured by the water contact angle (θ). For LDPE surfaces with θ > ∼60–65°, stronger adhesion forces were observed for bovine serum albumin, fibrinogen and human FXII than for the surfaces with θ < 60°. Smaller adhesion forces were observed for FXII than for the other two proteins on all surfaces although trends were identical. Increasing the contact time from 0 to 50 s for each protein–surface combination increased the adhesion force regardless of surface wettability. Time varying adhesion data was fit to an exponential model and free energies of protein unfolding were calculated. This data, viewed in light of previously published studies, suggests a 2-step model of protein denaturation, an early stage on the order of seconds to minutes where the outer surface of the protein interacts with the substrate and a second stage involving movement of hydrophobic amino acids from the protein core to the protein/surface interface. Impact statement The work described in this manuscript shows a stark transition between protein adherent and protein non-adherent materials in the range of water contact angles 60–65°, consistent with known changes in protein adsorption and activity. Time-dependent changes in adhesion force were used to calculate unfolding energies relating to protein–surface interactions. This analysis provides justification for a 2-step model of protein denaturation on surfaces. PMID:17466368

A mobile precursor determines protein resistance on nanostructured surfaces.

PubMed

Wang, Kang; Chen, Ye; Gong, Xiangjun; Xia, Jianlong; Zhao, Junpeng; Shen, Lei

2018-05-09

Biomaterials are often engineered with nanostructured surfaces to control interactions with proteins and thus regulate their biofunctions. However, the mechanism of how nanostructured surfaces resist or attract proteins together with the underlying design rules remains poorly understood at a molecular level, greatly limiting attempts to develop high-performance biomaterials and devices through the rational design of nanostructures. Here, we study the dynamics of nonspecific protein adsorption on block copolymer nanostructures of varying adhesive domain areas in a resistant matrix. Using surface plasmon resonance and single molecule tracking techniques, we show that weakly adsorbed proteins with two-dimensional diffusivity are critical precursors to protein resistance on nanostructured surfaces. The adhesive domain areas must be more than tens or hundreds of times those of the protein footprints to slow down the 2D-mobility of the precursor proteins for their irreversible adsorption. This precursor model can be used to quantitatively analyze the kinetics of nonspecific protein adsorption on nanostructured surfaces. Our method is applicable to precisely manipulate protein adsorption and resistance on various nanostructured surfaces, e.g., amphiphilic, low-surface-energy, and charged nanostructures, for the design of protein-compatible materials.

Surface Mediated Protein Disaggregation

NASA Astrophysics Data System (ADS)

Radhakrishna, Mithun; Kumar, Sanat K.

2014-03-01

Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

Dynamic nanoplatforms in biosensor and membrane constitutional systems.

PubMed

Mahon, Eugene; Aastrup, Teodor; Barboiu, Mihail

2012-01-01

Molecular recognition in biological systems occurs mainly at interfacial environments such as membrane surfaces, enzyme active sites, or the interior of the DNA double helix. At the cell membrane surface, carbohydrate-protein recognition principles apply to a range of specific non-covalent interactions including immune response, cell proliferation, adhesion and death, cell-cell interaction and communication. Protein-protein recognition meanwhile accounts for signalling processes and ion channel structure. In this chapter we aim to describe such constitutional dynamic interfaces for biosensing and membrane transport applications. Constitutionally adaptive interfaces may mimic the recognition capabilities intrinsic to natural recognition processes. We present some recent examples of 2D and 3D constructed sensors and membranes of this type and describe their sensing and transport capabilities.

The Arabidopsis SOS5 Locus Encodes a Putative Cell Surface Adhesion Protein and Is Required for Normal Cell Expansion

PubMed Central

Shi, Huazhong; Kim, YongSig; Guo, Yan; Stevenson, Becky; Zhu, Jian-Kang

2003-01-01

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein–like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf. PMID:12509519

Phospholipids as an alternative to direct covalent coupling: surface functionalization of nanoporous alumina for protein recognition and purification.

PubMed

Lazzara, Thomas D; Behn, Daniela; Kliesch, Torben-Tobias; Janshoff, Andreas; Steinem, Claudia

2012-01-15

Anodic aluminum oxide (AAO) substrates with aligned, cylindrical, non-intersecting pores with diameters of 75 nm and depths of 3.5 or 10 μm were functionalized with lipid monolayers harboring different receptor lipids. AAO was first functionalized with dodecyl-trichlorosilane, followed by fusion of small unilamellar vesicles (SUVs) forming a lipid monolayer. The SUVs' lipid composition was transferred onto the AAO surface, allowing us to control the surface receptor density. Owing to the optical transparency of the AAO, the overall vesicle spreading process and subsequent protein binding to the receptor-doped lipid monolayers could be investigated in situ by optical waveguide spectroscopy (OWS). SUV spreading occurred at the pore-rim interface, followed by lateral diffusion of lipids within the pore-interior surface until homogeneous coverage was achieved with a lipid monolayer. The functionality of the system was demonstrated through streptavidin binding onto a biotin-DOPE containing POPC membrane, showing maximum protein coverage at 10 mol% of biotin-DOPE. The system enabled us to monitor in real-time the selective extraction of two histidine-tagged proteins, PIGEA14 (14 kDa) and ezrin (70 kDa), directly from cell lysate solutions using a DOGS-NTA(Ni)/DOPC (1:9) membrane. The purification process including protein binding and elution was monitored by OWS and confirmed by SDS-PAGE. Copyright © 2011 Elsevier Inc. All rights reserved.

Exhaustively sampling peptide adsorption with metadynamics.

PubMed

Deighan, Michael; Pfaendtner, Jim

2013-06-25

Simulating the adsorption of a peptide or protein and obtaining quantitative estimates of thermodynamic observables remains challenging for many reasons. One reason is the dearth of molecular scale experimental data available for validating such computational models. We also lack simulation methodologies that effectively address the dual challenges of simulating protein adsorption: overcoming strong surface binding and sampling conformational changes. Unbiased classical simulations do not address either of these challenges. Previous attempts that apply enhanced sampling generally focus on only one of the two issues, leaving the other to chance or brute force computing. To improve our ability to accurately resolve adsorbed protein orientation and conformational states, we have applied the Parallel Tempering Metadynamics in the Well-Tempered Ensemble (PTMetaD-WTE) method to several explicitly solvated protein/surface systems. We simulated the adsorption behavior of two peptides, LKα14 and LKβ15, onto two self-assembled monolayer (SAM) surfaces with carboxyl and methyl terminal functionalities. PTMetaD-WTE proved effective at achieving rapid convergence of the simulations, whose results elucidated different aspects of peptide adsorption including: binding free energies, side chain orientations, and preferred conformations. We investigated how specific molecular features of the surface/protein interface change the shape of the multidimensional peptide binding free energy landscape. Additionally, we compared our enhanced sampling technique with umbrella sampling and also evaluated three commonly used molecular dynamics force fields.

Reversibly immobilized biological materials in monolayer films on electrodes

DOEpatents

Weaver, P.F.; Frank, A.J.

1993-05-04

Methods and techniques are described for reversibly binding charged biological particles in a fluid medium to an electrode surface. The methods are useful in a variety of applications. The biological materials may include microbes, proteins, and viruses. The electrode surface may consist of reversibly electroactive materials such as polyvinylferrocene, silicon-linked ferrocene or quinone.

Reversibly immobilized biological materials in monolayer films on electrodes

DOEpatents

Weaver, Paul F.; Frank, Arthur J.

1993-01-01

Methods and techniques are described for reversibly binding charged biological particles in a fluid medium to an electrode surface. The methods are useful in a variety of applications. The biological materials may include microbes, proteins, and viruses. The electrode surface may consist of reversibly electroactive materials such as polyvinylferrocene, silicon-linked ferrocene or quinone.

Optimization of implant/bone attachment: The effects of implant surface porosity, bioactive ceramic coatings, and delivery of adsorbed growth factors

NASA Astrophysics Data System (ADS)

Melican, Mora Carolynne

Various surface treatments and coating materials have been tested for use on metal alloy orthopaedic implants. Their purpose has been to enhance the bioactivity of the implant surfaces, and thus to increase the rate and degree of bony attachment in vivo in an attempt to hasten recovery time, increase implant service lifetime, and lessen pain associated with loosened orthopaedic implants. A series of in vivo and in vitro studies were performed to determine the influence of different implant surfaces including porous metal surfaces with varied porosity with depth, resorbable and non-resorbable plasma-sprayed hydroxyapatite (HA) coatings, and finally HA coatings with an adsorbed layer of human recombinant bone morphogenetic protein (rhBMP-2), an osteoinductive protein. Textured as-cast metal surfaces produced by investment casting in three dimensionally printed ceramic molds have exhibited superior bony ingrowth and attachment. Plasma-sprayed HA coatings have been shown to be appropriate substrates for osteoblast proliferation (particularly on highly crystalline HA) and stem cell proliferation (particularly on less crystalline HA). Less crystalline HA coatings have shown promise as delivery systems for different levels of rhBMP-2. The osteoinductive protein has been shown to remain active after delivery to the system, and was most effective when delivered in concentrations ranging from 30 to 50 ng/ml. Combinations of these surface treatments for metal implant surfaces warrant further investigation.

Hairy and Slippery Polyoxazoline-Based Copolymers on Model and Cartilage Surfaces.

PubMed

Morgese, Giulia; Ramakrishna, Shivaprakash N; Simic, Rok; Zenobi-Wong, Marcy; Benetti, Edmondo M

2018-02-12

Comb-like polymers presenting a hydroxybenzaldehyde (HBA)-functionalized poly(glutamic acid) (PGA) backbone and poly(2-methyl-2-oxazoline) (PMOXA) side chains chemisorb on aminolized substrates, including cartilage surfaces, forming layers that reduce protein contamination and provide lubrication. The structure, physicochemical, biopassive, and tribological properties of PGA-PMOXA-HBA films are finely determined by the copolymer architecture, its reactivity toward the surface, i.e. PMOXA side-chain crowding and HBA density, and by the copolymer solution concentration during assembly. Highly reactive species with low PMOXA content form inhomogeneous layers due to the limited possibility of surface rearrangements by strongly anchored copolymers, just partially protecting the functionalized surface from protein contamination and providing a relatively weak lubrication on cartilage. Biopassivity and lubrication can be improved by increasing copolymer concentration during assembly, leading to a progressive saturation of surface defects across the films. In a different way, less reactive copolymers presenting high PMOXA side-chain densities form uniform, biopassive, and lubricious films, both on model aminolized silicon oxide surfaces, as well as on cartilage substrates. When assembled at low concentrations these copolymers adopt a "lying down" conformation, i.e. adhering via their backbones onto the substrates, while at high concentrations they undergo a conformational transition, assuming a more densely packed, "standing up" structure, where they stretch perpendicularly from the substrate. This specific arrangement reduces protein contamination and improves lubrication both on model as well as on cartilage surfaces.

Adsorption of the natural protein surfactant Rsn-2 onto liquid interfaces.

PubMed

Brandani, Giovanni B; Vance, Steven J; Schor, Marieke; Cooper, Alan; Kennedy, Malcolm W; Smith, Brian O; MacPhee, Cait E; Cheung, David L

2017-03-22

To stabilize foams, droplets and films at liquid interfaces a range of protein biosurfactants have evolved in nature. Compared to synthetic surfactants, these combine surface activity with biocompatibility and low solution aggregation. One recently studied example is Rsn-2, a component of the foam nest of the frog Engystomops pustulosus, which has been predicted to undergo a clamshell-like opening transition at the air-water interface. Using atomistic molecular dynamics simulations and surface tension measurements we study the adsorption of Rsn-2 onto air-water and cyclohexane-water interfaces. The protein adsorbs readily at both interfaces, with adsorption mediated by the hydrophobic N-terminus. At the cyclohexane-water interface the clamshell opens, due to the favourable interaction between hydrophobic residues and cyclohexane molecules and the penetration of cyclohexane molecules into the protein core. Simulations of deletion mutants showed that removal of the N-terminus inhibits interfacial adsorption, which is consistent with the surface tension measurements. Deletion of the hydrophilic C-terminus also affects adsorption, suggesting that this plays a role in orienting the protein at the interface. The characterisation of the interfacial behaviour gives insight into the factors that control the interfacial adsorption of proteins, which may inform new applications of this and similar proteins in areas including drug delivery and food technology and may also be used in the design of synthetic molecules showing similar changes in conformation at interfaces.

Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: advances and applications.

PubMed

Hyeon, Jeong Eun; Jeon, Sang Duck; Han, Sung Ok

2013-11-01

The cellulosome is one of nature's most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. Each protein module in the cellulosome system is potentially useful in an advanced biotechnology application. The high-affinity interactions between the cohesin and dockerin domains can be used in protein-based biosensors to improve both sensitivity and selectivity. The scaffolding protein includes a carbohydrate-binding module (CBM) that attaches strongly to cellulose substrates and facilitates the purification of proteins fused with the dockerin module through a one-step CBM purification method. Although the surface layer homology (SLH) domain of CbpA is not present in other strains, replacement of the cell surface anchoring domain allows a foreign protein to be displayed on the surface of other strains. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. Thus, the development of various configurations of multi-functional protein complexes for use as tools in whole-cell biocatalyst systems has drawn considerable attention as an attractive strategy for bioprocess applications. This review provides a detailed summary of the current achievements in Clostridium-derived multi-functional complex development and the impact of these complexes in various areas of biotechnology. Copyright © 2013 Elsevier Inc. All rights reserved.

Heat shock proteins on the human sperm surface.

PubMed

Naaby-Hansen, Soren; Herr, John C

2010-01-01

The sperm plasma membrane is known to be critical to fertilization and to be highly regionalized into domains of head, mid- and principal pieces. However, the molecular composition of the sperm plasma membrane and its alterations during genital tract passage, capacitation and the acrosome reaction remains to be fully dissected. A two-dimensional gel-based proteomic study previously identified 98 human sperm proteins which were accessible for surface labelling with both biotin and radioiodine. In this report twelve dually labelled protein spots were excised from stained gels or PDVF membranes and analysed by mass spectrometry (MS) and Edman degradation. Seven members from four different heat shock protein (HSP) families were identified including HYOU1 (ORP150), HSPC1 (HSP86), HSPA5 (Bip), HSPD1 (HSP60), and several isoforms of the two testis-specific HSP70 chaperones HSPA2 and HSPA1L. An antiserum raised against the testis-specific HSPA2 chaperone reacted with three 65kDa HSPA2 isoforms and three high molecular weight surface proteins (78-79kDa, 84kDa and 90-93kDa). These proteins, together with seven 65kDa HSP70 forms, reacted with human anti-sperm IgG antibodies that blocked in vitro fertilization in humans. Three of these surface biotinylated human sperm antigens were immunoprecipitated with a rabbit antiserum raised against a linear peptide epitope in Chlamydia trachomatis HSP70. The results indicate diverse HSP chaperones are accessible for surface labelling on human sperm. Some of these share epitopes with C. trachomatis HSP70, suggesting an association between genital tract infection, immunity to HSP70 and reproductive failure. 2009 Elsevier Ireland Ltd. All rights reserved.

Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: mechanisms and mysteries.

PubMed

Billings, Paul C; Pacifici, Maurizio

2015-01-01

Heparan sulfate (HS) is a component of cell surface and matrix-associated proteoglycans (HSPGs) that, collectively, play crucial roles in many physiologic processes including cell differentiation, organ morphogenesis and cancer. A key function of HS is to bind and interact with signaling proteins, growth factors, plasma proteins, immune-modulators and other factors. In doing so, the HS chains and HSPGs are able to regulate protein distribution, bio-availability and action on target cells and can also serve as cell surface co-receptors, facilitating ligand-receptor interactions. These proteins contain an HS/heparin-binding domain (HBD) that mediates their association and contacts with HS. HBDs are highly diverse in sequence and predicted structure, contain clusters of basic amino acids (Lys and Arg) and possess an overall net positive charge, most often within a consensus Cardin-Weintraub (CW) motif. Interestingly, other domains and residues are now known to influence protein-HS interactions, as well as interactions with other glycosaminoglycans, such as chondroitin sulfate. In this review, we provide a description and analysis of HBDs in proteins including amphiregulin, fibroblast growth factor family members, heparanase, sclerostin and hedgehog protein family members. We discuss HBD structural and functional features and important roles carried out by other protein domains, and also provide novel conformational insights into the diversity of CW motifs present in Sonic, Indian and Desert hedgehogs. Finally, we review progress in understanding the pathogenesis of a rare pediatric skeletal disorder, Hereditary Multiple Exostoses (HME), characterized by HS deficiency and cartilage tumor formation. Advances in understanding protein-HS interactions will have broad implications for basic biology and translational medicine as well as for the development of HS-based therapeutics.

Human IgG1 Responses to Surface Localised Schistosoma mansoni Ly6 Family Members Drop following Praziquantel Treatment.

PubMed

Chalmers, Iain W; Fitzsimmons, Colin M; Brown, Martha; Pierrot, Christine; Jones, Frances M; Wawrzyniak, Jakub M; Fernandez-Fuentes, Narcis; Tukahebwa, Edridah M; Dunne, David W; Khalife, Jamal; Hoffmann, Karl F

2015-01-01

The heptalaminate-covered, syncytial tegument is an important anatomical adaptation that enables schistosome parasites to maintain long-term, intravascular residence in definitive hosts. Investigation of the proteins present in this surface layer and the immune responses elicited by them during infection is crucial to our understanding of host/parasite interactions. Recent studies have revealed a number of novel tegumental surface proteins including three (SmCD59a, SmCD59b and Sm29) containing uPAR/Ly6 domains (renamed SmLy6A SmLy6B and SmLy6D in this study). While vaccination with SmLy6A (SmCD59a) and SmLy6D (Sm29) induces protective immunity in experimental models, human immunoglobulin responses to representative SmLy6 family members have yet to be thoroughly explored. Using a PSI-BLAST-based search, we present a comprehensive reanalysis of the Schistosoma mansoni Ly6 family (SmLy6A-K). Our examination extends the number of members to eleven (including three novel proteins) and provides strong evidence that the previously identified vaccine candidate Sm29 (renamed SmLy6D) is a unique double uPAR/Ly6 domain-containing representative. Presence of canonical cysteine residues, signal peptides and GPI-anchor sites strongly suggest that all SmLy6 proteins are cell surface-bound. To provide evidence that SmLy6 members are immunogenic in human populations, we report IgG1 (as well as IgG4 and IgE) responses against two surface-bound representatives (SmLy6A and SmLy6B) within a cohort of S. mansoni-infected Ugandan males before and after praziquantel treatment. While pre-treatment IgG1 prevalence for SmLy6A and SmLy6B differs amongst the studied population (7.4% and 25.3% of the cohort, respectively), these values are both higher than IgG1 prevalence (2.7%) for a sub-surface tegumental antigen, SmTAL1. Further, post-treatment IgG1 levels against surface-associated SmLy6A and SmLy6B significantly drop (p = 0.020 and p < 0.001, respectively) when compared to rising IgG1 levels against sub-surface SmTAL1. Collectively, these results expand the number of SmLy6 proteins found within S. mansoni and specifically demonstrate that surface-associated SmLy6A and SmLy6B elicit immunological responses during infection in endemic communities.

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells.

PubMed

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A; Multhoff, Gabriele

2005-06-15

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

Heat Shock Protein 70 Surface-Positive Tumor Exosomes Stimulate Migratory and Cytolytic Activity of Natural Killer Cells

PubMed Central

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A.; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A.

2006-01-01

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo−/CX exosomes were Hsp70 membrane derived positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes. PMID:15958569

Dynamics simulations for engineering macromolecular interactions

NASA Astrophysics Data System (ADS)

Robinson-Mosher, Avi; Shinar, Tamar; Silver, Pamela A.; Way, Jeffrey

2013-06-01

The predictable engineering of well-behaved transcriptional circuits is a central goal of synthetic biology. The artificial attachment of promoters to transcription factor genes usually results in noisy or chaotic behaviors, and such systems are unlikely to be useful in practical applications. Natural transcriptional regulation relies extensively on protein-protein interactions to insure tightly controlled behavior, but such tight control has been elusive in engineered systems. To help engineer protein-protein interactions, we have developed a molecular dynamics simulation framework that simplifies features of proteins moving by constrained Brownian motion, with the goal of performing long simulations. The behavior of a simulated protein system is determined by summation of forces that include a Brownian force, a drag force, excluded volume constraints, relative position constraints, and binding constraints that relate to experimentally determined on-rates and off-rates for chosen protein elements in a system. Proteins are abstracted as spheres. Binding surfaces are defined radially within a protein. Peptide linkers are abstracted as small protein-like spheres with rigid connections. To address whether our framework could generate useful predictions, we simulated the behavior of an engineered fusion protein consisting of two 20 000 Da proteins attached by flexible glycine/serine-type linkers. The two protein elements remained closely associated, as if constrained by a random walk in three dimensions of the peptide linker, as opposed to showing a distribution of distances expected if movement were dominated by Brownian motion of the protein domains only. We also simulated the behavior of fluorescent proteins tethered by a linker of varying length, compared the predicted Förster resonance energy transfer with previous experimental observations, and obtained a good correspondence. Finally, we simulated the binding behavior of a fusion of two ligands that could simultaneously bind to distinct cell-surface receptors, and explored the landscape of linker lengths and stiffnesses that could enhance receptor binding of one ligand when the other ligand has already bound to its receptor, thus, addressing potential mechanisms for improving targeted signal transduction proteins. These specific results have implications for the design of targeted fusion proteins and artificial transcription factors involving fusion of natural domains. More broadly, the simulation framework described here could be extended to include more detailed system features such as non-spherical protein shapes and electrostatics, without requiring detailed, computationally expensive specifications. This framework should be useful in predicting behavior of engineered protein systems including binding and dissociation reactions.

Dynamics simulations for engineering macromolecular interactions.

PubMed

Robinson-Mosher, Avi; Shinar, Tamar; Silver, Pamela A; Way, Jeffrey

2013-06-01

The predictable engineering of well-behaved transcriptional circuits is a central goal of synthetic biology. The artificial attachment of promoters to transcription factor genes usually results in noisy or chaotic behaviors, and such systems are unlikely to be useful in practical applications. Natural transcriptional regulation relies extensively on protein-protein interactions to insure tightly controlled behavior, but such tight control has been elusive in engineered systems. To help engineer protein-protein interactions, we have developed a molecular dynamics simulation framework that simplifies features of proteins moving by constrained Brownian motion, with the goal of performing long simulations. The behavior of a simulated protein system is determined by summation of forces that include a Brownian force, a drag force, excluded volume constraints, relative position constraints, and binding constraints that relate to experimentally determined on-rates and off-rates for chosen protein elements in a system. Proteins are abstracted as spheres. Binding surfaces are defined radially within a protein. Peptide linkers are abstracted as small protein-like spheres with rigid connections. To address whether our framework could generate useful predictions, we simulated the behavior of an engineered fusion protein consisting of two 20,000 Da proteins attached by flexible glycine/serine-type linkers. The two protein elements remained closely associated, as if constrained by a random walk in three dimensions of the peptide linker, as opposed to showing a distribution of distances expected if movement were dominated by Brownian motion of the protein domains only. We also simulated the behavior of fluorescent proteins tethered by a linker of varying length, compared the predicted Förster resonance energy transfer with previous experimental observations, and obtained a good correspondence. Finally, we simulated the binding behavior of a fusion of two ligands that could simultaneously bind to distinct cell-surface receptors, and explored the landscape of linker lengths and stiffnesses that could enhance receptor binding of one ligand when the other ligand has already bound to its receptor, thus, addressing potential mechanisms for improving targeted signal transduction proteins. These specific results have implications for the design of targeted fusion proteins and artificial transcription factors involving fusion of natural domains. More broadly, the simulation framework described here could be extended to include more detailed system features such as non-spherical protein shapes and electrostatics, without requiring detailed, computationally expensive specifications. This framework should be useful in predicting behavior of engineered protein systems including binding and dissociation reactions.

Swarming: flexible roaming plans.

PubMed

Partridge, Jonathan D; Harshey, Rasika M

2013-03-01

Movement over an agar surface via swarming motility is subject to formidable challenges not encountered during swimming. Bacteria display a great deal of flexibility in coping with these challenges, which include attracting water to the surface, overcoming frictional forces, and reducing surface tension. Bacteria that swarm on "hard" agar surfaces (robust swarmers) display a hyperflagellated and hyperelongated morphology. Bacteria requiring a "softer" agar surface (temperate swarmers) do not exhibit such a dramatic morphology. For polarly flagellated robust swarmers, there is good evidence that restriction of flagellar rotation somehow signals the induction of a large number of lateral flagella, but this scenario is apparently not relevant to temperate swarmers. Swarming bacteria can be further subdivided by their requirement for multiple stators (Mot proteins) or a stator-associated protein (FliL), secretion of essential polysaccharides, cell density-dependent gene regulation including surfactant synthesis, a functional chemotaxis signaling pathway, appropriate cyclic (c)-di-GMP levels, induction of virulence determinants, and various nutritional requirements such as iron limitation or nitrate availability. Swarming strategies are as diverse as the bacteria that utilize them. The strength of these numerous designs stems from the vantage point they offer for understanding mechanisms for effective colonization of surface niches, acquisition of pathogenic potential, and identification of environmental signals that regulate swarming. The signature swirling and streaming motion within a swarm is an interesting phenomenon in and of itself, an emergent behavior with properties similar to flocking behavior in diverse systems, including birds and fish, providing a convenient new avenue for modeling such behavior.

Swarming: Flexible Roaming Plans

PubMed Central

Partridge, Jonathan D.

2013-01-01

Movement over an agar surface via swarming motility is subject to formidable challenges not encountered during swimming. Bacteria display a great deal of flexibility in coping with these challenges, which include attracting water to the surface, overcoming frictional forces, and reducing surface tension. Bacteria that swarm on “hard” agar surfaces (robust swarmers) display a hyperflagellated and hyperelongated morphology. Bacteria requiring a “softer” agar surface (temperate swarmers) do not exhibit such a dramatic morphology. For polarly flagellated robust swarmers, there is good evidence that restriction of flagellar rotation somehow signals the induction of a large number of lateral flagella, but this scenario is apparently not relevant to temperate swarmers. Swarming bacteria can be further subdivided by their requirement for multiple stators (Mot proteins) or a stator-associated protein (FliL), secretion of essential polysaccharides, cell density-dependent gene regulation including surfactant synthesis, a functional chemotaxis signaling pathway, appropriate cyclic (c)-di-GMP levels, induction of virulence determinants, and various nutritional requirements such as iron limitation or nitrate availability. Swarming strategies are as diverse as the bacteria that utilize them. The strength of these numerous designs stems from the vantage point they offer for understanding mechanisms for effective colonization of surface niches, acquisition of pathogenic potential, and identification of environmental signals that regulate swarming. The signature swirling and streaming motion within a swarm is an interesting phenomenon in and of itself, an emergent behavior with properties similar to flocking behavior in diverse systems, including birds and fish, providing a convenient new avenue for modeling such behavior. PMID:23264580

New methods for the analysis of the protein-solvent interface

NASA Astrophysics Data System (ADS)

Goodfellow, Julia M.; Pitt, William R.; Smart, Oliver S.; Williams, Mark A.

1995-09-01

The protein-solvent interface is complex and may include solvent channels and cavities as well as the normal surface water molecules. We describe several algorithms for investigating the intra- and inter-molecular interactions of proteins in general but with the aim of developing methods to accurately and definitively characterise the interactions of water and other small ligands with proteins. Specifically, we present the methods which underlie three programs (AQUARIUS2, HOLE and PRO_ACT) which can be used to to look at different aspects of these interactions.

Separation techniques: Chromatography

PubMed Central

Coskun, Ozlem

2016-01-01

Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification. PMID:28058406

Competitive binding effects on surface-enhanced Raman scattering of peptide molecules

NASA Astrophysics Data System (ADS)

Seballos, Leo; Richards, Nicole; Stevens, Daniel J.; Patel, Mira; Kapitzky, Laura; Lokey, Scott; Millhauser, Glenn; Zhang, Jin Z.

2007-10-01

Surface enhanced Raman scattering (SERS) has been conducted on tryptophan (W), proline (P) and tyrosine (Y) containing peptides that include W-P-Y, Y-P-W, W-P-P-P-Y, Y-P-P-P-W, W-P-P-P-P-P-Y, and Y-P-P-P-P-P-W to gain insight into molecular binding behavior on a metal substrate to eventually apply in protein SERS detection. The peptides are shown to bind through the molecule's carboxylic end, but the strong affinity of the tryptophan residue to the substrate surface, in conjunction with its large polarizability, dominates each molecule's SERS signal with the strong presence of its ring modes in all samples. These results are important for understanding SERS of protein molecules.

The synthesis and protein resistance of amphiphilic PDMS-b-(PDMS-g-cysteine) copolymers

NASA Astrophysics Data System (ADS)

Lei, Yufeng; Lin, Yaling; Zhang, Anqiang

2017-10-01

Zwitterionic polymers have been used to cope with nonspecific protein adsorption and bio-fouling problems for a wide range of materials, including biomedical devices, marine coatings and membrane separation. However, direct surface modification with highly water-soluble zwitterionic polymers is rather difficult due to their poor attachment to hydrophobic solid surfaces. In this work, we utilize the hydrophobic interaction to anchor zwitterionic polysiloxanes grafted with cysteine onto surfaces by adding an hydrophobic block of polydimethylsiloxanes, referred as PDMS-b-(PDMS-g-Cys)s. The synthesis involves only three steps of reactions, and the structures of each product were characterized using GPC, FT-IR and 1H NMR. The adsorption and protein resistance of PDMS-b-(PDMS-g-Cys)s on a gold surface are investigated with QCM-D. The results show that the hydrophobic interaction moieties of the additional PDMS blocks help the hydrophilic cysteine-grafted blocks stably attach and then function on the sensor. These findings suggest that the addition of hydrophobic moieties provides an effective approach to construct anti-fouling interfaces with zwitterionic polymers in aqueous solution.

A model of adsorption of albumin on the implant surface titanium and titanium modified carbon coatings (MWCNT-EPD). 2D correlation analysis

NASA Astrophysics Data System (ADS)

Wesełucha-Birczyńska, Aleksandra; Stodolak-Zych, Ewa; Piś, Wojciech; Długoń, Elżbieta; Benko, Aleksandra; Błażewicz, Marta

2016-11-01

Common materials used as orthopedic implants are titanium and its alloys. To improve its compatibility with the environment of a living organism titanium implant surfaces are covered with bioactive layers of MWCNT. During the insertion into a living organism such material is exposed to direct contact with the patient's blood, which includes proteins - eg. albumin. The adsorption of albumin may constitute one of the early stages of implant surface modification serving cell adhesion. An analysis of this phenomenon in terms of the kinetics of deposition of protein on the surface of the implant confirms its biocompatibility in vivo. The proposed working model of the adsorption of albumin allows for choosing the best of time for the protein to form a stable connection with the surface of the titanium implant. Traditional methods of materials engineering and chemistry allow for the obtaining of information about the presence of a protein on the surface (UV-Vis, the wettability). The application of 2D correlation analysis, in turn, gains insight into the dynamics of the changes associated with the deposition of protein (the formation of a uniform layer, the change in conformation). This analysis has allowed for the selection of an optimal time of protein adsorption to the surface of the implant. Better compatibility with the body of the implant provides its modification by introducing layers that accelerate the material-tissue interactions. Such a composition is a layer of carbon nanotubes (MWCNTs) deposited on titanium by the electrophoretic (EPD) method. Using Raman spectroscopy and analyzing the spectra with the 2D correlation method it is possible to gain insight into the molecular structure of this layer. Our studies indicate that albumin in contact with the surface of titanium has obtained stable conformation after 30 min (confirmed by: UV-Vis, Raman). Shifts of the CH2, CH3 stretching bands position as well as an analysis of the amide I band confirms this conformation. The dynamics of these changes are noticed as correlation peaks observed on 2D maps.

Shapes of embedded minimal surfaces

PubMed Central

Colding, Tobias H.; Minicozzi, William P.

2006-01-01

Surfaces that locally minimize area have been extensively used to model physical phenomena, including soap films, black holes, compound polymers, protein folding, etc. The mathematical field dates to the 1740s but has recently become an area of intense mathematical and scientific study, specifically in the areas of molecular engineering, materials science, and nanotechnology because of their many anticipated applications. In this work, we show that all minimal surfaces are built out of pieces of the surfaces in Figs. 1 and 2. PMID:16847265

Preferred orientation of albumin adsorption on a hydrophilic surface from molecular simulation.

PubMed

Hsu, Hao-Jen; Sheu, Sheh-Yi; Tsay, Ruey-Yug

2008-12-01

In general, non-specific protein adsorption follows a two-step procedure, i.e. first adsorption onto a surface in native form, and a subsequent conformational change on the surface. In order to predict the subsequent conformational change, it is important to determine the preferred orientation of an adsorbed protein in the first step of the adsorption. In this work, a method based on finding the global minimum of the interaction potential energy of an adsorbed protein has been developed to delineate the preferred orientations for the adsorption of human serum albumin (HSA) on a model surface with a hydrophilic self-assembled monolayer (SAM). For computational efficiency, solvation effects were greatly simplified by only including the dampening of electrostatic effects while neglecting contributions due to the competition of water molecules for the functional groups on the surface. A contour map obtained by systematic rotation of a molecule in conjunction with perpendicular motion to the surface gives the minimum interaction energy of the adsorbed molecule at various adsorption orientations. Simulation results show that for an -OH terminated SAM surface, a "back-on" orientation of HSA is the preferred orientation. The projection area of this adsorption orientation corresponds with the "triangular-side-on" adsorption of a heart shaped HSA molecule. The method proposed herein is able to provide results which are consistent with those predicted by Monte Carlo (MC) simulations with a substantially less computing cost. The high computing efficiency of the current method makes it possible to be implemented as a design tool for the control of protein adsorption on surfaces; however, before this can be fully realized, these methods must be further developed to enable interaction free energy to be calculated in place of potential energy, along with a more realistic representation of solvation effects.

Exploration of the Energy Landscape of Acetylcholinesterase by Molecular Dynamics Simulation.

NASA Astrophysics Data System (ADS)

McCammon, J. Andrew

2002-03-01

Proteins have rough energy landscapes. Often more states than just the ground state are occupied and have biological functions. It is essential to study these conformational substates and the dynamical transitions among them. Acetylcholinesterase (AChE) is an important enzyme that has biological functions including the termination of synaptic transmission signals. X-ray structures show that it has an active site that is accessible only via a long and narrow channel from its surface. Therefore the fact that acetylcholine and larger ligands can reach the active site is believed to reflect the protein's structural fluctuation. We carried out long molecular dynamics simulations to investigate the dynamics of AChE and its relation to biological function, and compared our results with experiments. The results reveal several "doors" that open intermittantly between the active site and the surface. Instead of having simple exponential decay correlation functions, the time series of these channels reveal complex, fractal gating between conformations. We also compared the AChE dynamics data with those from an AchE-fasciculin complex. (Fasciculin is a small protein that is a natural inhibitor of AChE.) The results show remarkable effects of the protein-protein interaction, including allosteric and dynamical inhibition by fasciculin besides direct steric blocking. More information and images can be found at http://mccammon.ucsd.edu

Hydrophobic potential of mean force as a solvation function for protein structure prediction.

PubMed

Lin, Matthew S; Fawzi, Nicolas Lux; Head-Gordon, Teresa

2007-06-01

We have developed a solvation function that combines a Generalized Born model for polarization of protein charge by the high dielectric solvent, with a hydrophobic potential of mean force (HPMF) as a model for hydrophobic interaction, to aid in the discrimination of native structures from other misfolded states in protein structure prediction. We find that our energy function outperforms other reported scoring functions in terms of correct native ranking for 91% of proteins and low Z scores for a variety of decoy sets, including the challenging Rosetta decoys. This work shows that the stabilizing effect of hydrophobic exposure to aqueous solvent that defines the HPMF hydration physics is an apparent improvement over solvent-accessible surface area models that penalize hydrophobic exposure. Decoys generated by thermal sampling around the native-state basin reveal a potentially important role for side-chain entropy in the future development of even more accurate free energy surfaces.

Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance

PubMed Central

Ritzefeld, Markus; Sewald, Norbert

2012-01-01

Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR. PMID:22500214

Water dynamics in protein hydration shells: the molecular origins of the dynamical perturbation.

PubMed

Fogarty, Aoife C; Laage, Damien

2014-07-17

Protein hydration shell dynamics play an important role in biochemical processes including protein folding, enzyme function, and molecular recognition. We present here a comparison of the reorientation dynamics of individual water molecules within the hydration shell of a series of globular proteins: acetylcholinesterase, subtilisin Carlsberg, lysozyme, and ubiquitin. Molecular dynamics simulations and analytical models are used to access site-resolved information on hydration shell dynamics and to elucidate the molecular origins of the dynamical perturbation of hydration shell water relative to bulk water. We show that all four proteins have very similar hydration shell dynamics, despite their wide range of sizes and functions, and differing secondary structures. We demonstrate that this arises from the similar local surface topology and surface chemical composition of the four proteins, and that such local factors alone are sufficient to rationalize the hydration shell dynamics. We propose that these conclusions can be generalized to a wide range of globular proteins. We also show that protein conformational fluctuations induce a dynamical heterogeneity within the hydration layer. We finally address the effect of confinement on hydration shell dynamics via a site-resolved analysis and connect our results to experiments via the calculation of two-dimensional infrared spectra.

Water Dynamics in Protein Hydration Shells: The Molecular Origins of the Dynamical Perturbation

PubMed Central

2014-01-01

Protein hydration shell dynamics play an important role in biochemical processes including protein folding, enzyme function, and molecular recognition. We present here a comparison of the reorientation dynamics of individual water molecules within the hydration shell of a series of globular proteins: acetylcholinesterase, subtilisin Carlsberg, lysozyme, and ubiquitin. Molecular dynamics simulations and analytical models are used to access site-resolved information on hydration shell dynamics and to elucidate the molecular origins of the dynamical perturbation of hydration shell water relative to bulk water. We show that all four proteins have very similar hydration shell dynamics, despite their wide range of sizes and functions, and differing secondary structures. We demonstrate that this arises from the similar local surface topology and surface chemical composition of the four proteins, and that such local factors alone are sufficient to rationalize the hydration shell dynamics. We propose that these conclusions can be generalized to a wide range of globular proteins. We also show that protein conformational fluctuations induce a dynamical heterogeneity within the hydration layer. We finally address the effect of confinement on hydration shell dynamics via a site-resolved analysis and connect our results to experiments via the calculation of two-dimensional infrared spectra. PMID:24479585

JET2 Viewer: a database of predicted multiple, possibly overlapping, protein-protein interaction sites for PDB structures.

PubMed

Ripoche, Hugues; Laine, Elodie; Ceres, Nicoletta; Carbone, Alessandra

2017-01-04

The database JET2 Viewer, openly accessible at http://www.jet2viewer.upmc.fr/, reports putative protein binding sites for all three-dimensional (3D) structures available in the Protein Data Bank (PDB). This knowledge base was generated by applying the computational method JET 2 at large-scale on more than 20 000 chains. JET 2 strategy yields very precise predictions of interacting surfaces and unravels their evolutionary process and complexity. JET2 Viewer provides an online intelligent display, including interactive 3D visualization of the binding sites mapped onto PDB structures and suitable files recording JET 2 analyses. Predictions were evaluated on more than 15 000 experimentally characterized protein interfaces. This is, to our knowledge, the largest evaluation of a protein binding site prediction method. The overall performance of JET 2 on all interfaces are: Sen = 52.52, PPV = 51.24, Spe = 80.05, Acc = 75.89. The data can be used to foster new strategies for protein-protein interactions modulation and interaction surface redesign. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase.

PubMed Central

Charbonneau, H; Tonks, N K; Walsh, K A; Fischer, E H

1988-01-01

A major protein tyrosine phosphatase (PTPase 1B) has been isolated in essentially homogeneous form from the soluble and particulate fractions of human placenta. Unexpectedly, partial amino acid sequences displayed no homology with the primary structures of the protein Ser/Thr phosphatases deduced from cDNA clones. However, the sequence is strikingly similar to the tandem C-terminal homologous domains of the leukocyte common antigen (CD45). A 157-residue segment of PTPase 1B displayed 40% and 33% sequence identity with corresponding regions from cytoplasmic domains I and II of human CD45. Similar degrees of identity have been observed among the catalytic domains of families of regulatory proteins such as protein kinases and cyclic nucleotide phosphodiesterases. On this basis, it is proposed that the CD45 family has protein tyrosine phosphatase activity and may represent a set of cell-surface receptors involved in signal transduction. This suggests that the repertoire of signal transduction mechanisms may include the direct control of an intracellular protein tyrosine phosphatase, offering the possibility of a regulatory balance with those protein tyrosine kinases that act at the internal surface of the membrane. Images PMID:2845400

Activation of cell-surface proteases promotes necroptosis, inflammation and cell migration.

PubMed

Cai, Zhenyu; Zhang, Anling; Choksi, Swati; Li, Weihua; Li, Tao; Zhang, Xue-Min; Liu, Zheng-Gang

2016-08-01

Necroptosis is a programmed, caspase-independent cell death that is morphologically similar to necrosis. TNF-induced necroptosis is mediated by receptor-interacting protein kinases, RIP1 and RIP3, and the mixed lineage kinase domain-like (MLKL). After being phosphorylated by RIP3, MLKL is translocated to the plasma membrane and mediates necroptosis. However, the execution of necroptosis and its role in inflammation and other cellular responses remain largely elusive. In this study, we report that MLKL-mediated activation of cell-surface proteases of the a disintegrin and metalloprotease (ADAM) family promotes necroptosis, inflammation and cell migration. ADAMs are specifically activated at the early stage of necroptosis when MLKL is phosphorylated and translocated to the cell plasma membrane. Activation of ADAMs induces ectodomain shedding of diverse cell-surface proteins including adhesion molecules, receptors, growth factors and cytokines. Importantly, the shedding of cell-surface proteins disrupts cell adhesion and accelerates necroptosis, while the soluble fragments of the cleaved proteins trigger the inflammatory responses. We also demonstrate that the shedding of E-cadherin ectodomain from necroptotic cells promotes cell migration. Thus, our study provides a novel mechanism of necroptosis-induced inflammation and new insights into the physiological and pathological functions of this unique form of cell death.

Activation of cell-surface proteases promotes necroptosis, inflammation and cell migration

PubMed Central

Cai, Zhenyu; Zhang, Anling; Choksi, Swati; Li, Weihua; Li, Tao; Zhang, Xue-Min; Liu, Zheng-Gang

2016-01-01

Necroptosis is a programmed, caspase-independent cell death that is morphologically similar to necrosis. TNF-induced necroptosis is mediated by receptor-interacting protein kinases, RIP1 and RIP3, and the mixed lineage kinase domain-like (MLKL). After being phosphorylated by RIP3, MLKL is translocated to the plasma membrane and mediates necroptosis. However, the execution of necroptosis and its role in inflammation and other cellular responses remain largely elusive. In this study, we report that MLKL-mediated activation of cell-surface proteases of the a disintegrin and metalloprotease (ADAM) family promotes necroptosis, inflammation and cell migration. ADAMs are specifically activated at the early stage of necroptosis when MLKL is phosphorylated and translocated to the cell plasma membrane. Activation of ADAMs induces ectodomain shedding of diverse cell-surface proteins including adhesion molecules, receptors, growth factors and cytokines. Importantly, the shedding of cell-surface proteins disrupts cell adhesion and accelerates necroptosis, while the soluble fragments of the cleaved proteins trigger the inflammatory responses. We also demonstrate that the shedding of E-cadherin ectodomain from necroptotic cells promotes cell migration. Thus, our study provides a novel mechanism of necroptosis-induced inflammation and new insights into the physiological and pathological functions of this unique form of cell death. PMID:27444869

Mapping Protein Surface Accessibility via an Electron Transfer Dissociation Selectively Cleavable Hydrazone Probe*

PubMed Central

Vasicek, Lisa; O'Brien, John P.; Browning, Karen S.; Tao, Zhihua; Liu, Hung-Wen; Brodbelt, Jennifer S.

2012-01-01

A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C. PMID:22393264

Lacritin and Other New Proteins of the Lacrimal Functional Unit

PubMed Central

McKown, Robert L.; Wang, Ningning; Raab, Ronald W.; Karnati, Roy; Zhang, Yinghui; Williams, Patricia B.; Laurie, Gordon W.

2009-01-01

The lacrimal functional unit (LFU) is defined by the 2007 International Dry Eye WorkShop as ‘an integrated system comprising the lacrimal glands, ocular surface (cornea, conjunctiva and meibomian glands) and lids, and the sensory and motor nerves that connect them’. The LFU maintains a healthy ocular surface primarily through a properly functioning tear film that provides protection, lubrication, and an environment for corneal epithelial cell renewal. LFU cells express thousands of proteins. Over two hundred new LFU proteins have been discovered in the last decade. Lacritin is a new LFU-specific growth factor in human tears that flows through ducts to target corneal epithelial cells on the ocular surface. When applied topically in rabbits, lacritin appears to increase the volume of basal tear secretion. Lacritin is one of only a handful of tear proteins preliminarily reported to be downregulated in blepharitis and in two dry eye syndromes. Computational analysis predicts an ordered C-terminal domain that binds the corneal epithelial cell surface proteoglycan syndecan-1 (SDC1) and is required for lacritin’s low nanomolar mitogenic activity. The lacritin binding site on the N-terminus of SDC1 is exposed by heparanase. Heparanase is constitutively expressed by the corneal epithelium and appears to be a normal constituent of tears. Binding triggers rapid signaling to downstream NFAT and mTOR. A wealth of other new proteins, originally designated as hypothetical when first identified by genomic sequencing, are expressed by the human LFU including: ALS2CL, ARHGEF19, KIAA1109, PLXNA1, POLG, WIPI1 and ZMIZ2. Their demonstrated or implied roles in human genetic disease or basic cellular functions are fuel for new investigation. Addressing topical areas in ocular surface physiology with new LFU proteins may reveal interesting new biological mechanisms and help get to the heart of ocular surface dysfunction. PMID:18840430

Nucleolin: acharan sulfate–binding protein on the surface of cancer cells

PubMed Central

Joo, Eun Ji; ten Dam, Gerdy B.; van Kuppevelt, Toin H.; Toida, Toshihiko; Linhardt, Robert J.; Kim, Yeong Shik

2005-01-01

Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure α-D-N-acetylglucosaminyl (1→4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid–Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 μg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS–PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight ~110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells. PMID:15329357

Life at the border: Adaptation of proteins to anisotropic membrane environment

PubMed Central

Pogozheva, Irina D; Mosberg, Henry I; Lomize, Andrei L

2014-01-01

This review discusses main features of transmembrane (TM) proteins which distinguish them from water-soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co-translational and post-translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large-scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen-bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region—between double bonds and carbonyl groups of lipids. These “midpolar” regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side-chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein–lipid binding. PMID:24947665

Deciphering fine molecular details of proteins' structure and function with a Protein Surface Topography (PST) method.

PubMed

Koromyslova, Anna D; Chugunov, Anton O; Efremov, Roman G

2014-04-28

Molecular surfaces are the key players in biomolecular recognition and interactions. Nowadays, it is trivial to visualize a molecular surface and surface-distributed properties in three-dimensional space. However, such a representation trends to be biased and ambiguous in case of thorough analysis. We present a new method to create 2D spherical projection maps of entire protein surfaces and manipulate with them--protein surface topography (PST). It permits visualization and thoughtful analysis of surface properties. PST helps to easily portray conformational transitions, analyze proteins' properties and their dynamic behavior, improve docking performance, and reveal common patterns and dissimilarities in molecular surfaces of related bioactive peptides. This paper describes basic usage of PST with an example of small G-proteins conformational transitions, mapping of caspase-1 intersubunit interface, and intrinsic "complementarity" in the conotoxin-acetylcholine binding protein complex. We suggest that PST is a beneficial approach for structure-function studies of bioactive peptides and small proteins.

Protein quantification on dendrimer-activated surfaces by using time-of-flight secondary ion mass spectrometry and principal component regression

NASA Astrophysics Data System (ADS)

Kim, Young-Pil; Hong, Mi-Young; Shon, Hyun Kyong; Chegal, Won; Cho, Hyun Mo; Moon, Dae Won; Kim, Hak-Sung; Lee, Tae Geol

2008-12-01

Interaction between streptavidin and biotin on poly(amidoamine) (PAMAM) dendrimer-activated surfaces and on self-assembled monolayers (SAMs) was quantitatively studied by using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The surface protein density was systematically varied as a function of protein concentration and independently quantified using the ellipsometry technique. Principal component analysis (PCA) and principal component regression (PCR) were used to identify a correlation between the intensities of the secondary ion peaks and the surface protein densities. From the ToF-SIMS and ellipsometry results, a good linear correlation of protein density was found. Our study shows that surface protein densities are higher on dendrimer-activated surfaces than on SAMs surfaces due to the spherical property of the dendrimer, and that these surface protein densities can be easily quantified with high sensitivity in a label-free manner by ToF-SIMS.

ABI domain containing proteins contribute to surface protein display and cell division in Staphylococcus aureus

PubMed Central

Frankel, Matthew B.; Wojcik, Brandon; DeDent, Andrea C.; Missiakas, Dominique M.; Schneewind, Olaf

2012-01-01

Summary The human pathogen Staphyloccocus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harbored transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross walls and in the relative abundance of staphylococci with cross walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. PMID:20923422

ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

PubMed

Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

2010-10-01

The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

Macrophage Biochemistry, Activation and Function

DTIC Science & Technology

1981-01-01

vacuolar apparatus become more abundant. Functional capabilities, including phagocytic activity, protein synthesis and surface receptors, also increase...properties of cell components of other tissues has led to the following assignment of marker enzymes to specific macrophage components. This assessment is...subfractions. The surface area of each histogram bar then gives the frac- tional amount of constituent present within each normalized fraction. Distribution

UBXD4, a UBX-containing protein, regulates the cell surface number and stability of alpha3-containing nicotinic acetylcholine receptors.

PubMed

Rezvani, Khosrow; Teng, Yanfen; Pan, Yaping; Dani, John A; Lindstrom, Jon; García Gras, Eduardo A; McIntosh, J Michael; De Biasi, Mariella

2009-05-27

Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the alpha3 and alpha4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with alpha3-containing nAChRs (alpha3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the alpha3beta2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of alpha3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the alpha3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of alpha3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the alpha3 subunit and, consequently, the number of receptors at the cell surface.

UBXD4, a UBX containing protein, regulates the cell surface number and the stability of α3-containing nicotinic acetylcholine receptors

PubMed Central

Rezvani, Khosrow; Teng, Yanfen; Pan, Yaping; Dani, John A.; Lindstrom, Jon.; Gras, Eduardo A. Garcáa; McIntosh, J. Michael; De Biasi, Mariella.

2010-01-01

Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the α3 and α4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with α3-containing nAChRs (α3* nAChRs) expressed in HEK293 cells, PC12 cells and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the α3β2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of α3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the α3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of α3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the α3 subunit and, consequently, the number of receptors at the cell surface. PMID:19474315

Solution structure of the phosphoryl transfer complex between the cytoplasmic A domain of the mannitol transporter IIMannitol and HPr of the Escherichia coli phosphotransferase system.

PubMed

Cornilescu, Gabriel; Lee, Byeong Ryong; Cornilescu, Claudia C; Wang, Guangshun; Peterkofsky, Alan; Clore, G Marius

2002-11-01

The solution structure of the complex between the cytoplasmic A domain (IIA(Mtl)) of the mannitol transporter II(Mannitol) and the histidine-containing phosphocarrier protein (HPr) of the Escherichia coli phosphotransferase system has been solved by NMR, including the use of conjoined rigid body/torsion angle dynamics, and residual dipolar couplings, coupled with cross-validation, to permit accurate orientation of the two proteins. A convex surface on HPr, formed by helices 1 and 2, interacts with a complementary concave depression on the surface of IIA(Mtl) formed by helix 3, portions of helices 2 and 4, and beta-strands 2 and 3. The majority of intermolecular contacts are hydrophobic, with a small number of electrostatic interactions at the periphery of the interface. The active site histidines, His-15 of HPr and His-65 of IIA(Mtl), are in close spatial proximity, and a pentacoordinate phosphoryl transition state can be readily accommodated with no change in protein-protein orientation and only minimal perturbations of the backbone immediately adjacent to the histidines. Comparison with two previously solved structures of complexes of HPr with partner proteins of the phosphotransferase system, the N-terminal domain of enzyme I (EIN) and enzyme IIA(Glucose) (IIA(Glc)), reveals a number of common features despite the fact that EIN, IIA(Glc), and IIA(Mtl) bear no structural resemblance to one another. Thus, entirely different underlying structural elements can form binding surfaces for HPr that are similar in terms of both shape and residue composition. These structural comparisons illustrate the roles of surface and residue complementarity, redundancy, incremental build-up of specificity and conformational side chain plasticity in the formation of transient specific protein-protein complexes in signal transduction pathways.

Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

PubMed

Dufrêne, Y F

2001-02-01

The application of atomic force microscopy (AFM) to probe the ultrastructure and physical properties of microbial cell surfaces is reviewed. The unique capabilities of AFM can be summarized as follows: imaging surface topography with (sub)nanometer lateral resolution; examining biological specimens under physiological conditions; measuring local properties and interaction forces. AFM is being used increasingly for: (i) visualizing the surface ultrastructure of microbial cell surface layers, including bacterial S-layers, purple membranes, porin OmpF crystals and fungal rodlet layers; (ii) monitoring conformational changes of individual membrane proteins; (iii) examining the morphology of bacterial biofilms, (iv) revealing the nanoscale structure of living microbial cells, including fungi, yeasts and bacteria, (v) mapping interaction forces at microbial surfaces, such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces; and (vi) probing the local mechanical properties of cell surface layers and of single cells.

Enhanced protein retention on poly(caprolactone) via surface initiated polymerization of acrylamide

NASA Astrophysics Data System (ADS)

Ma, Yuhao; Cai, Mengtan; He, Liu; Luo, Xianglin

2016-01-01

To enhance the biocompatibility or extend the biomedical application of poly(caprolactone) (PCL), protein retention on PCL surface is often required. In this study, poly(acrylamide) (PAAm) brushes were grown from PCL surface via surface-initiated atom transfer radical polymerization (SI-ATRP) and served as a protein-capturing platform. Grafted PAAm was densely packed on surface and exhibited superior protein retention ability. Captured protein was found to be resistant to washing under detergent environment. Furthermore, protein structure after being captured was investigated by circular dichroism (CD) spectroscopy, and the CD spectra verified that secondary structure of captured proteins was maintained, indicating no denaturation of protein happened for retention process.

Examination of Campylobacter jejuni putative adhesins leads to the identification of a new protein, designated FlpA, required for chicken colonization

USDA-ARS?s Scientific Manuscript database

Campylobacter jejuni colonization of chickens is dependent upon surface exposed proteins termed adhesins. Putative C. jejuni adhesins include CadF, CapA, JlpA, MOMP, PEB1, Cj1279c, and Cj1349c. We examined the genetic relatedness of ninety-seven C. jejuni isolates recovered from human, poultry, bo...

Soy-based Polymers for Surface Modification and Interactions with Lignocellulosic Materials

NASA Astrophysics Data System (ADS)

Salas Araujo, Carlos Luis

Recent environmental concerns about the use of synthetic materials that are often used to maintain our quality of life has triggered a significant amount of research to develop new technologies and to adopt sustainable, bio-based materials. Cellulose, lignin and other plant-derived macromolecules including proteins from soybeans have witnessed recent, renewed interest by the industrial and scientific communities. For example, soybean proteins have been proposed for a variety of applications, including wood adhesives, bio-plastics, composites and functional materials that may include synthetic polymers. Despite its importance in such systems or materials, very little is known about the fundamental nature of the interactions between soy proteins and other polymers. Therefore, this work addresses this issue by a systematic investigation of the interactions between soy proteins with the two most abundant macromolecules in the biosphere, namely, cellulose and lignin and with the most widely used synthetic polymer, polypropylene (PP). The adsorption of the main soy protein globulins, glycinin (11S) and beta-conglycinin (7S), was studied by using ultrathin films of cellulose, lignin and PP (as well as reference silica and organic self-assembled monolayers (SAMs) surfaces) that were used as substrates. The extent and dynamics of adsorption was monitored by using quartz crystal microgravimetry with dissipation (QCM-D), surface plasmon resonance (SPR) as well as complementary techniques including circular dichroism (CD) and atomic force microscopy (AFM). QCM-D experiments indicated that soy protein adsorption was strongly affected by changes in the physicochemical environment. An increased adsorption of glycinin on silica (by 13%) and cellulose (by 89%) was observed with the increased ionic strength of the aqueous solution, from 0 to 0.1 M NaCl. This highlights the relevance of electrostatic interactions in the adsorption process. In contrast, the adsorption of beta-conglycinin was reduced (by 25 and 57 % on silica and cellulose, respectively). Similarly, the addition of 10 mM of 2-mercaptoethanol (a denaturing agent) reduced the mass adsorbed for both proteins. The amounts of 11S and 7S adsorbed on lignin and self-assembled 1-dodecanethiol monolayers were higher when the protein was in the native state if compared to that after chemical denaturation (by using urea and 2-mercaptoethanol). Urea-denatured proteins adsorbed more extensively onto the hydrophobic SAM monolayes. The reduction in water contact angle after protein adsorption (≈40° and 35° for native 11S and 7S, respectively) suggests strong nonspecific interactions between the protein and the substrates, favoring conformational changes at the interface that contribute to exposure and rearrangement of hydrophobic and hydrophilic amino acid residues. The adsorption on polypropylene thin films and nonwovens of different grades of soy proteins in their native as well as thermally-denatured states, including purified glycinin and beta-conglycinin as well as commercial soy flour and isolate was investigated at 25 °C in PBS buffer (pH 7.4). It was found that application of a primer layer of a cationic surfactant, dioctadecyldimethylammonium bromide (DODA) dramatically enhanced protein adsorption, which resulted in fully wettable systems. Fluorescence imaging experiments with tagged proteins confirmed the contribution of a fully-covering layer facilitated by the cationic surfactant pre-treatment. Furthermore, complementary wicking tests indicated that the nonwoven fabrics absorbed a significant amount of water (≈25 times their weight) when the fibers carried pre-adsorbed proteins.

Elucidation of metal-ion accumulation induced by hydrogen bonds on protein surfaces by using porous lysozyme crystals containing Rh(III) ions as the model surfaces.

PubMed

Ueno, Takafumi; Abe, Satoshi; Koshiyama, Tomomi; Ohki, Takahiro; Hikage, Tatsuo; Watanabe, Yoshihito

2010-03-01

Metal-ion accumulation on protein surfaces is a crucial step in the initiation of small-metal clusters and the formation of inorganic materials in nature. This event is expected to control the nucleation, growth, and position of the materials. There remain many unknowns, as to how proteins affect the initial process at the atomic level, although multistep assembly processes of the materials formation by both native and model systems have been clarified at the macroscopic level. Herein the cooperative effects of amino acids and hydrogen bonds promoting metal accumulation reactions are clarified by using porous hen egg white lysozyme (HEWL) crystals containing Rh(III) ions, as model protein surfaces for the reactions. The experimental results reveal noteworthy implications for initiation of metal accumulation, which involve highly cooperative dynamics of amino acids and hydrogen bonds: i) Disruption of hydrogen bonds can induce conformational changes of amino-acid residues to capture Rh(III) ions. ii) Water molecules pre-organized by hydrogen bonds can stabilize Rh(III) coordination as aqua ligands. iii) Water molecules participating in hydrogen bonds with amino-acid residues can be replaced by Rh(III) ions to form polynuclear structures with the residues. iv) Rh(III) aqua complexes are retained on amino-acid residues through stabilizing hydrogen bonds even at low pH (approximately 2). These metal-protein interactions including hydrogen bonds may promote native metal accumulation reactions and also may be useful in the preparation of new inorganic materials that incorporate proteins.

Interaction of fibrinogen and albumin with titanium dioxide nanoparticles of different crystalline phases

NASA Astrophysics Data System (ADS)

Marucco, Arianna; Fenoglio, Ivana; Turci, Francesco; Fubini, Bice

2013-04-01

TiO2 nanoparticles (NPs) are contained in different kinds of industrial products including paints, self-cleaning glasses, sunscreens. TiO2 is also employed in photocatalysis and it has been proposed for waste water treatment. Micrometric TiO2 is generally considered a safe material, while there is concern on the possible health effects of nanometric titania. Due to their small size NPs may migrate within the human body possibly entering in the blood stream. Therefore studies on the interaction of NPs with plasma proteins are needed. In fact, the interaction with proteins is believed to ultimately influences the NPs biological fate. Fibrinogen and albumin are two of the most abundant plasma proteins. They are involved in several important physiological functions. Furthermore, fibrinogen is known to trigger platelet adhesion and inflammation. For these reasons the study of the interaction between these protein and nanoparticles is an important step toward the understanding of the behavior of NPs in the body. In this study we investigated the interaction of albumin and fibrinogen with TiO2 nanoparticles of different crystal phases (rutile and anatase) using an integrated set of techniques. The amount of adsorbed fibrinogen and albumin for each TiO2 surface was investigated by using the bicinchoninic acid assay (BCA). The variation of the surface charge of the NP-protein conjugates respect to the naked NPs was used to indirectly estimate both surface coverage and reversibility of the adsorption upon dilution. Surface charge was monitored by measuring the ζ potential with a conventional electrophoretic light scattering (ELS) system. The extent of protein deformation was evaluated by Raman Spectroscopy. We found that both proteins adsorb irreversibly against electrostatic repulsion, likely undergoing conformational changes or selective orientation upon adsorption. The size of primary particles and the particles aggregation rather than the crystal phase modulate the affinity of fibrinogen for the TiO2 surfaces.

JAM related proteins in mucosal homeostasis and inflammation

PubMed Central

Luissint, Anny-Claude; Nusrat, Asma; Parkos, Charles A.

2014-01-01

Mucosal surfaces are lined by epithelial cells that form a physical barrier protecting the body against external noxious substances and pathogens. At a molecular level, the mucosal barrier is regulated by tight junctions (TJs) that seal the paracellular space between adjacent epithelial cells. Transmembrane proteins within TJs include Junctional Adhesion Molecules (JAMs) that belong to the CTX (Cortical Thymocyte marker for Xenopus) family of proteins. JAM family encompasses three classical members (JAM-A, -B and –C) and related molecules including JAM4, JAM-Like protein (JAM-L), Coxsackie and Adenovirus Receptor (CAR), CAR-Like Membrane Protein (CLMP) and Endothelial cell-Selective Adhesion Molecule (ESAM). JAMs have multiple functions that include regulation of endothelial and epithelial paracellular permeability, leukocyte recruitment during inflammation, angiogenesis, cell migration and proliferation. In this review, we summarize the current knowledge regarding the roles of the JAM family members in the regulation of mucosal homeostasis and leukocyte trafficking with a particular emphasis on barrier function and its perturbation during pathological inflammation. PMID:24667924

Significance of surface charge and shell material of superparamagnetic iron oxide nanoparticle (SPION) based core/shell nanoparticles on the composition of the protein corona.

PubMed

Sakulkhu, Usawadee; Mahmoudi, Morteza; Maurizi, Lionel; Coullerez, Geraldine; Hofmann-Amtenbrink, Margarethe; Vries, Marcel; Motazacker, Mahdi; Rezaee, Farhad; Hofmann, Heinrich

2015-02-01

As nanoparticles (NPs) are increasingly used in many applications their safety and efficient applications in nanomedicine have become concerns. Protein coronas on nanomaterials' surfaces can influence how the cell "recognizes" nanoparticles, as well as the in vitro and in vivo NPs' behaviors. The SuperParamagnetic Iron Oxide Nanoparticle (SPION) is one of the most prominent agents because of its superparamagnetic properties, which is useful for separation applications. To mimic surface properties of different types of NPs, a core-shell SPION library was prepared by coating with different surfaces: polyvinyl alcohol polymer (PVA) (positive, neutral and negative), SiO2 (positive and negative), titanium dioxide and metal gold. The SPIONs with different surfaces were incubated at a fixed serum : nanoparticle surface ratio, magnetically trapped and washed. The tightly bound proteins were quantified and identified. The surface charge has a great impact on protein adsorption, especially on PVA and silica where proteins preferred binding to the neutral and positively charged surfaces. The importance of surface material on protein adsorption was also revealed by preferential binding on TiO2 and gold coated SPION, even negatively charged. There is no correlation between the protein net charge and the nanoparticle surface charge on protein binding, nor direct correlation between the serum proteins' concentration and the proteins detected in the coronas.

Cell Cycle-Dependent Phosphorylation of Theileria annulata Schizont Surface Proteins

PubMed Central

von Schubert, Conrad; Wastling, Jonathan M.; Heussler, Volker T.; Woods, Kerry L.

2014-01-01

The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr), serine (p-Ser) and threonine-proline (p-Thr-Pro) epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state. PMID:25077614

Dissecting Arabidopsis Gβ Signal Transduction on the Protein Surface1[W][OA

PubMed Central

Jiang, Kun; Frick-Cheng, Arwen; Trusov, Yuri; Delgado-Cerezo, Magdalena; Rosenthal, David M.; Lorek, Justine; Panstruga, Ralph; Booker, Fitzgerald L.; Botella, José Ramón; Molina, Antonio; Ort, Donald R.; Jones, Alan M.

2012-01-01

The heterotrimeric G-protein complex provides signal amplification and target specificity. The Arabidopsis (Arabidopsis thaliana) Gβ-subunit of this complex (AGB1) interacts with and modulates the activity of target cytoplasmic proteins. This specificity resides in the structure of the interface between AGB1 and its targets. Important surface residues of AGB1, which were deduced from a comparative evolutionary approach, were mutated to dissect AGB1-dependent physiological functions. Analysis of the capacity of these mutants to complement well-established phenotypes of Gβ-null mutants revealed AGB1 residues critical for specific AGB1-mediated biological processes, including growth architecture, pathogen resistance, stomata-mediated leaf-air gas exchange, and possibly photosynthesis. These findings provide promising new avenues to direct the finely tuned engineering of crop yield and traits. PMID:22570469

Protein recognition using synthetic small-molecular binders toward optical protein sensing in vitro and in live cells.

PubMed

Kubota, Ryou; Hamachi, Itaru

2015-07-07

Chemical sensing of amino acids, peptides, and proteins provides fruitful information to understand their biological functions, as well as to develop the medical and technological applications. To detect amino acids, peptides, and proteins in vitro and in vivo, vast kinds of chemical sensors including small synthetic binders/sensors, genetically-encoded fluorescent proteins and protein-based semisynthetic biosensors have been intensely investigated. This review deals with concepts, strategies, and applications of protein recognition and sensing using small synthetic binders/sensors, which are now actively studied but still in the early stage of investigation. The recognition strategies for peptides and proteins can be divided into three categories: (i) recognition of protein substructures, (ii) protein surface recognition, and (iii) protein sensing through protein-ligand interaction. Here, we overview representative examples of protein recognition and sensing, and discuss biological or diagnostic applications such as potent inhibitors/modulators of protein-protein interactions.

REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

PubMed Central

Ho, Vincent K.; Angelotti, Timothy

2013-01-01

Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins. Therefore, some REEPs can be further described as ER membrane shaping adapter proteins. PMID:24098485

3D-SURFER 2.0: web platform for real-time search and characterization of protein surfaces.

PubMed

Xiong, Yi; Esquivel-Rodriguez, Juan; Sael, Lee; Kihara, Daisuke

2014-01-01

The increasing number of uncharacterized protein structures necessitates the development of computational approaches for function annotation using the protein tertiary structures. Protein structure database search is the basis of any structure-based functional elucidation of proteins. 3D-SURFER is a web platform for real-time protein surface comparison of a given protein structure against the entire PDB using 3D Zernike descriptors. It can smoothly navigate the protein structure space in real-time from one query structure to another. A major new feature of Release 2.0 is the ability to compare the protein surface of a single chain, a single domain, or a single complex against databases of protein chains, domains, complexes, or a combination of all three in the latest PDB. Additionally, two types of protein structures can now be compared: all-atom-surface and backbone-atom-surface. The server can also accept a batch job for a large number of database searches. Pockets in protein surfaces can be identified by VisGrid and LIGSITE (csc) . The server is available at http://kiharalab.org/3d-surfer/.

The chaperone-histone partnership: for the greater good of histone traffic and chromatin plasticity.

PubMed

Hondele, Maria; Ladurner, Andreas G

2011-12-01

Histones are highly positively charged proteins that wrap our genome. Their surface properties also make them prone to nonspecific interactions and aggregation. A class of proteins known as histone chaperones is dedicated to safeguard histones by aiding their proper incorporation into nucleosomes. Histone chaperones facilitate ordered nucleosome assembly and disassembly reactions through the formation of semi-stable histone-chaperone intermediates without requiring ATP, but merely providing a complementary protein surface for histones to dynamically interact with. Recurrent 'chaperoning' mechanisms involve the masking of the histone's positive charge and the direct blocking of crucial histone surface sites, including those required for H3-H4 tetramerization or the binding of nucleosomal DNA. This shielding prevents histones from engaging in premature or unwanted interactions with nucleic acids and other cellular components. In this review, we analyze recent structural studies on chaperone-histone interactions and discuss the implications of this vital partnership for nucleosome assembly and disassembly pathways. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mechanisms of the epithelial-to-mesenchymal transition in sea urchin embryos

PubMed Central

Katow, Hideki

2015-01-01

Sea urchin mesenchyme is composed of the large micromere-derived spiculogenetic primary mesenchyme cells (PMC), veg2-tier macromere-derived non-spiculogenetic mesenchyme cells, the small micromere-derived germ cells, and the macro- and mesomere-derived neuronal mesenchyme cells. They are formed through the epithelial-to-mesenchymal transition (EMT) and possess multipotency, except PMCs that solely differentiate larval spicules. The process of EMT is associated with modification of epithelial cell surface property that includes loss of affinity to the apical and basal extracellular matrices, inter-epithelial cell adherens junctions and epithelial cell surface-specific proteins. These cell surface structures and molecules are endocytosed during EMT and utilized as initiators of cytoplasmic signaling pathways that often initiate protein phosphorylation to activate the gene regulatory networks. Acquisition of cell motility after EMT in these mesenchyme cells is associated with the expression of proteins such as Lefty, Snail and Seawi. Structural simplicity and genomic database of this model will further promote detailed EMT research. PMID:26716069

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform

PubMed Central

Hu, Che-Ming J.; Zhang, Li; Aryal, Santosh; Cheung, Connie; Fang, Ronnie H.; Zhang, Liangfang

2011-01-01

Efforts to extend nanoparticle residence time in vivo have inspired many strategies in particle surface modifications to bypass macrophage uptake and systemic clearance. Here we report a top-down biomimetic approach in particle functionalization by coating biodegradable polymeric nanoparticles with natural erythrocyte membranes, including both membrane lipids and associated membrane proteins for long-circulating cargo delivery. The structure, size and surface zeta potential, and protein contents of the erythrocyte membrane-coated nanoparticles were verified using transmission electron microscopy, dynamic light scattering, and gel electrophoresis, respectively. Mice injections with fluorophore-loaded nanoparticles revealed superior circulation half-life by the erythrocyte-mimicking nanoparticles as compared to control particles coated with the state-of-the-art synthetic stealth materials. Biodistribution study revealed significant particle retention in the blood 72 h following the particle injection. The translocation of natural cellular membranes, their associated proteins, and the corresponding functionalities to the surface of synthetic particles represents a unique approach in nanoparticle functionalization. PMID:21690347

Controlling Self-Assembly of Engineered Peptides on Graphite by Rational Mutation

PubMed Central

So, Christopher R.; Hayamizu, Yuhei; Yazici, Hilal; Gresswell, Carolyn; Khatayevich, Dmitriy; Tamerler, Candan; Sarikaya, Mehmet

2012-01-01

Self-assembly of proteins on surfaces is utilized in many fields to integrate intricate biological structures and diverse functions with engineered materials. Controlling proteins at bio-solid interfaces relies on establishing key correlations between their primary sequences and resulting spatial organizations on substrates. Protein self-assembly, however, remains an engineering challenge. As a novel approach, we demonstrate here that short dodecapeptides selected by phage display are capable of self-assembly on graphite and form long-range ordered biomolecular nanostructures. Using atomic force microscopy and contact angle studies, we identify three amino-acid domains along the primary sequence that steer peptide ordering and lead to nanostructures with uniformly displayed residues. The peptides are further engineered via simple mutations to control fundamental interfacial processes, including initial binding, surface aggregation and growth kinetics, and intermolecular interactions. Tailoring short peptides via their primary sequence offers versatile control over molecular self-assembly, resulting in well-defined surface properties essential in building engineered, chemically rich, bio-solid interfaces. PMID:22233341

A glycosylphosphatidylinositol anchor is required for membrane localization but dispensable for cell wall association of chitin deacetylase 2 in Cryptococcus neoformans.

PubMed

Gilbert, Nicole M; Baker, Lorina G; Specht, Charles A; Lodge, Jennifer K

2012-01-01

Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. The surface of a pathogenic microbe is a major interface with its host. In fungi, the outer surface consists of a complex matrix known as the cell wall, which includes polysaccharides, proteins, and other molecules. The mammalian host recognizes many of these surface molecules and mounts appropriate responses to combat the microbial infection. Cryptococcus neoformans is a serious fungal pathogen that kills over 600,000 people annually. It converts most of its chitin, a cell wall polysaccharide, to chitosan, which is necessary for virulence. Chitin deacetylase enzymes have been identified in the cell wall, and our studies were undertaken to understand how the deacetylase is linked to the wall and where it has activity. Our results have implications for the current model of chitosan biosynthesis and further challenge the paradigm of covalent linkages between cell wall proteins and polysaccharides through a lipid modification of the protein.

Identification of cell surface glycoprotein markers for glioblastoma-derived stem-like cells using a lectin microarray and LC-MS/MS approach

PubMed Central

He, Jintang; Liu, Yashu; Xie, Xiaolei; Zhu, Thant; Soules, Mary; DiMeco, Francesco; Vescovi, Angelo L.; Fan, Xing; Lubman, David M.

2010-01-01

Despite progress in the treatment of glioblastoma, more than 95% of patients suffering from this disease still die within two years. Recent findings support the belief that cancer stem-like cells are responsible for tumor formation and ongoing growth. Here a method combining lectin microarray and LC-MS/MS was used to discover the cell surface glycoprotein markers of a glioblastoma-derived stem-like cell line. Lectin microarray analysis of cell surface glycans showed that two galactose-specific lectins Trichosanthes kirilowii agglutinin (TKA) and Peanut agglutinin (PNA) could distinguish the stem-like glioblastoma neurosphere culture from a traditional adherent glioblastoma cell line. Agarose-bound TKA and PNA were used to capture the glycoproteins from the two cell cultures, which were analyzed by LC-MS/MS. The glycoproteins were quantified by spectral counting, resulting in the identification of 12 and 11 potential glycoprotein markers from the TKA and PNA captured fractions respectively. Almost all of these proteins were membrane proteins. Differential expression was verified by Western blotting analysis of 6 interesting proteins, including the up-regulated Receptor-type tyrosine-protein phosphatase zeta, Tenascin-C, Chondroitin sulfate proteoglycan NG2, Podocalyxin-like protein 1 and CD90, and the down-regulated CD44. An improved understanding of these proteins may be important for earlier diagnosis and better therapeutic targeting of glioblastoma. PMID:20235609

Plasmodium cysteine repeat modular proteins 1-4: complex proteins with roles throughout the malaria parasite life cycle.

PubMed

Thompson, Joanne; Fernandez-Reyes, Delmiro; Sharling, Lisa; Moore, Sally G; Eling, Wijnand M; Kyes, Sue A; Newbold, Christopher I; Kafatos, Fotis C; Janse, Chris J; Waters, Andrew P

2007-06-01

The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a >100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum blood-stage parasites and are exposed on the surface of haemolymph- and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.

Disturbed vesicular trafficking of membrane proteins in prion disease.

PubMed

Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

Identification and Characterization of the Novel LysM Domain-Containing Surface Protein Sep from Lactobacillus fermentum BR11 and Its Use as a Peptide Fusion Partner in Lactobacillus and Lactococcus

PubMed Central

Turner, Mark S.; Hafner, Louise M.; Walsh, Terry; Giffard, Philip M.

2004-01-01

Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria. PMID:15184172

Protein and Bacterial Antifouling Behavior of Melt-Coextruded Nanofiber Mats.

PubMed

Kim, Si-Eun; Zhang, Cong; Advincula, Abigail A; Baer, Eric; Pokorski, Jonathan K

2016-04-13

Antifouling surfaces are important for biomedical devices to prevent secondary infections and mitigate the effects of the foreign body response. Herein, we describe melt-coextruded poly(ε-caprolactone) (PCL) nanofiber mats grafted with antifouling polymers. Nonwoven PCL fiber mats are produced using a multilayered melt coextrusion process followed by high-pressure hydroentanglement to yield porous patches. The resulting fiber mats show submicrometer cross-sectional fiber dimensions and yield pore sizes that were nearly uniform, with a mean pore size of 1.6 ± 0.9 μm. Several antifouling polymers, including hydrophilic, zwitterionic, and amphipathic molecules, are grafted to the surface of the mats using a two-step procedure that includes photochemistry followed by the copper-catalyzed azide-alkyne cycloaddition reaction. Fiber mats are evaluated using separate adsorption tests for serum proteins and E. coli. The results indicate that poly(oligo(ethylene glycol) methyl ether methacrylate)-co-(trifluoroethyl methacrylate) (poly(OEGMEMA-co-TFEMA)) grafted mats exhibit approximately 85% less protein adhesion and 97% less E. coli adsorption when compared to unmodified PCL fibermats. In dynamic antifouling testing, the amphiphilic fluorous polymer surface shows the highest flux and highest rejection value of foulants. The work presented within has implications on the high-throughput production of antifouling microporous patches for medical applications.

Surface analysis of gold nanoparticles functionalized with thiol-modified glucose SAMs for biosensor applications.

NASA Astrophysics Data System (ADS)

Spampinato, Valentina; Parracino, Mariaantonietta; La Spina, Rita; Rossi, Francois; Ceccone, Giacomo

2016-02-01

In this work, Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS), Principal Component Analysis (PCA) and X-ray Photoelectron Spectroscopy (XPS) have been used to characterize the surface chemistry of gold substrates before and after functionalization with thiol-modified glucose self-assembled monolayers and subsequent biochemical specific recognition of maltose binding protein (MBP). The results indicate that the surface functionalization is achieved both on flat and nanoparticles gold substrates thus showing the potential of the developed system as biodetection platform. Moreover, the method presented here has been found to be a sound and valid approach to characterize the surface chemistry of nanoparticles functionalized with large molecules. Both techniques were proved to be very useful tools for monitoring all the functionalization steps, including the investigation of the biological behaviour of the glucose-modified particles in presence of the maltose binding protein.

Surface Analysis of Gold Nanoparticles Functionalized with Thiol-Modified Glucose SAMs for Biosensor Applications

PubMed Central

Spampinato, Valentina; Parracino, Maria Antonietta; La Spina, Rita; Rossi, Francois; Ceccone, Giacomo

2016-01-01

In this work, Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS), Principal Component Analysis (PCA) and X-ray Photoelectron Spectroscopy (XPS) have been used to characterize the surface chemistry of gold substrates before and after functionalization with thiol-modified glucose self-assembled monolayers and subsequent biochemical specific recognition of maltose binding protein (MBP). The results indicate that the surface functionalization is achieved both on flat and nanoparticles gold substrates thus showing the potential of the developed system as biodetection platform. Moreover, the method presented here has been found to be a sound and valid approach to characterize the surface chemistry of nanoparticles functionalized with large molecules. Both techniques were proved to be very useful tools for monitoring all the functionalization steps, including the investigation of the biological behavior of the glucose-modified particles in the presence of the maltose binding protein. PMID:26973830

Maleimide-activated aryl diazonium salts for electrode surface functionalization with biological and redox-active molecules.

PubMed

Harper, Jason C; Polsky, Ronen; Wheeler, David R; Brozik, Susan M

2008-03-04

A versatile and simple method is introduced for formation of maleimide-functionalized surfaces using maleimide-activated aryl diazonium salts. We show for the first time electrodeposition of N-(4-diazophenyl)maleimide tetrafluoroborate on gold and carbon electrodes which was characterized via voltammetry, grazing angle FTIR, and ellipsometry. Electrodeposition conditions were used to control film thickness and yielded submonolayer-to-multilayer grafting. The resulting phenylmaleimide surfaces served as effective coupling agents for electrode functionalization with ferrocene and the redox-active protein cytochrome c. The utility of phenylmaleimide diazonium toward formation of a diazonium-activated conjugate, followed by direct electrodeposition of the diazonium-modified DNA onto the electrode surface, was also demonstrated. Effective electron transfer was obtained between immobilized molecules and the electrodes. This novel application of N-phenylmaleimide diazonium may facilitate the development of bioelectronic devices including biofuel cells, biosensors, and DNA and protein microarrays.

Plasma protein adsorption to zwitterionic poly (carboxybetaine methacrylate) modified surfaces: chain chemistry and end-group effects on protein adsorption kinetics, adsorbed amounts and immunoblots.

PubMed

Abraham, Sinoj; Bahniuk, Markian S; Unsworth, Larry D

2012-12-01

Protein-surface interactions are crucial to the overall biocompatability of biomaterials, and are thought to be the impetus towards the adverse host responses such as blood coagulation and complement activation. Only a few studies hint at the ultra-low fouling potential of zwitterionic poly(carboxybetaine methacrylate) (PCBMA) grafted surfaces and, of those, very few systematically investigate their non-fouling behavior. In this work, single protein adsorption studies as well as protein adsorption from complex solutions (i.e. human plasma) were used to evaluate the non-fouling potential of PCBMA grafted silica wafers prepared by nitroxide-mediated free radical polymerization. PCBMAs used for surface grafting varied in charge separating spacer groups that influence the overall surface charges, and chain end-groups that influence the overall hydrophilicity, thereby, allows a better understanding of these effects towards the protein adsorption for these materials. In situ ellipsometry was used to quantify the adsorbed layer thickness and adsorption kinetics for the adsorption of four proteins from single protein buffer solutions, viz, lysozyme, α-lactalbumin, human serum albumin and fibrinogen. Total amount of protein adsorbed on surfaces differed as a function of surface properties and protein characteristics. Finally, immunoblots results showed that human plasma protein adsorption to these surfaces resulted, primarily, in the adsorption of human serum albumin, with total protein adsorbed amounts being the lowest for PCBMA-3 (TEMPO). It was apparent that surface charge and chain hydrophilicity directly influenced protein adsorption behavior of PCBMA systems and are promising materials for biomedical applications.

Precise control of surface electrostatic forces on polymer brush layers with opposite charges for resistance to protein adsorption.

PubMed

Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

2016-10-01

Various molecular interaction forces are generated during protein adsorption process on material surfaces. Thus, it is necessary to control them to suppress protein adsorption and the subsequent cell and tissue responses. A series of binary copolymer brush layers were prepared via surface-initiated atom transfer radical polymerization, by mixing the cationic monomer unit and anionic monomer unit randomly in various ratios. Surface characterization revealed that the constructed copolymer brush layers exhibited an uniform super-hydrophilic nature and different surface potentials. The strength of the electrostatic interaction forces operating on these mixed-charge copolymer brush surfaces was evaluated quantitatively using force-versus-distance (f-d) curve measurements by atomic force microscopy (AFM) and probes modified by negatively charged carboxyl groups or positively charged amino groups. The electrostatic interaction forces were determined based on the charge ratios of the copolymer brush layers. Notably, the surface containing equivalent cationic/anionic monomer units hardly interacted with both the charged groups. Furthermore, the protein adsorption force and the protein adsorption mass on these surfaces were examined by AFM f-d curve measurement and surface plasmon resonance measurement, respectively. To clarify the influence of the electrostatic interaction on the protein adsorption behavior on the surface, three kinds of proteins having negative, positive, and relatively neutral net charges under physiological conditions were used in this study. We quantitatively demonstrated that the amount of adsorbed proteins on the surfaces would have a strong correlation with the strength of surface-protein interaction forces, and that the strength of surface-protein interaction forces would be determined from the combination between the properties of the electrostatic interaction forces on the surfaces and the charge properties of the proteins. Especially, the copolymer brush surface composed of equivalent cationic/anionic monomer units exhibited no significant interaction forces, and dramatically suppressed the adsorption of proteins regardless of their charge properties. We conclude that the established methodology could elucidate relationship between the protein adsorption behavior and molecular interaction, especially the electrostatic interaction forces, and demonstrated that the suppression of the electrostatic interactions with the ionic functional groups would be important for the development of new polymeric biomaterials with a high repellency of protein adsorption. Copyright © 2016 Elsevier Ltd. All rights reserved.

The role played by alternative splicing in antigenic variability in human endo-parasites.

PubMed

Hull, Rodney; Dlamini, Zodwa

2014-01-28

Endo-parasites that affect humans include Plasmodium, the causative agent of malaria, which remains one of the leading causes of death in human beings. Despite decades of research, vaccines to this and other endo-parasites remain elusive. This is in part due to the hyper-variability of the parasites surface proteins. Generally these surface proteins are encoded by a large family of genes, with only one being dominantly expressed at certain life stages. Another layer of complexity can be introduced through the alternative splicing of these surface proteins. The resulting isoforms may differ from each other with regard to cell localisation, substrate affinities and functions. They may even differ in structure to the extent that they are no longer recognised by the host's immune system. In many cases this leads to changes in the N terminus of these proteins. The geographical localisation of endo-parasitic infections around the tropics and the highest incidences of HIV-1 infection in the same areas, adds a further layer of complexity as parasitic infections affect the host immune system resulting in higher HIV infection rates, faster disease progression, and an increase in the severity of infections and complications in HIV diagnosis. This review discusses some examples of parasite surface proteins that are alternatively spliced in trypanosomes, Plasmodium and the parasitic worm Schistosoma as well as what role alternate splicing may play in the interaction between HIV and these endo-parasites.

The "Sticky Patch" Model of Crystallization and Modification of Proteins for Enhanced Crystallizability.

PubMed

Derewenda, Zygmunt S; Godzik, Adam

2017-01-01

Crystallization of macromolecules has long been perceived as a stochastic process, which cannot be predicted or controlled. This is consistent with another popular notion that the interactions of molecules within the crystal, i.e., crystal contacts, are essentially random and devoid of specific physicochemical features. In contrast, functionally relevant surfaces, such as oligomerization interfaces and specific protein-protein interaction sites, are under evolutionary pressures so their amino acid composition, structure, and topology are distinct. However, current theoretical and experimental studies are significantly changing our understanding of the nature of crystallization. The increasingly popular "sticky patch" model, derived from soft matter physics, describes crystallization as a process driven by interactions between select, specific surface patches, with properties thermodynamically favorable for cohesive interactions. Independent support for this model comes from various sources including structural studies and bioinformatics. Proteins that are recalcitrant to crystallization can be modified for enhanced crystallizability through chemical or mutational modification of their surface to effectively engineer "sticky patches" which would drive crystallization. Here, we discuss the current state of knowledge of the relationship between the microscopic properties of the target macromolecule and its crystallizability, focusing on the "sticky patch" model. We discuss state-of-the-art in silico methods that evaluate the propensity of a given target protein to form crystals based on these relationships, with the objective to design variants with modified molecular surface properties and enhanced crystallization propensity. We illustrate this discussion with specific cases where these approaches allowed to generate crystals suitable for structural analysis.

Unraveling Gardnerella vaginalis Surface Proteins Using Cell Shaving Proteomics.

PubMed

Marín, Elvira; Haesaert, Annelies; Padilla, Laura; Adán, Jaume; Hernáez, María L; Monteoliva, Lucía; Gil, Concha

2018-01-01

Gardnerella vaginalis is one of the main etiologic agents of bacterial vaginosis (BV). This infection is responsible for a wide range of public health costs and is associated with several adverse outcomes during pregnancy. Improving our understanding of G. vaginalis protein cell surface will assist in BV diagnosis. This study represents the first proteomic approach that has analyzed the exposed proteins on G. vaginalis cell surface using a shaving approach. The 261 G. vaginalis proteins identified using this approach were analyzed with bioinformatic tools to detect characteristic motifs from surface-exposed proteins, such as signal peptides (36 proteins), lipobox domains (17 proteins), LPXTG motifs (5 proteins) and transmembrane alpha-helices (66 proteins). One third of the identified proteins were found to have at least one typical motif of surface-exposed proteins. Furthermore, the subcellular location was examined using two predictors (PSORT and Gpos-mPLoc). These bioinformatic tools classified 17% of the identified proteins as surface-associated proteins. Interestingly, we identified 13 members of the ATP-binding cassette (ABC) superfamily, which were mainly involved in the translocation of various substrates across membranes. To validate the location of the G. vaginalis surface-exposed proteins, an immunofluorescence assay with antibodies against Escherichia coli GroEL was performed to reveal the extracellular location of the moonlighting GroEL. In addition, monoclonal antibodies (mAb) against G. vaginalis Cna protein were produced and used to validate the location of Cna on the surface of the G. vaginalis . These high affinity anti-Cna mAb represent a useful tool for the study of this pathogenic microorganism and the BV.

Unraveling Gardnerella vaginalis Surface Proteins Using Cell Shaving Proteomics

PubMed Central

Marín, Elvira; Haesaert, Annelies; Padilla, Laura; Adán, Jaume; Hernáez, María L.; Monteoliva, Lucía; Gil, Concha

2018-01-01

Gardnerella vaginalis is one of the main etiologic agents of bacterial vaginosis (BV). This infection is responsible for a wide range of public health costs and is associated with several adverse outcomes during pregnancy. Improving our understanding of G. vaginalis protein cell surface will assist in BV diagnosis. This study represents the first proteomic approach that has analyzed the exposed proteins on G. vaginalis cell surface using a shaving approach. The 261 G. vaginalis proteins identified using this approach were analyzed with bioinformatic tools to detect characteristic motifs from surface-exposed proteins, such as signal peptides (36 proteins), lipobox domains (17 proteins), LPXTG motifs (5 proteins) and transmembrane alpha-helices (66 proteins). One third of the identified proteins were found to have at least one typical motif of surface-exposed proteins. Furthermore, the subcellular location was examined using two predictors (PSORT and Gpos-mPLoc). These bioinformatic tools classified 17% of the identified proteins as surface-associated proteins. Interestingly, we identified 13 members of the ATP-binding cassette (ABC) superfamily, which were mainly involved in the translocation of various substrates across membranes. To validate the location of the G. vaginalis surface-exposed proteins, an immunofluorescence assay with antibodies against Escherichia coli GroEL was performed to reveal the extracellular location of the moonlighting GroEL. In addition, monoclonal antibodies (mAb) against G. vaginalis Cna protein were produced and used to validate the location of Cna on the surface of the G. vaginalis. These high affinity anti-Cna mAb represent a useful tool for the study of this pathogenic microorganism and the BV. PMID:29867878

Fundamentals of nanoscale polymer-protein interactions and potential contributions to solid-state nanobioarrays.

PubMed

Hahm, Jong-in

2014-08-26

Protein adsorption onto polymer surfaces is a very complex, ubiquitous, and integrated process, impacting essential areas of food processing and packaging, health devices, diagnostic tools, and medical products. The nature of protein-surface interactions is becoming much more complicated with continuous efforts toward miniaturization, especially for the development of highly compact protein detection and diagnostic devices. A large body of literature reports on protein adsorption from the perspective of ensemble-averaged behavior on macroscopic, chemically homogeneous, polymeric surfaces. However, protein-surface interactions governing the nanoscale size regime may not be effectively inferred from their macroscopic and microscopic characteristics. Recently, research efforts have been made to produce periodically arranged, nanoscopic protein patterns on diblock copolymer surfaces solely through self-assembly. Intriguing protein adsorption phenomena are directly probed on the individual biomolecule level for a fundamental understanding of protein adsorption on nanoscale surfaces exhibiting varying degrees of chemical heterogeneity. Insight gained from protein assembly on diblock copolymers can be effectively used to control the surface density, conformation, orientation, and biofunctionality of prebound proteins in highly miniaturized applications, now approaching the nanoscale. This feature article will highlight recent experimental and theoretical advances made on these fronts while focusing on single-biomolecule-level investigations of protein adsorption behavior combined with surface chemical heterogeneity on the length scale commensurate with a single protein. This article will also address advantages and challenges of the self-assembly-driven patterning technology used to produce protein nanoarrays and its implications for ultrahigh density, functional, and quantifiable protein detection in a highly miniaturized format.

Efficient protein-repelling thin films regulated by chain mobility of low-Tg polymers with increased stability via crosslinking

NASA Astrophysics Data System (ADS)

Zhang, Jinghui; Huang, Zhiwei; Liu, Dan

2017-12-01

Polymer thin films are generally employed as coatings on implants to prevent protein adsorption. Polymer chain mobility and surface softness have been found to contribute to the protein resistance, but also bring film instability in a liquid protein medium. We investigated the protein resistance ability of three low-Tg polymers, including hydrophobic polymers polyisoprene (PI), poly(n-butyl methacrylate) (PnBMA) and hydrophilic polyethylene oxide (PEO), by overcoming the instability issue with crosslinking. We found that the Tgs of PI and PEO can be increased to around 0 °C after crosslinking. The remained strong chain mobility of both films can still resist protein adsorption regardless the hydrophobicity, yet greatly increases the film stability under an aqueous circumstance. The PnBMA film increased its Tg to around room temperature after crosslinking, which deteriorated the protein-resistance ability having the surface covered by BSA molecules. Our results support that the chain mobility of a polymer film plays an important role in resisting protein adsorption due to the increased entropy associated with more mobile polymer chains. By tune the degree of crosslinking, the stability of polymer in aqueous environment can be increased while the protein resistant ability can be remained. Our results provide a new strategy to design polymer materials for effective antifouling.

A polyvalent hybrid protein elicits antibodies against the diverse allelic types of block 2 in Plasmodium falciparum merozoite surface protein 1.

PubMed

Tetteh, Kevin K A; Conway, David J

2011-10-13

Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice. The full hybrid protein induced high titre antibodies against each of the major block 2 allelic types expressed as separate recombinant proteins and against a wide range of allelic types naturally expressed by a panel of diverse P. falciparum isolates, while the sub-component modules had partial antigenic coverage as expected. This encourages further development and evaluation of the full MSP1 block 2 polyvalent hybrid protein as a candidate blood-stage component of a malaria vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

PubMed

Crowdus, Carolyn A; Marsh, Antoinette E; Saville, Willliam J; Lindsay, David S; Dubey, J P; Granstrom, David E; Howe, Daniel K

2008-11-25

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

Medical implants and methods of making medical implants

DOEpatents

Shaw, Wendy J; Yonker, Clement R; Fulton, John L; Tarasevich, Barbara J; McClain, James B; Taylor, Doug

2014-09-16

A medical implant device having a substrate with an oxidized surface and a silane derivative coating covalently bonded to the oxidized surface. A bioactive agent is covalently bonded to the silane derivative coating. An implantable stent device including a stent core having an oxidized surface with a layer of silane derivative covalently bonded thereto. A spacer layer comprising polyethylene glycol (PEG) is covalently bonded to the layer of silane derivative and a protein is covalently bonded to the PEG. A method of making a medical implant device including providing a substrate having a surface, oxidizing the surface and reacting with derivitized silane to form a silane coating covalently bonded to the surface. A bioactive agent is then covalently bonded to the silane coating. In particular instances, an additional coating of bio-absorbable polymer and/or pharmaceutical agent is deposited over the bioactive agent.

SALSA-A dance on a slippery floor with changing partners.

PubMed

Reichhardt, M P; Holmskov, U; Meri, S

2017-09-01

It is becoming increasingly clear that the connections between our immune system and the microbiota colonizing us have a tremendous impact on human health. A number of innate molecular defence mechanisms cooperate to selectively target unwanted microorganisms at the mucosal surfaces. Amongst others these include the complement system, IgA and the SALSA molecule. The salivary scavenger and agglutinin (SALSA), also known as deleted in malignant brain tumors 1 (DMBT1), salivary agglutinin (SAG) or gp340 is a multifunctional molecule with important functions in innate immunity, inflammation and epithelial homeostasis. The SALSA protein is expressed at most mucosal surfaces, where it is one of the most abundant proteins. In the fetal meconium and infant intestine it may constitute even up to 10% of the total protein amount. SALSA is found either directly associated with the epithelial surface or secreted into the lining fluids. In the fluid-phase SALSA interacts with a number of bacterial and viral organisms, as well as with endogenous ligands, including IgA, lactoferrin, surfactant proteins and complement components. While complement has been shown to impact the mucosal environment, this remains an area of limited research. The multiple interactions of the SALSA molecule provide a scaffold, where this potent defence system may engage in cooperative microbial clearance together with corresponding mucosal host ligands. With its high abundance, and multiple effects on both host and microbes, the SALSA molecule is a key player in maintaining the immunological balance at the mucosal surfaces. This is further supported by observations linking the expression of different SALSA isoforms to the development of chronic inflammatory conditions, such as Crohn's disease and ulcerative colitis. This review describes the latest advances in understanding functions of SALSA and its different isoforms. Recently recognized functions are related to complement activation and regulation, endothelial development and epithelial homeostasis. In addition, we suggest mechanisms how SALSA regulates inflammation at the mucosal surfaces. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation and Characterization of Adhesive Secretion from Cuvierian Tubules of Sea Cucumber Holothuria forskåli (Echinodermata: Holothuroidea)

PubMed Central

Baranowska, Malgorzata; Schloßmacher, Ute; McKenzie, J. Douglas; Müller, Werner E. G.; Schröder, Heinz C.

2011-01-01

The sea cucumber Holothuria forskåli possesses a specialized system called Cuvierian tubules. During mechanical stimulation white filaments (tubules) are expelled and become sticky upon contact with any object. We isolated a protein with adhesive properties from protein extracts of Cuvierian tubules from H. forskåli. This protein was identified by antibodies against recombinant precollagen D which is located in the byssal threads of the mussel Mytilus galloprovincialis. To find out the optimal procedure for extraction and purification, the identified protein was isolated by several methods, including electroelution, binding to glass beads, immunoprecipitation, and gel filtration. Antibodies raised against the isolated protein were used for localization of the adhesive protein in Cuvierian tubules. Immunostaining and immunogold electron microscopical studies revealed the strongest immunoreactivity in the mesothelium; this tissue layer is involved in adhesion. Adhesion of Cuvierian tubule extracts was measured on the surface of various materials. The extracted protein showed the strongest adhesion to Teflon surface. Increased adhesion was observed in the presence of potassium and EDTA, while cadmium caused a decrease in adhesion. Addition of antibodies and trypsin abolished the adhesive properties of the extract. PMID:22013488

Host Immune Evasion by Lyme and Relapsing Fever Borreliae: Findings to Lead Future Studies for Borrelia miyamotoi

PubMed Central

Stone, Brandee L.; Brissette, Catherine A.

2017-01-01

The emerging pathogen, Borrelia miyamotoi, is a relapsing fever spirochete vectored by the same species of Ixodes ticks that carry the causative agents of Lyme disease in the US, Europe, and Asia. Symptoms caused by infection with B. miyamotoi are similar to a relapsing fever infection. However, B. miyamotoi has adapted to different vectors and reservoirs, which could result in unique physiology, including immune evasion mechanisms. Lyme Borrelia utilize a combination of Ixodes-produced inhibitors and native proteins [i.e., factor H-binding proteins (FHBPs)/complement regulator-acquiring surface proteins, p43, BBK32, BGA66, BGA71, CD59-like protein] to inhibit complement, while some relapsing fever spirochetes use C4b-binding protein and likely Ornithodoros-produced inhibitors. To evade the humoral response, Borrelia utilize antigenic variation of either outer surface proteins (Osps) and the Vmp-like sequences (Vls) system (Lyme borreliae) or variable membrane proteins (Vmps, relapsing fever borreliae). B. miyamotoi possesses putative FHBPs and antigenic variation of Vmps has been demonstrated. This review summarizes and compares the common mechanisms utilized by Lyme and relapsing fever spirochetes, as well as the current state of understanding immune evasion by B. miyamotoi. PMID:28154563

Host Immune Evasion by Lyme and Relapsing Fever Borreliae: Findings to Lead Future Studies for Borrelia miyamotoi.

PubMed

Stone, Brandee L; Brissette, Catherine A

2017-01-01

The emerging pathogen, Borrelia miyamotoi , is a relapsing fever spirochete vectored by the same species of Ixodes ticks that carry the causative agents of Lyme disease in the US, Europe, and Asia. Symptoms caused by infection with B. miyamotoi are similar to a relapsing fever infection. However, B. miyamotoi has adapted to different vectors and reservoirs, which could result in unique physiology, including immune evasion mechanisms. Lyme Borrelia utilize a combination of Ixodes -produced inhibitors and native proteins [i.e., factor H-binding proteins (FHBPs)/complement regulator-acquiring surface proteins, p43, BBK32, BGA66, BGA71, CD59-like protein] to inhibit complement, while some relapsing fever spirochetes use C4b-binding protein and likely Ornithodoros -produced inhibitors. To evade the humoral response, Borrelia utilize antigenic variation of either outer surface proteins (Osps) and the Vmp-like sequences (Vls) system (Lyme borreliae) or variable membrane proteins (Vmps, relapsing fever borreliae). B. miyamotoi possesses putative FHBPs and antigenic variation of Vmps has been demonstrated. This review summarizes and compares the common mechanisms utilized by Lyme and relapsing fever spirochetes, as well as the current state of understanding immune evasion by B. miyamotoi .

Three steps to gold: mechanism of protein adsorption revealed by Brownian and molecular dynamics simulations.

PubMed

Ozboyaci, M; Kokh, D B; Wade, R C

2016-04-21

The addition of three N-terminal histidines to β-lactamase inhibitor protein was shown experimentally to increase its binding potency to an Au(111) surface substantially but the binding mechanism was not resolved. Here, we propose a complete adsorption mechanism for this fusion protein by means of a multi-scale simulation approach and free energy calculations. We find that adsorption is a three-step process: (i) recognition of the surface predominantly by the histidine fusion peptide and formation of an encounter complex facilitated by a reduced dielectric screening of water in the interfacial region, (ii) adsorption of the protein on the surface and adoption of a specific binding orientation, and (iii) adaptation of the protein structure on the metal surface accompanied by induced fit. We anticipate that the mechanistic features of protein adsorption to an Au(111) surface revealed here can be extended to other inorganic surfaces and proteins and will therefore aid the design of specific protein-surface interactions.

The Application of Ligand-Mapping Molecular Dynamics Simulations to the Rational Design of Peptidic Modulators of Protein-Protein Interactions.

PubMed

Tan, Yaw Sing; Spring, David R; Abell, Chris; Verma, Chandra S

2015-07-14

A computational ligand-mapping approach to detect protein surface pockets that interact with hydrophobic moieties is presented. In this method, we incorporated benzene molecules into explicit solvent molecular dynamics simulations of various protein targets. The benzene molecules successfully identified the binding locations of hydrophobic hot-spot residues and all-hydrocarbon cross-links from known peptidic ligands. They also unveiled cryptic binding sites that are occluded by side chains and the protein backbone. Our results demonstrate that ligand-mapping molecular dynamics simulations hold immense promise to guide the rational design of peptidic modulators of protein-protein interactions, including that of stapled peptides, which show promise as an exciting new class of cell-penetrating therapeutic molecules.

Regulation of AMPA receptor localization in lipid rafts

PubMed Central

Hou, Qingming; Huang, Yunfei; Amato, Stephen; Snyder, Solomon H.; Huganir, Richard L.; Man, Heng-Ye

2009-01-01

Lipid rafts are special microdomains enriched in cholesterol, sphingolipids and certain proteins, and play important roles in a variety of cellular functions including signal transduction and protein trafficking. We report that in cultured cortical and hippocampal neurons the distribution of lipid rafts is development-dependent. Lipid rafts in mature neurons exist on the entire cell-surface and display a high degree of mobility. AMPA receptors co-localize and associate with lipid rafts in the plasma membrane. The association of AMPARs with rafts is under regulation; through the NOS–NO pathway, NMDA receptor activity increases AMPAR localization in rafts. During membrane targeting, AMPARs insert into or at close proximity of the surface raft domains. Perturbation of lipid rafts dramatically suppresses AMPA receptor exocytosis, resulting in significant reduction in AMPAR cell-surface expression. PMID:18411055

Competitive adsorption in model charged protein mixtures: Equilibrium isotherms and kinetics behavior

NASA Astrophysics Data System (ADS)

Fang, F.; Szleifer, I.

2003-07-01

The competitive adsorption of proteins of different sizes and charges is studied using a molecular theory. The theory enables the study of charged systems explicitly including the size, shape, and charge distributions in all the molecular species in the mixture. Thus, this approach goes beyond the commonly used Poisson-Boltzmann approximation. The adsorption isotherms of the protein mixtures are studied for mixtures of two proteins of different size and charge. The amount of proteins adsorbed and the fraction of each protein is calculated as a function of the bulk composition of the solution and the amount of salt in the system. It is found that the total amount of proteins adsorbed is a monotonically decreasing function of the fraction of large proteins on the bulk solution and for fixed protein composition of the salt concentration. However, the composition of the adsorbed layer is a complicated function of the bulk composition and solution ionic strength. The structure of the adsorb layer depends upon the bulk composition and salt concentration. In general, there are multilayers adsorbed due to the long-range character of the electrostatic interactions. When the composition of large proteins in bulk is in very large excess it is found that the structure of the adsorb multilayer is such that the layer in contact with the surface is composed by a mixture of large and small proteins. However, the second and third layers are almost exclusively composed of large proteins. The theory is also generalized to study the time-dependent adsorption. The approach is based on separation of time scales into fast modes for the ions from the salt and the solvent and slow for the proteins. The dynamic equations are written for the slow modes, while the fast ones are obtained from the condition of equilibrium constrained to the distribution of proteins given by the slow modes. Two different processes are presented: the adsorption from a homogeneous solution to a charged surface at low salt concentration, and large excess of the large proteins in bulk. The second process is the kinetics of structural and adsorption change by changing the salt concentration of the bulk solution from low to high. The first process shows a large overshoot of the large proteins on the surface due to their excess in solution, followed by a surface replacement by the smaller molecules. The second process shows a very fast desorption of the large proteins followed by adsorption at latter stages. This process is found to be driven by large electrostatic repulsions induced by the fast ions from the salt approaching the surface. The relevance of the theoretical predictions to experimental system and possible directions for improvements of the theory are discussed.

Silane surface modification for improved bioadhesion of esophageal stents

NASA Astrophysics Data System (ADS)

Karakoy, Mert; Gultepe, Evin; Pandey, Shivendra; Khashab, Mouen A.; Gracias, David H.

2014-08-01

Stent migration occurs in 10-40% of patients who undergo placement of esophageal stents, with higher migration rates seen in those treated for benign esophageal disorders. This remains a major drawback of esophageal stent therapy. In this paper, we propose a new surface modification method to increase the adhesion between self-expandable metallic stents (SEMS) and tissue while preserving their removability. Taking advantage of the well-known affinity between epoxide and amine terminated silane coupling agents with amine and carboxyl groups that are abundant in proteins and related molecules in the human body; we modified the surfaces of silicone coated esophageal SEMS with these adhesive self-assembled monolayers (SAMs). We utilized vapor phase silanization to modify the surfaces of different substrates including PDMS strips and SEMS, and measured the force required to slide these substrates on a tissue piece. Our results suggest that surface modification of esophageal SEMS via covalent attachment of protein-binding coupling agents improves adhesion to tissue and could offer a solution to reduce SEMS migration while preserving their removability.

The electrokinetic behavior of calcium oxalate monohydrate in macromolecular solutions

NASA Technical Reports Server (NTRS)

Curreri, P. A.; Onoda, G. Y., Jr.; Finlayson, B.

1988-01-01

Electrophoretic mobilities were measured for calcium oxalate monohydrate (COM) in solutions containing macromolecules. Two mucopolysaccharides (sodium heparin and chrondroitin sulfate) and two proteins (positively charged lysozyme and negatively charged bovine serum albumin) were studied as adsorbates. The effects of pH, calcium oxalate surface charge (varied by calcium or oxalate ion activity), and citrate concentration were investigated. All four macromolecules showed evidence for chemical adsorption. The macromolecule concentrations needed for reversing the surface charge indicated that the mucopopolysacchrides have greater affinity for the COM surface than the proteins. The amount of proteins that can chemically adsorb appears to be limited to approximately one monomolecular layer. When the surface charge is high, an insufficient number of proteins can chemically adsorb to neutralize or reverse the surface charge. The remaining surface charge is balanced by proteins held near the surface by longer range electrostatic forces only. Citrate ions at high concentrations appear to compete effectively with the negative protein for surface sites but show no evidence for competing with the positively charged protein.

A coarse grain model for protein-surface interactions

NASA Astrophysics Data System (ADS)

Wei, Shuai; Knotts, Thomas A.

2013-09-01

The interaction of proteins with surfaces is important in numerous applications in many fields—such as biotechnology, proteomics, sensors, and medicine—but fundamental understanding of how protein stability and structure are affected by surfaces remains incomplete. Over the last several years, molecular simulation using coarse grain models has yielded significant insights, but the formalisms used to represent the surface interactions have been rudimentary. We present a new model for protein surface interactions that incorporates the chemical specificity of both the surface and the residues comprising the protein in the context of a one-bead-per-residue, coarse grain approach that maintains computational efficiency. The model is parameterized against experimental adsorption energies for multiple model peptides on different types of surfaces. The validity of the model is established by its ability to quantitatively and qualitatively predict the free energy of adsorption and structural changes for multiple biologically-relevant proteins on different surfaces. The validation, done with proteins not used in parameterization, shows that the model produces remarkable agreement between simulation and experiment.

Binding ligand prediction for proteins using partial matching of local surface patches.

PubMed

Sael, Lee; Kihara, Daisuke

2010-01-01

Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

Binding Ligand Prediction for Proteins Using Partial Matching of Local Surface Patches

PubMed Central

Sael, Lee; Kihara, Daisuke

2010-01-01

Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group. PMID:21614188

Noninvasive noble metal nanoparticle arrays for surface-enhanced Raman spectroscopy of proteins

NASA Astrophysics Data System (ADS)

Inya-Agha, Obianuju; Forster, Robert J.; Keyes, Tia E.

2007-02-01

Noble metal nanoparticles arrays are well established substrates for surface enhanced Raman spectroscopy (SERS). Their ability to enhance optical fields is based on the interaction of their surface valence electrons with incident electromagnetic radiation. In the array configuration, noble metal nanoparticles have been used to produce SER spectral enhancements of up to 10 8 orders of magnitude, making them useful for the trace analysis of physiologically relevant analytes such as proteins and peptides. Electrostatic interactions between proteins and metal surfaces result in the preferential adsorption of positively charged protein domains onto metal surfaces. This preferential interaction has the effect of disrupting the native conformation of the protein fold, with a concomitant loss of protein function. A major historic advantage of Raman microspectroscopy has been is its non-invasive nature; protein denaturation on the metal surfaces required for SER spectroscopy renders it a much more invasive technique. Further, part of the analytical power of Raman spectroscopy lies in its use as a secondary conformation probe. The protein structural loss which occurs on the metal surface results in secondary conformation readings which are not true to the actual native state of the analyte. This work presents a method for chemical fabrication of noble metal SERS arrays with surface immobilized layers which can protect protein native conformation without excessively mitigating the electromagnetic enhancements of spectra. Peptide analytes are used as model systems for proteins. Raman spectra of alpha lactalbumin on surfaces and when immobilized on these novel arrays are compared. We discuss the ability of the surface layer to protect protein structure whilst improving signal intensity.

Carboxylator: incorporating solvent-accessible surface area for identifying protein carboxylation sites

NASA Astrophysics Data System (ADS)

Lu, Cheng-Tsung; Chen, Shu-An; Bretaña, Neil Arvin; Cheng, Tzu-Hsiu; Lee, Tzong-Yi

2011-10-01

In proteins, glutamate (Glu) residues are transformed into γ-carboxyglutamate (Gla) residues in a process called carboxylation. The process of protein carboxylation catalyzed by γ-glutamyl carboxylase is deemed to be important due to its involvement in biological processes such as blood clotting cascade and bone growth. There is an increasing interest within the scientific community to identify protein carboxylation sites. However, experimental identification of carboxylation sites via mass spectrometry-based methods is observed to be expensive, time-consuming, and labor-intensive. Thus, we were motivated to design a computational method for identifying protein carboxylation sites. This work aims to investigate the protein carboxylation by considering the composition of amino acids that surround modification sites. With the implication of a modified residue prefers to be accessible on the surface of a protein, the solvent-accessible surface area (ASA) around carboxylation sites is also investigated. Radial basis function network is then employed to build a predictive model using various features for identifying carboxylation sites. Based on a five-fold cross-validation evaluation, a predictive model trained using the combined features of amino acid sequence (AA20D), amino acid composition, and ASA, yields the highest accuracy at 0.874. Furthermore, an independent test done involving data not included in the cross-validation process indicates that in silico identification is a feasible means of preliminary analysis. Additionally, the predictive method presented in this work is implemented as Carboxylator (http://csb.cse.yzu.edu.tw/Carboxylator/), a web-based tool for identifying carboxylated proteins with modification sites in order to help users in investigating γ-glutamyl carboxylation.

Reversible Immobilization of Proteins in Sensors and Solid-State Nanopores.

PubMed

Ananth, Adithya; Genua, María; Aissaoui, Nesrine; Díaz, Leire; Eisele, Nico B; Frey, Steffen; Dekker, Cees; Richter, Ralf P; Görlich, Dirk

2018-05-01

The controlled functionalization of surfaces with proteins is crucial for many analytical methods in life science research and biomedical applications. Here, a coating for silica-based surfaces is established which enables stable and selective immobilization of proteins with controlled orientation and tunable surface density. The coating is reusable, retains functionality upon long-term storage in air, and is applicable to surfaces of complex geometry. The protein anchoring method is validated on planar surfaces, and then a method is developed to measure the anchoring process in real time using silicon nitride solid-state nanopores. For surface attachment, polyhistidine tags that are site specifically introduced into recombinant proteins are exploited, and the yeast nucleoporin Nsp1 is used as model protein. Contrary to the commonly used covalent thiol chemistry, the anchoring of proteins via polyhistidine tag is reversible, permitting to take proteins off and replace them by other ones. Such switching in real time in experiments on individual nanopores is monitored using ion conductivity. Finally, it is demonstrated that silica and gold surfaces can be orthogonally functionalized to accommodate polyhistidine-tagged proteins on silica but prevent protein binding to gold, which extends the applicability of this surface functionalization method to even more complex sensor devices. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acrolein Microspheres Are Bonded To Large-Area Substrates

NASA Technical Reports Server (NTRS)

Rembaum, Alan; Yen, Richard C. K.

1988-01-01

Reactive cross-linked microspheres produced under influence of ionizing radiation in aqueous solutions of unsaturated aldehydes, such as acrolein, with sodium dodecyl sulfate. Diameters of spheres depend on concentrations of ingredients. If polystyrene, polymethylmethacrylate, or polypropylene object immersed in solution during irradiation, microspheres become attached to surface. Resulting modified surface has grainy coating with reactivity similar to free microspheres. Aldehyde-substituted-functional microspheres react under mild conditions with number of organic reagents and with most proteins. Microsphere-coated macrospheres or films used to immobilize high concentrations of proteins, enzymes, hormones, viruses, cells, and large number of organic compounds. Applications include separation techniques, clinical diagnostic tests, catalytic processes, and battery separators.

Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

PubMed Central

Liao, Sumei; Klein, Marlise I.; Heim, Kyle P.; Fan, Yuwei; Bitoun, Jacob P.; Ahn, San-Joon; Burne, Robert A.; Koo, Hyun; Brady, L. Jeannine

2014-01-01

Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA. PMID:24748612

Structural Study of the C-Terminal Domain of Nonstructural Protein 1 from Japanese Encephalitis Virus.

PubMed

Poonsiri, Thanalai; Wright, Gareth S A; Diamond, Michael S; Turtle, Lance; Solomon, Tom; Antonyuk, Svetlana V

2018-04-01

Japanese encephalitis virus (JEV) is a mosquito-transmitted flavivirus that is closely related to other emerging viral pathogens, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout Southeast Asia, the Pacific Islands, and the Far East, causing around 68,000 cases of infection worldwide each year. In this report, we present a 2.1-Å-resolution crystal structure of the C-terminal β-ladder domain of JEV nonstructural protein 1 (NS1-C). The surface charge distribution of JEV NS1-C is similar to those of WNV and ZIKV but differs from that of DENV. Analysis of the JEV NS1-C structure, with in silico molecular dynamics simulation and experimental solution small-angle X-ray scattering, indicates extensive loop flexibility on the exterior of the protein. This, together with the surface charge distribution, indicates that flexibility influences the protein-protein interactions that govern pathogenicity. These factors also affect the interaction of NS1 with the 22NS1 monoclonal antibody, which is protective against West Nile virus infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 mediates interaction with membranes and that sulfate binding sites common to NS1 structures are not glycosaminoglycan binding interfaces. This report highlights several differences between flavivirus NS1 proteins and contributes to our understanding of their structure-pathogenic function relationships. IMPORTANCE JEV is a major cause of viral encephalitis in Asia. Despite extensive vaccination, epidemics still occur. Nonstructural protein 1 (NS1) plays a role in viral replication, and, because it is secreted, it can exhibit a wide range of interactions with host proteins. NS1 sequence and protein folds are conserved within the Flavivirus genus, but variations in NS1 protein-protein interactions among viruses likely contribute to differences in pathogenesis. Here, we compared characteristics of the C-terminal β-ladder domain of NS1 between flaviviruses, including surface charge, loop flexibility, epitope cross-reactivity, membrane adherence, and glycosaminoglycan binding. These structural features are central to NS1 functionality and may provide insight into the development of diagnostic tests and therapeutics. Copyright © 2018 American Society for Microbiology.

Impact of hydrophilic and hydrophobic functionalization of flat TiO2/Ti surfaces on proteins adsorption

NASA Astrophysics Data System (ADS)

Fabre, Héloïse; Mercier, Dimitri; Galtayries, Anouk; Portet, David; Delorme, Nicolas; Bardeau, Jean-François

2018-02-01

Controlling adsorption of proteins onto medical devices is a key issue for implant-related infections. As self-assembled monolayers (SAMs) on titanium oxide represent a good model to study the surface-protein interactions, TiO2 surface properties were modified by grafting bisphosphonate molecules terminated with hydrophilic poly(ethylene glycol) groups and hydrophobic perfluoropolyether ones, respectively. Characterisation of the surface chemistry and surface topography of the modified surfaces was performed using XPS and atomic force microscopy (AFM). Quartz-crystal microbalance with dissipation (QCM-D) was used to determine the mass of adsorbed proteins as well as its kinetics. Poly(ethylene glycol)-terminated SAMs were the most effective surfaces to limit the adsorption of both BSA and fibrinogen in comparison to perfluorinated-terminated SAMs and non-modified TiO2 surfaces, as expected. The adsorption was not reversible in the case of BSA, while a partial reversibility was observed with Fg, most probably due to multilayers of proteins. The grafted surfaces adsorbed about the same quantity of proteins in terms of molecules per surface area, most probably in monolayer or island-like groups of adsorbed proteins. The adsorption on pristine TiO2 reveals a more important, non-specific adsorption of proteins.

Design of Nanostructured Biological Materials Through Self-Assembly of Peptides and Proteins

DTIC Science & Technology

2002-01-01

of applications, including scaffolding for tissue repair in regenerative medicine, drug delivery and biological surface engineering. Tirrell and...colleagues [2] designed artificial proteins that undergo self-assembly to form hydrogels responsive to pH and other environmental changes. Ghadiri and...showed that other β-sheet peptide systems can also undergo self-assembly into regular nanofiber structures. Although they share no sequence

Selective binding of proteins on functional nanoparticles via reverse charge parity model: an in vitro study

NASA Astrophysics Data System (ADS)

Ghosh, Goutam; Panicker, Lata; Barick, K. C.

2014-03-01

The conformation of proteins absorbed on nanoparticles surface plays a crucial role in applications of nanoparticles in biomedicine. The surface protein conformation depends on several factors, namely, nature of protein-nanoparticles interaction, chemical composition of the surface of nanoparticles etc. A model of the electrostatic binding of proteins on charged surface nanoparticles has been proposed earlier (Ghosh et al 2013 Colloids Surf. B 103 267). Also, the irreversible denaturation of the protein conformation due to binding of counterions was reported. In this paper, we have used this model, involving reverse charge parity, to show selective binding of proteins on charged surface iron oxide nanoparticles (IONPs). IONPs were surface functionalized with cetylpyridinium chloride (CPC), cetyl(trimethyl)ammonium bromide (CTAB) and cetylpyridinium iodide (CPI). The effect of counterions (Cl-, Br- and I-) on protein conformation has also been investigated. Several proteins such as α-lactalbumin (ALA), β-lactoglobulin (BLG), ovalbumin (OVA), bovin serum albumin (BSA) and HEWL were chosen for this investigation.

Concordance of preclinical and clinical pharmacology and toxicology of therapeutic monoclonal antibodies and fusion proteins: cell surface targets

PubMed Central

Bugelski, Peter J; Martin, Pauline L

2012-01-01

Monoclonal antibodies (mAbs) and fusion proteins directed towards cell surface targets make an important contribution to the treatment of disease. The purpose of this review was to correlate the clinical and preclinical data on the 15 currently approved mAbs and fusion proteins targeted to the cell surface. The principal sources used to gather data were: the peer reviewed Literature; European Medicines Agency ‘Scientific Discussions’; and the US Food and Drug Administration ‘Pharmacology/Toxicology Reviews’ and package inserts (United States Prescribing Information). Data on the 15 approved biopharmaceuticals were included: abatacept; abciximab; alefacept; alemtuzumab; basiliximab; cetuximab; daclizumab; efalizumab; ipilimumab; muromonab; natalizumab; panitumumab; rituximab; tocilizumab; and trastuzumab. For statistical analysis of concordance, data from these 15 were combined with data on the approved mAbs and fusion proteins directed towards soluble targets. Good concordance with human pharmacodynamics was found for mice receiving surrogates or non-human primates (NHPs) receiving the human pharmaceutical. In contrast, there was poor concordance for human pharmacodynamics in genetically deficient mice and for human adverse effects in all three test systems. No evidence that NHPs have superior predictive value was found. PMID:22168282

Evidence that Surface Proteins Sn14 and Sn16 of Sarcocystis neurona Merozoites Are Involved in Infection and Immunity†

PubMed Central

Liang, Fang Ting; Granstrom, David E.; Zhao, Xiao Min; Timoney, John F.

1998-01-01

Sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis (EPM). Based on an analysis of 25,000 equine serum and cerebrospinal fluid (CSF) samples, including samples from horses with neurologic signs typical of EPM or with histologically or parasitologically confirmed EPM, four major immunoblot band patterns have been identified. Twenty-three serum and CSF samples representing each of the four immunoblot patterns were selected from 220 samples from horses with neurologic signs resembling EPM and examined for inhibitory effects on the infectivity of S. neurona by an in vitro neutralization assay. A high correlation between immunoblot band pattern and neutralizing activity was detected. Two proteins, Sn14 and Sn16 (14 and 16 kDa, respectively), appeared to be important for in vitro infection. A combination of the results of surface protein labeling, immunoprecipitation, Western blotting, and trypsin digestion suggests that these molecules are surface proteins and may be useful components of a vaccine against S. neurona infection. Although S. neurona is an obligate intracellular parasite, it is potentially a target for specific antibodies which may lyse merozoites via complement or inhibit their attachment and penetration to host cells. PMID:9573058

An adhesin from hydrogen-utilizing rumen methanogen Methanobrevibacter ruminantium M1 binds a broad range of hydrogen-producing microorganisms.

PubMed

Ng, Filomena; Kittelmann, Sandra; Patchett, Mark L; Attwood, Graeme T; Janssen, Peter H; Rakonjac, Jasna; Gagic, Dragana

2016-09-01

Symbiotic associations are ubiquitous in the microbial world and have a major role in shaping the evolution of both partners. One of the most interesting mutualistic relationships exists between protozoa and methanogenic archaea in the fermentative forestomach (rumen) of ruminant animals. Methanogens reside within and on the surface of protozoa as symbionts, and interspecies hydrogen transfer is speculated to be the main driver for physical associations observed between the two groups. In silico analyses of several rumen methanogen genomes have previously shown that up to 5% of genes encode adhesin-like proteins, which may be central to rumen interspecies attachment. We hypothesized that adhesin-like proteins on methanogen cell surfaces facilitate attachment to protozoal hosts. Using phage display technology, we have identified a protein (Mru_1499) from Methanobrevibacter ruminantium M1 as an adhesin that binds to a broad range of rumen protozoa (including the genera Epidinium and Entodinium). This unique adhesin also binds the cell surface of the bacterium Butyrivibrio proteoclasticus, suggesting a broad adhesion spectrum for this protein. © 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Coating gigaporous polystyrene microspheres with cross-linked poly(vinyl alcohol) hydrogel as a rapid protein chromatography matrix.

PubMed

Qu, Jian-Bo; Huan, Guan-Sheng; Chen, Yan-Li; Zhou, Wei-Qing; Liu, Jian-Guo; Huang, Fang

2014-08-13

Gigaporous polystyrene (PS) microspheres were hydrophilized by in situ polymerization to give a stable cross-linked poly(vinyl alcohol) (PVA) hydrogel coating, which can shield proteins from the hydrophobic PS surface underneath. The amination of microspheres (PS-NH2) was first carried out through acetylization, oximation and reduction, and then 4,4'-azobis (4-cyanovaleric acid) (ACV), a polymerization initiator, was covalently immobilized on PS-NH2 through amide bond formation, and the cross-linked poly(vinyl acetate) (PVAc) was prepared by radical polymerization at the surfaces of ACV-immobilized PS microspheres (PS-ACV). Finally, the cross-linked PVA hydrogel coated gigaporous PS microspheres (PS-PVA) was easily achieved through alcoholysis of PVAc. Results suggested that the PS microspheres were effectively coated with cross-linked PVA hydrogel, where the gigaporrous structure remained under optimal conditions. After hydrophilic modification (PS-PVA), the protein-resistant ability of microspheres was greatly improved. The hydroxyl-rich PS-PVA surface can be easily derivatized by classical chemical methods. Performance advantages of the PS-PVA column in flow experiment include good permeability, low backpressure, and mechanical stability. These results indicated that PS-PVA should be promising in rapid protein chromatography.

Development of exosome surface display technology in living human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stickney, Zachary, E-mail: zstickney@scu.edu; Losacco, Joseph, E-mail: jlosacco@scu.edu; McDevitt, Sophie, E-mail: smmcdevitt@scu.edu

Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell–cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated themore » successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.« less

Identification of malaria parasite-infected red blood cell surface aptamers by inertial microfluidic SELEX (I-SELEX)

NASA Astrophysics Data System (ADS)

Birch, Christina M.; Hou, Han Wei; Han, Jongyoon; Niles, Jacquin C.

2015-07-01

Plasmodium falciparum malaria parasites invade and remodel human red blood cells (RBCs) by trafficking parasite-synthesized proteins to the RBC surface. While these proteins mediate interactions with host cells that contribute to disease pathogenesis, the infected RBC surface proteome remains poorly characterized. Here we use a novel strategy (I-SELEX) to discover high affinity aptamers that selectively recognize distinct epitopes uniquely present on parasite-infected RBCs. Based on inertial focusing in spiral microfluidic channels, I-SELEX enables stringent partitioning of cells (efficiency ≥ 106) from unbound oligonucleotides at high volume throughput (~2 × 106 cells min-1). Using an RBC model displaying a single, non-native antigen and live malaria parasite-infected RBCs as targets, we establish suitability of this strategy for de novo aptamer selections. We demonstrate recovery of a diverse set of aptamers that recognize distinct, surface-displayed epitopes on parasite-infected RBCs with nanomolar affinity, including an aptamer against the protein responsible for placental sequestration, var2CSA. These findings validate I-SELEX as a broadly applicable aptamer discovery platform that enables identification of new reagents for mapping the parasite-infected RBC surface proteome at higher molecular resolution to potentially contribute to malaria diagnostics, therapeutics and vaccine efforts.

TMEM2: A missing link in hyaluronan catabolism identified?

PubMed

Yamaguchi, Yu; Yamamoto, Hayato; Tobisawa, Yuki; Irie, Fumitoshi

2018-03-27

Hyaluronan (HA) is a glycosaminoglycan (GAG) composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is an extremely long, unbranched polymer, which often exceeds 10 6  Da and sometimes reaches 10 7  Da. A feature that epitomizes HA is its rapid turnover; one-third of the total body HA is turned over daily. The current model of HA catabolism postulates that high-molecular weight HA in the extracellular space is first cleaved into smaller fragments by a hyaluronidase(s) that resides at the cell surface, followed by internalization of fragments and their degradation into monosaccharides in lysosomes. Over the last decade, considerable research has shown that the HYAL family of hyaluronidases plays significant roles in HA catabolism. Nonetheless, the identity of a hyaluronidase responsible for the initial step of HA cleavage on the cell surface remains elusive, as biochemical and enzymological properties of HYAL proteins are not entirely consistent with those expected of cell surface hyaluronidases. Recent identification of transmembrane 2 (TMEM2) as a cell surface protein that possesses potent hyaluronidase activity suggests that it may be the "missing" cell surface hyaluronidase, and that novel models of HA catabolism should include this protein. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Surface modification of protein enhances encapsulation in chitosan nanoparticles

NASA Astrophysics Data System (ADS)

Koyani, Rina D.; Andrade, Mariana; Quester, Katrin; Gaytán, Paul; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

2018-04-01

Chitosan nanoparticles have a huge potential as nanocarriers for environmental and biomedical purposes. Protein encapsulation in nano-sized chitosan provides protection against inactivation, proteolysis, and other alterations due to environmental conditions, as well as the possibility to be targeted to specific tissues by ligand functionalization. In this work, we demonstrate that the chemical modification of the protein surface enhances the protein loading in chitosan nanocarriers. Encapsulation of green fluorescent protein and the cytochrome P450 was studied. The increase of electrostatic interactions between the free amino groups of chitosan and the increased number of free carboxylic groups in the protein surface enhance the protein loading, protein retention, and, thus, the enzymatic activity of chitosan nanoparticles. The chemical modification of protein surface with malonic acid moieties reduced drastically the protein isoelectric point increasing the protein interaction with the polycationic biomaterial and chitosan. The chemical modification of protein does not alter the morphology of chitosan nanoparticles that showed an average diameter of 18 nm, spheroidal in shape, and smooth surfaced. The strategy of chemical modification of protein surface, shown here, is a simple and efficient technique to enhance the protein loading in chitosan nanoparticles. This technique could be used for other nanoparticles based on polycationic or polyanionic materials. The increase of protein loading improves, doubtless, the performance of protein-loaded chitosan nanoparticles for biotechnological and biomedical applications.

Presence of closely spaced protein thiols on the surface of mammalian cells.

PubMed Central

Donoghue, N.; Yam, P. T.; Jiang, X. M.; Hogg, P. J.

2000-01-01

It has been proposed that certain cell-surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane-impermeable trivalent arsenical to identify closely spaced thiols in cell-surface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell-surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the gamma-glutamyl residue of GSAO (GSAO-B). Incorporation of GSAO-B into proteins was assessed by measuring the biotin using streptavidin-peroxidase. Up to 12 distinct proteins were labeled with GSAO-B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO-B. These findings demonstrate that the cell-surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active. PMID:11206065

The effect of polymer surface modification on polymer-protein interaction via interfacial polymerization and hydrophilic polymer grafting

USDA-ARS?s Scientific Manuscript database

Protein membrane separation is prone to fouling on the membrane surface resulting from protein adsorption onto the surface. Surface modification of synthetic membranes is one way to reduce fouling. We investigated surface modification of polyethersulfone (PES) as a way of improving hydrophilicity ...

Dynamics simulations for engineering macromolecular interactions

PubMed Central

Robinson-Mosher, Avi; Shinar, Tamar; Silver, Pamela A.; Way, Jeffrey

2013-01-01

The predictable engineering of well-behaved transcriptional circuits is a central goal of synthetic biology. The artificial attachment of promoters to transcription factor genes usually results in noisy or chaotic behaviors, and such systems are unlikely to be useful in practical applications. Natural transcriptional regulation relies extensively on protein-protein interactions to insure tightly controlled behavior, but such tight control has been elusive in engineered systems. To help engineer protein-protein interactions, we have developed a molecular dynamics simulation framework that simplifies features of proteins moving by constrained Brownian motion, with the goal of performing long simulations. The behavior of a simulated protein system is determined by summation of forces that include a Brownian force, a drag force, excluded volume constraints, relative position constraints, and binding constraints that relate to experimentally determined on-rates and off-rates for chosen protein elements in a system. Proteins are abstracted as spheres. Binding surfaces are defined radially within a protein. Peptide linkers are abstracted as small protein-like spheres with rigid connections. To address whether our framework could generate useful predictions, we simulated the behavior of an engineered fusion protein consisting of two 20 000 Da proteins attached by flexible glycine/serine-type linkers. The two protein elements remained closely associated, as if constrained by a random walk in three dimensions of the peptide linker, as opposed to showing a distribution of distances expected if movement were dominated by Brownian motion of the protein domains only. We also simulated the behavior of fluorescent proteins tethered by a linker of varying length, compared the predicted Förster resonance energy transfer with previous experimental observations, and obtained a good correspondence. Finally, we simulated the binding behavior of a fusion of two ligands that could simultaneously bind to distinct cell-surface receptors, and explored the landscape of linker lengths and stiffnesses that could enhance receptor binding of one ligand when the other ligand has already bound to its receptor, thus, addressing potential mechanisms for improving targeted signal transduction proteins. These specific results have implications for the design of targeted fusion proteins and artificial transcription factors involving fusion of natural domains. More broadly, the simulation framework described here could be extended to include more detailed system features such as non-spherical protein shapes and electrostatics, without requiring detailed, computationally expensive specifications. This framework should be useful in predicting behavior of engineered protein systems including binding and dissociation reactions. PMID:23822508

Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor

PubMed Central

Li, Xueming; Song, Siyu; Shuai, Qi; Pei, Yihan; Aastrup, Teodor; Pei, Yuxin; Pei, Zhichao

2015-01-01

A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery. PMID:26369583

Human and Server Docking Prediction for CAPRI Round 30–35 Using LZerD with Combined Scoring Functions

PubMed Central

Peterson, Lenna X.; Kim, Hyungrae; Esquivel-Rodriguez, Juan; Roy, Amitava; Han, Xusi; Shin, Woong-Hee; Zhang, Jian; Terashi, Genki; Lee, Matt; Kihara, Daisuke

2016-01-01

We report the performance of protein-protein docking predictions by our group for recent rounds of the Critical Assessment of Prediction of Interactions (CAPRI), a community-wide assessment of state-of-the-art docking methods. Our prediction procedure uses a protein-protein docking program named LZerD developed in our group. LZerD represents a protein surface with 3D Zernike descriptors (3DZD), which are based on a mathematical series expansion of a 3D function. The appropriate soft representation of protein surface with 3DZD makes the method more tolerant to conformational change of proteins upon docking, which adds an advantage for unbound docking. Docking was guided by interface residue prediction performed with BindML and cons-PPISP as well as literature information when available. The generated docking models were ranked by a combination of scoring functions, including PRESCO, which evaluates the native-likeness of residues’ spatial environments in structure models. First, we discuss the overall performance of our group in the CAPRI prediction rounds and investigate the reasons for unsuccessful cases. Then, we examine the performance of several knowledge-based scoring functions and their combinations for ranking docking models. It was found that the quality of a pool of docking models generated by LZerD, i.e. whether or not the pool includes near-native models, can be predicted by the correlation of multiple scores. Although the current analysis used docking models generated by LZerD, findings on scoring functions are expected to be universally applicable to other docking methods. PMID:27654025

Surface properties of adipocyte lipid-binding protein: Response to lipid binding, and comparison with homologous proteins.

PubMed

LiCata, V J; Bernlohr, D A

1998-12-01

Adipocyte lipid-binding protein (ALBP) is one of a family of intracellular lipid-binding proteins (iLBPs) that bind fatty acids, retinoids, and other hydrophobic ligands. The different members of this family exhibit a highly conserved three-dimensional structure; and where structures have been determined both with (holo) and without (apo) bound lipid, observed conformational changes are extremely small (Banaszak, et al., 1994, Adv. Prot. Chem. 45, 89; Bernlohr, et al., 1997, Annu. Rev. Nutr. 17, 277). We have examined the electrostatic, hydrophobic, and water accessible surfaces of ALBP in the apo form and of holo forms with a variety of bound ligands. These calculations reveal a number of previously unrecognized changes between apo and holo ALBP, including: 1) an increase in the overall protein surface area when ligand binds, 2) expansion of the binding cavity when ligand is bound, 3) clustering of individual residue exposure increases in the area surrounding the proposed ligand entry portal, and 4) ligand-binding dependent variation in the topology of the electrostatic potential in the area surrounding the ligand entry portal. These focused analyses of the crystallographic structures thus reveal a number of subtle but consistent conformational and surface changes that might serve as markers for differential targeting of protein-lipid complexes within the cell. Most changes are consistent from ligand to ligand, however there are some ligand-specific changes. Comparable calculations with intestinal fatty-acid-binding protein and other vertebrate iLBPs show differences in the electrostatic topology, hydrophobic topology, and in localized changes in solvent exposure near the ligand entry portal. These results provide a basis toward understanding the functional and mechanistic differences among these highly structurally homologous proteins. Further, they suggest that iLBPs from different tissues exhibit one of two predominant end-state structural distributions of the ligand entry portal.

Fundamentals of Nanoscale Polymer–Protein Interactions and Potential Contributions to Solid-State Nanobioarrays

PubMed Central

2015-01-01

Protein adsorption onto polymer surfaces is a very complex, ubiquitous, and integrated process, impacting essential areas of food processing and packaging, health devices, diagnostic tools, and medical products. The nature of protein–surface interactions is becoming much more complicated with continuous efforts toward miniaturization, especially for the development of highly compact protein detection and diagnostic devices. A large body of literature reports on protein adsorption from the perspective of ensemble-averaged behavior on macroscopic, chemically homogeneous, polymeric surfaces. However, protein–surface interactions governing the nanoscale size regime may not be effectively inferred from their macroscopic and microscopic characteristics. Recently, research efforts have been made to produce periodically arranged, nanoscopic protein patterns on diblock copolymer surfaces solely through self-assembly. Intriguing protein adsorption phenomena are directly probed on the individual biomolecule level for a fundamental understanding of protein adsorption on nanoscale surfaces exhibiting varying degrees of chemical heterogeneity. Insight gained from protein assembly on diblock copolymers can be effectively used to control the surface density, conformation, orientation, and biofunctionality of prebound proteins in highly miniaturized applications, now approaching the nanoscale. This feature article will highlight recent experimental and theoretical advances made on these fronts while focusing on single-biomolecule-level investigations of protein adsorption behavior combined with surface chemical heterogeneity on the length scale commensurate with a single protein. This article will also address advantages and challenges of the self-assembly-driven patterning technology used to produce protein nanoarrays and its implications for ultrahigh density, functional, and quantifiable protein detection in a highly miniaturized format. PMID:24456577

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

PubMed

Lee, Eun Jeong; Yun, Un-Jung; Koo, Kyung Hee; Sung, Jee Young; Shim, Jaegal; Ye, Sang-Kyu; Hong, Kyeong-Man; Kim, Yong-Nyun

2014-01-01

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function. © 2013.

Protein nanoparticles as drug delivery carriers for cancer therapy.

PubMed

Lohcharoenkal, Warangkana; Wang, Liying; Chen, Yi Charlie; Rojanasakul, Yon

2014-01-01

Nanoparticles have increasingly been used for a variety of applications, most notably for the delivery of therapeutic and diagnostic agents. A large number of nanoparticle drug delivery systems have been developed for cancer treatment and various materials have been explored as drug delivery agents to improve the therapeutic efficacy and safety of anticancer drugs. Natural biomolecules such as proteins are an attractive alternative to synthetic polymers which are commonly used in drug formulations because of their safety. In general, protein nanoparticles offer a number of advantages including biocompatibility and biodegradability. They can be prepared under mild conditions without the use of toxic chemicals or organic solvents. Moreover, due to their defined primary structure, protein-based nanoparticles offer various possibilities for surface modifications including covalent attachment of drugs and targeting ligands. In this paper, we review the most significant advancements in protein nanoparticle technology and their use in drug delivery arena. We then examine the various sources of protein materials that have been used successfully for the construction of protein nanoparticles as well as their methods of preparation. Finally, we discuss the applications of protein nanoparticles in cancer therapy.

Targeting kinase signaling pathways with constrained peptide scaffolds

PubMed Central

Hanold, Laura E.; Fulton, Melody D.; Kennedy, Eileen J.

2017-01-01

Kinases are amongst the largest families in the human proteome and serve as critical mediators of a myriad of cell signaling pathways. Since altered kinase activity is implicated in a variety of pathological diseases, kinases have become a prominent class of proteins for targeted inhibition. Although numerous small molecule and antibody-based inhibitors have already received clinical approval, several challenges may still exist with these strategies including resistance, target selection, inhibitor potency and in vivo activity profiles. Constrained peptide inhibitors have emerged as an alternative strategy for kinase inhibition. Distinct from small molecule inhibitors, peptides can provide a large binding surface area that allows them to bind shallow protein surfaces rather than defined pockets within the target protein structure. By including chemical constraints within the peptide sequence, additional benefits can be bestowed onto the peptide scaffold such as improved target affinity and target selectivity, cell permeability and proteolytic resistance. In this review, we highlight examples of diverse chemistries that are being employed to constrain kinase-targeting peptide scaffolds and highlight their application to modulate kinase signaling as well as their potential clinical implications. PMID:28185915

Label-Free Quantitative Immunoassay of Fibrinogen in Alzheimer Disease Patient Plasma Using Fiber Optical Surface Plasmon Resonance

NASA Astrophysics Data System (ADS)

Kim, Jisoo; Kim, SeJin; Nguyen, Tan Tai; Lee, Renee; Li, Tiehua; Yun, Changhyun; Ham, Youngeun; An, Seong Soo A.; Ju, Heongkyu

2016-05-01

We present a real-time quantitative immunoassay to detect fibrinogen in the blood plasma of Alzheimer's disease patients using multimode fiber optical sensors in which surface plasmon resonance (SPR) was employed. Nanometer-thick bimetals including silver and aluminum were coated onto the core surface of the clad-free part (5 cm long) of the fiber for SPR excitation at the He-Ne laser wavelength of 632.8 nm. The histidine-tagged peptide was then coated on the metal surface to immobilize the fibrinogen antibody for the selective capture of fibrinogen among the proteins in the patient blood plasma. The SPR fiber optical sensor enabled quantitative detection of concentrations of fibrinogen from the different human patient blood at a detection limit of ˜20 ng/ml. We also observed a correlation in the fibrinogen concentration measurement between enzyme-linked immunosorbent assay and our SPR fiber-based sensors. This suggests that the presented SPR fiber-based sensors that do not rely on the use of labels such as fluorophores can be used for a real-time quantitative assay of a specific protein such as fibrinogen in a human blood that is known to contain many other kinds of proteins together.

Surfing the Protein-Protein Interaction Surface Using Docking Methods: Application to the Design of PPI Inhibitors.

PubMed

Sable, Rushikesh; Jois, Seetharama

2015-06-23

Blocking protein-protein interactions (PPI) using small molecules or peptides modulates biochemical pathways and has therapeutic significance. PPI inhibition for designing drug-like molecules is a new area that has been explored extensively during the last decade. Considering the number of available PPI inhibitor databases and the limited number of 3D structures available for proteins, docking and scoring methods play a major role in designing PPI inhibitors as well as stabilizers. Docking methods are used in the design of PPI inhibitors at several stages of finding a lead compound, including modeling the protein complex, screening for hot spots on the protein-protein interaction interface and screening small molecules or peptides that bind to the PPI interface. There are three major challenges to the use of docking on the relatively flat surfaces of PPI. In this review we will provide some examples of the use of docking in PPI inhibitor design as well as its limitations. The combination of experimental and docking methods with improved scoring function has thus far resulted in few success stories of PPI inhibitors for therapeutic purposes. Docking algorithms used for PPI are in the early stages, however, and as more data are available docking will become a highly promising area in the design of PPI inhibitors or stabilizers.

Computational and theoretical approaches for studies of a lipid recognition protein on biological membranes

PubMed Central

Yamamoto, Eiji

2017-01-01

Many cellular functions, including cell signaling and related events, are regulated by the association of peripheral membrane proteins (PMPs) with biological membranes containing anionic lipids, e.g., phosphatidylinositol phosphate (PIP). This association is often mediated by lipid recognition modules present in many PMPs. Here, I summarize computational and theoretical approaches to investigate the molecular details of the interactions and dynamics of a lipid recognition module, the pleckstrin homology (PH) domain, on biological membranes. Multiscale molecular dynamics simulations using combinations of atomistic and coarse-grained models yielded results comparable to those of actual experiments and could be used to elucidate the molecular mechanisms of the formation of protein/lipid complexes on membrane surfaces, which are often difficult to obtain using experimental techniques. Simulations revealed some modes of membrane localization and interactions of PH domains with membranes in addition to the canonical binding mode. In the last part of this review, I address the dynamics of PH domains on the membrane surface. Local PIP clusters formed around the proteins exhibit anomalous fluctuations. This dynamic change in protein-lipid interactions cause temporally fluctuating diffusivity of proteins, i.e., the short-term diffusivity of the bound protein changes substantially with time, and may in turn contribute to the formation/dissolution of protein complexes in membranes. PMID:29159013

Identification of a polymorphic collagen-like protein in the crustacean bacteria Pasteuria ramosa.

PubMed

Mouton, Laurence; Traunecker, Emmanuel; McElroy, Kerensa; Du Pasquier, Louis; Ebert, Dieter

2009-12-01

Pasteuria ramosa is a spore-forming bacterium that infects Daphnia species. Previous results demonstrated a high specificity of host clone/parasite genotype interactions. Surface proteins of bacteria often play an important role in attachment to host cells prior to infection. We analyzed surface proteins of P. ramosa spores by two-dimensional gel electrophoresis. For the first time, we prove that two isolates selected for their differences in infectivity reveal few but clear-cut differences in protein patterns. Using internal sequencing and LC/MS/MS, we identified a collagen-like protein named Pcl1a (Pasteuria collagen-like protein 1a). This protein, reconstructed with the help of Pasteuria genome sequences, contains three domains: a 75-amino-acid amino-terminal domain with a potential transmembrane helix domain, a central collagen-like region (CLR) containing Gly-Xaa-Yaa (GXY) repeats, and a 7-amino-acid carboxy-terminal domain. The CLR region is polymorphic among the two isolates with amino-acid substitutions and a variable number of GXY triplets. Collagen-like proteins are rare in prokaryotes, although they have been described in several pathogenic bacteria, including Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis, closely related to Pasteuria species, in which they could be involved in the adherence of bacteria to host cells.

Role of fibronectin in intravesical BCG therapy for superficial bladder cancer.

PubMed

Ratliff, T L; Kavoussi, L R; Catalona, W J

1988-02-01

Intravesical bacillus Calmette-Guerin (BCG) has been demonstrated to be effective both for prophylaxis and treatment of superficial bladder cancer. In order to identify the progression of events that result in BCG-mediated antitumor activity, studies were performed to evaluate the mechanism of binding of BCG within the bladder. Histological and quantitative studies in a mouse model revealed that BCG attached to the bladder wall only in areas of urothelial damage. Preliminary in vitro data showed that BCG attached to surfaces coated with extracellular matrix proteins. Further studies were then performed using purified extracellular matrix proteins to identify the proteins responsible for attachment. BCG were observed to attach to surfaces coated only with purified fibronectin (FN) but not to other purified proteins including laminin, collagen or fibrinogen. The attachment of BCG to purified FN in vitro was dose dependent and was inhibited by anti-FN antibodies. Moreover, BCG attachment in vivo to bladders with damaged urothelial surfaces was inhibited more than 95% by anti-FN antibodies, but binding was not affected by anti-laminin antibodies or preimmune serum. A survey of commercially available BCG vaccines (Pasteur, Tice, Glaxo, Connaught) showed that only Glaxo BCG did not attach to FN-coated surfaces. Glaxo BCG also was shown to express inferior antitumor activity suggesting that the absence of FN binding by Glaxo may have been associated with the absence of antitumor activity of the vaccine.

Comparison of the surface coat proteins of the pine wood nematode appeared during host pine infection and in vitro culture by a proteomic approach.

PubMed

Shinya, Ryoji; Morisaka, Hironobu; Takeuchi, Yuko; Ueda, Mitsuyoshi; Futai, Kazuyoshi

2010-12-01

Pine wilt disease, caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has become of worldwide quarantine concern in recent years. Here, we disclosed the surface coat (SC) proteins of the PWN which are thought to be one of the key components in pine wilt development. This is the first report that focused on the SC proteins and thoroughly identified those proteins of a plant-parasitic nematode using the proteomic approach. In this study, SC protein profiles were compared for PWNs grown on the fungus Botrytis cinerea and in host pine seedlings. The results demonstrated that the gross amount of PWN SC proteins drastically increased during infection of the host pine. Thirty-seven protein bands showed significant quantity differences between fungus-grown and host-origin PWNs, and were used for identification by matrix-assisted laser desorption ionization time of flight mass spectrometry analysis. These included several proteins that are presumed to be involved in the host immune response; for example, regulators of reactive oxygen species (ROS) and a ROS scavenger. These results might suggest that the PWN SC proteins are crucial in modulating or evading host immune response. Our data provide a new insight into the mechanism of pine wilt disease and the biological role of the SC proteins of plant-parasitic nematodes.

The identification of foam-forming soluble proteins from wheat (Triticum aestivum) dough.

PubMed

Salt, Louise J; Robertson, James A; Jenkins, John A; Mulholland, Francis; Mills, E N Clare

2005-04-01

Proteomic methods have been used to identify foam-forming soluble proteins from dough that may play an important role in stabilising gas bubbles in dough, and hence influence the crumb structure of bread. Proteins from a soluble fraction of dough (dough liquor) or dough liquor foam have been separated by two-dimensional gel electrophoresis, and 42 identified using a combination of matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight analyses. Major polypeptide components included beta-amylase, tritin and serpins, with members of the alpha-amylase/trypsin inhibitor family being particularly abundant. Neither prolamin seed storage proteins nor the surface-active protein puroindoline were found. Commonly used dough ingredients (NaCl, Na L-ascorbate) had only a minor effect on the 2-DE protein profiles of dough liquor, of which one of the more significant was the loss of 9 kDa nonspecific lipid transfer protein. Many proteins were lost in dough liquor foam, particularly tritin, whilst a number of alpha-amylase inhibitors were more dominant, suggesting that these are amongst the most strongly surface-active proteins in dough liquor. Such proteins may play a role determining the ability of the aqueous phase of doughs, as represented by dough liquor, to form an elastic interface lining the bubbles, and hence maintain their integrity during dough proving.

Self-generated covalent cross-links in the cell-surface adhesins of Gram-positive bacteria.

PubMed

Baker, Edward N; Squire, Christopher J; Young, Paul G

2015-10-01

The ability of bacteria to adhere to other cells or to surfaces depends on long, thin adhesive structures that are anchored to their cell walls. These structures include extended protein oligomers known as pili and single, multi-domain polypeptides, mostly based on multiple tandem Ig-like domains. Recent structural studies have revealed the widespread presence of covalent cross-links, not previously seen within proteins, which stabilize these domains. The cross-links discovered so far are either isopeptide bonds that link lysine side chains to the side chains of asparagine or aspartic acid residues or ester bonds between threonine and glutamine side chains. These bonds appear to be formed by spontaneous intramolecular reactions as the proteins fold and are strategically placed so as to impart considerable mechanical strength. © 2015 Authors; published by Portland Press Limited.

Correlation between surface morphology and surface forces of protein A adsorbed on mica.

PubMed Central

Ohnishi, S; Murata, M; Hato, M

1998-01-01

We have investigated the morphology and surface forces of protein A adsorbed on mica surface in the protein solutions of various concentrations. The force-distance curves, measured with a surface force apparatus (SFA), were interpreted in terms of two different regimens: a "large-distance" regimen in which an electrostatic double-layer force dominates, and an "adsorbed layer" regimen in which a force of steric origin dominates. To further clarify the forces of steric origin, the surface morphology of the adsorbed protein layer was investigated with an atomic force microscope (AFM) because the steric repulsive forces are strongly affected by the adsorption mode of protein A molecules on mica. At lower protein concentrations (2 ppm, 10 ppm), protein A molecules were adsorbed "side-on" parallel to the mica surfaces, forming a monolayer of approximately 2.5 nm. AFM images at higher concentrations (30 ppm, 100 ppm) showed protruding structures over the monolayer, which revealed that the adsorbed protein A molecules had one end oriented into the solution, with the remainder of each molecule adsorbed side-on to the mica surface. These extending ends of protein A overlapped each other and formed a "quasi-double layer" over the mica surface. These AFM images proved the existence of a monolayer of protein A molecules at low concentrations and a "quasi-double layer" with occasional protrusions at high concentrations, which were consistent with the adsorption mode observed in the force-distance curves. PMID:9449346

Correlation of cell surface proteins of distinct Beauveria bassiana cell types and adaption to varied environment and interaction with the host insect.

PubMed

Yang, Zhi; Jiang, Hongyan; Zhao, Xin; Lu, Zhuoyue; Luo, Zhibing; Li, Xuebing; Zhao, Jing; Zhang, Yongjun

2017-02-01

The insect fungal pathogen Beauveria bassiana produces a number of distinct cell types that include aerial conidia, blastospores and haemolymph-derived cells, termed hyphal bodies, to adapt varied environment niches and within the host insect. These cells display distinct biochemical properties and surface structures, and a highly ordered outermost brush-like structure uniquely present on hyphal bodies, but not on any in vitro cells. Here, we found that the outermost structure on the hyphal bodies mainly consisted of proteins associated to structural wall components in that most of it could be removed by dithiothreitol (DTT) or proteinase K. DTT-treatment also caused delayed germination, decreased tolerance to ultraviolet irradiation and virulence of conidia or blastospores, with decreased adherence and alternated carbohydrate epitopes, suggesting involvement in fungal development, stress responses and virulence. To characterize these cell surface molecules, proteins were released from the living cells using DTT, and identified and quantitated using label-free quantitative mass spectrometry. Thereafter, a series of bioinformatics programs were used to predict cell surface-associated proteins (CSAPs), and 96, 166 and 54 CSAPs were predicted from the identified protein pools of conidia, blastospores and hyphal bodies, respectively, which were involved in utilization of carbohydrate, nitrogen, and lipid, detoxification, pathogen-host interaction, and likely other cellular processes. Thirteen, sixty-nine and six CSAPs were exclusive in conidia, blastospores and hyphal bodies, respectively, which were verified by eGFP-tagged proteins at their N-terminus. Our data provide a crucial cue to understand mechanism of B. bassiana to adapt to varied environment and interaction with insect host. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B

PubMed Central

Wang, Guirong; Taneva, Svetla; Keough, Kevin M.W.; Floros, Joanna

2010-01-01

Summary Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A2) and SP-A2 (1A0), and the coexpressed SP-A1/SP-A2 (6A2/1A0) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A0) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A2) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A2/1A0) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity. PMID:17678872

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii.

PubMed

Schmid-Siegert, Emanuel; Richard, Sophie; Luraschi, Amanda; Mühlethaler, Konrad; Pagni, Marco; Hauser, Philippe M

2017-11-07

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different. IMPORTANCE Pneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens. Copyright © 2017 Schmid-Siegert et al.

Protein Structural Analysis via Mass Spectrometry-Based Proteomics

PubMed Central

Artigues, Antonio; Nadeau, Owen W.; Rimmer, Mary Ashley; Villar, Maria T.; Du, Xiuxia; Fenton, Aron W.; Carlson, Gerald M.

2017-01-01

Modern mass spectrometry (MS) technologies have provided a versatile platform that can be combined with a large number of techniques to analyze protein structure and dynamics. These techniques include the three detailed in this chapter: 1) hydrogen/deuterium exchange (HDX), 2) limited proteolysis, and 3) chemical crosslinking (CX). HDX relies on the change in mass of a protein upon its dilution into deuterated buffer, which results in varied deuterium content within its backbone amides. Structural information on surface exposed, flexible or disordered linker regions of proteins can be achieved through limited proteolysis, using a variety of proteases and only small extents of digestion. CX refers to the covalent coupling of distinct chemical species and has been used to analyze the structure, function and interactions of proteins by identifying crosslinking sites that are formed by small multi-functional reagents, termed crosslinkers. Each of these MS applications is capable of revealing structural information for proteins when used either with or without other typical high resolution techniques, including NMR and X-ray crystallography. PMID:27975228

Characterization of Medium Conditioned by Irradiated Cells Using Proteome-Wide, High-Throughput Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Springer, David L.; Ahram, Mamoun; Adkins, Joshua N.

Shedding, the release of cell surface proteins by regulated proteolysis, is a general cellular response to injury and is responsible for generating numerous bioactive molecules including growth factors and cytokines. The purpose of our work is to determine whether low doses of low-linear energy transfer (LET) radiation induce shedding of bioactive molecules. Using a mass spectrometry-based global proteomics method, we tested this hypothesis by analyzing for shed proteins in medium from irradiated human mammary epithelial cells (HMEC). Several hundred proteins were identified, including transforming growth factor beta (TGFB); however, no changes in protein abundances attributable to radiation exposure, based onmore » immunoblotting methods, were observed. These results demonstrate that our proteomic-based approach has the sensitivity to identify the kinds of proteins believed to be released after low-dose radiation exposure but that improvements in mass spectrometry-based protein quantification will be required to detect the small changes in abundance associated with this type of insult.« less

Quantification of the Influence of Protein-Protein Interactions on Adsorbed Protein Structure and Bioactivity

PubMed Central

Wei, Yang; Thyparambil, Aby A.; Latour, Robert A.

2013-01-01

While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-white lysozyme (HEWL) adsorbed on silica glass, poly(methyl methacrylate), and polyethylene as our model systems. In order to vary protein-protein interaction effects over a wide range, HEWL was first adsorbed to each surface type under widely varying protein solution concentrations for 2 h to saturate the surface, followed by immersion in pure buffer solution for 15 h to equilibrate the adsorbed protein layers in the absence of additionally adsorbing protein. Periodic measurements were made at selected time points of the areal density of the adsorbed protein layer as an indicator of the level of protein-protein interaction effects within the layer, and these values were then correlated with measurements of the adsorbed protein’s secondary structure and bioactivity. The results from these studies indicate that protein-protein interaction effects help stabilize the structure of HEWL adsorbed on silica glass, have little influence on the structural behavior of HEWL on HDPE, and actually serve to destabilize HEWL’s structure on PMMA. The bioactivity of HEWL on silica glass and HDPE was found to decrease in direct proportion to the degree of adsorption-induce protein unfolding. A direct correlation between bioactivity and the conformational state of adsorbed HEWL was less apparent on PMMA, thus suggesting that other factors influenced HEWL’s bioactivity on this surface, such as the accessibility of HEWL’s bioactive site being blocked by neighboring proteins or the surface itself. The developed methods provide an effective means to characterize the influence of protein-protein interaction effects and provide new molecular-level insights into how protein-protein interaction effects combine with protein-surface interaction and internal protein stability effects to influence the structure and bioactivity of adsorbed protein. PMID:23751416

Protein Surface Mimetics: Understanding How Ruthenium Tris(Bipyridines) Interact with Proteins.

PubMed

Hewitt, Sarah H; Filby, Maria H; Hayes, Ed; Kuhn, Lars T; Kalverda, Arnout P; Webb, Michael E; Wilson, Andrew J

2017-01-17

Protein surface mimetics achieve high-affinity binding by exploiting a scaffold to project binding groups over a large area of solvent-exposed protein surface to make multiple cooperative noncovalent interactions. Such recognition is a prerequisite for competitive/orthosteric inhibition of protein-protein interactions (PPIs). This paper describes biophysical and structural studies on ruthenium(II) tris(bipyridine) surface mimetics that recognize cytochrome (cyt) c and inhibit the cyt c/cyt c peroxidase (CCP) PPI. Binding is electrostatically driven, with enhanced affinity achieved through enthalpic contributions thought to arise from the ability of the surface mimetics to make a greater number of noncovalent interactions than CCP with surface-exposed basic residues on cyt c. High-field natural abundance 1 H, 15 N HSQC NMR experiments are consistent with surface mimetics binding to cyt c in similar manner to CCP. This provides a framework for understanding recognition of proteins by supramolecular receptors and informing the design of ligands superior to the protein partners upon which they are inspired. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein composition of the hepatitis A virus quasi-envelope.

PubMed

McKnight, Kevin L; Xie, Ling; González-López, Olga; Rivera-Serrano, Efraín E; Chen, Xian; Lemon, Stanley M

2017-06-20

The Picornaviridae are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered "nonenveloped," recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasi-enveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation. Amino acid-coded mass tagging (AACT) with stable isotopes followed by tandem mass spectrometry sequencing and AACT quantitation of peptides provided unambiguous identification of proteins associated with eHAV versus unrelated extracellular vesicles with similar buoyant density. Multiple peptides were identified from HAV capsid proteins (53.7% coverage), but none from nonstructural proteins, indicating capsids are packaged as cargo into eHAV vesicles via a highly specific sorting process. Other eHAV-associated proteins ( n = 105) were significantly enriched for components of the endolysosomal system (>60%, P < 0.001) and included many common exosome-associated proteins such as the tetraspanin CD9 and dipeptidyl peptidase 4 (DPP4) along with multiple endosomal sorting complex required for transport III (ESCRT-III)-associated proteins. Immunoprecipitation confirmed that DPP4 is displayed on the surface of eHAV produced in cell culture or present in sera from humans with acute hepatitis A. No LC3-related peptides were identified by mass spectrometry. RNAi depletion studies confirmed that ESCRT-III proteins, particularly CHMP2A, function in eHAV biogenesis. In addition to identifying surface markers of eHAV vesicles, the results support an exosome-like mechanism of eHAV egress involving endosomal budding of HAV capsids into multivesicular bodies.

Human and server docking prediction for CAPRI round 30-35 using LZerD with combined scoring functions.

PubMed

Peterson, Lenna X; Kim, Hyungrae; Esquivel-Rodriguez, Juan; Roy, Amitava; Han, Xusi; Shin, Woong-Hee; Zhang, Jian; Terashi, Genki; Lee, Matt; Kihara, Daisuke

2017-03-01

We report the performance of protein-protein docking predictions by our group for recent rounds of the Critical Assessment of Prediction of Interactions (CAPRI), a community-wide assessment of state-of-the-art docking methods. Our prediction procedure uses a protein-protein docking program named LZerD developed in our group. LZerD represents a protein surface with 3D Zernike descriptors (3DZD), which are based on a mathematical series expansion of a 3D function. The appropriate soft representation of protein surface with 3DZD makes the method more tolerant to conformational change of proteins upon docking, which adds an advantage for unbound docking. Docking was guided by interface residue prediction performed with BindML and cons-PPISP as well as literature information when available. The generated docking models were ranked by a combination of scoring functions, including PRESCO, which evaluates the native-likeness of residues' spatial environments in structure models. First, we discuss the overall performance of our group in the CAPRI prediction rounds and investigate the reasons for unsuccessful cases. Then, we examine the performance of several knowledge-based scoring functions and their combinations for ranking docking models. It was found that the quality of a pool of docking models generated by LZerD, that is whether or not the pool includes near-native models, can be predicted by the correlation of multiple scores. Although the current analysis used docking models generated by LZerD, findings on scoring functions are expected to be universally applicable to other docking methods. Proteins 2017; 85:513-527. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haarmeyer, Carolyn N.; Smith, Matthew D.; Chundawat, Shishir P. S.

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterizedmore » 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Altogether, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases.« less

Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein

DOE PAGES

Haarmeyer, Carolyn N.; Smith, Matthew D.; Chundawat, Shishir P. S.; ...

2016-10-17

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterizedmore » 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Altogether, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases.« less

Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein.

PubMed

Haarmeyer, Carolyn N; Smith, Matthew D; Chundawat, Shishir P S; Sammond, Deanne; Whitehead, Timothy A

2017-04-01

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue toward energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28-0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases. Biotechnol. Bioeng. 2017;114: 740-750. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Exploring Molecular-Biomembrane Interactions with Surface Plasmon Resonance and Dual Polarization Interferometry Technology: Expanding the Spotlight onto Biomembrane Structure.

PubMed

Lee, Tzong-Hsien; Hirst, Daniel J; Kulkarni, Ketav; Del Borgo, Mark P; Aguilar, Marie-Isabel

2018-06-13

The molecular analysis of biomolecular-membrane interactions is central to understanding most cellular systems but has emerged as a complex technical challenge given the complexities of membrane structure and composition across all living cells. We present a review of the application of surface plasmon resonance and dual polarization interferometry-based biosensors to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. We first describe the optical principals and instrumentation of surface plasmon resonance, including both linear and extraordinary transmission modes and dual polarization interferometry. We then describe the wide range of model membrane systems that have been developed for deposition on the chips surfaces that include planar, polymer cushioned, tethered bilayers, and liposomes. This is followed by a description of the different chemical immobilization or physisorption techniques. The application of this broad range of engineered membrane surfaces to biomolecular-membrane interactions is then overviewed and how the information obtained using these techniques enhance our molecular understanding of membrane-mediated peptide and protein function. We first discuss experiments where SPR alone has been used to characterize membrane binding and describe how these studies yielded novel insight into the molecular events associated with membrane interactions and how they provided a significant impetus to more recent studies that focus on coincident membrane structure changes during binding of peptides and proteins. We then discuss the emerging limitations of not monitoring the effects on membrane structure and how SPR data can be combined with DPI to provide significant new information on how a membrane responds to the binding of peptides and proteins.

AFM study of adsorption of protein A on a poly(dimethylsiloxane) surface

NASA Astrophysics Data System (ADS)

Yu, Ling; Lu, Zhisong; Gan, Ye; Liu, Yingshuai; Li, Chang Ming

2009-07-01

In this paper, the morphology and kinetics of adsorption of protein A on a PDMS surface is studied by AFM. The results of effects of pH, protein concentration and contact time of the adsorption reveal that the morphology of adsorbed protein A is significantly affected by pH and adsorbed surface concentration, in which the pH away from the isoelectric point (IEP) of protein A could produce electrical repulsion to change the protein conformation, while the high adsorbed surface protein volume results in molecular networks. Protein A can form an adsorbed protein film on PDMS with a maximum volume of 2.45 × 10-3 µm3. This work enhances our fundamental understanding of protein A adsorption on PDMS, a frequently used substrate component in miniaturized immunoassay devices.

Proteins at the Biomaterial Electrolyte Interface

NASA Astrophysics Data System (ADS)

Tengvall, Pentti

2005-03-01

Proteins adsorb rapidly onto solid and polymeric surfaces because the association process is in the vast majority of cases energetically favourable, i.e. exothermic. The most common exceptions to this rule are hydrophilic interfaces with low net charge and high mobility, e.g. immobilized PEGs. Current research in the research area tries to understand and control unwanted and wanted adsorption by studying the adsorption kinetics, protein surface binding specificity, protein exchange at interfaces, and surface protein repulsion mechanisms. In blood plasma model systems humoral cascade reactions such as surface mediated coagulation and immune complement raise considerable interest due to the immediate association to blood compatibility, and in tissue applications the binding between surfaces and membrane receptors in cells and tissues. Thus, the understanding of interfacial events at the protein level is of large importance in applications such as blood and tissue contacting biomaterials, in vitro medical and biological diagnostics, food industry and in marine anti-fouling technology. Well described consequences of adsorption are a lowered system energy, increased system entropy, irreversible binding, conformational changes, specific surface/protein interactions, and in biomedical materials applications surface opsonization followed by cell-surface interactions and a host tissue response. This lecture will deal with some mechanisms known to be of importance for the adsorption processes, such as the influence of surface chemistry and surface energy, the composition of the protein solution, the Vroman effect, and residence time. Examples will be shown from ellipsometric experiments using different model surfaces in single/few protein solutions, and specific attention be given to blood serum and plasma experiments on coagulation and immune complement at interfaces.

Molecular characterization of calreticulin from Anopheles stephensi midgut cells and functional assay of the recombinant calreticulin with Plasmodium berghei ookinetes.

PubMed

Borhani Dizaji, Nahid; Basseri, Hamid Reza; Naddaf, Saied Reza; Heidari, Mansour

2014-10-25

Transmission blocking vaccines (TBVs) that target the antigens on the midgut epithelium of Anopheles mosquitoes are among the promising tools for the elimination of the malaria parasite. Characterization and analysis of effective antigens is the first step to design TBVs. Calreticulin (CRT), a lectin-like protein, from Anopheles albimanus midgut, has shown antigenic features, suggesting a promising and novel TBV target. CRT is a highly conserved protein with similar features in vertebrates and invertebrates including anopheline. We cloned the full-length crt gene from malaria vector, Anopheles stephensi (AsCrt) and explored the interaction of recombinant AsCrt protein, expressed in a prokaryotic system (pGEX-6p-1), with surface proteins of Plasmodium berghei ookinetes by immunofluorescence assay. The cellular localization of AsCrt was determined using the baculovirus expression system. Sequence analysis of the whole cDNA of AsCrt revealed that AsCrt contains an ORF of 1221 bp. The amino acid sequence of AsCrt protein obtained in this study showed 64% homology with similar protein in human. The AsCrt shares the most common features of CRTs from other species. This gene encodes a 406 amino-acid protein with a molecular mass of 46 kDa, which contains a predicted 16 amino-acid signal peptides, conserved cysteine residues, a proline-rich region, and highly acidic C-terminal domain with endoplasmic reticulum retrieval sequence HDEL. The production of GST-AsCrt recombinant protein was confirmed by Western blot analysis using an antibody against the GST protein. The FITC-labeled GST-AsCrt exhibited a significant interaction with P. berghei ookinete surface proteins. Purified recombinant GST-AsCrt, labeled with FITC, displayed specific binding to the surface of P. berghei ookinetes in comparison with control. Moreover, the expression of AsCrt in baculovirus expression system indicated that AsCrt was localized on the surface of Sf9 cells. Our results suggest that AsCrt could be utilized as a potential target for future studies in TBV area for malaria control. Copyright © 2014 Elsevier B.V. All rights reserved.

Enhancement of surface graft density of MPEG on alginate/chitosan hydrogel microcapsules for protein repellency.

PubMed

Zheng, Jiani; Xie, Hongguo; Yu, Weiting; Tan, Mingqian; Gong, Faquan; Liu, Xiudong; Wang, Feng; Lv, Guojun; Liu, Wanfa; Zheng, Guoshuang; Yang, Yan; Xie, Weiyang; Ma, Xiaojun

2012-09-18

Alginate/chitosan/alginate (ACA) hydrogel microcapsules were modified with methoxy poly(ethylene glycol) (MPEG) to improve protein repellency and biocompatibility. Increased MPEG surface graft density (n(S)) on hydrogel microcapsules was achieved by controlling the grafting parameters including the buffer layer substrate, membrane thickness, and grafting method. X-ray photoelectron spectroscopy (XPS) model was employed to quantitatively analyze n(S) on this three-dimensional (3D) hydrogel network structure. Our results indicated that neutralizing with alginate, increasing membrane thickness, and in situ covalent grafting could increase n(S) effectively. ACAC(PEG) was more promising than ACC(PEG) in protein repellency because alginate supplied more -COO(-) negative binding sites and prevented MPEG from diffusing. The n(S) increased with membrane thickness, showing better protein repellency. Moreover, the in situ covalent grafting provided an effective way to enhance n(S), and 1.00 ± 0.03 chains/nm(2) was achieved, exhibiting almost complete immunity to protein adsorption. This antifouling hydrogel biomaterial is expected to be useful in transplantation in vivo.

GPI-anchored protein organization and dynamics at the cell surface

PubMed Central

Saha, Suvrajit; Anilkumar, Anupama Ambika; Mayor, Satyajit

2016-01-01

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite. PMID:26394904

GPI-anchored protein organization and dynamics at the cell surface.

PubMed

Saha, Suvrajit; Anilkumar, Anupama Ambika; Mayor, Satyajit

2016-02-01

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Salvage of failed protein targets by reductive alkylation.

PubMed

Tan, Kemin; Kim, Youngchang; Hatzos-Skintges, Catherine; Chang, Changsoo; Cuff, Marianne; Chhor, Gekleng; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw; An, Hao; Babnigg, Gyorgy; Bigelow, Lance; Joachimiak, Grazyna; Li, Hui; Mack, Jamey; Makowska-Grzyska, Magdalena; Maltseva, Natalia; Mulligan, Rory; Tesar, Christine; Zhou, Min; Joachimiak, Andrzej

2014-01-01

The growth of diffraction-quality single crystals is of primary importance in protein X-ray crystallography. Chemical modification of proteins can alter their surface properties and crystallization behavior. The Midwest Center for Structural Genomics (MCSG) has previously reported how reductive methylation of lysine residues in proteins can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. Recently, this approach has been expanded to include ethylation and isopropylation in the MCSG protein crystallization pipeline. Applying standard methods, 180 unique proteins were alkylated and screened using standard crystallization procedures. Crystal structures of 12 new proteins were determined, including the first ethylated and the first isopropylated protein structures. In a few cases, the structures of native and methylated or ethylated states were obtained and the impact of reductive alkylation of lysine residues was assessed. Reductive methylation tends to be more efficient and produces the most alkylated protein structures. Structures of methylated proteins typically have higher resolution limits. A number of well-ordered alkylated lysine residues have been identified, which make both intermolecular and intramolecular contacts. The previous report is updated and complemented with the following new data; a description of a detailed alkylation protocol with results, structural features, and roles of alkylated lysine residues in protein crystals. These contribute to improved crystallization properties of some proteins.

Salvage of Failed Protein Targets by Reductive Alkylation

PubMed Central

Tan, Kemin; Kim, Youngchang; Hatzos-Skintges, Catherine; Chang, Changsoo; Cuff, Marianne; Chhor, Gekleng; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw; An, Hao; Babnigg, Gyorgy; Bigelow, Lance; Joachimiak, Grazyna; Li, Hui; Mack, Jamey; Makowska-Grzyska, Magdalena; Maltseva, Natalia; Mulligan, Rory; Tesar, Christine; Zhou, Min; Joachimiak, Andrzej

2014-01-01

The growth of diffraction-quality single crystals is of primary importance in protein X-ray crystallography. Chemical modification of proteins can alter their surface properties and crystallization behavior. The Midwest Center for Structural Genomics (MCSG) has previously reported how reductive methylation of lysine residues in proteins can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. Recently, this approach has been expanded to include ethylation and isopropylation in the MCSG protein crystallization pipeline. Applying standard methods, 180 unique proteins were alkylated and screened using standard crystallization procedures. Crystal structures of 12 new proteins were determined, including the first ethylated and the first isopropylated protein structures. In a few cases, the structures of native and methylated or ethylated states were obtained and the impact of reductive alkylation of lysine residues was assessed. Reductive methylation tends to be more efficient and produces the most alkylated protein structures. Structures of methylated proteins typically have higher resolution limits. A number of well-ordered alkylated lysine residues have been identified, which make both intermolecular and intramolecular contacts. The previous report is updated and complemented with the following new data; a description of a detailed alkylation protocol with results, structural features, and roles of alkylated lysine residues in protein crystals. These contribute to improved crystallization properties of some proteins. PMID:24590719

Magnetic nanotubes

DOEpatents

Matsui, Hiroshi; Matsunaga, Tadashi

2010-11-16

A magnetic nanotube includes bacterial magnetic nanocrystals contacted onto a nanotube which absorbs the nanocrystals. The nanocrystals are contacted on at least one surface of the nanotube. A method of fabricating a magnetic nanotube includes synthesizing the bacterial magnetic nanocrystals, which have an outer layer of proteins. A nanotube provided is capable of absorbing the nanocrystals and contacting the nanotube with the nanocrystals. The nanotube is preferably a peptide bolaamphiphile. A nanotube solution and a nanocrystal solution including a buffer and a concentration of nanocrystals are mixed. The concentration of nanocrystals is optimized, resulting in a nanocrystal to nanotube ratio for which bacterial magnetic nanocrystals are immobilized on at least one surface of the nanotubes. The ratio controls whether the nanocrystals bind only to the interior or to the exterior surfaces of the nanotubes. Uses include cell manipulation and separation, biological assay, enzyme recovery, and biosensors.

A magnetic-dependent protein corona of tailor-made superparamagnetic iron oxides alters their biological behaviors

NASA Astrophysics Data System (ADS)

Liu, Ziyao; Zhan, Xiaohui; Yang, Minggang; Yang, Qi; Xu, Xianghui; Lan, Fang; Wu, Yao; Gu, Zhongwei

2016-03-01

In recent years, it is becoming increasingly evident that once nanoparticles come into contact with biological fluids, a protein corona surely forms and critically affects the biological behaviors of nanoparticles. Herein, we investigate whether the formation of protein corona on the surface of superparamagnetic iron oxides (SPIOs) is influenced by static magnetic field. Under static magnetic field, there is no obvious variation in the total amount of protein adsorption, but the proportion of adsorbed proteins significantly changes. Noticeably, certain proteins including apolipoproteins, complement system proteins and acute phase proteins, increase in the protein corona of SPIOs in the magnetic field. More importantly, the magnetic-dependent protein corona of SPIOs enhances the cellular uptake of SPIOs into the normal cell line (3T3 cells) and tumor cell line (HepG2 cells), due to increased adsorption of apolipoprotein. In addition, SPIOs with the magnetic-dependent protein corona cause high cytotoxicity to 3T3 cells and HepG2 cells. This work discloses that superparamagnetism as a key feature of SPIOs affects the composition of protein corona to a large extent, which further alters the biological behaviors of SPIOs.In recent years, it is becoming increasingly evident that once nanoparticles come into contact with biological fluids, a protein corona surely forms and critically affects the biological behaviors of nanoparticles. Herein, we investigate whether the formation of protein corona on the surface of superparamagnetic iron oxides (SPIOs) is influenced by static magnetic field. Under static magnetic field, there is no obvious variation in the total amount of protein adsorption, but the proportion of adsorbed proteins significantly changes. Noticeably, certain proteins including apolipoproteins, complement system proteins and acute phase proteins, increase in the protein corona of SPIOs in the magnetic field. More importantly, the magnetic-dependent protein corona of SPIOs enhances the cellular uptake of SPIOs into the normal cell line (3T3 cells) and tumor cell line (HepG2 cells), due to increased adsorption of apolipoprotein. In addition, SPIOs with the magnetic-dependent protein corona cause high cytotoxicity to 3T3 cells and HepG2 cells. This work discloses that superparamagnetism as a key feature of SPIOs affects the composition of protein corona to a large extent, which further alters the biological behaviors of SPIOs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08447d

Role of Transbilayer Distribution of Lipid Molecules on the Structure and Protein-Lipid Interaction of an Amyloidogenic Protein on the Membrane Surface

NASA Astrophysics Data System (ADS)

Cheng, Kwan; Cheng, Sara

We used molecular dynamics simulations to examine the effects of transbilayer distribution of lipid molecules, particularly anionic lipids with negatively charged headgroups, on the structure and binding kinetics of an amyloidogenic protein on the membrane surface and subsequent protein-induced structural disruption of the membrane. Our systems consisted of a model beta-sheet rich dimeric protein absorbed on asymmetric bilayers with neutral and anionic lipids and symmetric bilayers with neutral lipids. We observed larger folding, domain aggregation, and tilt angle of the absorbed protein on the asymmetric bilayer surfaces. We also detected more focused bilayer thinning in the asymmetric bilayer due to weak lipid-protein interactions. Our results support the mechanism that the higher lipid packing in the protein-contacting lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions of an amyloidogenic protein on the membrane surface. We speculate that the observed surface-induced structural and protein-lipid interaction of our model amyloidogenic protein may play a role in the early membrane-associated amyloid cascade pathway that leads to membrane structural damage of neurons in Alzheimer's disease. NSF ACI-1531594.

α-Crystallins Are Small Heat Shock Proteins: Functional and Structural Properties.

PubMed

Tikhomirova, T S; Selivanova, O M; Galzitskaya, O V

2017-02-01

During its life cycle, a cell can be subjected to various external negative effects. Many proteins provide cell protection, including small heat shock proteins (sHsp) that have chaperone-like activity. These proteins have several important functions involving prevention of apoptosis and retention of cytoskeletal integrity; also, sHsp take part in the recovery of enzyme activity. The action mechanism of sHsp is based on the binding of hydrophobic regions exposed to the surface of a molten globule. α-Crystallins presented in chordate cells as two αA- and αB-isoforms are the most studied small heat shock proteins. In this review, we describe the main functions of α-crystallins, features of their secondary and tertiary structures, and examples of their partners in protein-protein interactions.

Electron Tomography Imaging of Surface Glycoproteins on Human Parainfluenza Virus 3: Association of Receptor Binding and Fusion Proteins before Receptor Engagement

PubMed Central

Gui, Long; Jurgens, Eric M.; Ebner, Jamie L.

2015-01-01

ABSTRACT In order to deliver their genetic material to host cells during infection, enveloped viruses use specialized proteins on their surfaces that bind cellular receptors and induce fusion of the viral and host membranes. In paramyxoviruses, a diverse family of single-stranded RNA (ssRNA) viruses, including several important respiratory pathogens, such as parainfluenza viruses, the attachment and fusion machinery is composed of two separate proteins: a receptor binding protein (hemagglutinin-neuraminidase [HN]) and a fusion (F) protein that interact to effect membrane fusion. Here we used negative-stain and cryo-electron tomography to image the 3-dimensional ultrastructure of human parainfluenza virus 3 (HPIV3) virions in the absence of receptor engagement. We observed that HN exists in at least two organizations. The first were arrays of tetrameric HN that lacked closely associated F proteins: in these purely HN arrays, HN adopted a “heads-down” configuration. In addition, we observed regions of complex surface density that contained HN in an apparently extended “heads-up” form, colocalized with prefusion F trimers. This colocalization with prefusion F prior to receptor engagement supports a model for fusion in which HN in its heads-up state and F may interact prior to receptor engagement without activating F, and that interaction with HN in this configuration is not sufficient to activate F. Only upon receptor engagement by HN’s globular head does HN transmit its activating signal to F. PMID:25691596

Dissecting binding of a β-barrel membrane protein by phage display.

PubMed

Meneghini, Luz M; Tripathi, Sarvind; Woodworth, Marcus A; Majumdar, Sudipta; Poulos, Thomas L; Weiss, Gregory A

2017-07-25

Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal. Here, we apply a new approach for the expedited protein characterization of the TBDT ShuA from Shigella dysenteriae, and elucidate the protein's initial steps during heme-uptake. ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable MP variants for rapid characterization of binding to various ligands. Site-directed mutagenesis studies targeting each extracellular loop region of ShuA demonstrate no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. The results identify a loop susceptible to antibody binding, and also a small molecule motif capable of disrupting ShuA from S. dysenteriae. The approach is generalizable to the dissection of other phage-displayed TBDTs and MPs.

Titanium Surface Roughing Treatments contribute to Higher Interaction with Salivary Proteins MG2 and Lactoferrin.

PubMed

Cavalcanti, Yuri Wanderley; Soare, Rodrigo Villamarim; Leite Assis, Marina Araújo; Zenóbio, Elton Gonçalves; Girundi, Francisco Mauro da Silva

2015-02-01

Some surface treatments performed on titanium can alter the composition of salivary pellicle formed on this abiotic surface. Such treatments modify the titanium's surface properties and can promote higher adsorption of proteins, which allow better integration of titanium to the biotic system. This study aimed to evaluate the interactions between salivary proteins and titanium disks with different surface treatments. Machined titanium disks (n = 48) were divided into four experimental groups (n = 12), according to their surface treatments: surface polishing (SP); acid etching (A); spot-blasting plus acid etching (SB-A); spot-blasting followed by acid etching and nano-functionalization (SB-A-NF). Titanium surfaces were characterized by surface roughness and scanning electron microscopy (SEM). Specimens were incubated with human saliva extracted from submandibular and sublingual glands. Total salivary protein adsorbed to titanium was quantified and samples were submitted to western blotting for mucin glycoprotein 2 (MG2) and lactoferrin identification. Surface roughness was statistically higher for SB-A and SB-A-NF groups. Scanning electron microscopy images confirmed that titanium surface treatments increased surface roughness with higher number of porous and scratches for SB-A and SB-A-NF groups. Total protein adsorption was significantly higher for SB-A and SB-A-NF groups (p < 0.05), which also presented higher interactions with MG2 and lactoferrin proteins. The roughing of titanium surface by spot-blasting plus acid etching treatments contribute to higher interaction with salivary proteins, such as MG2 and lactoferrin. Titanium surface roughing increases the interactions of the substratum with salivary proteins, which can influence the integration of dental implants and their components to the oral environment. However, those treatments should be used carefully intraorally, avoiding increase biofilm formation.

Targeted Proteomics and Absolute Protein Quantification for the Construction of a Stoichiometric Host-Pathogen Surface Density Model*

PubMed Central

Sjöholm, Kristoffer; Kilsgård, Ola; Teleman, Johan; Happonen, Lotta; Malmström, Lars; Malmström, Johan

2017-01-01

Sepsis is a systemic immune response responsible for considerable morbidity and mortality. Molecular modeling of host-pathogen interactions in the disease state represents a promising strategy to define molecular events of importance for the transition from superficial to invasive infectious diseases. Here we used the Gram-positive bacterium Streptococcus pyogenes as a model system to establish a mass spectrometry based workflow for the construction of a stoichiometric surface density model between the S. pyogenes surface, the surface virulence factor M-protein, and adhered human blood plasma proteins. The workflow relies on stable isotope labeled reference peptides and selected reaction monitoring mass spectrometry analysis of a wild-type strain and an M-protein deficient mutant strain, to generate absolutely quantified protein stoichiometry ratios between S. pyogenes and interacting plasma proteins. The stoichiometry ratios in combination with a novel targeted mass spectrometry method to measure cell numbers enabled the construction of a stoichiometric surface density model using protein structures available from the protein data bank. The model outlines the topology and density of the host-pathogen protein interaction network on the S. pyogenes bacterial surface, revealing a dense and highly organized protein interaction network. Removal of the M-protein from S. pyogenes introduces a drastic change in the network topology, validated by electron microscopy. We propose that the stoichiometric surface density model of S. pyogenes in human blood plasma represents a scalable framework that can continuously be refined with the emergence of new results. Future integration of new results will improve the understanding of protein-protein interactions and their importance for bacterial virulence. Furthermore, we anticipate that the general properties of the developed workflow will facilitate the production of stoichiometric surface density models for other types of host-pathogen interactions. PMID:28183813

A Stable Human-Cell System Overexpressing Cystic Fibrosis Transmembrane Conductance Regulator Recombinant Protein at the Cell Surface

PubMed Central

Dai, Qun; Aleksandrov, Andrei A.; Bajrami, Bekim; Diego, Pamela Ann; Wu, Xing; Ray, Marjorie; Naren, Anjaparavanda P.; Riordan, John R.; Yao, Xudong; DeLucas, Lawrence J.; Urbatsch, Ina L.; Kappes, John C.

2015-01-01

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, or an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTRFLAG-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTRFLAG-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment futrure CF drug discovery efforts, including biophysical and structural studies. PMID:25577540

Anions in Electrothermal Supercharging of Proteins with Electrospray Ionization Follow a Reverse Hofmeister Series

PubMed Central

2015-01-01

The effects of different anions on the extent of electrothermal supercharging of proteins from aqueous ammonium and sodium salt solutions were investigated. Sulfate and hydrogen phosphate are the most effective anions at producing high charge state protein ions from buffered aqueous solution, whereas iodide and perchlorate are ineffective with electrothermal supercharging. The propensity for these anions to produce high charge state protein ions follows the following trend: sulfate > hydrogen phosphate > thiocyanate > bicarbonate > chloride > formate ≈ bromide > acetate > iodide > perchlorate. This trend correlates with the reverse Hofmeister series over a wide range of salt concentrations (1 mM to 2 M) and with several physical properties, including solvent surface tension, anion viscosity B-coefficient, and anion surface/bulk partitioning coefficient, all of which are related to the Hofmeister series. The effectiveness of electrothermal supercharging does not depend on bubble formation, either from thermal degradation of the buffer or from coalescence of dissolved gas. These results provide evidence that the effect of different ions in the formation of high charge state ions by electrothermal supercharging is largely a result of Hofmeister effects on protein stability leading to protein unfolding in the heated ESI droplet. PMID:24410546

Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

PubMed Central

Austin, Pamela; Heller, Markus; Williams, David E.; McIntosh, Lawrence P.; Vogl, A. Wayne; Foster, Leonard J.; Andersen, Raymond J.; Roberge, Michel; Roskelley, Calvin D.

2010-01-01

Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface. PMID:20520768

Controlling adsorption and passivation properties of bovine serum albumin on silica surfaces by ionic strength modulation and cross-linking.

PubMed

Park, Jae Hyeon; Sut, Tun Naw; Jackman, Joshua A; Ferhan, Abdul Rahim; Yoon, Bo Kyeong; Cho, Nam-Joon

2017-03-29

Understanding the physicochemical factors that influence protein adsorption onto solid supports holds wide relevance for fundamental insights into protein structure and function as well as for applications such as surface passivation. Ionic strength is a key parameter that influences protein adsorption, although how its modulation might be utilized to prepare well-coated protein adlayers remains to be explored. Herein, we investigated how ionic strength can be utilized to control the adsorption and passivation properties of bovine serum albumin (BSA) on silica surfaces. As protein stability in solution can influence adsorption kinetics, the size distribution and secondary structure of proteins in solution were first characterized by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and circular dichroism (CD) spectroscopy. A non-monotonic correlation between ionic strength and protein aggregation was observed and attributed to colloidal agglomeration, while the primarily α-helical character of the protein in solution was maintained in all cases. Quartz crystal microbalance-dissipation (QCM-D) experiments were then conducted in order to track protein adsorption onto silica surfaces as a function of ionic strength, and the measurement responses indicated that total protein uptake at saturation coverage is lower with increasing ionic strength. In turn, the QCM-D data and the corresponding Voigt-Voinova model analysis support that the surface area per bound protein molecule is greater with increasing ionic strength. While higher protein uptake under lower ionic strengths by itself did not result in greater surface passivation under subsequent physiologically relevant conditions, the treatment of adsorbed protein layers with a gluteraldehyde cross-linking agent stabilized the bound protein in this case and significantly improved surface passivation. Collectively, our findings demonstrate that ionic strength modulation influences BSA adsorption uptake on account of protein spreading and can be utilized in conjunction with covalent cross-linking strategies to prepare well-coated protein adlayers for improved surface passivation.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp.

PubMed

Souza, Natalie M; Vieira, Monica L; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2012-09-01

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K(D)) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. Copyright © 2012 Elsevier Ltd. All rights reserved.

Increasing the affinity of selective bZIP-binding peptides through surface residue redesign.

PubMed

Kaplan, Jenifer B; Reinke, Aaron W; Keating, Amy E

2014-07-01

The coiled-coil dimer is a prevalent protein interaction motif that is important for many cellular processes. The basic leucine-zipper (bZIP) transcription factors are one family of proteins for which coiled-coil mediated dimerization is essential for function, and misregulation of bZIPs can lead to disease states including cancer. This makes coiled coils attractive protein-protein interaction targets to disrupt using engineered molecules. Previous work designing peptides to compete with native coiled-coil interactions focused primarily on designing the core residues of the interface to achieve affinity and specificity. However, folding studies on the model bZIP GCN4 show that coiled-coil surface residues also contribute to binding affinity. Here we extend a prior study in which peptides were designed to bind tightly and specifically to representative members of each of 20 human bZIP families. These "anti-bZIP" peptides were designed with an emphasis on target-binding specificity, with contributions to design-target specificity and affinity engineered considering only the coiled-coil core residues. High-throughput testing using peptide arrays indicated many successes. We have now measured the binding affinities and specificities of anti-bZIPs that bind to FOS, XBP1, ATF6, and CREBZF in solution and tested whether redesigning the surface residues can increase design-target affinity. Incorporating residues that favor helix formation into the designs increased binding affinities in all cases, providing low-nanomolar binders of each target. However, changes in surface electrostatic interactions sometimes changed the binding specificity of the designed peptides. © 2014 The Protein Society.

The ‘Sticky Patch’ Model of Crystallization and Modification of Proteins for Enhanced Crystallizability

PubMed Central

Derewenda, Zygmunt S.; Godzik, Adam

2017-01-01

Crystallization of macromolecules has long been perceived as a stochastic process, which cannot be predicted or controlled. This is consistent with another popular notion that the interactions of molecules within the crystal, i.e. crystal contacts, are essentially random and devoid of specific physicochemical features. In contrast, functionally relevant surfaces, such as oligomerization interfaces and specific protein-protein interaction sites, are under evolutionary pressures so their amino acid composition, structure and topology are distinct. However, current theoretical and experimental studies are significantly changing our understanding of the nature of crystallization. The increasingly popular ‘sticky patch’ model, derived from soft matter physics, describes crystallization as a process driven by interactions between select, specific surface patches, with properties thermodynamically favorable for cohesive interactions. Independent support for this model comes from various sources including structural studies and bioinformatics. Proteins that are recalcitrant to crystallization can be modified for enhanced crystallizability through chemical or mutational modification of their surface to effectively engineer ‘sticky patches’ which would drive crystallization. Here, we discuss the current state of knowledge of the relationship between the microscopic properties of the target macromolecule and its crystallizability, focusing on the ‘sticky patch’ model. We discuss state-of-art in silico methods that evaluate the propensity of a given target protein to form crystals based on these relationships, with the objective to design of variants with modified molecular surface properties and enhanced crystallization propensity. We illustrate this discussion with specific cases where these approaches allowed to generate crystals suitable for structural analysis. PMID:28573570

Smart polymers as surface modifiers for bioanalytical devices and biomaterials: theory and practice

NASA Astrophysics Data System (ADS)

Ivanov, A. E.; Zubov, V. P.

2016-06-01

Smart, or responsive polymers can reversibly change their state of aggregation, thus switching from water-soluble to insoluble state, in response to minor changes in temperature, pH or solvent composition. Grafting of these polymers to solid surfaces imparts the surfaces with controllable wettability and adsorption behaviour. The review summarizes the theoretical models and the results of physical measurements of the conformational transitions in grafted polymer chains and polymer brushes. Primary attention is paid to the grafting density and the length and spatial arrangement of grafted chains, the role of polystyrene, organosilane or alkanethiol sublayers and their effects on adsorption of proteins and adhesion of cells. The key applications of grafted smart polymers such as cell culture and tissue engineering, cell and protein separation, biosensing and targeted drug delivery are surveyed. The bibliography includes 174 references.

Self-assembled tethered bimolecular lipid membranes.

PubMed

Sinner, Eva-Kathrin; Ritz, Sandra; Naumann, Renate; Schiller, Stefan; Knoll, Wolfgang

2009-01-01

This chapter describes some of the strategies developed in our group for designing, constructing and structurally and functionally characterizing tethered bimolecular lipid membranes (tBLM). We introduce this platform as a novel model membrane system that complements the existing ones, for example, Langmuir monolayers, vesicular liposomal dispersions and bimolecular ("black") lipid membranes. Moreover, it offers the additional advantage of allowing for studies of the influence of membrane structure and order on the function of integral proteins, for example, on how the composition and organization of lipids in a mixed membrane influence the ion translocation activity of integral channel proteins. The first strategy that we introduce concerns the preparation of tethered monolayers by the self-assembly of telechelics. Their molecular architecture with a headgroup, a spacer unit (the "tether") and the amphiphile that mimics the lipid molecule allows them to bind specifically to the solid support thus forming the proximal layer of the final architecture. After fusion of vesicles that could contain reconstituted proteins from a liposomal dispersion in contact to this monolayer the tethered bimolecular lipid membrane is obtained. This can then be characterized by a broad range of surface analytical techniques, including surface plasmon spectroscopies, the quartz crystal microbalance, fluorescence and IR spectroscopies, and electrochemical techniques, to mention a few. It is shown that this concept allows for the construction of tethered lipid bilayers with outstanding electrical properties including resistivities in excess of 10 MOmega cm2. A modified strategy uses the assembly of peptides as spacers that couple covalently via their engineered sulfhydryl or lipoic acid groups at the N-terminus to the employed gold substrate, while their C-terminus is being activated afterward for the coupling of, for example, dimyristoylphosphatidylethanol amine (DMPE) lipid molecules via the NH2 moiety of their headgroups. It is demonstrated that these membranes are well suited for the in situ synthesis of membrane protein by a cell-free expression approach. The vectorial integration of an in vitro synthesized odorant receptor, OR5 from the rat, is demonstrated by means of antibodies that specifically bind to a tag at the N-terminus of the receptor and is read out by surface plasmon fluorescence spectroscopy. A completely different strategy employs his-tagged membrane proteins in their solubilized form binding to a surface-attached Ni(+)-NTA monolayer generating a well-oriented protein layer the density of which can be easily controlled by online monitoring the binding (assembly) step by surface plasmon spectroscopy. Moreover, the attachment of the his-tag to either the C- or the N-terminus allows for the complete control of the protein orientation. After the exchange of the detergent micelle by a lipid bilayer via a surface dialysis procedure an electrically very well isolating protein-tethered membrane is formed. We show that this "wiring" of the functional units allows for the (external) manipulation of the oxidation state of the redox-protein cytochrome c Oxidase by the control of the potential applied to the gold substrate which is used as the working electrode in an electrochemical attachment.

Optical monitoring of proteins at solid interfaces

NASA Astrophysics Data System (ADS)

Dunne, G.; McDonnell, L.; Miller, R.; McMillan, N. D.; O'Rourke, B.; Mitchell, C. I.

2005-06-01

The adsorption properties of polymers are of great importance for implant studies. A better understanding of these properties can lead to improved implant materials. In this study the surface energy of different polymers was derived from contact angle measurements taken using profile analysis tensiometry (PAT) of sessile drops of water. The contact angles were measured for advancing and receding water drops on polished polymer surfaces and also on polymer surfaces modified by adsorbing protein to the surface prior to analysis of the sessile drop. The protein used was bovine serum albumin (BSA) and the surfaces were poly-methylmethacrylate (PMMA), poly-ether-ether-ketone (PEEK) and stainless steel. The polymer surfaces were also studied using atomic force microscopy (AFM). Images of the surfaces were taken in different states: rough, smooth and with albumin adsorbed. As a method to identify the proteins on the surface easier, anti-albumin antibodies with 30nm nano gold particles attached were adsorbed to the albumin on the surfaces. Using nano gold particles made the imaging more straightforward and thus made identification of the protein on the surface easier. The results from this work show the differing hydrophobicities of polymer surfaces under different conditions and a new nanotechnological method of protein identification.

The interactions of peripheral membrane proteins with biological membranes

DOE PAGES

Johs, Alexander; Whited, A. M.

2015-07-29

The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

Auto-immune encephalitis as differential diagnosis of infectious encephalitis

PubMed Central

Armangue, Thaís; Leypoldt, Frank; Dalmau, Josep

2014-01-01

Purpose of review To describe the main types of autoimmune encephalitis with special emphasis on those associated with antibodies against neuronal cell surface or synaptic proteins, and the differential diagnosis with infectious encephalitis. Recent findings There is a continuous expansion of the number of cell surface or synaptic proteins that are targets of autoimmunity. The most recently identified include the mGluR5, DPPX, and the GABAAR. In these and previously known autoimmune encephalitis (NMDAR, AMPAR, GABABR, LGI1, CASPR2), the prodromal symptoms or types of presentations often suggest a viral encephalitis. We review here clues that help in the differential diagnosis with infectious encephalitis. Moreover, recent investigations indicate that viral encephalitis (e.g., herpes simplex) can trigger synaptic autoimmunity. In all these disorders immunotherapy is usually effective. Summary Autoimmune encephalitis comprises an expanding group of potentially treatable disorders that should be included in the differential diagnosis of any type of encephalitis. PMID:24792345

Effects of carbohydrate/protein ratio on the microstructure and the barrier and sorption properties of wheat starch-whey protein blend edible films.

PubMed

Basiak, Ewelina; Lenart, Andrzej; Debeaufort, Frédéric

2017-02-01

Starch and whey protein isolate and their mixtures were used for making edible films. Moisture sorption isotherms, water vapour permeability, sorption of aroma compounds, microstructure, water contact angle and surface properties were investigated. With increasing protein content, the microstructure changes became more homogeneous. The water vapour permeability increases with both the humidity gradient and the starch content. For all films, the hygroscopicity increases with starch content. Surface properties change according to the starch/whey protein ratio and are mainly related to the polar component of the surface tension. Films composed of 80% starch and 20% whey proteins have more hydrophobic surfaces than the other films due to specific interactions. The effect of carbohydrate/protein ratio significantly influences the microstructure, the surface wettability and the barrier properties of wheat starch-whey protein blend films. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

The Sus operon: a model system for starch uptake by the human gut Bacteroidetes

PubMed Central

Foley, Matthew H.; Cockburn, Darrell W.; Koropatkin, Nicole M.

2016-01-01

Resident bacteria in the densely populated human intestinal tract must efficiently compete for carbohydrate nutrition. The Bacteroidetes, a dominant bacterial phylum in the mammalian gut, encode a plethora of discrete polysaccharide utilization loci (PULs) that are selectively activated to facilitate glycan capture at the cell surface. The most well-studied PUL-encoded glycan-up-take system is the starch utilization system (Sus) of Bacteroides thetaiotaomicron. The Sus includes the requisite proteins for binding and degrading starch at the surface of the cell preceding oligosaccharide transport across the outer membrane for further depolymerization to glucose in the periplasm. All mammalian gut Bacteroidetes possess analogous Sus-like systems that target numerous diverse glycans. In this review, we discuss what is known about the eight Sus proteins of B. thetaiotaomicron that define the Sus-like paradigm of nutrient acquisition that is exclusive to the Gram-negative Bacteroidetes. We emphasize the well-characterized outer membrane proteins SusDEF and the α-amylase SusG, each of which have unique structural features that allow them to interact with starch on the cell surface. Despite the apparent redundancy in starch-binding sites among these proteins, each has a distinct role during starch catabolism. Additionally, we consider what is known about how these proteins dynamically interact and cooperate in the membrane and propose a model for the formation of the Sus outer membrane complex. PMID:27137179

A novel Pfs38 protein complex on the surface of Plasmodium falciparum blood-stage merozoites.

PubMed

Paul, Gourab; Deshmukh, Arunaditya; Kaur, Inderjeet; Rathore, Sumit; Dabral, Surbhi; Panda, Ashutosh; Singh, Susheel Kumar; Mohmmed, Asif; Theisen, Michael; Malhotra, Pawan

2017-02-16

The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface. Recombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-1 65 were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte binding assay with 6-cys proteins were undertaken to find out their possible role in host-parasite infection and seropositivity was assessed using Indian and Liberian sera. Immunoprecipitation of parasite-derived polypeptides, followed by LC-MS/MS analysis, identified a large Pfs38 complex comprising of 6-cys proteins: Pfs41, Pfs38, Pfs12 and other merozoite surface proteins: GLURP, SERA5 and MSP-1. The existence of such a complex was further corroborated by several protein-protein interaction tools, co-localization and co-sedimentation analysis. Pfs38 protein of Pfs38 complex binds to host red blood cells (RBCs) directly via glycophorin A as a receptor. Seroprevalence analysis showed that of the six antigens, prevalence varied from 40 to 99%, being generally highest for MSP-1 65 and GLURP proteins. Together the data show the presence of a large Pfs38 protein-associated complex on the parasite surface which is involved in RBC binding. These results highlight the complex molecular interactions among the P. falciparum merozoite surface proteins and advocate the development of a multi-sub-unit malaria vaccine based on some of these protein complexes on merozoite surface.

Crystal Nucleation Using Surface-Energy-Modified Glass Substrates.

PubMed

Nordquist, Kyle A; Schaab, Kevin M; Sha, Jierui; Bond, Andrew H

2017-08-02

Systematic surface energy modifications to glass substrates can induce nucleation and improve crystallization outcomes for small molecule active pharmaceutical ingredients (APIs) and proteins. A comparatively broad probe for function is presented in which various APIs, proteins, organic solvents, aqueous media, surface energy motifs, crystallization methods, form factors, and flat and convex surface energy modifications were examined. Replicate studies ( n ≥ 6) have demonstrated an average reduction in crystallization onset times of 52(4)% (alternatively 52 ± 4%) for acetylsalicylic acid from 91% isopropyl alcohol using two very different techniques: bulk cooling to 0 °C using flat surface energy modifications or microdomain cooling to 4 °C from the interior of a glass capillary having convex surface energy modifications that were immersed in the solution. For thaumatin and bovine pancreatic trypsin, a 32(2)% reduction in crystallization onset times was demonstrated in vapor diffusion experiments ( n ≥ 15). Nucleation site arrays have been engineered onto form factors frequently used in crystallization screening, including microscope slides, vials, and 96- and 384-well high-throughput screening plates. Nucleation using surface energy modifications on the vessels that contain the solutes to be crystallized adds a layer of useful variables to crystallization studies without requiring significant changes to workflows or instrumentation.

Up-regulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 (HCN3) by Specific Interaction with K+ Channel Tetramerization Domain-containing Protein 3 (KCTD3)*

PubMed Central

Cao-Ehlker, Xiaochun; Zong, Xiangang; Hammelmann, Verena; Gruner, Christian; Fenske, Stefanie; Michalakis, Stylianos; Wahl-Schott, Christian; Biel, Martin

2013-01-01

Most ion channels consist of the principal ion-permeating core subunit(s) and accessory proteins that are assembled with the channel core. The biological functions of the latter proteins are diverse and include the regulation of the biophysical properties of the ion channel, its connection to signaling pathways and the control of its cell surface expression. There is recent evidence that native hyperpolarization-activated cyclic nucleotide-gated channel complexes (HCN1–4) also contain accessory subunits, among which TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) has been most extensively studied. Here, we identify KCTD3, a so far uncharacterized member of the potassium channel tetramerization-domain containing (KCTD) protein family as an HCN3-interacting protein. KCTD3 is widely expressed in brain and some non-neuronal tissues and colocalizes with HCN3 in specific regions of the brain including hypothalamus. Within the HCN channel family, KCTD3 specifically binds to HCN3 and leads to a profound up-regulation of cell surface expression and current density of this channel. HCN3 can also functionally interact with TRIP8b; however, we found no evidence for channel complexes containing both TRIP8b and KCTD3. The C terminus of HCN3 is crucially required for functional interaction with KCTD3. Replacement of the cytosolic C terminus of HCN2 by the corresponding domain of HCN3 renders HCN2 sensitive to regulation by KCTD3. The C-terminal-half of KCTD3 is sufficient for binding to HCN3. However, the complete protein including the N-terminal tetramerization domain is needed for HCN3 current up-regulation. Together, our experiments indicate that KCTD3 is an accessory subunit of native HCN3 complexes. PMID:23382386

Effect of mechanical denaturation on surface free energy of protein powders.

PubMed

Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T; Blagbrough, Ian S; Conway, Barbara R

2016-10-01

Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms. Copyright © 2016 Elsevier B.V. All rights reserved.

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components

PubMed Central

Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, UA

2014-01-01

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and Impact of the Study Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. PMID:24935714

EzMol: A Web Server Wizard for the Rapid Visualization and Image Production of Protein and Nucleic Acid Structures.

PubMed

Reynolds, Christopher R; Islam, Suhail A; Sternberg, Michael J E

2018-01-31

EzMol is a molecular visualization Web server in the form of a software wizard, located at http://www.sbg.bio.ic.ac.uk/ezmol/. It is designed for easy and rapid image manipulation and display of protein molecules, and is intended for users who need to quickly produce high-resolution images of protein molecules but do not have the time or inclination to use a software molecular visualization system. EzMol allows the upload of molecular structure files in PDB format to generate a Web page including a representation of the structure that the user can manipulate. EzMol provides intuitive options for chain display, adjusting the color/transparency of residues, side chains and protein surfaces, and for adding labels to residues. The final adjusted protein image can then be downloaded as a high-resolution image. There are a range of applications for rapid protein display, including the illustration of specific areas of a protein structure and the rapid prototyping of images. Copyright © 2018. Published by Elsevier Ltd.

Druggable protein interaction sites are more predisposed to surface pocket formation than the rest of the protein surface.

PubMed

Johnson, David K; Karanicolas, John

2013-01-01

Despite intense interest and considerable effort via high-throughput screening, there are few examples of small molecules that directly inhibit protein-protein interactions. This suggests that many protein interaction surfaces may not be intrinsically "druggable" by small molecules, and elevates in importance the few successful examples as model systems for improving our fundamental understanding of druggability. Here we describe an approach for exploring protein fluctuations enriched in conformations containing surface pockets suitable for small molecule binding. Starting from a set of seven unbound protein structures, we find that the presence of low-energy pocket-containing conformations is indeed a signature of druggable protein interaction sites and that analogous surface pockets are not formed elsewhere on the protein. We further find that ensembles of conformations generated with this biased approach structurally resemble known inhibitor-bound structures more closely than equivalent ensembles of unbiased conformations. Collectively these results suggest that "druggability" is a property encoded on a protein surface through its propensity to form pockets, and inspire a model in which the crude features of the predisposed pocket(s) restrict the range of complementary ligands; additional smaller conformational changes then respond to details of a particular ligand. We anticipate that the insights described here will prove useful in selecting protein targets for therapeutic intervention.

A synergistic approach to protein crystallization: Combination of a fixed-arm carrier with surface entropy reduction

PubMed Central

Moon, Andrea F; Mueller, Geoffrey A; Zhong, Xuejun; Pedersen, Lars C

2010-01-01

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail. PMID:20196072

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy.

PubMed

Das, Priyadip; Duanias-Assaf, Tal; Reches, Meital

2017-03-06

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Morphological and proteomic analysis of biofilms from the Antarctic archaeon, Halorubrum lacusprofundi.

PubMed

Liao, Y; Williams, T J; Ye, J; Charlesworth, J; Burns, B P; Poljak, A; Raftery, M J; Cavicchioli, R

2016-11-22

Biofilms enhance rates of gene exchange, access to specific nutrients, and cell survivability. Haloarchaea in Deep Lake, Antarctica, are characterized by high rates of intergenera gene exchange, metabolic specialization that promotes niche adaptation, and are exposed to high levels of UV-irradiation in summer. Halorubrum lacusprofundi from Deep Lake has previously been reported to form biofilms. Here we defined growth conditions that promoted the formation of biofilms and used microscopy and enzymatic digestion of extracellular material to characterize biofilm structures. Extracellular DNA was found to be critical to biofilms, with cell surface proteins and quorum sensing also implicated in biofilm formation. Quantitative proteomics was used to define pathways and cellular processes involved in forming biofilms; these included enhanced purine synthesis and specific cell surface proteins involved in DNA metabolism; post-translational modification of cell surface proteins; specific pathways of carbon metabolism involving acetyl-CoA; and specific responses to oxidative stress. The study provides a new level of understanding about the molecular mechanisms involved in biofilm formation of this important member of the Deep Lake community.

Morphological and proteomic analysis of biofilms from the Antarctic archaeon, Halorubrum lacusprofundi

PubMed Central

Liao, Y.; Williams, T. J.; Ye, J.; Charlesworth, J.; Burns, B. P.; Poljak, A.; Raftery, M. J.; Cavicchioli, R.

2016-01-01

Biofilms enhance rates of gene exchange, access to specific nutrients, and cell survivability. Haloarchaea in Deep Lake, Antarctica, are characterized by high rates of intergenera gene exchange, metabolic specialization that promotes niche adaptation, and are exposed to high levels of UV-irradiation in summer. Halorubrum lacusprofundi from Deep Lake has previously been reported to form biofilms. Here we defined growth conditions that promoted the formation of biofilms and used microscopy and enzymatic digestion of extracellular material to characterize biofilm structures. Extracellular DNA was found to be critical to biofilms, with cell surface proteins and quorum sensing also implicated in biofilm formation. Quantitative proteomics was used to define pathways and cellular processes involved in forming biofilms; these included enhanced purine synthesis and specific cell surface proteins involved in DNA metabolism; post-translational modification of cell surface proteins; specific pathways of carbon metabolism involving acetyl-CoA; and specific responses to oxidative stress. The study provides a new level of understanding about the molecular mechanisms involved in biofilm formation of this important member of the Deep Lake community. PMID:27874045

Regulation of Macrophage Recognition through the Interplay of Nanoparticle Surface Functionality and Protein Corona.

PubMed

Saha, Krishnendu; Rahimi, Mehran; Yazdani, Mahdieh; Kim, Sung Tae; Moyano, Daniel F; Hou, Singyuk; Das, Ridhha; Mout, Rubul; Rezaee, Farhad; Mahmoudi, Morteza; Rotello, Vincent M

2016-04-26

Using a family of cationic gold nanoparticles (NPs) with similar size and charge, we demonstrate that proper surface engineering can control the nature and identity of protein corona in physiological serum conditions. The protein coronas were highly dependent on the hydrophobicity and arrangement of chemical motifs on NP surface. The NPs were uptaken in macrophages in a corona-dependent manner, predominantly through recognition of specific complement proteins in the NP corona. Taken together, this study shows that surface functionality can be used to tune the protein corona formed on NP surface, dictating the interaction of NPs with macrophages.

The Staphylococcus aureus Global Regulator MgrA Modulates Clumping and Virulence by Controlling Surface Protein Expression

PubMed Central

Crosby, Heidi A.; Schlievert, Patrick M.; Merriman, Joseph A.; King, Jessica M.; Salgado-Pabón, Wilmara; Horswill, Alexander R.

2016-01-01

Staphylococcus aureus is a human commensal and opportunistic pathogen that causes devastating infections in a wide range of locations within the body. One of the defining characteristics of S. aureus is its ability to form clumps in the presence of soluble fibrinogen, which likely has a protective benefit and facilitates adhesion to host tissue. We have previously shown that the ArlRS two-component regulatory system controls clumping, in part by repressing production of the large surface protein Ebh. In this work we show that ArlRS does not directly regulate Ebh, but instead ArlRS activates expression of the global regulator MgrA. Strains lacking mgrA fail to clump in the presence of fibrinogen, and clumping can be restored to an arlRS mutant by overexpressing either arlRS or mgrA, indicating that ArlRS and MgrA constitute a regulatory pathway. We used RNA-seq to show that MgrA represses ebh, as well as seven cell wall-associated proteins (SraP, Spa, FnbB, SasG, SasC, FmtB, and SdrD). EMSA analysis showed that MgrA directly represses expression of ebh and sraP. Clumping can be restored to an mgrA mutant by deleting the genes for Ebh, SraP and SasG, suggesting that increased expression of these proteins blocks clumping by steric hindrance. We show that mgrA mutants are less virulent in a rabbit model of endocarditis, and virulence can be partially restored by deleting the genes for the surface proteins ebh, sraP, and sasG. While mgrA mutants are unable to clump, they are known to have enhanced biofilm capacity. We demonstrate that this increase in biofilm formation is partially due to up-regulation of SasG, a surface protein known to promote intercellular interactions. These results confirm that ArlRS and MgrA constitute a regulatory cascade, and that they control expression of a number of genes important for virulence, including those for eight large surface proteins. PMID:27144398

Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding.

PubMed

Jolly, Amber L; Agarwal, Paresh; Metruccio, Matteo M E; Spiciarich, David R; Evans, David J; Bertozzi, Carolyn R; Fleiszig, Suzanne M J

2017-06-01

Cell surface glycosylation is thought to be involved in barrier function against microbes at mucosal surfaces. Previously we showed that the epithelium of healthy mouse corneas becomes vulnerable to Pseudomonas aeruginosa adhesion if it lacks the innate defense protein MyD88 (myeloid differentiation primary response gene 88), or after superficial injury by blotting with tissue paper. Here we explored their effect on corneal surface glycosylation using a metabolic label, tetra-acetylated N -azidoacetylgalactosamine (Ac 4 GalNAz). Ac 4 GalNAz treatment labeled the surface of healthy mouse corneas, leaving most cells viable, and bacteria preferentially associated with GalNAz-labeled regions. Surprisingly, corneas from MyD88 -/- mice displayed similar GalNAz labeling to wild-type corneas, but labeling was reduced and patchy on IL-1 receptor (IL-1R)-knockout mouse corneas ( P < 0.05, ANOVA). Tissue paper blotting removed GalNAz-labeled surface cells, causing DAPI labeling (permeabilization) of underlying cells. MS of material collected on the tissue paper blots revealed 67 GalNAz-labeled proteins, including intracellular proteins. These data show that the normal distribution of surface glycosylation requires IL-1R, but not MyD88, and is not sufficient to prevent bacterial binding. They also suggest increased P. aeruginosa adhesion to MyD88 -/- and blotted corneas is not due to reduction in total surface glycosylation, and for tissue paper blotting is likely due to cell permeabilization.-Jolly, A. L., Agarwal, P., Metruccio, M. M. E., Spiciarich, D. R., Evans, D. J., Bertozzi, C. R., Fleiszig, S. M. J. Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding. © FASEB.

SFG analysis of surface bound proteins: a route towards structure determination.

PubMed

Weidner, Tobias; Castner, David G

2013-08-14

The surface of a material is rapidly covered with proteins once that material is placed in a biological environment. The structure and function of these bound proteins play a key role in the interactions and communications of the material with the biological environment. Thus, it is crucial to gain a molecular level understanding of surface bound protein structure. While X-ray diffraction and solution phase NMR methods are well established for determining the structure of proteins in the crystalline or solution phase, there is not a corresponding single technique that can provide the same level of structural detail about proteins at surfaces or interfaces. However, recent advances in sum frequency generation (SFG) vibrational spectroscopy have significantly increased our ability to obtain structural information about surface bound proteins and peptides. A multi-technique approach of combining SFG with (1) protein engineering methods to selectively introduce mutations and isotopic labels, (2) other experimental methods such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) and near edge X-ray absorption fine structure (NEXAFS) to provide complementary information, and (3) molecular dynamic (MD) simulations to extend the molecular level experimental results is a particularly promising route for structural characterization of surface bound proteins and peptides. By using model peptides and small proteins with well-defined structures, methods have been developed to determine the orientation of both backbone and side chains to the surface.

SFG analysis of surface bound proteins: A route towards structure determination

PubMed Central

Weidner, Tobias; Castner, David G.

2013-01-01

The surface of a material is rapidly covered with proteins once that material is placed in a biological environment. The structure and function of these bound proteins play a key role in the interactions and communications of the material with the biological environment. Thus, it is crucial to gain a molecular level understanding of surface bound protein structure. While X-ray diffraction and solution phase NMR methods are well established for determining the structure of proteins in the crystalline or solution phase, there is not a corresponding single technique that can provide the same level of structural detail about proteins at surfaces or interfaces. However, recent advances in sum frequency generation (SFG) vibrational spectroscopy have significantly increased our ability to obtain structural information about surface bound proteins and peptides. A multi-technique approach of combining SFG with (1) protein engineering methods to selectively introduce mutations and isotopic labels, (2) other experimental methods such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) and near edge x-ray absorption fine structure (NEXAFS) to provide complementary information, and (3) molecular dynamic (MD) simulations to extend the molecular level experimental results is a particularly promising route for structural characterization of surface bound proteins and peptides. By using model peptides and small proteins with well-defined structures, methods have been developed to determine the orientation of both backbone and side chains to the surface. PMID:23727992

Improving biocompatibility by surface modification techniques on implantable bioelectronics.

PubMed

Lin, Peter; Lin, Chii-Wann; Mansour, Raafat; Gu, Frank

2013-09-15

For implantable bioelectronic devices, the interface between the device and the biological environment requires significant attention as it dictates the device performance in vivo. Non-specific protein adsorption onto the device surface is the initial stage of many degradation mechanisms that will ultimately compromise the functionality of the device. In order to preserve the functionality of any implanted bioelectronics overtime, protein adsorption must be controlled. This review paper outlines two major approaches to minimize protein adsorption onto the surface of implantable electronics. The first approach is surface coating, which minimizes close proximity interactions between proteins and device surfaces by immobilizing electrically neutral hydrophilic polymers as surface coating. These coatings reduce protein fouling by steric repulsion and formation of a hydration layer which acts as both a physical and energetic barrier that minimize protein adsorption onto the device. Relevant performances of various conventional hydrophilic coatings are discussed. The second approach is surface patterning using arrays of hydrophobic nanostructures through photolithography techniques. By establishing a large slip length via super hydrophobic surfaces, the amount of proteins adsorbed to the surface of the device can be reduced. The last section discusses emerging surface coating techniques utilizing zwitterionic polymers where ultralow-biofouling surfaces have been demonstrated. These surface modification techniques may significantly improve the long-term functionality of implantable bioelectronics, thus allowing researchers to overcome challenges to diagnose and treat chronic neurological and cardiovascular diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Water organization between oppositely charged surfaces: Implications for protein sliding along DNA a)

NASA Astrophysics Data System (ADS)

Marcovitz, Amir; Naftaly, Aviv; Levy, Yaakov

2015-02-01

Water molecules are abundant in protein-DNA interfaces, especially in their nonspecific complexes. In this study, we investigated the organization and energetics of the interfacial water by simplifying the geometries of the proteins and the DNA to represent them as two equally and oppositely charged planar surfaces immersed in water. We found that the potential of mean force for bringing the two parallel surfaces into close proximity comprises energetic barriers whose properties strongly depend on the charge density of the surfaces. We demonstrated how the organization of the water molecules into discretized layers and the corresponding energetic barriers to dehydration can be modulated by the charge density on the surfaces, salt, and the structure of the surfaces. The 1-2 layers of ordered water are tightly bound to the charged surfaces representing the nonspecific protein-DNA complex. This suggests that water might mediate one-dimensional diffusion of proteins along DNA (sliding) by screening attractive electrostatic interactions between the positively charged molecular surface on the protein and the negatively charged DNA backbone and, in doing so, reduce intermolecular friction in a manner that smoothens the energetic landscape for sliding, and facilitates the 1D diffusion of the protein.

A role for surface hydrophobicity in protein-protein recognition.

PubMed Central

Young, L.; Jernigan, R. L.; Covell, D. G.

1994-01-01

The role of hydrophobicity as a determinant of protein-protein interactions is examined. Surfaces of apo-protein targets comprising 9 classes of enzymes, 7 antibody fragments, hirudin, growth hormone, and retinol-binding protein, and their associated ligands with available X-ray structures for their complexed forms, are scanned to determine clusters of surface-accessible amino acids. Clusters of surface residues are ranked on the basis of the hydrophobicity of their constituent amino acids. The results indicate that the location of the co-crystallized ligand is commonly found to correspond with one of the strongest hydrophobic clusters on the surface of the target molecule. In 25 of 38 cases, the correspondence is exact, with the position of the most hydrophobic cluster coinciding with more than one-third of the surface buried by the bound ligand. The remaining 13 cases demonstrate this correspondence within the top 6 hydrophobic clusters. These results suggest that surface hydrophobicity can be used to identify regions of a protein's surface most likely to interact with a binding ligand. This fast and simple procedure may be useful for identifying small sets of well-defined loci for possible ligand attachment. PMID:8061602

Ellipsometric studies of synthetic albumin-binding chitosan-derivatives and selected blood plasma proteins

NASA Astrophysics Data System (ADS)

Sarkar, Sabyasachi

This dissertation summarizes work on the synthesis of chitosan-derivatives and the development of ellipsometric methods to characterize materials of biological origin. Albumin-binding chitosan-derivatives were synthesized via addition reactions that involve amine groups naturally present in chitosan. These surfaces were shown to have an affinity towards human serum albumin via ELISA, UV spectroscopy and SDS PAGE. Modified surfaces were characterized with IR ellipsometry at various stages of their synthesis using appropriate optical models. It was found that spin cast chitosan films were anisotropic in nature. All optical models used for characterizing chitosan-derivatives were thus anisotropic. Chemical signal dependence on molecular structure and composition was illustrated via IR spectroscopic ellipsometry (IRSE). An anisotropic optical model of an ensemble of Lorentz oscillators were used to approximate material behavior. The presence of acetic acid in spin-cast non-neutralized chitosan samples was thus shown. IRSE application to biomaterials was also demonstrated by performing a step-wise chemical characterizations during synthesis stages. Protein adsorbed from single protein solutions on these modified surfaces was monitored by visible in-situ variable wavelength ellipsometry. Based on adsorption profiles obtained from single protein adsorption onto silicon surfaces, lumped parameter kinetic models were developed. These models were used to fit experimental data of immunoglobulin-G of different concentrations and approximate conformational changes in fibrinogen adsorption. Biomaterial characterization by ellipsometry was further extended to include characterization of individual protein solutions in the IR range. Proteins in an aqueous environment were characterized by attenuated total internal reflection (ATR) IR ellipsometry using a ZnSe prism. Parameterized dielectric functions were created for individual proteins using Lorentz oscillators. These parameterized dielectric functions were then used to describe the growth and eventual enzymatic degradation of a multilayered IgG structure by dynamic ATR-IR ellipsometry measurements. ATR-IR ellipsometry was also used to observe the specificity of anti-bodies to antigens.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao, Jinhui; Buchko, Garry W.; Shaw, Wendy J.

The interactions between proteins and surfaces are critical to a number of important processes including biomineralization, the biocompatibility of biomaterials, and the function of biosensors. Although many proteins exist as monomers or small oligomers, amelogenin is a unique protein that self-assembles into supramolecular structures called “nanospheres,” aggregates of 100’s of monomers that are 20-60 nm in diameter. The nanosphere quaternary structure is observed in solution, however, the quaternary structure of amelogenin adsorbed onto hydroxyapatite (HAP) surfaces is not known even though it may be important to amelogenin’s function in forming highly elongated and intricately assembled HAP crystallites during enamel formation.more » We report studies of the interactions of the enamel protein, amelogenin (rpM179), with a well-defined (100) face prepared by synthesis of large crystals of HAP. High resolution, in-situ atomic force microscopy (AFM) was used to directly observe protein adsorption onto HAP at the molecular level within an aqueous solution environment. Our study shows that the amelogenin nanospheres disassemble onto the HAP surface, breaking down into oligomeric (25-mer) subunits of the larger nanosphere. In some cases, the disassembly event is directly observed by in situ imaging for the first time. Quantification of the adsorbate amounts by size analysis led to the determination of a protein binding energy (17.1 kbT) to a specific face of HAP (100). The kinetics of disassembly are greatly slowed in aged solutions, indicating there are time-dependent increases in oligomer-oligomer binding interactions within the nanosphere. A small change in the sequence of amelogenin by the attachment of a histidine tag to the N-terminus of rpM179 to form rp(H)M180 results in the adsorption of a complete second layer on top of the underlying first layer. Our research elucidates how supramolecular protein structures interact and break down at surfaces and how small changes in the primary sequence of amelogenin can affect the disassembly process.« less

The S-layer homology domain-containing protein SlhA from Paenibacillus alvei CCM 2051(T) is important for swarming and biofilm formation.

PubMed

Janesch, Bettina; Koerdt, Andrea; Messner, Paul; Schäffer, Christina

2013-01-01

Swarming and biofilm formation have been studied for a variety of bacteria. While this is well investigated for Gram-negative bacteria, less is known about Gram-positive bacteria, including Paenibacillus alvei, a secondary invader of diseased honeybee colonies infected with Melissococcus pluton, the causative agent of European foulbrood (EFB). Paenibacillus alvei CCM 2051(T) is a Gram-positive bacterium which was recently shown to employ S-layer homology (SLH) domains as cell wall targeting modules to display proteins on its cell surface. This study deals with the newly identified 1335-amino acid protein SlhA from P. alvei which carries at the C‑terminus three consecutive SLH-motifs containing the predicted binding sequences SRGE, VRQD, and LRGD instead of the common TRAE motif. Based on the proof of cell surface location of SlhA by fluorescence microscopy using a SlhA-GFP chimera, the binding mechanism was investigated in an in vitro assay. To unravel a putative function of the SlhA protein, a knockout mutant was constructed. Experimental data indicated that one SLH domain is sufficient for anchoring of SlhA to the cell surface, and the SLH domains of SlhA recognize both the peptidoglycan and the secondary cell wall polymer in vitro. This is in agreement with previous data from the S-layer protein SpaA, pinpointing a wider utilization of that mechanism for cell surface display of proteins in P. alvei. Compared to the wild-type bacterium ΔslhA revealed changed colony morphology, loss of swarming motility and impaired biofilm formation. The phenotype was similar to that of the flagella knockout Δhag, possibly due to reduced EPS production influencing the functionality of the flagella of ΔslhA. This study demonstrates the involvement of the SLH domain-containing protein SlhA in swarming and biofilm formation of P. alvei CCM 2051(T).

Aberrant expression of genes and proteins in pterygium and their implications in the pathogenesis

PubMed Central

Feng, Qing-Yang; Hu, Zi-Xuan; Song, Xi-Ling; Pan, Hong-Wei

2017-01-01

Pterygium is a common ocular surface disease induced by a variety of factors. The exact pathogenesis of pterygium remains unclear. Numbers of genes and proteins are discovered in pterygium and they function differently in the occurrence and development of this disease. We searched the Web of Science and PubMed throughout history for literatures about the subject. The keywords we used contain pterygium, gene, protein, angiogenesis, fibrosis, proliferation, inflammation, pathogenesis and therapy. In this review, we summarize the aberrant expression of a range of genes and proteins in pterygium compared with normal conjunctiva or cornea, including growth factors, matrix metalloproteinases and tissue inhibitors of metalloproteinases, interleukins, tumor suppressor genes, proliferation related proteins, apoptosis related proteins, cell adhesion molecules, extracellular matrix proteins, heat shock proteins and tight junction proteins. We illustrate their possible mechanisms in the pathogenesis of pterygium as well as the related intervention based on them for pterygium therapy. PMID:28730091

Fabrication of biofunctional nanomaterials via Escherichia coli OmpF protein air/water interface insertion/integration with copolymeric amphiphiles.

PubMed

Ho, Dean; Chang, Stacy; Montemagno, Carlo D

2006-06-01

Fabrication of next-generation biologically active materials will involve the integration of proteins with synthetic membrane materials toward a wide spectrum of applications in nanoscale medicine, including high-throughput drug testing, energy conversion for powering medical devices, and bio-cloaking films for mimicry of cellular membrane surfaces toward the enhancement of implant biocompatibility. We have used ABA triblock copolymer membranes (PMOXA-PDMS-PMOXA) of varied thicknesses as platform materials for Langmuir film-based functionalization with the OmpF pore protein from Escherichia coli by fabricating monolayers of copolymer amphiphile-protein complexes on the air/water interface. Here we demonstrate that the ability for protein insertion at the air/water interface during device fabrication is dependent upon the initial surface coverage with the copolymer as well as copolymer thickness. Methacrylate-terminated block copolymer structures that were 4 nm (4METH) and 8 nm (8METH) in length were used as the protein reconstitution matrix, whereas a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid (~4 nm thickness) was used as a comparison to demonstrate the effects of copolymer length on protein integration capabilities. Wilhemy surface pressure measurements (mN/m) revealed a greater protein insertion in the 4METH and POPC structures compared with the 8METH structure, indicating that shorter copolymer chains possess enhanced biomimicry of natural lipid-based membranes. In addition, comparisons between the isothermal characteristics of the 4METH, 8METH, and POPC membranes reveal that phase transitions of the 4METH resemble a blend of the 8METH and POPC materials, indicating that the 4METH chain may possess hybrid properties of both copolymers and lipids. Furthermore, we have shown that following the deposition of the amphiphilic materials on the air/water interface, the OmpF can be deposited directly on top of the amphiphiles (surface addition), thus effectively further enhancing protein insertion because of the buoying effects of the membranes. These characteristics of Langmuir-Blodgett-based fabrication of copolymer-biomolecule hybrids represent a synthesis strategy for next-generation biomedical materials.

Surface reengineering of RPA70N enables cocrystallization with an inhibitor of the replication protein A interaction motif of ATR interacting protein.

PubMed

Feldkamp, Michael D; Frank, Andreas O; Kennedy, J Phillip; Patrone, James D; Vangamudi, Bhavatarini; Waterson, Alex G; Fesik, Stephen W; Chazin, Walter J

2013-09-17

Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these protein-protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of protein-ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N-peptide fusion construct have been reported, RPA70N-inhibitor complexes have been recalcitrant to crystallization. Analysis of the P61 lattice of RPA70N crystals led us to hypothesize that the ligand-binding surface was occluded. Surface reengineering to alter key crystal lattice contacts led to the design of RPA70N E7R, E100R, and E7R/E100R mutants. These mutants crystallized in a P212121 lattice that clearly had significant solvent channels open to the critical basic cleft. Analysis of X-ray crystal structures, target peptide binding affinities, and (15)N-(1)H heteronuclear single-quantum coherence nuclear magnetic resonance spectra showed that the mutations do not result in perturbations of the RPA70N ligand-binding surface. The success of the design was demonstrated by determining the structure of RPA70N E7R soaked with a ligand discovered in a previously reported molecular fragment screen. A fluorescence anisotropy competition binding assay revealed this compound can inhibit the interaction of RPA70N with the peptide binding motif from the DNA damage response protein ATRIP. The implications of the results are discussed in the context of ongoing efforts to design RPA70N inhibitors.

Polymers and biopolymers at interfaces

NASA Astrophysics Data System (ADS)

Hall, A. R.; Geoghegan, M.

2018-03-01

This review updates recent progress in the understanding of the behaviour of polymers at surfaces and interfaces, highlighting examples in the areas of wetting, dewetting, crystallization, and ‘smart’ materials. Recent developments in analysis tools have yielded a large increase in the study of biological systems, and some of these will also be discussed, focussing on areas where surfaces are important. These areas include molecular binding events and protein adsorption as well as the mapping of the surfaces of cells. Important techniques commonly used for the analysis of surfaces and interfaces are discussed separately to aid the understanding of their application.

Surface plasmon resonance-enabled antibacterial digital versatile discs

NASA Astrophysics Data System (ADS)

Dou, Xuan; Chung, Pei-Yu; Jiang, Peng; Dai, Jianli

2012-02-01

We report the achievement of effective sterilization of exemplary bacteria including Escherichia coli and Geobacillus stearothermophilus spores on a digital versatile disc (DVD). The spiral arrangement of aluminum-covered pits generates strong surface plasmon resonance (SPR) absorption of near-infrared light, leading to high surface temperature that could even damage the DVD plastics. Localized protein denaturation and high sterilization efficiency have been demonstrated by using a fluorescence microscope and cell cultures. Numerical simulations have also been conducted to model the SPR properties and the surface temperature distribution of DVDs under laser illumination. The theoretical predictions agree reasonably well with the experimental results.

Membrane Fusion Promoted by Increasing Surface Densities of the Paramyxovirus F and HN Proteins: Comparison of Fusion Reactions Mediated by Simian Virus 5 F, Human Parainfluenza Virus Type 3 F, and Influenza Virus HA

PubMed Central

Dutch, Rebecca Ellis; Joshi, Sangeeta Bagai; Lamb, Robert A.

1998-01-01

The membrane fusion reaction promoted by the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) proteins and hemagglutinin-neuraminidase (HN) proteins was characterized when the surface densities of F and HN were varied. Using a quantitative content mixing assay, it was found that the extent of SV5 F-mediated fusion was dependent on the surface density of the SV5 F protein but independent of the density of SV5 HN protein, indicating that HN serves only a binding function in the reaction. However, the extent of HPIV-3 F protein promoted fusion reaction was found to be dependent on surface density of HPIV-3 HN protein, suggesting that the HPIV-3 HN protein is a direct participant in the fusion reaction. Analysis of the kinetics of lipid mixing demonstrated that both initial rates and final extents of fusion increased with rising SV5 F protein surface densities, suggesting that multiple fusion pores can be active during SV5 F protein-promoted membrane fusion. Initial rates and extent of lipid mixing were also found to increase with increasing influenza virus hemagglutinin protein surface density, suggesting parallels between the mechanism of fusion promoted by these two viral fusion proteins. PMID:9733810

Protein-surfactant interactions at hydrophobic interfaces studied with total internal reflection fluorescence correlation spectroscopy (TIR-FCS).

PubMed

Sonesson, Andreas W; Blom, Hans; Hassler, Kai; Elofsson, Ulla M; Callisen, Thomas H; Widengren, Jerker; Brismar, Hjalmar

2008-01-15

The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C12E6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C12E6/LAS concentration, the surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 microM C12E6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.

Proteomic Definitions of Mesenchymal Stem Cells

PubMed Central

Maurer, Martin H.

2011-01-01

Mesenchymal stem cells (MSCs) are pluripotent cells isolated from the bone marrow and various other organs. They are able to proliferate and self-renew, as well as to give rise to progeny of at least the osteogenic, chondrogenic, and adipogenic lineages. Despite this functional definition, MSCs can also be defined by their expression of a distinct set of cell surface markers. In the current paper, studies investigating the proteome of human MSCs are reviewed with the aim to identify common protein markers of MSCs. The proteomic analysis of MSCs revealed a distinct set of proteins representing the basic molecular inventory, including proteins for (i) cell surface markers, (ii) the responsiveness to growth factors, (iii) the reuse of developmental signaling cascades in adult stem cells, (iv) the interaction with molecules of the extracellular matrix, (v) the expression of genes regulating transcription and translation, (vi) the control of the cell number, and (vii) the protection against cellular stress. PMID:21437194

Plasma surface modification of nanofiltration (NF) thin-film composite (TFC) membranes to improve anti organic fouling

NASA Astrophysics Data System (ADS)

Kim, Eun-Sik; Yu, Qingsong; Deng, Baolin

2011-09-01

Commercial nanofiltration (NF) thin-film composite (TFC) membranes were treated by low-pressure NH3 plasma, and the effects of the plasma treatment were investigated in terms of the membrane hydrophilicity, pure water flux, salt rejection, protein adsorption, and humic acid fouling. Experimental results indicated that the membrane surface hydrophilicity was increased by the plasma treatment, and changes in the hydrophilicity as well as membrane performance including permeate flux and fouling varied with the original membrane characteristics (e.g., roughness and hydrophilicity). Water flux of plasma treated membranes was the highest with 10 min and 90 W of plasma treatment, and salt rejection was mainly affected by the intensity of the plasma power. Results of bovine serum albumin (BSA) adsorption demonstrated that the protein adsorption decreased with increasing plasma treatment time. The plasma treatment that resulted in more negatively charged surfaces could also better prevent Aldrich humic acid (AHA) attachment on the membrane surface.

Competitive Protein Adsorption on Polysaccharide and Hyaluronate Modified Surfaces

PubMed Central

Ombelli, Michela; Costello, Lauren; Postle, Corinne; Anantharaman, Vinod; Meng, Qing Cheng; Composto, Russell J.; Eckmann, David M.

2011-01-01

We measured adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) onto six distinct bare and dextran- and hyaluronate-modified silicon surfaces created using two dextran grafting densities and three hyaluronic acid (HA) sodium salts derived from human umbilical cord, rooster comb and streptococcus zooepidemicus. Film thickness and surface morphology depended on HA molecular weight and concentration. BSA coverage was enhanced on surfaces upon competitive adsorption of BSA:Fg mixtures. Dextranization differentially reduced protein adsorption onto surfaces based on oxidation state. Hyaluronization was demonstrated to provide the greatest resistance to protein coverage, equivalent to that of the most resistant dextranized surface. Resistance to protein adsorption was independent of the type of hyaluronic acid utilized. With changing bulk protein concentration from 20 to 40 µg ml−1 for each species, Fg coverage on silicon increased by 4×, whereas both BSA and Fg adsorption on dextran and HA were far less dependent of protein bulk concentration. PMID:21623481

Atomic force microscopy – looking at mechanosensors on the cell surface

PubMed Central

Heinisch, Jürgen J.; Lipke, Peter N.; Beaussart, Audrey; El Kirat Chatel, Sofiane; Dupres, Vincent; Alsteens, David; Dufrêne, Yves F.

2012-01-01

Summary Living cells use cell surface proteins, such as mechanosensors, to constantly sense and respond to their environment. However, the way in which these proteins respond to mechanical stimuli and assemble into large complexes remains poorly understood at the molecular level. In the past years, atomic force microscopy (AFM) has revolutionized the way in which biologists analyze cell surface proteins to molecular resolution. In this Commentary, we discuss how the powerful set of advanced AFM techniques (e.g. live-cell imaging and single-molecule manipulation) can be integrated with the modern tools of molecular genetics (i.e. protein design) to study the localization and molecular elasticity of individual mechanosensors on the surface of living cells. Although we emphasize recent studies on cell surface proteins from yeasts, the techniques described are applicable to surface proteins from virtually all organisms, from bacteria to human cells. PMID:23077172

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2)

PubMed Central

Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

2016-01-01

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

Activation of m1 muscarinic acetylcholine receptor induces surface transport of KCNQ channels through a CRMP-2-mediated pathway.

PubMed

Jiang, Ling; Kosenko, Anastasia; Yu, Clinton; Huang, Lan; Li, Xuejun; Hoshi, Naoto

2015-11-15

Neuronal excitability is strictly regulated by various mechanisms, including modulation of ion channel activity and trafficking. Stimulation of m1 muscarinic acetylcholine receptor (also known as CHRM1) increases neuronal excitability by suppressing the M-current generated by the Kv7/KCNQ channel family. We found that m1 muscarinic acetylcholine receptor stimulation also triggers surface transport of KCNQ subunits. This receptor-induced surface transport was observed with KCNQ2 as well as KCNQ3 homomeric channels, but not with Kv3.1 channels. Deletion analyses identified that a conserved domain in a proximal region of the N-terminal tail of KCNQ protein is crucial for this surface transport--the translocation domain. Proteins that bind to this domain were identified as α- and β-tubulin and collapsin response mediator protein 2 (CRMP-2; also known as DPYSL2). An inhibitor of casein kinase 2 (CK2) reduced tubulin binding to the translocation domain, whereas an inhibitor of glycogen synthase kinase 3 (GSK3) facilitated CRMP-2 binding to the translocation domain. Consistently, treatment with the GSK3 inhibitor enhanced receptor-induced KCNQ2 surface transport. M-current recordings from neurons showed that treatment with a GSK3 inhibitor shortened the duration of muscarinic suppression and led to over-recovery of the M-current. These results suggest that m1 muscarinic acetylcholine receptor stimulates surface transport of KCNQ channels through a CRMP-2-mediated pathway. © 2015. Published by The Company of Biologists Ltd.

Modulating the activity of protein conjugated to gold nanoparticles by site-directed orientation and surface density of bound protein.

PubMed

Liu, Feng; Wang, Lei; Wang, Hongwei; Yuan, Lin; Li, Jingwen; Brash, John Law; Chen, Hong

2015-02-18

The key property of protein-nanoparticle conjugates is the bioactivity of the protein. The ability to accurately modulate the activity of protein on the nanoparticles at the interfaces is important in many applications. In the work reported here, modulation of the activity of protein-gold nanoparticle (AuNP) conjugates by specifically orienting the protein and by varying the surface density of the protein was investigated. Different orientations were achieved by introducing cysteine (Cys) residues at specific sites for binding to gold. We chose Escherichia coli inorganic pyrophosphatase (PPase) as a model protein and used site-directed mutagenesis to generate two mutant types (MTs) with a single Cys residue on the surface: MT1 with Cys near the active center and MT2 with Cys far from the active center. The relative activities of AuNP conjugates with wild type (WT), MT1, and MT2 were found to be 44.8%, 68.8%, and 91.2% of native PPase in aqueous solution. Site-directed orientation with the binding site far from the active center thus allowed almost complete preservation of the protein activity. The relative activity of WT and MT2 conjugates did not change with the surface density of the protein, while that of MT1 increased significantly with increasing surface density. These results demonstrate that site-directed orientation and surface density can both modulate the activity of proteins conjugated to AuNP and that orientation has a greater effect than density. Furthermore, increasing the surface density of the specifically oriented protein MT2, while having no significant effect on the specific activity of the protein, still allowed increased protein loading on the AuNP and thus increased the total protein activity. This is of great importance in the study on the interface of protein and nanoparticle and the applications for enzyme immobilization, drug delivery, and biocatalysis.

Platelet Adhesion and Activation on Chiral Surfaces: The Influence of Protein Adsorption.

PubMed

Fan, Yonghong; Luo, Rifang; Han, Honghong; Weng, Yajun; Wang, Hong; Li, Jing'an; Yang, Ping; Wang, Yunbing; Huang, Nan

2017-10-03

Adsorbed proteins and their conformational change on blood-contacting biomaterials will determine their final hemocompatibility. It has frequently been reported that surface chirality of biomaterials may highly influence their protein adsorption behavior. Here, lysine and tartaric acid with different chirality were immobilized onto TiO 2 films respectively, and the influence of surface chirality on protein adsorption, platelet adhesion, and activation was also investigated. It showed that the l- and d-molecule grafted samples had almost the same grafting density, surface topography, chemical components, and hydrophilicity in this study. However, biological behaviors such as protein adsorption, platelet adhesion, and activation were quite different. The d-lysine grafted surface had a greater ability to inhibit both bovine serum albumin and fibrinogen adsorption, along with less degeneration of fibrinogen compared to the l-lysine anchored surface. However, the d-tartaric acid grafted surface adsorbed more protein but with less denatured fibrinogen compared to the l-tartaric acid grafted one. Further studies showed that the secondary structural change of the adsorbed albumin and fibrinogen on all surfaces with deduction of the α-helix content and increase of disordered structure, while the changing degree was apparently varied. As a result, the d-lysine immobilized surface absorbed less platelets and red blood cells and achieved slightly increased platelet activation. For tartaric acid anchored surfaces, a larger number of platelets adhered to the D-surface but were less activated compared to the L-surface. In conclusion, the surface chirality significantly influenced the adsorption and conformational change of blood plasma protein, which in turn influenced both platelet adhesion and activation.

Great interactions: How binding incorrect partners can teach us about protein recognition and function.

PubMed

Vamparys, Lydie; Laurent, Benoist; Carbone, Alessandra; Sacquin-Mora, Sophie

2016-10-01

Protein-protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications. The prediction of protein binding sites in particular is of paramount importance since proteins now represent a major class of therapeutic targets. Amongst others methods, docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on a protein surface. From the analysis of the protein interfaces generated by a massive cross-docking experiment using the 168 proteins of the Docking Benchmark 2.0, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is also possible to predict a protein's binding residues without having any prior knowledge regarding its potential interaction partners. Evaluating the performance of cross-docking predictions using the area under the specificity-sensitivity ROC curve (AUC) leads to an AUC value of 0.77 for the complete benchmark (compared to the 0.5 AUC value obtained for random predictions). Furthermore, a new clustering analysis performed on the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein's functional group. Finally, in several cases, the binding-site predictions resulting from the cross-docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Proteins 2016; 84:1408-1421. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Silane surface modification for improved bioadhesion of esophageal stents

PubMed Central

Karakoy, Mert; Gultepe, Evin; Pandey, Shivendra; Khashab, Mouen A.; Gracias, David H.

2014-01-01

Stent migration occurs in 10-40% of patients who undergo placement of esophageal stents, with higher migration rates seen in those treated for benign esophageal disorders. This remains a major drawback of esophageal stent therapy. In this paper, we propose a new surface modification method to increase the adhesion between self-expandable metallic stents (SEMS) and tissue while preserving their removability. Taking advantage of the well-known affinity between epoxide and amine terminated silane coupling agents with amine and carboxyl groups that are abundant in proteins and related molecules in the human body; we modified the surfaces of silicone coated esophageal SEMS with these adhesive self-assembled monolayers (SAMs). We utilized vapor phase silanization to modify the surfaces of different substrates including PDMS strips and SEMS, and measured the force required to slide these substrates on a tissue piece. Our results suggest that surface modification of esophageal SEMS via covalent attachment of protein-binding coupling agents improves adhesion to tissue and could offer a solution to reduce SEMS migration while preserving their removability. PMID:25663731

Poisson-Boltzmann model for protein-surface electrostatic interactions and grid-convergence study using the PyGBe code

NASA Astrophysics Data System (ADS)

Cooper, Christopher D.; Barba, Lorena A.

2016-05-01

Interactions between surfaces and proteins occur in many vital processes and are crucial in biotechnology: the ability to control specific interactions is essential in fields like biomaterials, biomedical implants and biosensors. In the latter case, biosensor sensitivity hinges on ligand proteins adsorbing on bioactive surfaces with a favorable orientation, exposing reaction sites to target molecules. Protein adsorption, being a free-energy-driven process, is difficult to study experimentally. This paper develops and evaluates a computational model to study electrostatic interactions of proteins and charged nanosurfaces, via the Poisson-Boltzmann equation. We extended the implicit-solvent model used in the open-source code PyGBe to include surfaces of imposed charge or potential. This code solves the boundary integral formulation of the Poisson-Boltzmann equation, discretized with surface elements. PyGBe has at its core a treecode-accelerated Krylov iterative solver, resulting in O(N log N) scaling, with further acceleration on hardware via multi-threaded execution on GPUs. It computes solvation and surface free energies, providing a framework for studying the effect of electrostatics on adsorption. We derived an analytical solution for a spherical charged surface interacting with a spherical dielectric cavity, and used it in a grid-convergence study to build evidence on the correctness of our approach. The study showed the error decaying with the average area of the boundary elements, i.e., the method is O(1 / N) , which is consistent with our previous verification studies using PyGBe. We also studied grid-convergence using a real molecular geometry (protein G B1 D4‧), in this case using Richardson extrapolation (in the absence of an analytical solution) and confirmed the O(1 / N) scaling. With this work, we can now access a completely new family of problems, which no other major bioelectrostatics solver, e.g. APBS, is capable of dealing with. PyGBe is open-source under an MIT license and is hosted under version control at https://github.com/barbagroup/pygbe. To supplement this paper, we prepared ;reproducibility packages; consisting of running and post-processing scripts in Python for replicating the grid-convergence studies, all the way to generating the final plots, with a single command.

Ultra-short wavelength x-ray system

DOEpatents

Umstadter, Donald [Ann Arbor, MI; He, Fei [Ann Arbor, MI; Lau, Yue-Ying [Potomac, MD

2008-01-22

A method and apparatus to generate a beam of coherent light including x-rays or XUV by colliding a high-intensity laser pulse with an electron beam that is accelerated by a synchronized laser pulse. Applications include x-ray and EUV lithography, protein structural analysis, plasma diagnostics, x-ray diffraction, crack analysis, non-destructive testing, surface science and ultrafast science.

Gold patterned biochips for on-chip immuno-MALDI-TOF MS: SPR imaging coupled multi-protein MS analysis.

PubMed

Kim, Young Eun; Yi, So Yeon; Lee, Chang-Soo; Jung, Yongwon; Chung, Bong Hyun

2012-01-21

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of immuno-captured target protein efficiently complements conventional immunoassays by offering rich molecular information such as protein isoforms or modifications. Direct immobilization of antibodies on MALDI solid support enables both target enrichment and MS analysis on the same plate, allowing simplified and potentially multiplexing protein MS analysis. Reliable on-chip immuno-MALDI-TOF MS for multiple biomarkers requires successful adaptation of antibody array biochips, which also must accommodate consistent reaction conditions on antibody arrays during immuno-capture and MS analysis. Here we developed a facile fabrication process of versatile antibody array biochips for reliable on-chip MALDI-TOF-MS analysis of multiple immuno-captured proteins. Hydrophilic gold arrays surrounded by super-hydrophobic surfaces were formed on a gold patterned biochip via spontaneous chemical or protein layer deposition. From antibody immobilization to MALDI matrix treatment, this hydrophilic/phobic pattern allowed highly consistent surface reactions on each gold spot. Various antibodies were immobilized on these gold spots both by covalent coupling or protein G binding. Four different protein markers were successfully analyzed on the present immuno-MALDI biochip from complex protein mixtures including serum samples. Tryptic digests of captured PSA protein were also effectively detected by on-chip MALDI-TOF-MS. Moreover, the present MALDI biochip can be directly applied to the SPR imaging system, by which antibody and subsequent antigen immobilization were successfully monitored.

Multifunctional Nature of the Arenavirus RING Finger Protein Z

PubMed Central

Fehling, Sarah Katharina; Lennartz, Frank; Strecker, Thomas

2012-01-01

Arenaviruses are a family of enveloped negative-stranded RNA viruses that can cause severe human disease ranging from encephalitis symptoms to fulminant hemorrhagic fever. The bi‑segmented RNA genome encodes four polypeptides: the nucleoprotein NP, the surface glycoprotein GP, the polymerase L, and the RING finger protein Z. Although it is the smallest arenavirus protein with a length of 90 to 99 amino acids and a molecular weight of approx. 11 kDa, the Z protein has multiple functions in the viral life cycle including (i) regulation of viral RNA synthesis, (ii) orchestration of viral assembly and budding, (iii) interaction with host cell proteins, and (iv) interferon antagonism. In this review, we summarize our current understanding of the structural and functional role of the Z protein in the arenavirus replication cycle. PMID:23202512

Studying protein structural changes based on surface plasmon resonance and surface-enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Hu, Wen-Pin; Chen, Shean-Jen; Yih, Jenq-Nan; Lin, G.-Y.; Chang, Guan L.

2004-06-01

The ability to recognize the conformational changes and structural variations of a protein when immobilized in a solid surface is of great importance in a variety of applications. Surface plasmon resonance (SPR) sensing is an appropriate technique for investigating interfacial phenomena, and enables the conformational changes of proteins to be monitored through the variation in the SPR angle shift. Meanwhile, the surface-enhanced Raman scattering (SERS) system can also assist in clarifying the changes in protein structure. The present study utilizes a 1 mM CrO3 phosphate buffer solution (PBS) to induce conformational changes of human serum albumin (HSA). Monitoring the corresponding SPR angle shifts and the SPR reflectivity spectrum enables the relationships between the conformational changes of the surface-immobilized protein and the thickness and dielectric constants of the protein layer to be estimated. The experimental SPR results indicate that the Cr6+ ions cause significant conformational change of the protein. It is established that the ions are not merely absorbed into the protein as a result of electrostatic forces, but that complex protein refolding events also take place. Furthermore, the data acquired from the SERS system yield valuable information regarding the changes which take place in the protein structure.

Temperature-Triggered Protein Adsorption on Polymer-Coated Nanoparticles in Serum.

PubMed

Koshkina, Olga; Lang, Thomas; Thiermann, Raphael; Docter, Dominic; Stauber, Roland H; Secker, Christian; Schlaad, Helmut; Weidner, Steffen; Mohr, Benjamin; Maskos, Michael; Bertin, Annabelle

2015-08-18

The protein corona, which forms on the nanoparticle's surface in most biological media, determines the nanoparticle's physicochemical characteristics. The formation of the protein corona has a significant impact on the biodistribution and clearance of nanoparticles in vivo. Therefore, the ability to influence the formation of the protein corona is essential to most biomedical applications, including drug delivery and imaging. In this study, we investigate the protein adsorption on nanoparticles with a hydrodynamic radius of 30 nm and a coating of thermoresponsive poly(2-isopropyl-2-oxazoline) in serum. Using multiangle dynamic light scattering (DLS) we demonstrate that heating of the nanoparticles above their phase separation temperature induces the formation of agglomerates, with a hydrodynamic radius of 1 μm. In serum, noticeably stronger agglomeration occurs at lower temperatures compared to serum-free conditions. Cryogenic transmission electron microscopy (cryo-TEM) revealed a high packing density of agglomerates when serum was not present. In contrast, in the presence of serum, agglomerated nanoparticles were loosely packed, indicating that proteins are intercalated between them. Moreover, an increase in protein content is observed upon heating, confirming that protein adsorption is induced by the alteration of the surface during phase separation. After cooling and switching the surface back, most of the agglomerates were dissolved and the main fraction returned to the original size of approximately 30 nm as shown by asymmetrical flow-field flow fractionation (AF-FFF) and DLS. Furthermore, the amounts of adsorbed proteins are similar before and after heating the nanoparticles to above their phase-separation temperature. Overall, our results demonstrate that the thermoresponsivity of the polymer coating enables turning the corona formation on nanoparticles on and off in situ. As the local heating of body areas can be easily done in vivo, the thermoresponsive coating could potentially be used to induce the agglomeration of nanoparticles and proteins and the accumulation of nanoparticles in a targeted body region.

Targeted Disruption of a Ring-infected Erythrocyte Surface Antigen (RESA)-like Export Protein Gene in Plasmodium falciparum Confers Stable Chondroitin 4-Sulfate Cytoadherence Capacity*

PubMed Central

Goel, Suchi; Muthusamy, Arivalagan; Miao, Jun; Cui, Liwang; Salanti, Ali; Winzeler, Elizabeth A.; Gowda, D. Channe

2014-01-01

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family proteins mediate the adherence of infected erythrocytes to microvascular endothelia of various organs, including the placenta, thereby contributing to cerebral, placental, and other severe malaria pathogenesis. Several parasite proteins, including KAHRP and PfEMP3, play important roles in the cytoadherence by mediating the clustering of PfEMP1 in rigid knoblike structures on the infected erythrocyte surface. The lack of a subtelomeric region of chromosome 2 that contains kahrp and pfemp3 causes reduced cytoadherence. In this study, microarray transcriptome analysis showed that the absence of a gene cluster, comprising kahrp, pfemp3, and four other genes, results in the loss of parasitized erythrocytes adhering to chondroitin 4-sulfate (C4S). The role of one of these genes, PF3D7_0201600/PFB0080c, which encodes PHISTb (Plasmodium helical interspersed subtelomeric b) domain-containing RESA-like protein 1 expressed on the infected erythrocyte surface, was investigated. Disruption of PFB0080c resulted in increased var2csa transcription and VAR2CSA surface expression, leading to higher C4S-binding capacity of infected erythrocytes. Further, PFB0080c-knock-out parasites stably maintained the C4S adherence through many generations of growth. Although the majority of PFB0080c-knock-out parasites bound to C4S even after culturing for 6 months, a minor population bound to both C4S and CD36. These results strongly suggest that the loss of PFB0080c markedly compromises the var gene switching process, leading to a marked reduction in the switching rate and additional PfEMP1 expression by a minor population of parasites. PFB0080c interacts with VAR2CSA and modulates knob-associated Hsp40 expression. Thus, PFB0080c may regulate VAR2CSA expression through these processes. Overall, we conclude that PFB0080c regulates PfEMP1 expression and the parasite's cytoadherence. PMID:25342752

Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon binding

PubMed Central

Sirois, Allison R.; Deny, Daniela A.; Baierl, Samantha R.; George, Katia S.

2018-01-01

Mesothelin is a cell surface protein that is overexpressed in numerous cancers, including breast, ovarian, lung, liver, and pancreatic tumors. Aberrant expression of mesothelin has been shown to promote tumor progression and metastasis through interaction with established tumor biomarker CA125. Therefore, molecules that specifically bind to mesothelin have potential therapeutic and diagnostic applications. However, no mesothelin-targeting molecules are currently approved for routine clinical use. While antibodies that target mesothelin are in development, some clinical applications may require a targeting molecule with an alternative protein fold. For example, non-antibody proteins are more suitable for molecular imaging and may facilitate diverse chemical conjugation strategies to create drug delivery complexes. In this work, we engineered variants of the fibronectin type III domain (Fn3) non-antibody protein scaffold to bind to mesothelin with high affinity, using directed evolution and yeast surface display. Lead engineered Fn3 variants were solubly produced and purified from bacterial culture at high yield. Upon specific binding to mesothelin on human cancer cell lines, the engineered Fn3 proteins internalized and co-localized to early endosomes. To our knowledge, this is the first report of non-antibody proteins engineered to bind mesothelin. The results validate that non-antibody proteins can be engineered to bind to tumor biomarker mesothelin, and encourage the continued development of engineered variants for applications such as targeted diagnostics and therapeutics. PMID:29738555

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases.

PubMed

Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

2012-08-01

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. Copyright © 2012 The Protein Society.

Identification of Group B Streptococcal Sip Protein, Which Elicits Cross-Protective Immunity

PubMed Central

Brodeur, Bernard R.; Boyer, Martine; Charlebois, Isabelle; Hamel, Josée; Couture, France; Rioux, Clément R.; Martin, Denis

2000-01-01

A protein of group B streptococci (GBS), named Sip for surface immunogenic protein, which is distinct from previously described surface proteins, was identified after immunological screening of a genomic library. Immunoblots using a Sip-specific monoclonal antibody indicated that a protein band with an approximate molecular mass of 53 kDa which did not vary in size was present in every GBS strain tested. Representatives of all nine GBS serotypes were included in the panel of strains. Cloning and sequencing of the sip gene revealed an open reading frame of 1,305 nucleotides coding for a polypeptide of 434 amino acid residues, with a calculated pI of 6.84 and molecular mass of 45.5 kDa. Comparison of the nucleotide sequences from six different strains confirmed with 98% identity that the sip gene is highly conserved among GBS isolates. N-terminal amino acid sequencing also indicated the presence of a 25-amino-acid signal peptide which is cleaved in the mature protein. More importantly, immunization with the recombinant Sip protein efficiently protected CD-1 mice against deadly challenges with six GBS strains of serotypes Ia/c, Ib, II/R, III, V, and VI. The data presented in this study suggest that this highly conserved protein induces cross-protective immunity against GBS infections and emphasize its potential as a universal vaccine candidate. PMID:10992461

Controlling Protein Surface Orientation by Strategic Placement of Oligo-Histidine Tags

PubMed Central

2017-01-01

We report oriented immobilization of proteins using the standard hexahistidine (His6)-Ni2+:NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magnitude for each additional His6-tag on the TagRFP proteins. All TagRFP variants with His6-tags located on only one side of the barrel-shaped protein yielded a 1.5 times higher surface coverage compared to variants with His6-tags on opposite sides of the so-called β-barrel. Time-resolved fluorescence anisotropy measurements supported by polarized infrared spectroscopy verified that the orientation (and thus coverage and functionality) of proteins on surfaces can be controlled by strategic placement of a His6-tag on the protein. Molecular dynamics simulations show how the differently tagged proteins reside at the surface in “end-on” and “side-on” orientations with each His6-tag contributing to binding. Also, not every dihistidine subunit in a given His6-tag forms a full coordination bond with the Ni2+:NTA SAMs, which varied with the position of the His6-tag on the protein. At equal valency but different tag positions on the protein, differences in binding were caused by probing for Ni2+:NTA moieties and by additional electrostatic interactions between different fractions of the β-barrel structure and charged NTA moieties. Potential of mean force calculations indicate there is no specific single-protein interaction mode that provides a clear preferential surface orientation, suggesting that the experimentally measured preference for the end-on orientation is a supra-protein, not a single-protein, effect. PMID:28850777

Preparation of High-Efficiency Cytochrome c-Imprinted Polymer on the Surface of Magnetic Carbon Nanotubes by Epitope Approach via Metal Chelation and Six-Membered Ring.

PubMed

Qin, Ya-Ping; Li, Dong-Yan; He, Xi-Wen; Li, Wen-You; Zhang, Yu-Kui

2016-04-27

A novel epitope molecularly imprinted polymer on the surface of magnetic carbon nanotubes (MCNTs@EMIP) was successfully fabricated to specifically recognize target protein cytochrome c (Cyt C) with high performance. The peptides sequences corresponding to the surface-exposed C-terminus domains of Cyt C was selected as epitope template molecule, and commercially available zinc acrylate and ethylene glycol dimethacrylate (EGDMA) were employed as functional monomer and cross-linker, respectively, to synthesize MIP via free radical polymerization. The epitope was immobilized via metal chelation and six-membered ring formed between the functional monomer and the hydroxyl and amino groups of the epitope. The resulting MCNTs@EMIP exhibited specific recognition ability toward target Cyt C including more satisfactory imprinting factor (about 11.7) than that of other reported imprinting methods. In addition, the MCNTs@EMIP demonstrated a high adsorption amount (about 780.0 mg g(-1)) and excellent selectivity. Besides, the magnetic property of the support material made the processes easy and highly efficient by assistance of an external magnetic field. High-performance liquid chromatography analysis of Cyt C in bovine blood real sample and protein mixture indicated that the specificity was not affected by other competitive proteins, which forcefully stated that the MCNTs@EMIP had potential to be applied in bioseparation area. In brief, this study provided a new protocol to detect target protein in complex sample via epitope imprinting approach and surface imprinting strategy.

Glutamate Induced Thermal Equilibrium Intermediate and Counteracting Effect on Chemical Denaturation of Proteins.

PubMed

Anumalla, Bramhini; Prabhu, N Prakash

2018-01-25

When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two-state transitions during chemical denaturation. The extent of stability exerted by Glu is higher for RNase A at higher temperature, whereas it provides more stability for α-LA at lower temperature. Thus, the experiments indicate that Glu induces a thermal equilibrium intermediate and increases the thermodynamic stability of proteins irrespective of their surface charges. The extent of stability varies between the proteins in a temperature-dependent manner.

Shape Complementarity of Protein-Protein Complexes at Multiple Resolutions

PubMed Central

Zhang, Qing; Sanner, Michel; Olson, Arthur J.

2010-01-01

Biological complexes typically exhibit intermolecular interfaces of high shape complementarity. Many computational docking approaches use this surface complementarity as a guide in the search for predicting the structures of protein-protein complexes. Proteins often undergo conformational changes in order to create a highly complementary interface when associating. These conformational changes are a major cause of failure for automated docking procedures when predicting binding modes between proteins using their unbound conformations. Low resolution surfaces in which high frequency geometric details are omitted have been used to address this problem. These smoothed, or blurred, surfaces are expected to minimize the differences between free and bound structures, especially those that are due to side chain conformations or small backbone deviations. In spite of the fact that this approach has been used in many docking protocols, there has yet to be a systematic study of the effects of such surface smoothing on the shape complementarity of the resulting interfaces. Here we investigate this question by computing shape complementarity of a set of 66 protein-protein complexes represented by multi-resolution blurred surfaces. Complexed and unbound structures are available for these protein-protein complexes. They are a subset of complexes from a non-redundant docking benchmark selected for rigidity (i.e. the proteins undergo limited conformational changes between their bound and unbound states). In this work we construct the surfaces by isocontouring a density map obtained by accumulating the densities of Gaussian functions placed at all atom centers of the molecule. The smoothness or resolution is specified by a Gaussian fall-off coefficient, termed “blobbyness”. Shape complementarity is quantified using a histogram of the shortest distances between two proteins' surface mesh vertices for both the crystallographic complexes and the complexes built using the protein structures in their unbound conformation. The histograms calculated for the bound complex structures demonstrate that medium resolution smoothing (blobbyness=−0.9) can reproduce about 88% of the shape complementarity of atomic resolution surfaces. Complexes formed from the free component structures show a partial loss of shape complementarity (more overlaps and gaps) with the atomic resolution surfaces. For surfaces smoothed to low resolution (blobbyness=−0.3), we find more consistency of shape complementarity between the complexed and free cases. To further reduce bad contacts without significantly impacting the good contacts we introduce another blurred surface, in which the Gaussian densities of flexible atoms are reduced. From these results we discuss the use of shape complementarity in protein-protein docking. PMID:18837463

Adsorption of enamel matrix proteins to a bovine-derived bone grafting material and its regulation of cell adhesion, proliferation, and differentiation.

PubMed

Miron, Richard J; Bosshardt, Dieter D; Hedbom, Erik; Zhang, Yufeng; Haenni, Beat; Buser, Daniel; Sculean, Anton

2012-07-01

The use of various combinations of enamel matrix derivative (EMD) and grafting materials has been shown to promote periodontal wound healing/regeneration. However, the downstream cellular behavior of periodontal ligament (PDL) cells and osteoblasts has not yet been studied. Furthermore, it is unknown to what extent the bleeding during regenerative surgery may influence the adsorption of exogenous proteins to the surface of bone grafting materials and the subsequent cellular behavior. In the present study, the aim is to test EMD adsorption to the surface of natural bone mineral (NBM) particles in the presence of blood and determine the effect of EMD coating to NBM particles on downstream cellular pathways, such as adhesion, proliferation, and differentiation of primary human osteoblasts and PDL cells. NBM particles were precoated in various settings with EMD or human blood and analyzed for protein adsorption patterns via fluorescent imaging and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using fluorescent double-stranded DNA-binding dye. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding runt-related transcription factor 2, alkaline phosphatase (ALP), osteocalcin (OC), and collagen1α1 (COL1A1), and mineralization was assessed using red dye staining. Analysis of cell attachment and cell proliferation revealed significantly higher osteoblast and PDL cell attachment on EMD-coated surfaces when compared with control and blood-coated surfaces. EMD also stimulated release of growth factors and cytokines, including bone morphogenetic protein 2 and transforming growth factor β1. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers, including COL1A1, ALP, and OC, in osteoblasts and PDL cells cultured on EMD-coated NBM particles. The present results suggest that 1) EMD enhances osteoblast and PDL cell attachment, proliferation, and differentiation on NBM particles, and 2) blood contamination of the grafting material before mixing with EMD may inhibit EMD adsorption.

Parasitization by Scleroderma guani influences protein expression in Tenebrio molitor pupae.

PubMed

Zhu, Jia-Ying; Wu, Guo-Xing; Ze, Sang-Zi; Stanley, David W; Yang, Bin

2014-07-01

Ectoparasitoid wasps deposit their eggs onto the surface and inject venom into their hosts. Venoms are chemically complex and they exert substantial impact on hosts, including permanent or temporary paralysis and developmental arrest. These visible venom effects are due to changes in expression of genes encoding physiologically relevant proteins. While the influence of parasitization on gene expression in several lepidopterans has been reported, the molecular details of parasitoid/beetle relationships remain mostly unknown. This shortcoming led us to pose the hypothesis that envenomation by the ectoparasitic ant-like bethylid wasp Scleroderma guani leads to changes in protein expression in the yellow mealworm beetle Tenebrio molitor. We tested our hypothesis by comparing the proteomes of non-parasitized and parasitized host pupae using iTRAQ-based proteomics. We identified 41 proteins that were differentially expressed (32↑- and 9↓-regulated) in parasitized pupae. We assigned these proteins to functional categories, including immunity, stress and detoxification, energy metabolism, development, cytoskeleton, signaling and others. We recorded parallel changes in mRNA levels and protein abundance in 14 selected proteins following parasitization. Our findings support our hypothesis by documenting changes in protein expression in parasitized hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

Surface derivatization strategy for combinatorial analysis of cell response to mixtures of protein domains.

PubMed

Chiang, Chunyi; Karuri, Stella W; Kshatriya, Pradnya P; Schwartz, Jeffrey; Schwarzbauer, Jean E; Karuri, Nancy W

2012-01-10

We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.

Effect of tissue scaffold topography on protein structure monitored by fluorescence spectroscopy.

PubMed

Portugal, Carla A M; Truckenmüller, Roman; Stamatialis, Dimitrios; Crespo, João G

2014-11-10

The impact of surface topography on the structure of proteins upon adhesion was assessed through non-invasive fluorescence monitoring. This study aimed at obtaining a better understanding about the role of protein structural status on cell-scaffold interactions. The changes induced upon adsorption of two model proteins with different geometries, trypsin (globular conformation) and fibrinogen (rod-shaped conformation) on poly-l-lactic acid (PLLA) scaffolds with different surface topographies, flat, fibrous and surfaces with aligned nanogrooves, were assessed by fluorescence spectroscopy monitoring, using tryptophan as structural probe. Hence, the maximum emission blue shift and the increase of fluorescence anisotropy observed after adsorption of globular and rod-like shaped proteins on surfaces with parallel nanogrooves were ascribed to more intense protein-surface interactions. Furthermore, the decrease of fluorescence anisotropy observed upon adsorption of proteins to scaffolds with fibrous morphology was more significant for rod-shaped proteins. This effect was associated to the ability of these proteins to adjust to curved surfaces. The additional unfolding of proteins induced upon adsorption on scaffolds with a fibrous morphology may be the reason for better cell attachment there, promoting an easier access of cell receptors to initially hidden protein regions (e.g. RGDS sequence), which are known to have a determinant role in cell attaching processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Druggable Protein Interaction Sites Are More Predisposed to Surface Pocket Formation than the Rest of the Protein Surface

PubMed Central

Johnson, David K.; Karanicolas, John

2013-01-01

Despite intense interest and considerable effort via high-throughput screening, there are few examples of small molecules that directly inhibit protein-protein interactions. This suggests that many protein interaction surfaces may not be intrinsically “druggable” by small molecules, and elevates in importance the few successful examples as model systems for improving our fundamental understanding of druggability. Here we describe an approach for exploring protein fluctuations enriched in conformations containing surface pockets suitable for small molecule binding. Starting from a set of seven unbound protein structures, we find that the presence of low-energy pocket-containing conformations is indeed a signature of druggable protein interaction sites and that analogous surface pockets are not formed elsewhere on the protein. We further find that ensembles of conformations generated with this biased approach structurally resemble known inhibitor-bound structures more closely than equivalent ensembles of unbiased conformations. Collectively these results suggest that “druggability” is a property encoded on a protein surface through its propensity to form pockets, and inspire a model in which the crude features of the predisposed pocket(s) restrict the range of complementary ligands; additional smaller conformational changes then respond to details of a particular ligand. We anticipate that the insights described here will prove useful in selecting protein targets for therapeutic intervention. PMID:23505360

Anomalous water dynamics at surfaces and interfaces: synergistic effects of confinement and surface interactions

NASA Astrophysics Data System (ADS)

Biswas, Rajib; Bagchi, Biman

2018-01-01

In nature, water is often found in contact with surfaces that are extended on the scale of molecule size but small on a macroscopic scale. Examples include lipid bilayers and reverse micelles as well as biomolecules like proteins, DNA and zeolites, to name a few. While the presence of surfaces and interfaces interrupts the continuous hydrogen bond network of liquid water, confinement on a mesoscopic scale introduces new features. Even when extended on a molecular scale, natural and biological surfaces often have features (like charge, hydrophobicity) that vary on the scale of the molecular diameter of water. As a result, many new and exotic features, which are not seen in the bulk, appear in the dynamics of water close to the surface. These different behaviors bear the signature of both water-surface interactions and of confinement. In other words, the altered properties are the result of the synergistic effects of surface-water interactions and confinement. Ultrafast spectroscopy, theoretical modeling and computer simulations together form powerful synergistic approaches towards an understanding of the properties of confined water in such systems as nanocavities, reverse micelles (RMs), water inside and outside biomolecules like proteins and DNA, and also between two hydrophobic walls. We shall review the experimental results and place them in the context of theory and simulations. For water confined within RMs, we discuss the possible interference effects propagating from opposite surfaces. Similar interference is found to give rise to an effective attractive force between two hydrophobic surfaces immersed and kept fixed at a separation of d, with the force showing an exponential dependence on this distance. For protein and DNA hydration, we shall examine a multitude of timescales that arise from frustration effects due to the inherent heterogeneity of these surfaces. We pay particular attention to the role of orientational correlations and modification of the same due to interaction with the surfaces.

Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.

PubMed

Shivange, Amol V; Hoeffken, Hans Wolfgang; Haefner, Stefan; Schwaneberg, Ulrich

2016-12-01

Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: ( i ) identification of conserved and highly variable regions; ( ii ) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; ( iii ) in vitro DNA recombination of synthetic genes; and ( iv ) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

Modeling the surface of Campylobacter fetus: protein surface layer stability and resistance to cationic antimicrobial peptides.

PubMed

Roberts, James M D; Graham, Lori L; Quinn, Bonnie; Pink, David A

2013-03-01

Campylobacter fetus is a Gram negative bacterium recognized for its virulence in animals and humans. This bacterium possesses a paracrystalline array of high molecular weight proteins known as surface-layer proteins covering its cell surface. A mathematical model has been made of the outer membrane of this bacterium, both with its surface-layer proteins (S+) and without (S-). Monte Carlo computer simulation was used to understand the stability of the surface-layer protein structure as a function of ionic concentration. The interactions of an electrically-charged antimicrobial agent, the cationic antimicrobial peptide protamine, with surface-layer proteins and with the lipopolysaccharides of the outer membrane were modeled and analyzed. We found that (1) divalent ions stabilize the surface-layer protein array by reducing the fluctuations perpendicular and parallel to the membrane plane thereby promoting adhesion to the LPS region. This was achieved via (2) divalent ions bridging the negatively-charged LPS Core. The effect of this bridging is to bring individual Core regions closer together so that the O-antigens can (3) increase their attractive van der Waals interactions and "collapse" to form a surface with reduced perpendicular fluctuations. These findings provide support for the proposal of Yang et al. [1]. (4) No evidence for a significant increase in Ca(2+) concentration in the region of the surface-layer protein subunits was observed in S+ simulations compared to S- simulations. (5) We predicted the trends of protamine MIC tests performed on C. fetus and these were in good agreement with our experimental results. Copyright © 2012 Elsevier B.V. All rights reserved.

Targeted Proteomics and Absolute Protein Quantification for the Construction of a Stoichiometric Host-Pathogen Surface Density Model.

PubMed

Sjöholm, Kristoffer; Kilsgård, Ola; Teleman, Johan; Happonen, Lotta; Malmström, Lars; Malmström, Johan

2017-04-01

Sepsis is a systemic immune response responsible for considerable morbidity and mortality. Molecular modeling of host-pathogen interactions in the disease state represents a promising strategy to define molecular events of importance for the transition from superficial to invasive infectious diseases. Here we used the Gram-positive bacterium Streptococcus pyogenes as a model system to establish a mass spectrometry based workflow for the construction of a stoichiometric surface density model between the S. pyogenes surface, the surface virulence factor M-protein, and adhered human blood plasma proteins. The workflow relies on stable isotope labeled reference peptides and selected reaction monitoring mass spectrometry analysis of a wild-type strain and an M-protein deficient mutant strain, to generate absolutely quantified protein stoichiometry ratios between S. pyogenes and interacting plasma proteins. The stoichiometry ratios in combination with a novel targeted mass spectrometry method to measure cell numbers enabled the construction of a stoichiometric surface density model using protein structures available from the protein data bank. The model outlines the topology and density of the host-pathogen protein interaction network on the S. pyogenes bacterial surface, revealing a dense and highly organized protein interaction network. Removal of the M-protein from S. pyogenes introduces a drastic change in the network topology, validated by electron microscopy. We propose that the stoichiometric surface density model of S. pyogenes in human blood plasma represents a scalable framework that can continuously be refined with the emergence of new results. Future integration of new results will improve the understanding of protein-protein interactions and their importance for bacterial virulence. Furthermore, we anticipate that the general properties of the developed workflow will facilitate the production of stoichiometric surface density models for other types of host-pathogen interactions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

S-layers: principles and applications

PubMed Central

Sleytr, Uwe B; Schuster, Bernhard; Egelseer, Eva-Maria; Pum, Dietmar

2014-01-01

Monomolecular arrays of protein or glycoprotein subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope components. S-layers are generally the most abundantly expressed proteins, have been observed in species of nearly every taxonomical group of walled bacteria, and represent an almost universal feature of archaeal envelopes. The isoporous lattices completely covering the cell surface provide organisms with various selection advantages including functioning as protective coats, molecular sieves and ion traps, as structures involved in surface recognition and cell adhesion, and as antifouling layers. S-layers are also identified to contribute to virulence when present as a structural component of pathogens. In Archaea, most of which possess S-layers as exclusive wall component, they are involved in determining cell shape and cell division. Studies on structure, chemistry, genetics, assembly, function, and evolutionary relationship of S-layers revealed considerable application potential in (nano)biotechnology, biomimetics, biomedicine, and synthetic biology. PMID:24483139

Improved delivery of the OVA-CD4 peptide to T helper cells by polymeric surface display on Salmonella

PubMed Central

2014-01-01

Background Autotransporter proteins represent a treasure trove for molecular engineers who modify Gram-negative bacteria for the export or secretion of foreign proteins across two membrane barriers. A particularly promising direction is the development of autotransporters as antigen display or secretion systems. Immunologists have been using ovalbumin as a reporter antigen for years and have developed sophisticated tools to detect specific T cells that respond to ovalbumin. Although ovalbumin-expressing bacteria are being used to trace T cell responses to colonizing or invading pathogens, current constructs for ovalbumin presentation have not been optimized. Results The activation of T helper cells in response to ovalbumin was improved by displaying the OVA-CD4 reporter epitope as a multimer on the surface of Salmonella and fused to the autotransporter MisL. Expression was optimized by including tandem in vivo promoters and two post-segregational killing systems for plasmid stabilization. Conclusions The use of an autotransporter protein to present relevant epitope repeats on the surface of bacteria, combined with additional techniques favoring stable and efficient in vivo transcription, optimizes antigen presentation to T cells. The technique of multimeric epitope surface display should also benefit the development of new Salmonella or other enterobacterial vaccines. PMID:24898796

A conserved fungal glycosyltransferase facilitates pathogenesis of plants by enabling hyphal growth on solid surfaces

PubMed Central

Plummer, Amy; Halsey, Kirstie; Lovegrove, Alison; Hammond-Kosack, Kim

2017-01-01

Pathogenic fungi must extend filamentous hyphae across solid surfaces to cause diseases of plants. However, the full inventory of genes which support this is incomplete and many may be currently concealed due to their essentiality for the hyphal growth form. During a random T-DNA mutagenesis screen performed on the pleomorphic wheat (Triticum aestivum) pathogen Zymoseptoria tritici, we acquired a mutant unable to extend hyphae specifically when on solid surfaces. In contrast “yeast-like” growth, and all other growth forms, were unaffected. The inability to extend surface hyphae resulted in a complete loss of virulence on plants. The affected gene encoded a predicted type 2 glycosyltransferase (ZtGT2). Analysis of >800 genomes from taxonomically diverse fungi highlighted a generally widespread, but discontinuous, distribution of ZtGT2 orthologues, and a complete absence of any similar proteins in non-filamentous ascomycete yeasts. Deletion mutants of the ZtGT2 orthologue in the taxonomically un-related fungus Fusarium graminearum were also severely impaired in hyphal growth and non-pathogenic on wheat ears. ZtGT2 expression increased during filamentous growth and electron microscopy on deletion mutants (ΔZtGT2) suggested the protein functions to maintain the outermost surface of the fungal cell wall. Despite this, adhesion to leaf surfaces was unaffected in ΔZtGT2 mutants and global RNAseq-based gene expression profiling highlighted that surface-sensing and protein secretion was also largely unaffected. However, ΔZtGT2 mutants constitutively overexpressed several transmembrane and secreted proteins, including an important LysM-domain chitin-binding virulence effector, Zt3LysM. ZtGT2 likely functions in the synthesis of a currently unknown, potentially minor but widespread, extracellular or outer cell wall polysaccharide which plays a key role in facilitating many interactions between plants and fungi by enabling hyphal growth on solid matrices. PMID:29020037

Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry.

PubMed

Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, Kathrin; Knobel, Sebastian; Dickinson, Anne M; Filby, Andrew; Wang, Xiao-Nong

2018-03-02

Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

Surface Adsorption in Nonpolarizable Atomic Models.

PubMed

Whitmer, Jonathan K; Joshi, Abhijeet A; Carlton, Rebecca J; Abbott, Nicholas L; de Pablo, Juan J

2014-12-09

Many ionic solutions exhibit species-dependent properties, including surface tension and the salting-out of proteins. These effects may be loosely quantified in terms of the Hofmeister series, first identified in the context of protein solubility. Here, our interest is to develop atomistic models capable of capturing Hofmeister effects rigorously. Importantly, we aim to capture this dependence in computationally cheap "hard" ionic models, which do not exhibit dynamic polarization. To do this, we have performed an investigation detailing the effects of the water model on these properties. Though incredibly important, the role of water models in simulation of ionic solutions and biological systems is essentially unexplored. We quantify this via the ion-dependent surface attraction of the halide series (Cl, Br, I) and, in so doing, determine the relative importance of various hypothesized contributions to ionic surface free energies. Importantly, we demonstrate surface adsorption can result in hard ionic models combined with a thermodynamically accurate representation of the water molecule (TIP4Q). The effect observed in simulations of iodide is commensurate with previous calculations of the surface potential of mean force in rigid molecular dynamics and polarizable density-functional models. Our calculations are direct simulation evidence of the subtle but sensitive role of water thermodynamics in atomistic simulations.

Surface grafted antibodies: controlled architecture permits enhanced antigen detection.

PubMed

Sebra, Robert P; Masters, Kristyn S; Bowman, Christopher N; Anseth, Kristi S

2005-11-22

The attachment of antibodies to substrate surfaces is useful for achieving specific detection of antigens and toxins associated with clinical and field diagnostics. Here, acrylated whole antibodies were produced through conjugation chemistry, with the goal of covalently photografting these proteins from surfaces in a controlled fashion, to facilitate rapid and sensitive antigenic detection. A living radical photopolymerization chemistry was used to graft the acrylated whole antibodies on polymer surfaces at controlled densities and spatial locations by controlling the exposure time and area, respectively. Copolymer grafts containing these antibodies were synthesized to demonstrate two principles. First, PEG functionalities were introduced to prevent nonspecific protein interactions and improve the reaction kinetics by increasing solvation and mobility of the antibody-containing chains. Both of these properties lead to sensitive (pM) and rapid (<20 min) detection of antigens with this surface modification technique. Second, graft composition was tailored to include multiple antibodies on the same grafted chains, establishing a means for simultaneously detecting multiple antigens on one grafted surface area. Finally, the addition of PEG spacers between the acrylate functionality and the pendant detection antibodies was tuned to enhance the detection of a short-half-life molecule, glucagon, in a complex biological environment, plasma.

A Universal Method for Fishing Target Proteins from Mixtures of Biomolecules using Isothermal Titration Calorimetry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, X.; Sun, Q; Kini, R

2008-01-01

The most challenging tasks in biology include the identification of (1) the orphan receptor for a ligand, (2) the ligand for an orphan receptor protein, and (3) the target protein(s) for a given drug or a lead compound that are critical for the pharmacological or side effects. At present, several approaches are available, including cell- or animal-based assays, affinity labeling, solid-phase binding assays, surface plasmon resonance, and nuclear magnetic resonance. Most of these techniques are not easy to apply when the target protein is unknown and the compound is not amenable to labeling, chemical modification, or immobilization. Here we demonstratemore » a new universal method for fishing orphan target proteins from a complex mixture of biomolecules using isothermal titration calorimetry (ITC) as a tracking tool. We took snake venom, a crude mixture of several hundred proteins/peptides, as a model to demonstrate our proposed ITC method in tracking the isolation and purification of two distinct target proteins, a major component and a minor component. Identities of fished out target proteins were confirmed by amino acid sequencing and inhibition assays. This method has the potential to make a significant advancement in the area of identifying orphan target proteins and inhibitor screening in drug discovery and characterization.« less

The impact of atomization on the surface composition of spray-dried milk droplets.

PubMed

Foerster, Martin; Gengenbach, Thomas; Woo, Meng Wai; Selomulya, Cordelia

2016-04-01

The dominant presence of fat at the surface of spray-dried milk powders has been widely reported in the literature and described as resulting in unfavourable powder properties. The mechanism(s) causing this phenomenon are yet to be clearly identified. A systematic investigation of the component distribution in atomized droplets and spray-dried particles consisting of model milk systems with different fat contents demonstrated that atomization strongly influences the final surface composition. Cryogenic flash-freezing of uniform droplets from a microfluidic jet nozzle directly after atomization helped to distinguish the influence of the atomization stage from the drying stage. It was confirmed that the overrepresentation of fat on the surface is independent of the atomization technique, including a pressure-swirl single-fluid spray nozzle and a pilot-scale rotary disk spray dryer commonly used in industry. It is proposed that during the atomization stage a disintegration mechanism along the oil-water interface of the fat globules causes the surface predominance of fat. X-ray photoelectron spectroscopic measurements detected the outermost fat layer and some adjacent protein present on both atomized droplets and spray-dried particles. Confocal laser scanning microscopy gave a qualitative insight into the protein and fat distribution throughout the cross-sections, and confirmed the presence of a fat film along the particle surface. The film remained on the surface in the subsequent drying stage, while protein accumulated underneath, driven by diffusion. The results demonstrated that atomization induces component segregation and fat-rich surfaces in spray-dried milk powders, and thus these cannot be prevented by adjusting the spray drying conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Modulation of Protein Fouling and Interfacial Properties at Carbon Surfaces via Immobilization of Glycans Using Aryldiazonium Chemistry

PubMed Central

Zen, Federico; Angione, M. Daniela; Behan, James A.; Cullen, Ronan J.; Duff, Thomas; Vasconcelos, Joana M.; Scanlan, Eoin M.; Colavita, Paula E.

2016-01-01

Carbon materials and nanomaterials are of great interest for biological applications such as implantable devices and nanoparticle vectors, however, to realize their potential it is critical to control formation and composition of the protein corona in biological media. In this work, protein adsorption studies were carried out at carbon surfaces functionalized with aryldiazonium layers bearing mono- and di-saccharide glycosides. Surface IR reflectance absorption spectroscopy and quartz crystal microbalance were used to study adsorption of albumin, lysozyme and fibrinogen. Protein adsorption was found to decrease by 30–90% with respect to bare carbon surfaces; notably, enhanced rejection was observed in the case of the tested di-saccharide vs. simple mono-saccharides for near-physiological protein concentration values. ζ-potential measurements revealed that aryldiazonium chemistry results in the immobilization of phenylglycosides without a change in surface charge density, which is known to be important for protein adsorption. Multisolvent contact angle measurements were used to calculate surface free energy and acid-base polar components of bare and modified surfaces based on the van Oss-Chaudhury-Good model: results indicate that protein resistance in these phenylglycoside layers correlates positively with wetting behavior and Lewis basicity. PMID:27108562

Modulation of Protein Fouling and Interfacial Properties at Carbon Surfaces via Immobilization of Glycans Using Aryldiazonium Chemistry

NASA Astrophysics Data System (ADS)

Zen, Federico; Angione, M. Daniela; Behan, James A.; Cullen, Ronan J.; Duff, Thomas; Vasconcelos, Joana M.; Scanlan, Eoin M.; Colavita, Paula E.

2016-04-01

Carbon materials and nanomaterials are of great interest for biological applications such as implantable devices and nanoparticle vectors, however, to realize their potential it is critical to control formation and composition of the protein corona in biological media. In this work, protein adsorption studies were carried out at carbon surfaces functionalized with aryldiazonium layers bearing mono- and di-saccharide glycosides. Surface IR reflectance absorption spectroscopy and quartz crystal microbalance were used to study adsorption of albumin, lysozyme and fibrinogen. Protein adsorption was found to decrease by 30-90% with respect to bare carbon surfaces; notably, enhanced rejection was observed in the case of the tested di-saccharide vs. simple mono-saccharides for near-physiological protein concentration values. ζ-potential measurements revealed that aryldiazonium chemistry results in the immobilization of phenylglycosides without a change in surface charge density, which is known to be important for protein adsorption. Multisolvent contact angle measurements were used to calculate surface free energy and acid-base polar components of bare and modified surfaces based on the van Oss-Chaudhury-Good model: results indicate that protein resistance in these phenylglycoside layers correlates positively with wetting behavior and Lewis basicity.

Construction of a cell-surface display system based on the N-terminal domain of ice nucleation protein and its application in identification of mycoplasma adhesion proteins.

PubMed

Bao, S; Yu, S; Guo, X; Zhang, F; Sun, Y; Tan, L; Duan, Y; Lu, F; Qiu, X; Ding, C

2015-07-01

To construct and demonstrate a surface display system that could be used to identify mycoplasma adhesion proteins. Using the N-terminal domain of InaZ (InaZN) as the anchoring motif and the enhanced green fluorescent protein (EGFP) as the reporter, the surface display system pET-InaZN-EGFP was constructed. Then, the mgc2 gene which encodes an adhesin and the holB gene which encodes DNA polymerase III subunit delta' (nonadhesin, negative control) of Mycoplasma gallisepticum were cloned into the pET-InaZN-EGFP respectively. The fusion proteins were expressed in Escherichia coli BL21 (DE3). The distribution of the fusion proteins in E. coli cells was determined using SDS-PAGE followed by Western blotting, based on cell fractionation. Escherichia coli cell surface display of the fusion protein was confirmed by immunofluorescence microscopy. The results indicated that the fusion proteins were not only anchored to the outer membrane fraction but also were successfully displayed on the surface of E. coli cells. Adhesion analysis of E. coli harbouring InaZN-EGFP-mgc2 to host cells showed that the MGC2-positive E. coli cells can effectively adhere to the surfaces of DF-1 cells. A surface display system using the InaZN as the anchoring motif and EGFP as the reporter was developed to identify putative adhesins of mycoplasma. Results indicated that adhesion by the cytadhesin-like protein MGC2 of mycoplasma can be reproduced using this surface display system. This is the first construction of surface display system which could be used to identify the adhesion proteins of mycoplasma. The method developed in this study can even be used to select and identify the adhesion proteins of other pathogens. © 2015 The Society for Applied Microbiology.

Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

PubMed

Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

2015-10-08

Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

Sensitive ionization of non-volatile analytes using protein solutions as spray liquid in desorption electrospray ionization mass spectrometry.

PubMed

Zhu, Zhiqiang; Han, Jing; Zhang, Yan; Zhou, Yafei; Xu, Ning; Zhang, Bo; Gu, Haiwei; Chen, Huanwen

2012-12-15

Desorption electrospray ionization (DESI) is the most popular ambient ionization technique for direct analysis of complex samples without sample pretreatment. However, for many applications, especially for trace analysis, it is of interest to improve the sensitivity of DESI-mass spectrometry (MS). In traditional DESI-MS, a mixture of methanol/water/acetic acid is usually used to generate the primary ions. In this article, dilute protein solutions were electrosprayed in the DESI method to create multiply charged primary ions for the desorption ionization of trace analytes on various surfaces (e.g., filter paper, glass, Al-foil) without any sample pretreatment. The analyte ions were then detected and structurally characterized using a LTQ XL mass spectrometer. Compared with the methanol/water/acetic acid (49:49:2, v/v/v) solution, protein solutions significantly increased the signal levels of non-volatile compounds such as benzoic acid, TNT, o-toluidine, peptide and insulin in either positive or negative ion detection mode. For all the analytes tested, the limits of detection (LODs) were reduced to about half of the original values which were obtained using traditional DESI. The results showed that the signal enhancement is highly correlated with the molecular weight of the proteins and the selected solid surfaces. The proposed DESI method is a universal strategy for rapid and sensitive detection of trace amounts of strongly bound and/or non-volatile analytes, including explosives, peptides, and proteins. The results indicate that the sensitivity of DESI can be further improved by selecting larger proteins and appropriate solid surfaces. Copyright © 2012 John Wiley & Sons, Ltd.

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers.

PubMed

Veith, Paul D; Glew, Michelle D; Gorasia, Dhana G; Reynolds, Eric C

2017-10-01

The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum. Proteins secreted by the T9SS have an N-terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C-terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C-terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components. © 2017 John Wiley & Sons Ltd.

Determination of conformation and orientation of immobilized peptides and proteins at buried interfaces

NASA Astrophysics Data System (ADS)

Shen, Lei; Ulrich, Nathan W.; Mello, Charlene M.; Chen, Zhan

2015-01-01

Surface immobilized peptides/proteins have important applications such as antimicrobial coating and biosensing. We report a study of such peptides/proteins using sum frequency generation vibrational spectroscopy and ATR-FTIR. Immobilization on surfaces via physical adsorption and chemical coupling revealed that structures of chemically immobilized peptides are determined by immobilization sites, chemical environments, and substrate surfaces. In addition, controlling enzyme orientation by engineering the surface immobilization site demonstrated that structures can be well-correlated to measured chemical activity. This research facilitates the development of immobilized peptides/proteins with improved activities by optimizing their surface orientation and structure.

Great interactions: How binding incorrect partners can teach us about protein recognition and function

PubMed Central

Vamparys, Lydie; Laurent, Benoist; Carbone, Alessandra

2016-01-01

ABSTRACT Protein–protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications. The prediction of protein binding sites in particular is of paramount importance since proteins now represent a major class of therapeutic targets. Amongst others methods, docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on a protein surface. From the analysis of the protein interfaces generated by a massive cross‐docking experiment using the 168 proteins of the Docking Benchmark 2.0, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is also possible to predict a protein's binding residues without having any prior knowledge regarding its potential interaction partners. Evaluating the performance of cross‐docking predictions using the area under the specificity‐sensitivity ROC curve (AUC) leads to an AUC value of 0.77 for the complete benchmark (compared to the 0.5 AUC value obtained for random predictions). Furthermore, a new clustering analysis performed on the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein's functional group. Finally, in several cases, the binding‐site predictions resulting from the cross‐docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Proteins 2016; 84:1408–1421. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:27287388

Electro-induced protein deposition on low-fouling surfaces

NASA Astrophysics Data System (ADS)

Cole, M. A.; Voelcker, N. H.; Thissen, H.

2007-12-01

Control over protein adsorption is a key issue for numerous biomedical applications ranging from diagnostic microarrays to tissue-engineered medical devices. Here, we describe a method for creating surfaces that prevent non-specific protein adsorption, which upon application of an external trigger can be transformed into surfaces showing high protein adsorption on demand. Silicon wafers were used as substrate materials upon which thin functional coatings were constructed by the deposition of an allylamine plasma polymer followed by high-density grafting of poly(ethylene oxide) aldehyde, resulting in a low-fouling surface. When the underlying highly doped silicon substrate was used as an electrode, the resulting electrostatic attraction between the electrode and charged proteins in solution induced protein deposition at the low-fouling interface. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were used to characterize the surface modifications. Controlled protein adsorption experiments were carried out using horseradish peroxidase. The amount of protein deposited at the surface was then investigated by means of a colorimetric assay. It is expected that the concept described here will find use in a variety of biotechnological and biomedical applications, particularly in the area of biochips.

Optimization of extraction parameters of PTP1β (protein tyrosine phosphatase 1β), inhibitory polyphenols, and anthocyanins from Zea mays L. using response surface methodology (RSM).

PubMed

Hwang, Seung Hwan; Kwon, Shin Hwa; Wang, Zhiqiang; Kim, Tae Hyun; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

2016-08-26

Protein tyrosine phosphatase expressed in insulin-sensitive tissues (such as liver, muscle, and adipose tissue) has a key role in the regulation of insulin signaling and pathway activation, making protein tyrosine phosphatase a promising target for the treatment of type 2 diabetes mellitus and obesity and response surface methodology (RSM) is an effective statistical technique for optimizing complex processes using a multi-variant approach. In this study, Zea mays L. (Purple corn kernel, PCK) and its constituents were investigated for protein tyrosine phosphatase 1β (PTP1β) inhibitory activity including enzyme kinetic study and to improve total yields of anthocyanins and polyphenols, four extraction parameters, including temperature, time, solid-liquid ratio, and solvent volume, were optimized by RSM. Isolation of seven polyphenols and five anthocyanins was achieved by PTP1β assay. Among them, cyanidin-3-(6"malonylglucoside) and 3'-methoxyhirsutrin showed the highest PTP1β inhibition with IC50 values of 54.06 and 64.04 μM, respectively and 4.52 mg gallic acid equivalent/g (GAE/g) of total polyphenol content (TPC) and 43.02 mg cyanidin-3-glucoside equivalent/100 g (C3GE/100g) of total anthocyanin content (TAC) were extracted at 40 °C for 8 h with a 33 % solid-liquid ratio and a 1:15 solvent volume. Yields were similar to predictions of 4.58 mg GAE/g of TPC and 42.28 mg C3GE/100 g of TAC. These results indicated that PCK and 3'-methoxyhirsutrin and cyanidin-3-(6"malonylglucoside) might be active natural compounds and could be apply by optimizing of extraction process using response surface methodology.

TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium.

PubMed

Froquet, Romain; le Coadic, Marion; Perrin, Jackie; Cherix, Nathalie; Cornillon, Sophie; Cosson, Pierre

2012-02-01

TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. In this study, we found that genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule in Dictyostelium amoebae. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits nine transmembrane domains and is essential for cellular adhesion. A contact site A (csA)-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of the Dictyostelium surface protein csA also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface.

Interactions of fat globule surface proteins during concentration of whole milk in a pilot-scale multiple-effect evaporator.

PubMed

Ye, Aiqian; Singh, Harjinder; Taylor, Michael W; Anema, Skelte G

2004-11-01

The changes in milk fat globules and fat globule surface proteins during concentration of whole milk using a pilot-scale multiple-effect evaporator were examined. The effects of heat treatment of milk at 95 degrees C for 20 s, prior to evaporation, on fat globule size and the milk fat globule membrane (MFGM) proteins were also determined. In both non-preheated and preheated whole milk, the size of milk fat globules decreased while the amount of total surface proteins at the fat globules increased as the milk passed through each effect of the evaporator. In non-preheated samples, the amount of caseins at the surface of fat globules increased markedly during evaporation with a relatively small increase in whey proteins. In preheated samples, both caseins and whey proteins were observed at the surface of fat globules and the amounts of these proteins increased during subsequent steps of evaporation. The major original MFGM proteins, xanthine oxidase, butyrophilin, PAS 6 and PAS 7, did not change during evaporation, however, PAS 6 and PAS 7 decreased during preheating. These results indicate that the proteins from the skim milk were adsorbed onto the fat globule surface when the milk fat globules were disrupted during evaporation.