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Sample records for immobilize membrane proteins

  1. A Novel Matrix for Immobilizing Protein: Supported Hybrid Nano C60-Lipid Membrane.

    PubMed

    He, Lulu; Yue, Qiulin; Zhang, Lele; Zhang, Xin

    2016-06-01

    Supported hybrid nano C60-lipid membrane based on cysteamine monolayer was made on gold electrode. Hemoglobin (Hb) could be immobilized in the membrane firmly because the membrane can supply a biological environment for Hb. The electrochemical behavior of Hb in the membrane was investigated by cyclic voltammetry. As a good electron mediator, C60 could make the electron transfer of the protein in hybrid C60-lipid membrane more accessible. PMID:27427649

  2. Influence of protein bulk properties on membrane surface coverage during immobilization.

    PubMed

    Militano, Francesca; Poerio, Teresa; Mazzei, Rosalinda; Piacentini, Emma; Gugliuzza, Annarosa; Giorno, Lidietta

    2016-07-01

    Biomolecules immobilization is a key factor for many biotechnological applications. For this purpose, the covalent immobilization of bovine serum albumin (BSA), lipase from Candida rugosa and protein G on differently functionalized regenerated cellulose membranes was investigated. Dynamic light scattering and electrophoresis measurements carried out on biomolecules in solution indicated the presence of monomers, dimers and trimers for both BSA and protein G, while large aggregates were observed for lipase. The immobilization rate and the surface coverage on functionalized regenerated cellulose membranes were studied as a function of biomolecule concentration. Results indicated that the saturation coverage of BSA and protein G was concentration independent (immobilized protein amount of 2.40±0.03mg/g and 2.65±0.07mg/g, respectively). Otherwise, a different immobilization kinetics trend was obtained for lipase, for which the immobilized amount increases as a function of time without reaching a saturation value. Atomic force microscopy (AFM) micrographs showed the formation of monolayers for both BSA and protein G on the membrane surface, while a multilayer structure is found for lipase, in agreement with the trends observed in the related immobilization kinetics. As a result, the morphology of the proteins layer on the membrane surface seems to be strictly dependent on the proteins behavior in solution. Besides, the surface coverage has been described for BSA and protein G by the pseudo second order models, the results indicating the surface reaction as the controlling step of immobilization kinetics. Finally, enzyme activity and binding capacity studies indicated the preservation of the biomolecule functional properties. PMID:27022871

  3. Separation, Immobilization, and Biocatalytic Utilization of Proteins by a Supramolecular Membrane

    PubMed Central

    Krieg, Elisha; Albeck, Shira; Weissman, Haim; Shimoni, Eyal; Rybtchinski, Boris

    2013-01-01

    Membrane separation of biomolecules and their application in biocatalysis is becoming increasingly important for biotechnology, demanding the development of new biocompatible materials with novel properties. In the present study, an entirely noncovalent water-based material is used as a membrane for size-selective separation, immobilization, and biocatalytic utilization of proteins. The membrane shows stable performance under physiological conditions, allowing filtration of protein mixtures with a 150 kDa molecular weight cutoff (∼8 nm hydrodynamic diameter cutoff). Due to the biocompatibility of the membrane, filtered proteins stay functionally active and retained proteins can be partially recovered. Upon filtration, large enzymes become immobilized within the membrane. They exhibit stable activity when subjected to a constant flux of substrates for prolonged periods of time, which can be used to carry out heterogeneous biocatalysis. The noncovalent membrane material can be easily disassembled, purified, reassembled, and reused, showing reproducible performance after recycling. The robustness, recyclability, versatility, and biocompatibility of the supramolecular membrane may open new avenues for manipulating biological systems. PMID:23675461

  4. Trypsin immobilization in ordered porous polymer membranes for effective protein digestion.

    PubMed

    Qiao, Juan; Kim, Jin Yong; Wang, Yuan Yuan; Qi, Li; Wang, Fu Yi; Moon, Myeong Hee

    2016-02-01

    Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores. PMID:26772135

  5. Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins

    PubMed Central

    Bon, Christel Le; Della Pia, Eduardo Antonio; Giusti, Fabrice; Lloret, Noémie; Zoonens, Manuela; Martinez, Karen L.; Popot, Jean-Luc

    2014-01-01

    Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8–35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting ‘OligAPol’ have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8–35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications. PMID:24744236

  6. Cell growth on immobilized cell growth factor. 7. Protein-free cell culture by using growth-factor-immobilized polymer membrane.

    PubMed

    Liu, S Q; Ito, Y; Imanishi, Y

    1993-02-01

    A protein-free culture of anchorage-dependent cells, mouse fibroblast cells, STO and 3T3-L1 and fibroic sarcoma cells, Swiss albino HSDM1C1, grown on a cell-growth protein, insulin, and/or a cell-adhesion protein, collagen, which are immobilized or coimmobilized on surface-hydrolyzed poly(methyl methacrylate) membrane, was investigated. By adding metal ions and lipids to the culture medium, a protein-free culture medium was composed, which was potent in promoting cell proliferation similarly to serum-containing culture medium. In particular, with insulin/collagen-coimmobilized membrane, a protein-free culture was established without detachment of growing cells over a long period. These protein-immobilized membranes could be used repeatedly. PMID:7763456

  7. Efficient protein immobilization on polyethersolfone electrospun nanofibrous membrane via covalent binding for biosensing applications.

    PubMed

    Mahmoudifard, Matin; Soudi, Sara; Soleimani, Masoud; Hosseinzadeh, Simzar; Esmaeili, Elaheh; Vossoughi, Manouchehr

    2016-01-01

    In this paper we introduce novel strategy for antibody immobilization using high surface area electrospun nanofibrous membrane based on ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. To present the high performance of proposed biosensors, anti-staphylococcus enterotoxin B (anti-SEB) was used as a model to demonstrate the utility of our proposed system. Polymer solution of polyethersolfone was used to fabricate fine nanofibrous membrane. Moreover, industrial polyvinylidene fluoride membrane and conventional microtiter plate were also used to compare the efficiency of antibody immobilization. Scanning electron microscopy images were taken to study the morphology of the membranes. The surface activation of nanofibrous membrane was done with the help of O2 plasma. PES nanofibrous membrane with carboxyl functional groups for covalent attachment of antibodies were treated by EDC/NHS coupling agent. The quantity of antibody immobilization was measured by enzyme-linked immuno sorbent assay (ELISA) method. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy was performed to confirm the covalent immobilization of antibody on membrane. Atomic force microscopy, scanning electron microscopy and invert fluorescence microscopy were used to analyze the antibody distribution pattern on solid surfaces. Results show that oxygen plasma treatment effectively increased the amount of antibody immobilization through EDC/NHS coupling chemistry. It was found that the use of nanofibrous membrane causes the improved detection signal of ELISA based biosensors in comparison to the standard assay carried out in the 96-well microtiter plate. This method has the potential to improve the ELISA-based biosensor and we believe that this technique can be used in various biosensing methods. PMID:26478348

  8. Dependence of protein binding capacity of dimethylamino-γ-butyric-acid (DMGABA)-immobilized porous membrane on composition of solvent used for DMGABA immobilization

    NASA Astrophysics Data System (ADS)

    Iwanade, Akio; Umeno, Daisuke; Saito, Kyoichi; Sugo, Takanobu

    2013-06-01

    Dimethylamino-γ-butyric acid (DMGABA) as an ampholite was reacted with the epoxy group of the poly-glycidyl methacrylate chain grafted onto the pore surface of a porous hollow-fiber polyethylene membrane by radiation-induced graft polymerization. DMGABA was dissolved in a mixture of dioxane and water at various dioxane volume fractions, defined by dividing the dioxane volume by the total volume. The equilibrium binding capacity (EBC) of the DMGABA-immobilized porous hollow-fiber membrane for lysozyme was evaluated in the permeation mode. The EBC was varied from a 1/50-fold monolayer binding capacity to a 10-fold monolayer binding capacity by controlling the composition of the solvent used for DMGABA immobilization and the molar conversion of the epoxy group into the DMGABA group.

  9. Immobilized fluid membranes for gas separation

    DOEpatents

    Liu, Wei; Canfield, Nathan L; Zhang, Jian; Li, Xiaohong Shari; Zhang, Jiguang

    2014-03-18

    Provided herein are immobilized liquid membranes for gas separation, methods of preparing such membranes and uses thereof. In one example, the immobilized membrane includes a porous metallic host matrix and an immobilized liquid fluid (such as a silicone oil) that is immobilized within one or more pores included within the porous metallic host matrix. The immobilized liquid membrane is capable of selective permeation of one type of molecule (such as oxygen) over another type of molecule (such as water). In some examples, the selective membrane is incorporated into a device to supply oxygen from ambient air to the device for electrochemical reactions, and at the same time, to block water penetration and electrolyte loss from the device.

  10. Denitrification using a membrane-immobilized biofilm

    SciTech Connect

    McCleaf, P.R. ); Schroeder, E.D. . Dept. of Civil and Environmental Engineering)

    1995-03-01

    Immobilized bacterial cell technology was applied, on a bench scale, to the selective removal of nitrate from contaminated water, together with the segregation of denitrifying bacteria and the carbon energy source from the treated water. The two-chambered reactor, with a microporous membrane for bacterial cell immobilization, performed at an average denitrification rate of 5,800 mg nitrate-nitrogen (NO[sub 3][sup [minus

  11. Mitigated membrane fouling of anammox membrane bioreactor by microbiological immobilization.

    PubMed

    Zhang, Zuotao; Liu, Sitong; Miyoshi, Taro; Matsuyama, Hideto; Ni, Jinren

    2016-02-01

    In this study, membrane fouling behavior of anammox MBR with or without carriers made by magnetic porous carbon microspheres was investigated. The results show that Trans Membrane Pressure was an order of magnitude lower after 50days due to use of carriers, which did not directly contact with membrane surface. Scanning Electron Microscope analysis indicates that abundance of anammox bacteria formed biofilm on membrane surface. Fourier transform infrared spectroscopy combined with amino acids contents analysis for membrane surface deposition show that metabolite released by anammox bacteria contains more hydrophobic groups than hydrophilic, which was considered as important reason for its abundant existence on hydrophobic membrane surface. Microbiological immobilization not only reduces biological membrane fouling, but also mitigates organic fouling including organic matter containing COO, hydrophobic groups (CH3, CH2 and CH etc), as well as inorganic deposition. Our finding provides an effective method for mitigating MBR membrane fouling in anammox process. PMID:26687491

  12. 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

  13. Chitosan-tethered poly(acrylonitrile-co-maleic acid) hollow fiber membrane for lipase immobilization.

    PubMed

    Ye, Peng; Xu, Zhi-Kang; Che, Ai-Fu; Wu, Jian; Seta, Patrick

    2005-11-01

    A protocol was used to prepare a dual-layer biomimetic membrane as support for enzyme immobilization by tethering chitosan on the surface of poly(acrylonitrile-co-maleic acid) (PANCMA) ultrafiltration hollow fiber membrane in the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxylsuccin-imide (NHS). The chemical change of the chitosan-modified PANCMA membrane surface was confirmed with Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Lipase from Candida rugosa was immobilized on this dual-layer biomimetic membrane using glutaraldehyde (GA), and on the nascent PANCMA membrane using EDC/NHS as coupling agent. The properties of the immobilized enzymes were assayed and compared with those of the free one. It was found that both the activity retention of the immobilized lipase and the amount of bound protein on the dual-layer biomimetic membrane (44.5% and 66.5 mg/m2) were higher than those on the nascent PANCMA membrane (33.9% and 53.7 mg/m2). The kinetic parameters of the free and immobilized lipases, Km and Vmax, were also assayed. The Km values were similar for the immobilized lipases, while the Vmax value of the immobilized lipase on the dual-layer biomimetic membrane was higher than that on the nascent PANCMA membrane. Results indicated that the pH and thermal stabilities of lipase increased upon immobilization. The residual activity of the immobilized lipase after 10 uses was 53% on the dual-layer biomimetic membrane and 62% on the nascent PANCMA membrane. PMID:15919112

  14. Drugging Membrane Protein Interactions.

    PubMed

    Yin, Hang; Flynn, Aaron D

    2016-07-11

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind cells to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally "undruggable" regions of membrane proteins, enabling modulation of protein-protein, protein-lipid, and protein-nucleic acid interactions. In this review, we survey the state of the art of high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  15. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  16. Immobilization of the urease on eggshell membrane and its application in biosensor.

    PubMed

    D'Souza, S F; Kumar, Jitendra; Jha, Sandeep Kumar; Kubal, B S

    2013-03-01

    Eggshell membrane is a natural material, essentially made up of protein fibers having flexibility in the aqueous solution and possessing gas and water permeability. It is used as a biomembrane for immobilization of urease for the development of a potentiometric urea biosensor. Eggshell membrane was treated with polyethyleneimine (PEI) to impart polycation characteristics. Urease was immobilized on the PEI treated eggshell membrane through adsorption. SEM study was carried out to observe the changes in surface morphology after immobilization. FTIR study of membrane was carried out to observe the changes in IR spectra after immobilization of enzyme. Immobilized membrane was associated with ammonium ion selective electrode. Biosensor exhibited sigmoidal responses for the urea concentration range from 0.5 to 10mM. The response time of the biosensor was 120 s. A single membrane was reused for 270 reactions without loss of activity. The urease-eggshell membranes were stable for 2 months when stored in buffer even at room temperature. PMID:25427497

  17. The immobilization of lipase on PVDF-co-HFP membrane

    NASA Astrophysics Data System (ADS)

    Kayhan, Naciye; Eyüpoǧlu, Volkan; Adem, Şevki

    2016-04-01

    Lipase is an enzyme having a lot of different industrial applications such as biodiesel production, biopolymer synthesis, enantiopure pharmaceutical productions, agrochemicals, etc. Its immobilized form on different substances is more conventional and useful than its free form. Supporting material was prepared using PVDF-co-HFP in laboratory conditions and attached 1,4-diaminobutane (DA) and epichlorohydrin (EPI) ligands to the membrane to immobilize lipase enzyme. The immobilization conditions such as enzyme amount, pH, the concentration of salt, thermal stability and activity were stabilized for our experimental setup. Then, biochemical characterizations were performed on immobilized lipase PVDF-co-HFP regarding optimal pH activity, temperature and thermal stability. Also, the desorption ratios of immobilized enzyme in two different pathway were investigated to confirm immobilization stability for 24 hours.

  18. Immobilization of enzyme onto poly(ethylene-vinyl alcohol) membrane

    SciTech Connect

    Imai, K.; Shiomi, T.; Uchida, K.; Miya, M.

    1986-02-01

    Invertase was ionically bound to the poly(ethylene-vinyl alcohol) membrane surface modified with two aminoacetals with different molecular length, 2-dimethyl-aminoacetoaldehyde dimethylacetal (AAA) and 3-(N,N-dimethylamino-n-propanediamine) propionaldehyde dimethylacetal (APA). Immobilization conditions were determined with respect to enzyme concentration in solution, pH value, ionic strength in immobilization solution, and immobilization time. Various properties of immobilized invertase were evaluated, and thermal stability was found especially to be improved by immobilization. The apparent Michaelis constant, Km, was smaller for invertase bound by APA with longer molecular lengths than for invertase bound by AAA. We attempted to bind glucoamylase of Rhizopus delemarorigin in the same way. The amount and activity of immobilized glucoamylase were much less than those of invertase. 16 references.

  19. Biomolecular membrane protein crystallization

    NASA Astrophysics Data System (ADS)

    Reddy Bolla, Jani; Su, Chih-Chia; Yu, Edward W.

    2012-07-01

    Integral membrane proteins comprise approximately 30% of the sequenced genomes, and there is an immediate need for their high-resolution structural information. Currently, the most reliable approach to obtain these structures is X-ray crystallography. However, obtaining crystals of membrane proteins that diffract to high resolution appears to be quite challenging, and remains a major obstacle in structural determination. This brief review summarizes a variety of methodologies for use in crystallizing these membrane proteins. Hopefully, by introducing the available methods, techniques, and providing a general understanding of membrane proteins, a rational decision can be made about now to crystallize these complex materials.

  20. Lipid membranes for membrane proteins.

    PubMed

    Kukol, Andreas

    2015-01-01

    The molecular dynamics (MD) simulation of membrane proteins requires the setup of an accurate representation of lipid bilayers. This chapter describes the setup of a lipid bilayer system from scratch using generally available tools, starting with a definition of the lipid molecule POPE, generation of a lipid bilayer, energy minimization, MD simulation, and data analysis. The data analysis includes the calculation of area and volume per lipid, deuterium order parameters, self-diffusion constant, and the electron density profile. PMID:25330959

  1. Solid-state NMR and Membrane Proteins

    PubMed Central

    Opella, Stanley J.

    2015-01-01

    The native environment for a membrane protein is a phospholipid bilayer. Because the protein is immobilized on NMR timescales by the interactions within a bilayer membrane, solid-state NMR methods are essential to obtain high-resolution spectra. Approaches have been developed for both unoriented and oriented samples, however, they all rest on the foundation of the most fundamental aspects solid-state NMR, and the chemical shift and homo- and hetero-nuclear dipole-dipole interactions. Solid-state NMR has advanced sufficiently to enable the structures of membrane proteins to be determined under near-native conditions in phospholipid bilayers. PMID:25681966

  2. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  3. Structures of membrane proteins

    PubMed Central

    Vinothkumar, Kutti R.; Henderson, Richard

    2010-01-01

    In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class. PMID:20667175

  4. Outer membrane protein purification.

    PubMed

    Arigita, C; Jiskoot, W; Graaf, M R; Kersten, G F

    2001-01-01

    The major outer membrane proteins (OMPs) from Neisseria meningitidis, which are expressed at high levels, are subdivided in five classes based on molecular weight (1,2) (see Table 1). Table 1 Major Meningococcal Outer-Membrane Proteins Outer-membrane proteins Name Molecular maass Function/characteristics Class 1 PorA 44-47 kDa Porin Class 2/3 PorB 37-42 kDa Porin Class 4 Rmp Reductionmodifiableprotein, unknown Class 5 Opa 26-30 kDa Adhesion,opacity protein Opc 25 kDa Invasion, opacity protein Iron-regulated proteins Mirp 37 kDa Iron acquisition (?);majoriron-regulatedprotein FrpB 70 kDa Ferric enterobactin receptor (also FetA) Adapted from ref. (1). PMID:21336748

  5. [sup 31]P NMR study of immobilized artificial membrane surfaces. Structure and dynamics of immobilized phospholipids

    SciTech Connect

    Qiu, X.; Pidgeon, C. )

    1993-11-25

    Chromatography surfaces were prepared by immobilizing a single-chain ether phospholipid at approximately a monolayer density on silica particles. The chromatography particles are denoted as [sup ether]IAM.PC[sup C10/C3], and they are stable to all solvents. The structure and dynamics of the interphase created by immobilizing phospholipids on silica particles were studied by [sup 31]P NMR methods. [sup ether]IAM.PC[sup C10/C3] spontaneously wets when suspended in both aqueous and organic solvents, and [sup 31]P NMR spectra were obtained in water, methanol, chloroform, acetonitrile, and acetone. [sup 31]P NMR spectra were subjected to line-shape analysis. From line-shape analysis, the correlation times for rapid internal motion ([tau]-PLL) and wobbling ([tau]-PRP) of the phospholipid headgroup were calculated for each solvent. Immobilized phospholipid headgroups comprising the IAM interfacial region undergo rapid reorientation similar to the case of the phospholipids forming liposome membranes with [tau]-PLL approximately 1 ns. Phospholipids in liposome membranes exhibit slower wobbling motion ([tau]-PRP approximately 1 ms) in the plane of the membrane. However, the immobilized phospholipids on [sup ether]IAM.PC[sup C10/C3] surfaces wobble with correlation times [tau]-PRP that depend on the solvent bathing the [sup ether]IAM.PC[sup C10/C3] surface. 41 refs., 9 figs., 2 tabs.

  6. Protein mediated membrane adhesion

    NASA Astrophysics Data System (ADS)

    Carlson, Andreas; Mahadevan, L.

    2015-05-01

    Adhesion in the context of mechanical attachment, signaling, and movement in cellular dynamics is mediated by the kinetic interactions between membrane-embedded proteins in an aqueous environment. Here, we present a minimal theoretical framework for the dynamics of membrane adhesion that accounts for the kinetics of protein binding, the elastic deformation of the membrane, and the hydrodynamics of squeeze flow in the membrane gap. We analyze the resulting equations using scaling estimates to characterize the spatiotemporal features of the adhesive patterning and corroborate them using numerical simulations. In addition to characterizing aspects of cellular dynamics, our results might also be applicable to a range of phenomena in physical chemistry and materials science where flow, deformation, and kinetics are coupled to each other in slender geometries.

  7. Real-time Monitoring of Intermediates Reveals the Reaction Pathway in the Thiol-Disulfide Exchange between Disulfide Bond Formation Protein A (DsbA) and B (DsbB) on a Membrane-immobilized Quartz Crystal Microbalance (QCM) System*

    PubMed Central

    Yazawa, Kenjiro; Furusawa, Hiroyuki; Okahata, Yoshio

    2013-01-01

    Disulfide bond formation protein B (DsbBS-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbASH,SH). The oxidized DsbAS-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbASH,SH and DsbBS-S,S-S. We immobilized DsbBS-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (kf, kr, and kcat, respectively) indicated that the two pairs of cysteine residues in DsbBS-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbBS-S,S-S. Our data suggested that the reaction pathway of almost all DsbASH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone. PMID:24145032

  8. Real-time monitoring of intermediates reveals the reaction pathway in the thiol-disulfide exchange between disulfide bond formation protein A (DsbA) and B (DsbB) on a membrane-immobilized quartz crystal microbalance (QCM) system.

    PubMed

    Yazawa, Kenjiro; Furusawa, Hiroyuki; Okahata, Yoshio

    2013-12-13

    Disulfide bond formation protein B (DsbBS-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbASH,SH). The oxidized DsbAS-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbASH,SH and DsbBS-S,S-S. We immobilized DsbBS-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (kf, kr, and kcat, respectively) indicated that the two pairs of cysteine residues in DsbBS-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbBS-S,S-S. Our data suggested that the reaction pathway of almost all DsbASH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone. PMID:24145032

  9. Chiral separation of amino acids in ultrafiltration through DNA-immobilized cellulose membranes

    NASA Astrophysics Data System (ADS)

    Higuchi, Akon; Hayashi, Akiyuki; Kanda, Naoki; Sanui, Kohei; Kitamura, Hanako

    2005-04-01

    Ultrafiltration experiments for the chiral separation of racemic tryptophan, phenylglycine and phenylalanine were investigated through immobilized DNA membranes having various pore sizes. L-tryptophan preferentially permeated through immobilized DNA membranes with a pore size<2.0 nm (molecular weight cut-off (MWCO)<5000) while D-tryptophan preferentially permeated through immobilized DNA membranes with a pore size>2.0 nm (MWCO>5000). These results are completely opposite tendency in the ultrafiltration of racemic phenylalanine through the immobilized DNA membranes. This may be originated from the different interaction between DNA and tryptophan compared to that between DNA and phenylalanine. However, in both cases the pore size of the immobilized DNA membranes regulated preferential permeation of the enantiomer through the membranes. The immobilized DNA membranes are categorized as channel type membranes and not as affinity membranes. Chiral separation models were proposed from using the chiral separation results of racemic amino acids, preferential adsorption of amino acid enantiomers and EPMA results.

  10. High Performance Immobilized Liquid Membrane for Carbon Dioxide Separations

    NASA Technical Reports Server (NTRS)

    Parrish, Clyde F. (Inventor)

    2005-01-01

    An immobilized liquid membrane has a substrate. A plurality of capsules is disposed on the substrate. Each of the capsules is permeable to a first gas of a mixture of gases comprising the st gas and a second gas. Each of the capsules is substantially impermeable to the second gas. A liquid is disposed in each of the capsules that is permeable to the first gas and substantially impermeable to the second gas.

  11. Membrane Protein Prediction Methods

    PubMed Central

    Punta, Marco; Forrest, Lucy R.; Bigelow, Henry; Kernytsky, Andrew; Liu, Jinfeng; Rost, Burkhard

    2007-01-01

    We survey computational approaches that tackle membrane protein structure and function prediction. While describing the main ideas that have led to the development of the most relevant and novel methods, we also discuss pitfalls, provide practical hints and highlight the challenges that remain. The methods covered include: sequence alignment, motif search, functional residue identification, transmembrane segment and protein topology predictions, homology and ab initio modeling. Overall, predictions of functional and structural features of membrane proteins are improving, although progress is hampered by the limited amount of high-resolution experimental information available. While predictions of transmembrane segments and protein topology rank among the most accurate methods in computational biology, more attention and effort will be required in the future to ameliorate database search, homology and ab initio modeling. PMID:17367718

  12. Membrane Bending by Protein Crowding

    NASA Astrophysics Data System (ADS)

    Stachowiak, Jeanne

    2014-03-01

    From endosomes and synaptic vesicles to the cristae of the mitochondria and the annulus of the nuclear pore, highly curved membranes are fundamental to the structure and physiology of living cells. The established view is that specific families of proteins are able to bend membranes by binding to them. For example, inherently curved proteins are thought to impose their structure on the membrane surface, while membrane-binding proteins with hydrophobic motifs are thought to insert into the membrane like wedges, driving curvature. However, computational models have recently revealed that these mechanisms would require specialized membrane-bending proteins to occupy nearly 100% of a curved membrane surface, an improbable physiological situation given the immense density and diversity of membrane-bound proteins, and the low expression levels of these specialized proteins within curved regions of the membrane. How then does curvature arise within the complex and crowded environment of cellular membranes? Our recent work using proteins involved in clathrin-mediated endocytosis, as well as engineered protein-lipid interactions, has suggested a new hypothesis - that lateral pressure generated by collisions between membrane-bound proteins can drive membrane bending. Specifically, by correlating membrane bending with quantitative optical measurements of protein density on synthetic membrane surfaces and simple physical models of collisions among membrane-bound proteins, we have demonstrated that protein-protein steric interactions can drive membrane curvature. These findings suggest that a simple imbalance in the concentration of membrane-bound proteins across a membrane surface can drive a membrane to bend, providing an efficient mechanism by which essentially any protein can contribute to shaping membranes.

  13. Glasslike Membrane Protein Diffusion in a Crowded Membrane.

    PubMed

    Munguira, Ignacio; Casuso, Ignacio; Takahashi, Hirohide; Rico, Felix; Miyagi, Atsushi; Chami, Mohamed; Scheuring, Simon

    2016-02-23

    Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion. PMID:26859708

  14. Prediction of protein orientation upon immobilization on biological and nonbiological surfaces.

    PubMed

    Talasaz, AmirAli H; Nemat-Gorgani, Mohsen; Liu, Yang; Ståhl, Patrik; Dutton, Robert W; Ronaghi, Mostafa; Davis, Ronald W

    2006-10-01

    We report on a rapid simulation method for predicting protein orientation on a surface based on electrostatic interactions. New methods for predicting protein immobilization are needed because of the increasing use of biosensors and protein microarrays, two technologies that use protein immobilization onto a solid support, and because the orientation of an immobilized protein is important for its function. The proposed simulation model is based on the premise that the protein interacts with the electric field generated by the surface, and this interaction defines the orientation of attachment. Results of this model are in agreement with experimental observations of immobilization of mitochondrial creatine kinase and type I hexokinase on biological membranes. The advantages of our method are that it can be applied to any protein with a known structure; it does not require modeling of the surface at atomic resolution and can be run relatively quickly on readily available computing resources. Finally, we also propose an orientation of membrane-bound cytochrome c, a protein for which the membrane orientation has not been unequivocally determined. PMID:17001006

  15. Prediction of protein orientation upon immobilization on biological and nonbiological surfaces

    NASA Astrophysics Data System (ADS)

    Talasaz, Amirali H.; Nemat-Gorgani, Mohsen; Liu, Yang; Ståhl, Patrik; Dutton, Robert W.; Ronaghi, Mostafa; Davis, Ronald W.

    2006-10-01

    We report on a rapid simulation method for predicting protein orientation on a surface based on electrostatic interactions. New methods for predicting protein immobilization are needed because of the increasing use of biosensors and protein microarrays, two technologies that use protein immobilization onto a solid support, and because the orientation of an immobilized protein is important for its function. The proposed simulation model is based on the premise that the protein interacts with the electric field generated by the surface, and this interaction defines the orientation of attachment. Results of this model are in agreement with experimental observations of immobilization of mitochondrial creatine kinase and type I hexokinase on biological membranes. The advantages of our method are that it can be applied to any protein with a known structure; it does not require modeling of the surface at atomic resolution and can be run relatively quickly on readily available computing resources. Finally, we also propose an orientation of membrane-bound cytochrome c, a protein for which the membrane orientation has not been unequivocally determined. electric double layer | electrostatic simulations | orientation flexibility

  16. Tracking Membrane Protein Association in Model Membranes

    PubMed Central

    Reffay, Myriam; Gambin, Yann; Benabdelhak, Houssain; Phan, Gilles; Taulier, Nicolas; Ducruix, Arnaud; Hodges, Robert S.; Urbach, Wladimir

    2009-01-01

    Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue. We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well. After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 Å, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We

  17. Proteins causing membrane fouling in membrane bioreactors.

    PubMed

    Miyoshi, Taro; Nagai, Yuhei; Aizawa, Tomoyasu; Kimura, Katsuki; Watanabe, Yoshimasa

    2015-01-01

    In this study, the details of proteins causing membrane fouling in membrane bioreactors (MBRs) treating real municipal wastewater were investigated. Two separate pilot-scale MBRs were continuously operated under significantly different operating conditions; one MBR was a submerged type whereas the other was a side-stream type. The submerged and side-stream MBRs were operated for 20 and 10 days, respectively. At the end of continuous operation, the foulants were extracted from the fouled membranes. The proteins contained in the extracted foulants were enriched by using the combination of crude concentration with an ultrafiltration membrane and trichloroacetic acid precipitation, and then separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The N-terminal amino acid sequencing analysis of the proteins which formed intensive spots on the 2D-PAGE gels allowed us to partially identify one protein (OmpA family protein originated from genus Brevundimonas or Riemerella anatipestifer) from the foulant obtained from the submerged MBR, and two proteins (OprD and OprF originated from genus Pseudomonas) from that obtained from the side-stream MBR. Despite the significant difference in operating conditions of the two MBRs, all proteins identified in this study belong to β-barrel protein. These findings strongly suggest the importance of β-barrel proteins in developing membrane fouling in MBRs. PMID:26360742

  18. Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins.

    PubMed

    Eisenbach, L; Ramanathan, R; Nelson, D L

    1983-11-01

    The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross-reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i-antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential. PMID:6415066

  19. Microtechnologies for membrane protein studies

    PubMed Central

    Suzuki, Hiroaki

    2008-01-01

    Despite the rapid and enormous progress in biotechnologies, the biochemical analysis of membrane proteins is still a difficult task. The presence of the large hydrophobic region buried in the lipid bilayer membrane (transmembrane domain) makes it difficult to analyze membrane proteins in standard assays developed for water-soluble proteins. To handle membrane proteins, the lipid bilayer membrane may be used as a platform to sustain their functionalities. Relatively slow progress in developing micro total analysis systems (μTAS) for membrane protein analysis directly reflects the difficulty of handling lipid membranes, which is a common problem in bulk measurement technologies. Nonetheless, researchers are continuing to develop efficient and sensitive analytical microsystems for the study of membrane proteins. Here, we review the latest developments, which enable detection of events caused by membrane proteins, such as ion channel current, membrane transport, and receptor/ligand interaction, by utilizing microfabricated structures. High-throughput and highly sensitive detection systems for membrane proteins are now becoming a realistic goal. Although most of these systems are still in the early stages of development, we believe this field will become one of the most important applications of μTAS for pharmaceutical and clinical screenings as well as for basic biochemical research. PMID:18335213

  20. Affinity Separation of Lectins Using Porous Membranes Immobilized with Glycopolymer Brushes Containing Mannose or N-Acetyl-d-Glucosamine

    PubMed Central

    Ogata, Yutaro; Seto, Hirokazu; Murakami, Tatsuya; Hoshino, Yu; Miura, Yoshiko

    2013-01-01

    Porous membranes with glycopolymer brushes were prepared as biomaterials for affinity separation. Glycopolymer brushes contained acrylic acid and D-mannose or N-acetyl-D-glucosamine, and were formed on substrates by surface-initiated atom transfer radical polymerization. The presence of glycopolymer brush was confirmed by X-ray photoelectron spectroscopy, contact angle, and ellipsometry measurements. The interaction between lectin and the glycopolymer immobilized on glass slides was confirmed using fluorescent-labeled proteins. Glycopolymer-immobilized surfaces exhibited specific adsorption of the corresponding lectin, compared with bovine serum albumin. Lectins were continuously rejected by the glycopolymer-immobilized membranes. When the protein solution was permeated through the glycopolymer-immobilized membrane, bovine serum albumin was not adsorbed on the membrane surface. In contrast, concanavalin A and wheat germ agglutinin were rejected by membranes incorporating D-mannose or N-acetyl-D-glucosamine, respectively. The amounts of adsorbed concanavalin A and wheat germ agglutinin was increased five- and two-fold that of adsorbed bovine serum albumin, respectively. PMID:24956944

  1. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    NASA Astrophysics Data System (ADS)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  2. Rapid protein immobilization for thin film continuous flow biocatalysis.

    PubMed

    Britton, Joshua; Raston, Colin L; Weiss, Gregory A

    2016-08-01

    A versatile enzyme immobilization strategy for thin film continuous flow processing is reported. Here, non-covalent and glutaraldehyde bioconjugation are used to immobilize enzymes on the surfaces of borosilicate reactors. This approach requires only ng of protein per reactor tube, with the stock protein solution readily recycled to sequentially coat >10 reactors. Confining reagents to thin films during immobilization reduced the amount of protein, piranha-cleaning solution, and other reagents by ∼96%. Through this technique, there was no loss of catalytic activity over 10 h processing. The results reported here combines the benefits of thin film flow processing with the mild conditions of biocatalysis. PMID:27461146

  3. Gluconic acid production in bioreactor with immobilized glucose oxidase plus catalase on polymer membrane adjacent to anion-exchange membrane.

    PubMed

    Godjevargova, Tzonka; Dayal, Rajeshwar; Turmanova, Sevdalina

    2004-10-20

    Gluconic acid was obtained in the permeate side of the bioreactor with glucose oxidase (GOD) immobilized onto anion-exchange membrane (AEM) of low-density polyethylene grafted with 4-vinylpiridine. The electric resistance of the anion-exchange membranes was increased after the enzyme immobilization on the membrane. The gluconic acid productions were relatively low with the GOD immobilized by any method on the AEM. To increase the enzyme reaction efficiency, GOD was immobilized on membrane of AN copolymer (PAN) adjacent to an anion-exchange membrane in bioreactor. Uses of anion-exchange membrane led to selective removal of the gluconic acid from the glucose solution and reduce the gluconic acid inhibition. The amount of gluconic acid obtained in the permeate side of the bioreactor with the GOD immobilized on the PAN membrane adjacent to the AEM under electrodialysis was about 30 times higher than that obtained with enzyme directly bound to the AEM. The optimal substrate concentration in the feed side was found to be about 1 g/l. Further experiments were carried out with the co-immobilized GOD plus Catalase (CAT) on the PAN membrane adjacent to the AEM to improve the efficiency of the immobilize system. The yield of this process was at least 95%. The storage stability of the co-immobilized GOD and CAT was studied (lost 20% of initial activity for 90 d). The results obtained clearly showed the higher potential of the dual membrane bioreactor with GOD plus CAT bound to ultrafiltration polymer membrane adjacent to the AEM. Storage stability of GOD activity in GOD plus CAT immobilized on PAN//AEM membranes and on AEM. PMID:15497133

  4. Dielectrophoretic immobilization of proteins: Quantification by atomic force microscopy.

    PubMed

    Laux, Eva-Maria; Knigge, Xenia; Bier, Frank F; Wenger, Christian; Hölzel, Ralph

    2015-09-01

    The combination of alternating electric fields with nanometer-sized electrodes allows the permanent immobilization of proteins by dielectrophoretic force. Here, atomic force microscopy is introduced as a quantification method, and results are compared with fluorescence microscopy. Experimental parameters, for example the applied voltage and duration of field application, are varied systematically, and the influence on the amount of immobilized proteins is investigated. A linear correlation to the duration of field application was found by atomic force microscopy, and both microscopical methods yield a square dependence of the amount of immobilized proteins on the applied voltage. While fluorescence microscopy allows real-time imaging, atomic force microscopy reveals immobilized proteins obscured in fluorescence images due to low S/N. Furthermore, the higher spatial resolution of the atomic force microscope enables the visualization of the protein distribution on single nanoelectrodes. The electric field distribution is calculated and compared to experimental results with very good agreement to atomic force microscopy measurements. PMID:26010162

  5. Membrane Protein Assembly into Nanodiscs

    PubMed Central

    Bayburt, Timothy H.; Sligar, Stephen G.

    2016-01-01

    Nanodiscs are soluble nanoscale phospholipid bilayers which can self-assemble integral membrane proteins for biophysical, enzymatic or structural investigations. This means for rendering membrane proteins soluble at the single molecule level offers advantages over liposomes or detergent micelles in terms of size, stability, ability to add genetically modifiable features to the Nanodisc structure and ready access to both sides of the phospholipid bilayer domain. Thus the Nanodisc system provides a novel platform for understanding membrane protein function. We provide an overview of the Nanodisc approach and document through several examples many of the applications to the study of the structure and function of integral membrane proteins. PMID:19836392

  6. Photo selective protein immobilization using bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Kim, Wan-Joong; Kim, Ansoon; Huh, Chul; Park, Chan Woo; Ah, Chil Seong; Kim, Bong Kyu; Yang, Jong-Heon; Chung, Kwang Hyo; Choi, Yo Han; Hong, Jongcheol; Sung, Gun Yong

    2012-11-01

    A simple and selective technique which immobilizes protein onto a solid substrate by using UV illumination has been developed. In protein immobilization, a Bovine serum albumin (BSA) performed bifunctional role as a cross-linker between substrate and proteins and as a blocker inhibiting a nonspecific protein adsorption. A new photo-induced protein immobilization process has been investigated at each step by fluorescence microscopy, ellipsometry, and Fourier transform infrared (FT-IR) spectroscopy. A UV photomask has been used to induce selective protein immobilization on target regions of the surface of the SiO2 substrates under UV illumination with negligible nonspecific binding. The UV illumination also showed improved photostability than the conventional methods which employed bifunctional photo-crosslinker molecules of photo-reactive diazirine. This new UV illumination-based photo-addressable protein immobilization provides a new approach for developing novel protein microarrays for multiplexed sensing as well as other types of bio-immobilization in biomedical devices and biotechnologies.

  7. Protein immobilization onto electrochemically synthesized CoFe nanowires

    PubMed Central

    Torati, Sri Ramulu; Reddy, Venu; Yoon, Seok Soo; Kim, CheolGi

    2015-01-01

    CoFe nanowires have been synthesized by the electrodeposition technique into the pores of a polycarbonate membrane with a nominal pore diameter of 50 nm, and the composition of CoFe nanowires varying by changing the source concentration of iron. The synthesized nanowire surfaces were functionalized with amine groups by treatment with aminopropyltriethoxysilane (APTES) linker, and then conjugated with streptavidin-Cy3 protein via ethyl (dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide coupling chemistry. The oxide surface of CoFe nanowire is easily modified with aminopropyltriethoxysilane to form an amine terminating group, which is covalently bonded to streptavidin-Cy3 protein. The physicochemical properties of the nanowires were analyzed through different characterization techniques such as scanning electron microscope, energy dispersive spectroscopy, and vibrating sample magnetometer. Fluorescence microscopic studies and Fourier transform infrared studies confirmed the immobilization of protein on the nanowire surface. In addition, the transmission electron microscope analysis reveals the thin protein layer which is around 12–15 nm on the nanowire surfaces. PMID:25609966

  8. Structure Prediction of Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Hu, Xiche

    Membrane proteins play a central role in many cellular and physiological processes. It is estimated that integral membrane proteins make up about 20-30% of the proteome (Krogh et al., 2001b; Stevens and Arkin, 2000; von Heijne, 1999). They are essential mediators of material and information transfer across cell membranes. Their functions include active and passive transport of molecules into and out of cells and organelles; transduction of energy among various forms (light, electrical, and chemical energy); as well as reception and transduction of chemical and electrical signals across membranes (Avdonin, 2005; Bockaert et al., 2002; Pahl, 1999; Rehling et al., 2004; Stack et al., 1995). Identifying these transmembrane (TM) proteins and deciphering their molecular mechanisms, then, is of great importance, particularly as applied to biomedicine. Membrane proteins are the targets of a large number of pharmacologically and toxicologically active substances, and are directly involved in their uptake, metabolism, and clearance (Bettler et al., 1998; Cohen, 2002; Heusser and Jardieu, 1997; Tibes et al., 2005; Xu et al., 2005). Despite the importance of membrane proteins, the knowledge of their high-resolution structures and mechanisms of action has lagged far behind in comparison to that of water-soluble proteins: less than 1% of all three-dimensional structures deposited in the Protein Data Bank are of membrane proteins. This unfortunate disparity stems from difficulties in overexpression and the crystallization of membrane proteins (Grisshammer and Tate, 1995; Michel, 1991).

  9. Nanofibrous poly(acrylonitrile-co-maleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization.

    PubMed

    Ye, Peng; Xu, Zhi-Kang; Wu, Jian; Innocent, Christophe; Seta, Patrick

    2006-08-01

    Nanofibrous membranes with an average diameter of 100 and 180 nm were fabricated from poly(acrylonitrile-co-maleic acid) (PANCMA) by the electrospinning process. These nanofibrous membranes contain reactive groups which can be used to covalently immobilize biomacromolecules. Two natural macromolecules, chitosan and gelatin, were tethered on these nanofibrous membranes to fabricate dual-layer biomimetic supports for enzyme immobilization in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS). Lipase from Candida rugosa was then immobilized on these dual-layer biomimetic supports using glutaraldehyde (GA), and on the nascent PANCMA fibrous membrane using EDC/NHS as coupling agent, respectively. The properties of the immobilized lipases were assayed. It was found that there is an increase of the activity retention of the immobilized lipase on the chitosan-modified nanofibrous membrane (45.6+/-1.8%) and on the gelatin-modified one (49.7+/-1.8%), compared to that on the nascent one (37.6+/-1.8%). The kinetic parameters of the free and immobilized lipases, K(m) and V(max), were also assayed. In comparison with the immobilized lipase on the nascent nanofibrous membrane, there is an increase of the V(max) value for the immobilized lipases on the chitosan- and gelatin-modified nanofibrous membranes. Results also indicate that the pH and thermal stabilities of lipases increase upon immobilization. The residual activities of the immobilized lipases are 55% on the chitosan-modified nanofibrous membrane and 60% on the gelatin-modified one, after 10 uses. PMID:16584770

  10. Enhanced starch hydrolysis using α-amylase immobilized on cellulose ultrafiltration affinity membrane.

    PubMed

    Konovalova, Viktoriia; Guzikevich, Kateryna; Burban, Anatoliy; Kujawski, Wojciech; Jarzynka, Karolina; Kujawa, Joanna

    2016-11-01

    In order to prepare ultrafiltration membranes possessing biocatalytic properties, α-amylase has been immobilized on cellulose membranes. Enzyme immobilization was based on a covalent bonding between chitosan and a surface of cellulose membrane, followed by an attachment of Cibacron Blue F3G-A dye as affinity ligand. Various factors affecting the immobilization process, such as enzyme concentration, pH of modifying solution, zeta-potential of membrane surface, and stability of immobilized enzyme were studied. The applicability of immobilized α-amylase has been investigated in ultrafiltration processes. The immobilization of α-amylase on membrane surface allows to increase the value of mass transfer coefficient and to decrease the concentration polarization effect during ultrafiltration of starch solutions. The enzyme layer on the membrane surface prevents a rapid increase of starch concentration due to the amylase hydrolysis of starch in the boundary layer. The presented affinity immobilization technique allows also for the regeneration of membranes from inactivated enzyme. PMID:27516322

  11. Aligned Immobilization of Proteins Using AC Electric Fields.

    PubMed

    Laux, Eva-Maria; Knigge, Xenia; Bier, Frank F; Wenger, Christian; Hölzel, Ralph

    2016-03-01

    Protein molecules are aligned and immobilized from solution by AC electric fields. In a single-step experiment, the enhanced green fluorescent proteins are immobilized on the surface as well as at the edges of planar nanoelectrodes. Alignment is found to follow the molecules' geometrical shape with their longitudinal axes parallel to the electric field. Simultaneous dielectrophoretic attraction and AC electroosmotic flow are identified as the dominant forces causing protein movement and alignment. Molecular orientation is determined by fluorescence microscopy based on polarized excitation of the proteins' chromophores. The chromophores' orientation with respect to the whole molecule supports X-ray crystal data. PMID:26779699

  12. Site-selective protein immobilization by covalent modification of GST fusion proteins.

    PubMed

    Zhou, Yiqing; Guo, Tianlin; Tang, Guanghui; Wu, Hui; Wong, Nai-Kei; Pan, Zhengying

    2014-11-19

    The immobilization of functional proteins onto solid supports using affinity tags is an attractive approach in recent development of protein microarray technologies. Among the commonly used fusion protein tags, glutathione S-transferase (GST) proteins have been indispensable tools for protein-protein interaction studies and have extensive applications in recombinant protein purification and reversible protein immobilization. Here, by utilizing pyrimidine-based small-molecule probes with a sulfonyl fluoride reactive group, we report a novel and general approach for site-selective immobilization of Schistosoma japonicum GST (sjGST) fusion proteins through irreversible and specific covalent modification of the tyrosine-111 residue of the sjGST tag. As demonstrated by sjGST-tagged eGFP and sjGST-tagged kinase activity assays, this immobilization approach offers the advantages of high immobilization efficiency and excellent retention of protein structure and activity. PMID:25340706

  13. Acetylcholinesterase immobilization on polyacrylamide/functionalized multi-walled carbon nanotube nanocomposite nanofibrous membrane.

    PubMed

    Amini, Navid; Mazinani, Saeedeh; Ranaei-Siadat, Seyed-Omid; Kalaee, Mohammad Reza; Hormozi, Saeed; Niknam, Kaveh; Firouzian, Nasrin

    2013-05-01

    In this work, polyacrylamide/multi-walled carbon nanotubes (MWCNT) solution is electrospun to nanocomposite nanofibrous membranes for acetylcholinesterase enzyme immobilization. A new method for enzyme immobilization is proposed, and the results of analysis show successful covalent bonding of enzymes on electrospun membrane surface besides their non-covalent entrapment. Fourier transform infrared spectroscopy, mechanical and thermal investigations of nanofibrous membrane approve successful cross-linking and enzyme immobilization. The enzyme relative activity and kinetic on both pure and nanocomposite membranes is investigated, and the results show proper performance of designed membrane to even improve the enzyme activity followed by immobilization compared to free enzyme. Scanning electron microscopy images show nanofibrous web of 3D structure with a low shrinkage and hydrogel structure followed by enzyme immobilization and cross-linking. Moreover, the important role of functionalized carbon nanotubes on final nanofibrous membrane functionality as a media for enzyme immobilization is investigated. The results show that MWCNT could act effectively for enzyme immobilization improvement via both physical (enhanced fibers' morphology and conductivity) and chemical (enzyme entrapment) methods. PMID:23475318

  14. Fourier transform infrared assay of membrane lipids immobilized to silica: leaching and stability of immobilized artificial membrane-bonded phases.

    PubMed

    Markovich, R J; Stevens, J M; Pidgeon, C

    1989-11-01

    A nondestructive, sensitive assay to monitor the hydrocarbon content of silica-based chromatography particles has been developed. The assay requires a microscope accessory interfaced with a Fourier transform infrared (FTIR) spectrometer. For determining hydrocarbon content, undiluted alkyl-silica-bonded phases were pressed into a thin wafer. Hydrocarbon content was quantitated using the integrated hydrocarbon band intensity between 2995 and 2825 cm-1 [i.e., band area C-H] and the integrated silica oxide band intensity between 1945 and 1780 cm-1 [i.e., band area Si-O]. Plotting the [band area C-H]/[band area Si-O] ratio vs the carbon content determined by elemental analysis gave a correlation coefficient of r = 0.997. The FTIR assay was validated on 5-, 7-, and 12-microns silica particles using three different immobilized artificial membrane (IAM) silica-bonded phases. The utility of the FTIR assay in determining hydrocarbon content was demonstrated by evaluating hydrocarbon leaching from IAM phases exposed to mobile-phase solvents. The ability of organic solvents to leach hydrocarbon from IAM phases containing phosphatidylcholine (PC) as the immobilized ligand was chloroform greater than ethanol approximately methanol greater than ethyl acetate greater than methylene chloride greater than acetonitrile greater than acetone. Acetone and acetonitrile cause very little hydrocarbon leaching from HPLC-IAM.PC columns. When challenged with different mobile phases, IAM.PC columns perfused with mobile phase are more stable than IAM.PC-bonded phases stirred in mobile phases. IAM.PC contains lecithin linked to silica by amide bonds.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2558589

  15. Molecular dynamics of membrane proteins.

    SciTech Connect

    Woolf, Thomas B.; Crozier, Paul Stewart; Stevens, Mark Jackson

    2004-10-01

    Understanding the dynamics of the membrane protein rhodopsin will have broad implications for other membrane proteins and cellular signaling processes. Rhodopsin (Rho) is a light activated G-protein coupled receptor (GPCR). When activated by ligands, GPCRs bind and activate G-proteins residing within the cell and begin a signaling cascade that results in the cell's response to external stimuli. More than 50% of all current drugs are targeted toward G-proteins. Rho is the prototypical member of the class A GPCR superfamily. Understanding the activation of Rho and its interaction with its Gprotein can therefore lead to a wider understanding of the mechanisms of GPCR activation and G-protein activation. Understanding the dark to light transition of Rho is fully analogous to the general ligand binding and activation problem for GPCRs. This transition is dependent on the lipid environment. The effect of lipids on membrane protein activity in general has had little attention, but evidence is beginning to show a significant role for lipids in membrane protein activity. Using the LAMMPS program and simulation methods benchmarked under the IBIG program, we perform a variety of allatom molecular dynamics simulations of membrane proteins.

  16. Immobilization of Mucor miehei Lipase onto Macroporous Aminated Polyethersulfone Membrane for Enzymatic Reactions

    PubMed Central

    Handayani, Nurrahmi; Loos, Katja; Wahyuningrum, Deana; Buchari; Zulfikar, Muhammad Ali

    2012-01-01

    Immobilization of enzymes is one of the most promising methods in enzyme performance enhancement, including stability, recovery, and reusability. However, investigation of suitable solid support in enzyme immobilization is still a scientific challenge. Polyethersulfone (PES) and aminated PES (PES–NH2) were successfully synthesized as novel materials for immobilization. Membranes with various pore sizes (from 10–600 nm) based on synthesized PES and PES–NH2 polymers were successfully fabricated to be applied as bioreactors to increase the immobilized lipase performances. The influence of pore sizes, concentration of additives, and the functional groups that are attached on the PES backbone on enzyme loading and enzyme activity was studied. The largest enzyme loading was obtained by Mucor miehei lipase immobilized onto a PES–NH2 membrane composed of 10% of PES–NH2, 8% of dibutyl phthalate (DBP), and 5% of polyethylene glycol (PEG) (872.62 µg/cm2). Hydrolytic activity of the immobilized lipases indicated that the activities of biocatalysts are not significantly decreased by immobilization. From the reusability test, the lipase immobilized onto PES–NH2 showed a better constancy than the lipase immobilized onto PES (the percent recovery of the activity of the lipases immobilized onto PES–NH2 and PES are 97.16% and 95.37%, respectively), which indicates that this novel material has the potential to be developed as a bioreactor for enzymatic reactions. PMID:24958172

  17. Monte Carlo simulations of protein micropatterning in biomembranes: effects of immobile sticky obstacles

    NASA Astrophysics Data System (ADS)

    Arnold, Andreas M.; Sevcsik, Eva; Schütz, Gerhard J.

    2016-09-01

    Single molecule trajectories of lipids and proteins can yield valuable information about the nanoscopic organization of the plasma membrane itself. The interpretation of such trajectories, however, is complicated, as the mobility of molecules can be affected by the presence of immobile obstacles, and the transient binding of the tracers to these obstacles. We have previously developed a micropatterning approach that allows for immobilizing a plasma membrane protein and probing the diffusional behavior of a putative interaction partner in living cells. Here, we provide guidelines on how this micropatterning approach can be extended to quantify interaction parameters between plasma membrane constituents in their natural environment. We simulated a patterned membrane system and evaluated the effect of different surface densities of patterned immobile obstacles on the relative mobility as well as the surface density of diffusing tracers. In the case of inert obstacles, the size of the obstacle can be assessed from its surface density at the percolation threshold, which in turn can be extracted from the diffusion behavior of the tracer. For sticky obstacles, 2D dissociation constants can be determined from the tracer diffusion or surface density.

  18. [X-ray microanalysis of the activity of immobilized urease on chitosan membrane].

    PubMed

    Ma, Xiao-li; Yao, Zi-hua

    2005-03-01

    The localization of the activity of immobilized urease on chitosan membrane was studied by X-ray microanalysis. BaCl2 and urea were selected as the capture and substrate respectively. The substrate was hydrolyzed by immobilized urease to produce NH3 and CO2 in Tris-HCl buffer (pH 7.0), and the latter was captured by BaCl2 to form precipitate. The precipite was deposited on the active site of immobilized urease. It is shown that the method is practicable and reliable. The optimum condition for the localization of activity of immobilized urease was studied. PMID:16013332

  19. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    PubMed

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  20. Immobilization and activity assay of cytochrome P450 on patterned lipid membranes

    SciTech Connect

    Ueda, Yoshihiro; Morigaki, Kenichi . E-mail: morigaki-kenichi@aist.go.jp; Tatsu, Yoshiro; Yumoto, Noboru; Imaishi, Hiromasa . E-mail: himaish@kobe-u.ac.jp

    2007-04-20

    We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.

  1. Immobilization and activity assay of cytochrome P450 on patterned lipid membranes.

    PubMed

    Ueda, Yoshihiro; Morigaki, Kenichi; Tatsu, Yoshiro; Yumoto, Noboru; Imaishi, Hiromasa

    2007-04-20

    We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s. PMID:17335776

  2. The interactions of peripheral membrane proteins with biological membranes

    SciTech Connect

    Johs, Alexander; Whited, A. M.

    2015-01-01

    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 approaches 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.

  3. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGESBeta

    Johs, Alexander; Whited, A. M.

    2015-01-01

    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

  4. Affinity-Driven Immobilization of Proteins to Hematite Nanoparticles.

    PubMed

    Zare-Eelanjegh, Elaheh; Bora, Debajeet K; Rupper, Patrick; Schrantz, Krisztina; Thöny-Meyer, Linda; Maniura-Weber, Katharina; Richter, Michael; Faccio, Greta

    2016-08-10

    Functional nanoparticles are valuable materials for energy production, bioelectronics, and diagnostic devices. The combination of biomolecules with nanosized material produces a new hybrid material with properties that can exceed the ones of the single components. Hematite is a widely available material that has found application in various sectors such as in sensing and solar energy production. We report a single-step immobilization process based on affinity and achieved by genetically engineering the protein of interest to carry a hematite-binding peptide. Fabricated hematite nanoparticles were then investigated for the immobilization of the two biomolecules C-phycocyanin (CPC) and laccase from Bacillus pumilus (LACC) under mild conditions. Genetic engineering of biomolecules with a hematite-affinity peptide led to a higher extent of protein immobilization and enhanced the catalytic activity of the enzyme. PMID:27429157

  5. Site-specific, reversible and fluorescent immobilization of proteins on CrAsH-modified surfaces for microarray analytics.

    PubMed

    Schulte-Zweckel, Janine; Rosi, Federica; Sreenu, Domalapally; Schröder, Hendrik; Niemeyer, Christof M; Triola, Gemma

    2014-10-28

    A novel technique for protein immobilization onto CrAsH-modified surfaces is presented. This approach enables an efficient, reversible and fluorogenic immobilization of proteins. Moreover, expressed proteins can also be directly immobilized from cellular lysates without prior purification. The immobilized proteins are suitable for protein-protein interaction studies and the fluorescence enhancement upon immobilization can be employed for the direct detection of the immobilized protein without the need for secondary detection methods. PMID:25207673

  6. Preparation of Coaxial-Electrospun Poly[bis(p-methylphenoxy)]phosphazene Nanofiber Membrane for Enzyme Immobilization

    PubMed Central

    Wang, Shu-Gen; Jiang, Xin; Chen, Peng-Cheng; Yu, An-Guo; Huang, Xiao-Jun

    2012-01-01

    A core/sheath nanofiber membrane with poly[bis(p-methylphenoxy)]phosphazene (PMPPh) as the sheath and easily spinnable polyacrylonitrile (PAN) as the core was prepared via a coaxial electrospinning process. Field-emission scanning electron microscopy and transmission electron microscopy were used to characterize the morphology of the nanofiber membrane. It was found that the concentration of the PAN spinning solution and the ratio of the core/sheath solution flow rates played a decisive role in the coaxial electrospinning process. In addition, the stabilized core/sheath PMPPh nanofiber membrane was investigated as a support for enzyme immobilization because of its excellent biocompatibility, high surface/volume ratio, and large porosity. Lipase from Candida rugosa was immobilized on the nanofiber membrane by adsorption. The properties of the immobilized lipase on the polyphosphazene nanofiber membrane were studied and compared with those of a PAN nanofiber membrane. The results showed that the adsorption capacity (20.4 ± 2.7 mg/g) and activity retention (63.7%) of the immobilized lipase on the polyphosphazene nanofiber membrane were higher than those on the PAN membrane. PMID:23203055

  7. Enzymatic removal of paracetamol from aqueous phase: horseradish peroxidase immobilized on nanofibrous membranes.

    PubMed

    Xu, Ran; Si, Yifang; Li, Fengting; Zhang, Bingru

    2015-03-01

    Paracetamol is a widely used as an analgesic and an antipyretic that can easily accumulate in aquatic environments. This study aimed to enhance paracetamol removal efficiency from water by combining the biocatalytic activity of horseradish peroxidase (HRP) with the adsorption of nanofibrous membrane. Poly(vinyl alcohol)/poly(acrylic acid)/SiO2 electrospinning nanofibrous membrane was prepared with fiber diameters of 200 to 300 nm. The membrane was made insoluble by the thermal cross-linking process. HRP, which was previously activated by 1,1'-carbonyldiimidazole, was covalently immobilized on the surface of nanofibers. Immobilized HRP retained 79.4 % of the activity of free HRP. The physical, chemical, and biochemical properties of the immobilized HRP and its application in paracetamol removal were comprehensively investigated. Immobilized HRP showed better storage capability and higher tolerance to the changes in pH and temperature than free HRP. Paracetamol removal rate by immobilized HRP (83.5 %) was similar to that of free HRP (84.4 %), but immobilized HRP showed excellent reusability. The results signify that enzyme immobilized on nanofibers has great application potential in water treatment. PMID:25269844

  8. Characterization of protein-immobilized polystyrene nanoparticles using impedance spectroscopy.

    PubMed

    Park, Soo-In; Lee, Sang-Yup

    2014-10-01

    A novel approach for characterization of non-conductive protein-immobilized nanoparticles using AC impedance spectroscopy combined with conductive atomic force microscopy was examined. As AC impedance spectroscopy can provide information on diverse electrical properties such as capacitance and inductance, it is applicable to the characterization of non-conductive substances. Several non-conductive protein-immobilized polystyrene nanoparticles were analyzed using AC impedance spectroscopy, and their impedance spectra were used as markers for nanoparticle identification. Analyses of impedance signals using an electrical circuit model established that the capacitance and inductance of each nanoparticle changed with the adsorbed protein and that impedance spectral differences were characteristic properties of the proteins. From this study, AC impedance spectroscopy was shown to be a useful tool for characterization of non-conductive nanoparticles and is expected to be applicable to the development of sensors for nanomaterials. PMID:25942903

  9. Targeting membrane proteins for antibody discovery using phage display

    PubMed Central

    Jones, Martina L.; Alfaleh, Mohamed A.; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W.; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B.; Chin, David Y.; Mahler, Stephen M.

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  10. Targeting membrane proteins for antibody discovery using phage display.

    PubMed

    Jones, Martina L; Alfaleh, Mohamed A; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B; Chin, David Y; Mahler, Stephen M

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  11. Protein synthesis rates in atrophied gastrocnemius muscles after limb immobilization

    NASA Technical Reports Server (NTRS)

    Tucker, K. R.; Seider, M. J.; Booth, F. W.

    1981-01-01

    Noting that protein synthesis declines in the gastrocnemius 6 hr after immobilization, the study sought to detect an increase of protein synthesis when the limb was freed, and to examine the effects of exercise on the rate of increase. Rats were used as subjects, with their hind legs in plaster of Paris in plantar flexion to eliminate strain on the gastrocnemius. Periods of immobilization were varied and samples of blood from the muscle were taken to track protein synthesis rates for different groups in immobilization and exercise regimens (running and weightlifting). Synthesis rates declined 3.6% during time in the cast, then increased 6.3%/day after the casts were removed. Both running and weightlifting were found to increase the fractional rate of protein formation in the gastrocnemius muscle when compared with contralateral muscles that were not exercised and were used as controls, suggesting that the mechanism controlling protein synthesis in skeletal muscles is rapidly responsive to changes in muscular contractile activity.

  12. Adaptable Lipid Matrix Promotes Protein-Protein Association in Membranes.

    PubMed

    Kuznetsov, Andrey S; Polyansky, Anton A; Fleck, Markus; Volynsky, Pavel E; Efremov, Roman G

    2015-09-01

    The cell membrane is "stuffed" with proteins, whose transmembrane (TM) helical domains spontaneously associate to form functionally active complexes. For a number of membrane receptors, a modulation of TM domains' oligomerization has been shown to contribute to the development of severe pathological states, thus calling for detailed studies of the atomistic aspects of the process. Despite considerable progress achieved so far, several crucial questions still remain: How do the helices recognize each other in the membrane? What is the driving force of their association? Here, we assess the dimerization free energy of TM helices along with a careful consideration of the interplay between the structure and dynamics of protein and lipids using atomistic molecular dynamics simulations in the hydrated lipid bilayer for three different model systems - TM fragments of glycophorin A, polyalanine and polyleucine peptides. We observe that the membrane driven association of TM helices exhibits a prominent entropic character, which depends on the peptide sequence. Thus, a single TM peptide of a given composition induces strong and characteristic perturbations in the hydrophobic core of the bilayer, which may facilitate the initial "communication" between TM helices even at the distances of 20-30 Å. Upon tight helix-helix association, the immobilized lipids accommodate near the peripheral surfaces of the dimer, thus disturbing the packing of the surrounding. The dimerization free energy of the modeled peptides corresponds to the strength of their interactions with lipids inside the membrane being the lowest for glycophorin A and similarly higher for both homopolymers. We propose that the ability to accommodate lipid tails determines the dimerization strength of TM peptides and that the lipid matrix directly governs their association. PMID:26575933

  13. Active surfaces engineered by immobilizing protein-polymer nanoreactors for selectively detecting sugar alcohols.

    PubMed

    Zhang, Xiaoyan; Lomora, Mihai; Einfalt, Tomaz; Meier, Wolfgang; Klein, Noreen; Schneider, Dirk; Palivan, Cornelia G

    2016-05-01

    We introduce active surfaces generated by immobilizing protein-polymer nanoreactors on a solid support for sensitive sugar alcohols detection. First, such selective nanoreactors were engineered in solution by simultaneous encapsulation of specific enzymes in copolymer polymersomes, and insertion of membrane proteins for selective conduct of sugar alcohols. Despite the artificial surroundings, and the thickness of the copolymer membrane, functionality of reconstituted Escherichia coli glycerol facilitator (GlpF) was preserved, and allowed selective diffusion of sugar alcohols to the inner cavity of the polymersome, where encapsulated ribitol dehydrogenase (RDH) enzymes served as biosensing entities. Ribitol, selected as a model sugar alcohol, was detected quantitatively by the RDH-nanoreactors with GlpF-mediated permeability in a concentration range of 1.5-9 mM. To obtain "active surfaces" for detecting sugar alcohols, the nanoreactors optimized in solution were then immobilized on a solid support: aldehyde groups exposed at the compartment external surface reacted via an aldehyde-amino reaction with glass surfaces chemically modified with amino groups. The nanoreactors preserved their architecture and activity after immobilization on the glass surface, and represent active biosensing surfaces for selective detection of sugar alcohols, with high sensitivity. PMID:26950167

  14. Selective Immobilization of Proteins onto Solid Supports Through Split-Intein Mediated Protein Trans-Splicing

    SciTech Connect

    Kwon, Y; Coleman, M A; Camarero, J A

    2005-08-23

    Protein microarrays have emerged as important tools for screening protein-protein interactions and hold great potential for various applications including proteomics research, drug discovery, and diagnostics. This work describes a novel method for the traceless immobilization of proteins to a solid support through split-intein mediated protein trans-splicing. This method has been successfully used for the immobilization of biologically active proteins from very diluted samples ({approx}1{micro}M) and it does not require the purification of the protein to be attached. This makes possible the direct immobilization of proteins from complex mixtures such as cellular lysates and it can also be easily interfaced with cell-free expression systems for high-throughput production of protein microarrays.

  15. 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.

  16. Protein immobilization and fluorescence quenching on polydopamine thin films.

    PubMed

    Chen, Daqun; Zhao, Lei; Hu, Weihua

    2016-09-01

    Mussel inspired polydopamine (PDA) film has attracted great interest as a versatile functional coating for biomolecule immobilization in various bio-related devices. However, the details regarding the interaction between a protein and PDA film remain unclear. Particularly, there is very limited knowledge regarding the protein immobilization on PDA film, even though it is of essential importance in various fields. The situation is even more complicated if considering the fact that quite a number of approaches (e.g., different oxidizing reagent, buffer pH, grown time, grown media, etc.) have been developed to grow PDA films. In this work, protein attachment on PDA film was systematically investigated by using the real-time and label-free surface plasmon resonance (SPR) technique. The kinetics of protein-PDA interaction was explored and the influence of buffer pH and deposition media on the protein attachment was studied. Fluorescent protein microarray was further printed on PDA-coated glass slides for quantitative investigations and together with SPR data, the interesting fluorescence quenching phenomenon of PDA film was revealed. This work may deepen our understanding on the PDA-protein interaction and offer a valuable guide for efficient protein attachment on PDA film in various bio-related applications. PMID:27254254

  17. Enzymatic activation of cellulose acetate membrane for reducing of protein fouling.

    PubMed

    Koseoglu-Imer, Derya Y; Dizge, Nadir; Koyuncu, Ismail

    2012-04-01

    In this study, the surface of cellulose acetate (CA) ultrafiltration membrane was activated with serine protease (Savinase) enzyme to reduce protein fouling. Enzyme molecules were covalently immobilized with glutaraldehyde (cross-linking agent) onto the surface of CA membranes. The membrane activation was verified using filtration experiments and morphological analysis. Scanning electron microscopy (SEM) images and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy of the activated membrane when compared with raw membrane were confirmed that the enzyme was immobilized onto the membrane surface. The immobilization efficiencies changed from 13.2 to 41.2% according to the enzyme ratios from 2.5 to 10.0 mg/mL. However, the permeability values decreased from 232±6 to 121±4 L/m(2) h bar with increasing enzyme concentration from 2.5 to 10.0 mg/mL. In fouling experiments, bovine serum albumin (BSA) was used as the protein model solution and activated sludge was used as the model biological sludge. Enzyme-activated membranes exhibited good filtration performances and protein rejection efficiencies were compared with raw CA membrane. Also the relative flux reduction (RFR) ratios of membranes were calculated as 97% and 88% for raw CA and enzyme-activated membranes (5 mg/mL savinase), respectively. The membrane activated with Savinase enzyme could be proposed as a surface treatment method before filtration to mitigate protein fouling. PMID:22218336

  18. Multiscale Simulation of Protein Mediated Membrane Remodeling

    PubMed Central

    Ayton, Gary S.; Voth, Gregory A.

    2009-01-01

    Proteins interacting with membranes can result in substantial membrane deformations and curvatures. This effect is known in its broadest terms as membrane remodeling. This review article will survey current multiscale simulation methodologies that have been employed to examine protein-mediated membrane remodeling. PMID:19922811

  19. Influences of Membrane Mimetic Environments on Membrane Protein Structures

    PubMed Central

    Zhou, Huan-Xiang; Cross, Timothy A.

    2013-01-01

    The number of membrane protein structures in the Protein Data Bank is becoming significant and growing. Here, the transmembrane domain structures of the helical membrane proteins are evaluated to assess the influences of the membrane mimetic environments. Toward this goal, many of the biophysical properties of membranes are discussed and contrasted with those of the membrane mimetics commonly used for structure determination. Although the mimetic environments can perturb the protein structures to an extent that potentially gives rise to misinterpretation of functional mechanisms, there are also many structures that have a native-like appearance. From this assessment, an initial set of guidelines is proposed for distinguishing native-like from nonnative-like membrane protein structures. With experimental techniques for validation and computational methods for refinement and quality assessment and enhancement, there are good prospects for achieving native-like structures for these very important proteins. PMID:23451886

  20. Computational modeling of membrane proteins

    PubMed Central

    Leman, Julia Koehler; Ulmschneider, Martin B.; Gray, Jeffrey J.

    2014-01-01

    The determination of membrane protein (MP) structures has always trailed that of soluble proteins due to difficulties in their overexpression, reconstitution into membrane mimetics, and subsequent structure determination. The percentage of MP structures in the protein databank (PDB) has been at a constant 1-2% for the last decade. In contrast, over half of all drugs target MPs, only highlighting how little we understand about drug-specific effects in the human body. To reduce this gap, researchers have attempted to predict structural features of MPs even before the first structure was experimentally elucidated. In this review, we present current computational methods to predict MP structure, starting with secondary structure prediction, prediction of trans-membrane spans, and topology. Even though these methods generate reliable predictions, challenges such as predicting kinks or precise beginnings and ends of secondary structure elements are still waiting to be addressed. We describe recent developments in the prediction of 3D structures of both α-helical MPs as well as β-barrels using comparative modeling techniques, de novo methods, and molecular dynamics (MD) simulations. The increase of MP structures has (1) facilitated comparative modeling due to availability of more and better templates, and (2) improved the statistics for knowledge-based scoring functions. Moreover, de novo methods have benefitted from the use of correlated mutations as restraints. Finally, we outline current advances that will likely shape the field in the forthcoming decade. PMID:25355688

  1. Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

    PubMed Central

    Shen, Hsin-Hui; Lithgow, Trevor; Martin, Lisandra L.

    2013-01-01

    The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. PMID:23344058

  2. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    DOEpatents

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  3. Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

    PubMed Central

    Tonnesen, Asger; Christensen, Sune M.; Tkach, Vadym; Stamou, Dimitrios

    2014-01-01

    Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm−1) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 kBT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general. PMID:24411252

  4. Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor

    PubMed Central

    Bono, Petri; Rubin, Kristofer; Higgins, Jonathan M. G.; Hynes, Richard O.

    2001-01-01

    The actin cytoskeleton plays a significant role in changes of cell shape and motility, and interactions between the actin filaments and the cell membrane are crucial for a variety of cellular processes. Several adaptor proteins, including talin, maintain the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. To facilitate studies of layilin's function, we have generated a layilin-Fc fusion protein comprising the extracellular part of layilin joined to human immunoglobulin G heavy chain and used this chimera to identify layilin ligands. Here, we demonstrate that layilin-Fc fusion protein binds to hyaluronan immobilized to Sepharose. Microtiter plate-binding assays, coprecipitation experiments, and staining of sections predigested with different glycosaminoglycan-degrading enzymes and cell adhesion assays all revealed that layilin binds specifically to hyaluronan but not to other tested glycosaminoglycans. Layilin's ability to bind hyaluronan, a ubiquitous extracellular matrix component, reveals an interesting parallel between layilin and CD44, because both can bind to cytoskeleton-membrane linker proteins through their cytoplasmic domains and to hyaluronan through their extracellular domains. This parallelism suggests a role for layilin in cell adhesion and motility. PMID:11294894

  5. Membrane proteins: always an insoluble problem?

    PubMed

    Rawlings, Andrea E

    2016-06-15

    Membrane proteins play crucial roles in cellular processes and are often important pharmacological drug targets. The hydrophobic properties of these proteins make full structural and functional characterization challenging because of the need to use detergents or other solubilizing agents when extracting them from their native lipid membranes. To aid membrane protein research, new methodologies are required to allow these proteins to be expressed and purified cheaply, easily, in high yield and to provide water soluble proteins for subsequent study. This mini review focuses on the relatively new area of water soluble membrane proteins and in particular two innovative approaches: the redesign of membrane proteins to yield water soluble variants and how adding solubilizing fusion proteins can help to overcome these challenges. This review also looks at naturally occurring membrane proteins, which are able to exist as stable, functional, water soluble assemblies with no alteration to their native sequence. PMID:27284043

  6. Membrane proteins: always an insoluble problem?

    PubMed Central

    Rawlings, Andrea E.

    2016-01-01

    Membrane proteins play crucial roles in cellular processes and are often important pharmacological drug targets. The hydrophobic properties of these proteins make full structural and functional characterization challenging because of the need to use detergents or other solubilizing agents when extracting them from their native lipid membranes. To aid membrane protein research, new methodologies are required to allow these proteins to be expressed and purified cheaply, easily, in high yield and to provide water soluble proteins for subsequent study. This mini review focuses on the relatively new area of water soluble membrane proteins and in particular two innovative approaches: the redesign of membrane proteins to yield water soluble variants and how adding solubilizing fusion proteins can help to overcome these challenges. This review also looks at naturally occurring membrane proteins, which are able to exist as stable, functional, water soluble assemblies with no alteration to their native sequence. PMID:27284043

  7. Ultrafast permeation of water through protein-based membranes.

    PubMed

    Peng, Xinsheng; Jin, Jian; Nakamura, Yoshimichi; Ohno, Takahisa; Ichinose, Izumi

    2009-06-01

    Pressure-driven filtration by porous membranes is widely used in the production of drinking water from ground and surface water. Permeation theory predicts that filtration rate is proportional to the pressure difference across the filtration membrane and inversely proportional to the thickness of the membrane. However, these membranes need to be able to withstand high water fluxes and pressures, which means that the active separation layers in commercial filtration systems typically have a thickness of a few tens to several hundreds of nanometres. Filtration performance might be improved by the use of ultrathin porous silicon membranes or carbon nanotubes immobilized in silicon nitride or polymer films, but these structures are difficult to fabricate. Here, we report a new type of filtration membrane made of crosslinked proteins that are mechanically robust and contain channels with diameters of less than 2.2 nm. We find that a 60-nm-thick membrane can concentrate aqueous dyes from fluxes up to 9,000 l h(-1) m(-2) bar(-1), which is approximately 1,000 times higher than the fluxes that can be withstood by commercial filtration membranes with similar rejection properties. Based on these results and molecular dynamics simulations, we propose that protein-surrounded channels with effective lengths of less than 5.8 nm can separate dye molecules while allowing the ultrafast permeation of water at applied pressures of less than 1 bar. PMID:19498395

  8. Oriented immobilization of proteins on grafted porous polymers

    NASA Astrophysics Data System (ADS)

    Carbajal, M. Laura; Smolko, Eduardo E.; Grasselli, Mariano

    2003-08-01

    The modification of polymers by radiation grafting has been utilized for several decades. The penetrability of gamma rays allows to modify the internal surfaces of porous materials retaining its mechanical properties. In recent years applications of these materials to obtain chromatographic supports and biocatalysts have been reported. In this work, we described the grafting of glycidyl methacrylate (GMA) onto a macroporous polysulfone polymer. Reproducible amount of grafting, from 10% to 60% was obtained by choosing favourable monomer concentration and gamma radiation doses from 6 kGy up. Afterwards, iminodiacetic acid (IDA) and amino phenyl arsine oxide (PAO) were covalently attached to the grafted polyGMA, in correspondence with the grafting degree. Later on, a recombinant histidin-patch thioredoxin protein (HP-rTrx) was immobilized onto this surface by two different ways, involving specific protein orientations. The first one involves an IDA-Ni 2+ complex and three HP-rTrx's histidines and the other one involves a co-ordination site between PAO and two proximal HP-rTrx's cysteines, which corresponds to the active site of the enzyme. Specific polyclonal antibodies recognize HP-rTrx on the polymer. Proper orientation of the protein was confirmed by HP-rTrx activity measurements. The described procedure allows the successful oriented immobilization of a protein onto a macroporous polysulfone material.

  9. Membrane tension and peripheral protein density mediate membrane shape transitions

    PubMed Central

    Shi, Zheng; Baumgart, Tobias

    2015-01-01

    Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation, and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature coupling proteins and therefore a wide range of endocytic proteins. PMID:25569184

  10. Membrane tension and peripheral protein density mediate membrane shape transitions.

    PubMed

    Shi, Zheng; Baumgart, Tobias

    2015-01-01

    Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here, we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins, such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature-coupling proteins and therefore a wide range of endocytic proteins. PMID:25569184

  11. Membrane tension and peripheral protein density mediate membrane shape transitions

    NASA Astrophysics Data System (ADS)

    Shi, Zheng; Baumgart, Tobias

    2015-01-01

    Endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. The mechanism of the coupling between these two processes to enable homeostasis is not well understood. Recently, an ultrafast endocytosis (UFE) pathway was revealed with a speed significantly exceeding classical clathrin-mediated endocytosis (CME). Membrane tension reduction is a potential mechanism by which endocytosis can be rapidly activated at remote sites. Here, we provide experimental evidence for a mechanism whereby membrane tension reduction initiates membrane budding and tubulation mediated by endocytic proteins, such as endophilin A1. We find that shape instabilities occur at well-defined membrane tensions and surface densities of endophilin. From our data, we obtain a membrane shape stability diagram that shows remarkable consistency with a quantitative model. This model applies to all laterally diffusive curvature-coupling proteins and therefore a wide range of endocytic proteins.

  12. [Immobilization and characterization of carbonic anhydrase on the surface of hollow fiber membrane of polymethyl pentene].

    PubMed

    Wang, Qinmei; Zhang, Dihua; Zhang, Jingxia

    2009-07-01

    We immobilized carbonic anhydrase (CA) onto the surface of membrane oxygenator of polymethyl pentene (PMP) to enhance the removal of carbon dioxide in blood by two steps. We first introduced hydroxyl groups onto PMP surface by water plasma treatment, and then coupled CA onto PMP surface by using cyanate bromide (CNBr) as a crosslinker. After plasma treatment, the contact angle with water and chemical composition of PMP surface were characterized by analysis system of surface contact angle and XPS. Using p-nitrophenyl acetate (p-NPA) as a substrate, the activity, concentration, storage stability and re-usability of immobilized CA on PMP hollow fibers were studied by ultraviolet spectrophotometer. The preliminary data showed that hydroxyl groups could be introduced on the surface of PMP by water plasma treatment, and CA with catalysis activity could be successfully introduced onto PMP surface in high immobilization efficiency. The activity of covalently immobilized CA increased with the increase of concentration of CNBr, and the maximum was 73% of the theoretical activity of CA spread on PMP surface in monolayer in studied range. Covalently immobilized CA showed higher reusability compared to physically adsorbed CA, and higher storage stability compared to free CA in solution at 37 degrees C. The method would be used potentially in the membrane oxygenator to improve the capacity of removal of carbon dioxide in blood in the future. PMID:19835148

  13. Immobilized-cell membrane bioreactor for high-strength phenol wastewater

    SciTech Connect

    Loh, K.C.; Chung, T.S.; Ang, W.F.

    2000-01-01

    An immobilized-cell membrane bioreactor was fabricated to investigate degradation of phenol at high concentrations using Pseudomonas putida American Type Culture Collection 49451. In the case of suspension cultures, P. putida utilized phenol at concentrations below 1,000 mg/L, but experienced substrate inhibition at higher concentrations. On the other hand, cells immobilized in 25% by weight polysulfone fibers degraded phenol at concentrations above 1,000 mg/L. At an initial phenol concentration of 1,200 mg/L, phenol was fully degraded within 95 h in the immobilized system, whereas no cell growth and phenol degradation were observed in the free suspension system at 1,000 mg/L phenol. In the immobilized system, it was observed that cells diffused from the membranes when phenol concentration reached noninhibitory levels in a few experiments. In such cases, the time taken for complete degradation was shorter with cell diffusion because suspensions cells were responsible for the rapid phenol degradation. Further biodegradation studies at phenol concentrations of 2,000 and 3,500 mg/L were also performed to evaluate the effectiveness of cell immobilization for delaying the effects of substrate inhibition. Phenol could be completely degraded at both high concentrations.

  14. Dual matrix-based immobilized trypsin for complementary proteolytic digestion and fast proteomics analysis with higher protein sequence coverage.

    PubMed

    Fan, Chao; Shi, Zhaomei; Pan, Yiting; Song, Zifeng; Zhang, Wanjun; Zhao, Xinyuan; Tian, Fang; Peng, Bo; Qin, Weijie; Cai, Yun; Qian, Xiaohong

    2014-02-01

    In an age of whole-genome analysis, the mass spectrometry-based bottom-up strategy is now considered to be the most powerful method for in-depth proteomics analysis. As part of this strategy, highly efficient and complete proteolytic digestion of proteins into peptides is crucial for successful proteome profiling with deep coverage. To achieve this goal, prolonged digestion time and the use of multiple proteases have been adopted. The long digestion time required and tedious sample treatment steps severely limit the sample processing throughput. Though utilization of immobilized protease greatly reduces the digestion time, highly efficient proteolysis of extremely complex proteomic samples remains a challenging task. Here, we propose a dual matrix-based complementary digestion method using two types of immobilized trypsin with opposite matrix hydrophobicity prepared by attaching trypsin on hydrophobic or hydrophilic polymer-brush-modified nanoparticles. The polymer brushes on the nanoparticles serve as three-dimensional supports for a large amount of trypsin immobilization and lead to ultrafast and highly efficient protein digestion. More importantly, the two types of immobilized trypsin show high complementarity in protein digestion with only ∼60% overlap in peptide identification for yeast and membrane protein of mouse liver. Complementary digestion by applying these two types of immobilized trypsin together leads to obviously enhanced protein and peptide identification. Furthermore, the dual matrix-based complementary digestion shows particular advantage in the digestion of membrane proteins, as twice the number of identified peptides is obtained compared with solution digestion using free proteases, demonstrating its potential as a promising alternative to promote proteomics analysis with higher protein sequence coverage. PMID:24447065

  15. Immobilization of L-lysine on microporous PVDF membranes for neuron culture.

    PubMed

    Young, Tai-Horng; Lin, Ui-Hsiang; Lin, Dar-Jong; Chang, Hsu-Hsien; Cheng, Liao-Ping

    2009-01-01

    Microporous poly(vinylidene fluoride) (PVDF) membranes with dense or porous surface were prepared by immersion precipitation of PVDF/TEP solutions in coagulation baths containing different amounts of water. Onto the membrane surface, poly(glycidyl methacrylate) (PGMA) was grafted by plasma-induced free radical polymerization. Then, L-lysine was covalently bonded to the as-grafted PGMA through ring-opening reactions between epoxide and amine to form amino alcohol. The highest attainable graft density of PGMA on a PVDF membrane was 0.293 mg/cm2. This was obtained when the reaction was carried out on a porous surface under an optimized reaction condition. For immobilization of L-lysine, the yield was found to depend on the reaction temperature and L-lysine concentration. The maximal yield was 0.226 mg/cm2, a value considerably higher than reported in the literature using other immobilization methods. Furthermore, neurons were cultured on L-lysine-immobilized PVDF membranes. The results indicated that these membrane surfaces were suited to the growth of neurons, with a MTT value higher than that of the standard culture dish. PMID:19323885

  16. Protein-Induced Membrane Curvature Alters Local Membrane Tension

    PubMed Central

    Rangamani, Padmini; Mandadap, Kranthi K.; Oster, George

    2014-01-01

    Adsorption of proteins onto membranes can alter the local membrane curvature. This phenomenon has been observed in biological processes such as endocytosis, tubulation, and vesiculation. However, it is not clear how the local surface properties of the membrane, such as membrane tension, change in response to protein adsorption. In this article, we show that the partial differential equations arising from classical elastic model of lipid membranes, which account for simultaneous changes in shape and membrane tension due to protein adsorption in a local region, cannot be solved for nonaxisymmetric geometries using straightforward numerical techniques; instead, a viscous-elastic formulation is necessary to fully describe the system. Therefore, we develop a viscous-elastic model for inhomogeneous membranes of the Helfrich type. Using the newly available viscous-elastic model, we find that the lipids flow to accommodate changes in membrane curvature during protein adsorption. We show that, at the end of protein adsorption process, the system sustains a residual local tension to balance the difference between the actual mean curvature and the imposed spontaneous curvature. We also show that this change in membrane tension can have a functional impact such as altered response to pulling forces in the presence of proteins. PMID:25099814

  17. Interaction of immobilized phosphofructokinase with soluble muscle proteins.

    PubMed

    Gerlach, G; Hofer, H W

    1986-05-01

    Selected glycolytic enzymes (including phosphoglucose isomerase, aldolase, glyceraldehyde phosphate dehydrogenase, enolase, pyruvate kinase and lactate dehydrogenase), as well as glycogen phosphorylase, creatine kinase, and adenylate kinase, bound to phosphofructokinase immobilized on an agarose gel. The affinity of phosphofructokinase to these various proteins differed, with phosphorylase exhibiting the strongest binding. Binding was reversed either by: (1) elution with high-ionic-strength buffer (0.4 M KCl); (2) the addition of a 5-10 mM concentration of ATP; or (3) high concentrations of fructose 6-phosphate (5 mM). PMID:2938635

  18. Targeting of Rough Endoplasmic Reticulum Membrane Proteins and Ribosomes in Invertebrate Neurons

    PubMed Central

    Rolls, Melissa M.; Hall, David H.; Victor, Martin; Stelzer, Ernst H. K.; Rapoport, Tom A.

    2002-01-01

    The endoplasmic reticulum (ER) is divided into rough and smooth domains (RER and SER). The two domains share most proteins, but RER is enriched in some membrane proteins by an unknown mechanism. We studied RER protein targeting by expressing fluorescent protein fusions to ER membrane proteins in Caenorhabditis elegans. In several cell types RER and general ER proteins colocalized, but in neurons RER proteins were concentrated in the cell body, whereas general ER proteins were also found in neurites. Surprisingly RER membrane proteins diffused rapidly within the cell body, indicating they are not localized by immobilization. Ribosomes were also concentrated in the cell body, suggesting they may be in part responsible for targeting RER membrane proteins. PMID:12006669

  19. A chemoenzymatic approach to protein immobilization onto crystalline cellulose nanoscaffolds.

    PubMed

    Uth, Christina; Zielonka, Stefan; Hörner, Sebastian; Rasche, Nicolas; Plog, Andreas; Orelma, Hannes; Avrutina, Olga; Zhang, Kai; Kolmar, Harald

    2014-11-10

    The immobilization of bioactive molecules onto nanocellulose leads to constructs that combine the properties of the grafted compounds with the biocompatibility and low cytotoxicity of cellulose carriers and the advantages given by their nanometer dimensions. However, the methods commonly used for protein grafting suffer from lack of selectivity, long reaction times, nonphysiological pH ranges and solvents, and the necessity to develop a tailor-made reaction strategy for each individual case. To overcome these restrictions, a generic two-step procedure was developed that takes advantage of the highly efficient oxime ligation combined with enzyme-mediated protein coupling onto the surface of peptide-modified crystalline nanocellulose. The described method is based on efficient and orthogonal transformations, requires no organic solvents, and takes place under physiological conditions. Being site-directed and regiospecific, it could be applied to a vast number of functional proteins. PMID:25070515

  20. Solid State NMR and Protein-Protein Interactions in Membranes

    PubMed Central

    Miao, Yimin; Cross, Timothy A.

    2013-01-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water soluble proteins and other membrane proteins. PMID:24034903

  1. Solid state NMR and protein-protein interactions in membranes.

    PubMed

    Miao, Yimin; Cross, Timothy A

    2013-12-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high-resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water-soluble proteins and other membrane proteins. PMID:24034903

  2. Artificial membranes for membrane protein purification, functionality and structure studies.

    PubMed

    Parmar, Mayuriben J; Lousa, Carine De Marcos; Muench, Stephen P; Goldman, Adrian; Postis, Vincent L G

    2016-06-15

    Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method. PMID:27284055

  3. A capture coupling method for the covalent immobilization of hexahistidine tagged proteins for surface plasmon resonance.

    PubMed

    Kimple, Adam J; Muller, Robin E; Siderovski, David P; Willard, Francis S

    2010-01-01

    Surface plasmon resonance (SPR) is a robust method to detect and quantify macromolecular interactions; however, to measure binding interactions, one component must be immobilized on a sensor surface. This is typically achieved using covalent immobilization via free amines or thiols, or noncovalent immobilization using high-affinity interactions such as biotin/streptavidin or antibody/antigen. In this chapter we describe a robust method to covalently immobilize His(6) fusion proteins on the sensor surface for SPR analysis. PMID:20217615

  4. A capture coupling method for the covalent immobilization of hexahistidine tagged proteins for surface plasmon resonance

    PubMed Central

    Kimple, Adam J.; Muller, Robin E.; Siderovski, David P.; Willard, Francis S.

    2011-01-01

    i. Summary Surface Plasmon Resonance (SPR) is a robust method to detect and quantify macromolecular interactions; however, to measure binding interactions, one component must be immobilized on a sensor surface. This is typically achieved using covalent immobilization via free amines or thiols, or noncovalent immobilization using high affinity interactions such as biotin/streptavidin or antibody/antigen. In this Chapter we describe a robust method to covalently immobilize His6 fusion proteins on the sensor surface for SPR analysis. PMID:20217615

  5. Protein Homeostasis at the Plasma Membrane

    PubMed Central

    2014-01-01

    The plasma membrane (PM) and endocytic protein quality control (QC) in conjunction with the endosomal sorting machinery either repairs or targets conformationally damaged membrane proteins for lysosomal/vacuolar degradation. Here, we provide an overview of emerging aspects of the underlying mechanisms of PM QC that fulfill a critical role in preserving cellular protein homeostasis in health and diseases. PMID:24985330

  6. Optimizing immobilization of avidin on surface-modified magnetic nanoparticles: characterization and application of protein-immobilized nanoparticles.

    PubMed

    Yang, Tao; Sun, Shuguo; Ma, Meihu; Lin, Qinlu; Zhang, Lin; Li, Yan; Luo, Feijun

    2015-10-01

    A simple optimization method of immobilization of avidin on magnetic nanoparticles (MNPs)' surface was proposed in this study. The avidin-immobilized MNPs were then developed and used to immobilize a model enzyme [Horseradish peroxidase (HRP)]. The loading capacity (LC) and activity of avidin-immobilized MNPs were optimized through selecting the most appropriate nanoparticle's size and shape, glutaraldehyde concentration, cross-linking reaction time, ultrasonic processing time, and initial concentration of avidin. The LC under optimized conditions was 63.37 ± 1.29 mg avidin/g MNPs, and the immobilized protein was still able to maintain its high biological activity of 10.86 ± 0.13 U/mg (biotin-binding activity of nature avidin was 14.1 U/mg) and better thermal stability compared to free avidin. A highly reusable, stable, and easily recovered immobilized HRP was obtained using MNPs as carriers. The immobilized HRP was reused repeatedly more than 9 times and retained more than 65 % of its original activity. PMID:26224655

  7. Crystal Dehydration in Membrane Protein Crystallography.

    PubMed

    Sanchez-Weatherby, Juan; Moraes, Isabel

    2016-01-01

    Crystal dehydration has been successfully implemented to facilitate the structural solution of a number of soluble and membrane protein structures over the years. This chapter will present the currently available tools to undertake controlled crystal dehydration, focusing on some successful membrane protein cases. Also discussed here will be some practical considerations regarding membrane protein crystals and the relationship between different techniques in order to help researchers to select the most suitable technique for their projects. PMID:27553236

  8. Serial Femtosecond Crystallography of Membrane Proteins.

    PubMed

    Zhu, Lan; Weierstall, Uwe; Cherezov, Vadim; Liu, Wei

    2016-01-01

    Membrane proteins, including G protein-coupled receptors (GPCRs), constitute the most important drug targets. The increasing number of targets requires new structural information, which has proven tremendously challenging due to the difficulties in growing diffraction-quality crystals. Recent developments of serial femtosecond crystallography at X-ray free electron lasers combined with the use of membrane-mimetic gel-like matrix of lipidic cubic phase (LCP-SFX) for crystal growth and delivery hold significant promise to accelerate structural studies of membrane proteins. This chapter describes the development and current status of the LCP-SFX technology and elaborates its future role in structural biology of membrane proteins. PMID:27553241

  9. Class II virus membrane fusion proteins

    SciTech Connect

    Kielian, Margaret . E-mail: kielian@aecom.yu.edu

    2006-01-05

    Enveloped animal viruses fuse their membrane with a host cell membrane, thus delivering the virus genetic material into the cytoplasm and initiating infection. This critical membrane fusion reaction is mediated by a virus transmembrane protein known as the fusion protein, which inserts its hydrophobic fusion peptide into the cell membrane and refolds to drive the fusion reaction. This review describes recent advances in our understanding of the structure and function of the class II fusion proteins of the alphaviruses and flaviviruses. Inhibition of the fusion protein refolding reaction confirms its importance in fusion and suggests new antiviral strategies for these medically important viruses.

  10. Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions

    PubMed Central

    Minsky, Burcu Baykal; Antoni, Christiane H.; Boehm, Heike

    2016-01-01

    Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end. PMID:26883791

  11. Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions

    NASA Astrophysics Data System (ADS)

    Minsky, Burcu Baykal; Antoni, Christiane H.; Boehm, Heike

    2016-02-01

    Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end.

  12. Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Bobak, D A; Moss, J; Vaughan, M

    1989-09-19

    Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator. PMID:2514798

  13. Mapping membrane protein structure with fluorescence

    PubMed Central

    Taraska, Justin W.

    2012-01-01

    Membrane proteins regulate many cellular processes including signaling cascades, ion transport, membrane fusion, and cell-to-cell communications. Understanding the architecture and conformational fluctuations of these proteins is critical to understanding their regulation and functions. Fluorescence methods including intensity mapping, fluorescence resonance energy transfer, and photo-induced electron transfer, allow for targeted measurements of domains within membrane proteins. These methods can reveal how a protein is structured and how it transitions between different conformational states. Here, I will review recent work done using fluorescence to map the structures of membrane proteins, focusing on how each of these methods can be applied to understanding the dynamic nature of individual membrane proteins and protein complexes. PMID:22445227

  14. Thermodynamics and kinetics of protein incorporation into membranes.

    PubMed Central

    Jähnig, F

    1983-01-01

    The free energy and enthalpy of protein incorporation into membranes are calculated with special emphasis on the hitherto neglected effects of immobilization of protein and perturbation of lipid order in the membrane. The free energy change is found to be determined by the hydrophobic effect as the driving force for incorporation and the protein immobilization effect which leads to a considerable reduction of the free energy gained from the hydrophobic effect. For incorporation of a hydrophobic, bilayer-spanning alpha-helix, the free energy change obtained is of the order of -15 kcal/mol (1 cal = 4.184 J) in agreement with experimental results. The lipid perturbation effect yields only a small contribution to the free energy change due to an energy/entropy compensation inherent in lipid order. This effect dominates the enthalpy change, giving rise to values on the order of 100 kcal/mol with a pronounced temperature dependence around the lipid phase transition as observed experimentally. The kinetics of protein incorporation are even more strongly affected by the lipid perturbation effect, leading to an abrupt decrease of the rate of incorporation below the lipid phase transition. PMID:6574506

  15. Membrane Protein Insertion at the Endoplasmic Reticulum

    PubMed Central

    Shao, Sichen; Hegde, Ramanujan S.

    2014-01-01

    Integral membrane proteins of the cell surface and most intracellular compartments of eukaryotic cells are assembled at the endoplasmic reticulum. Two highly conserved and parallel pathways mediate membrane protein targeting to and insertion into this organelle. The classical cotranslational pathway, utilized by most membrane proteins, involves targeting by the signal recognition particle followed by insertion via the Sec61 translocon. A more specialized posttranslational pathway, employed by many tail-anchored membrane proteins, is composed of entirely different factors centered around a cytosolic ATPase termed TRC40 or Get3. Both of these pathways overcome the same biophysical challenges of ferrying hydrophobic cargo through an aqueous milieu, selectively delivering it to one among several intracellular membranes and asymmetrically integrating its transmembrane domain(s) into the lipid bilayer. Here, we review the conceptual and mechanistic themes underlying these core membrane protein insertion pathways, the complexities that challenge our understanding, and future directions to over-come these obstacles. PMID:21801011

  16. Unlocking the eukaryotic membrane protein structural proteome

    PubMed Central

    Lee, John Kyongwon; Stroud, Robert Michael

    2012-01-01

    Summary Most of the 231 unique membrane protein structures (as of 3/2010) are of bacterial membrane proteins (MPs) expressed in bacteria, or eukaryotic MPs from natural sources. However eukaryotic membrane proteins, especially those with more than three membrane crossings rarely succumb to any suitable expression in bacterial cells. They typically require expression in eukaryotic cells that can provide appropriate endoplasmic reticulum, chaperones, targeting and post-translational processing. In evidence, only ~20 eukaryotic MP structures have resulted from heterologous expression. This is required for a general approach to target particular human or pathogen membrane proteins of importance to human health. The first of these appeared in 2005. Our review addresses the special issues that pertain to the expression of eukaryotic and human membrane proteins, and recent advances in the tool kit for crystallization and structure determination. PMID:20739007

  17. Novel humic acid-bonded magnetite nanoparticles for protein immobilization.

    PubMed

    Bayrakci, Mevlut; Gezici, Orhan; Bas, Salih Zeki; Ozmen, Mustafa; Maltas, Esra

    2014-09-01

    The present paper is the first report that introduces (i) a useful methodology for chemical immobilization of humic acid (HA) to aminopropyltriethoxysilane-functionalized magnetite iron oxide nanoparticles (APS-MNPs) and (ii) human serum albumin (HSA) binding to the obtained material (HA-APS-MNPs). The newly prepared magnetite nanoparticle was characterized by using Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and elemental analysis. Results indicated that surface modification of the bare magnetite nanoparticles (MNPs) with aminopropyltriethoxysilane (APS) and HA was successfully performed. The protein binding studies that were evaluated in batch mode exhibited that HA-APS-MNPs could be efficiently used as a substrate for the binding of HSA from aqueous solutions. Usually, recovery values higher than 90% were found to be feasible by HA-APS-MNPs, while that value was around 2% and 70% in the cases of MNPs and APS-MNPs, respectively. Hence, the capacity of MNPs was found to be significantly improved by immobilization of HA. Furthermore, thermal degradation of HA-APS-MNPs and HSA bonded HA-APS-MNPs was evaluated in terms of the Horowitz-Metzger equation in order to determine kinetic parameters for thermal decomposition. Activation energies calculated for HA-APS-MNPs (20.74 kJmol(-1)) and HSA bonded HA-APS-MNPs (33.42 kJmol(-1)) implied chemical immobilization of HA to APS-MNPs, and tight interactions between HA and HA-APS-MNPs. PMID:25063152

  18. Surface Modification of Polypropylene Microporous Membrane by Atmospheric-Pressure Plasma Immobilization of N,N-dimethylamino Ethyl Methacrylate

    NASA Astrophysics Data System (ADS)

    Zhong, Shaofeng

    2010-10-01

    Surface modification of polypropylene microporous membrane (PPMM) was performed by atmospheric pressure dielectric barrier discharge plasma immobilization of N,N-dimethylamino ethyl methacrylate (DMAEMA). Structural and morphological changes on the membrane surface were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (FT-IR/ATR), X-ray photoelectron spectroscope (XPS) and field emission scanning electron microscopy (FE-SEM). Water contact angles of the membrane surfaces were also measured by the sessile drop method. Results reveal that both the plasma-treating conditions and the adsorbed DMAEMA amount have remarkable effects on the immobilization degree of DMAEMA. Peroxide determination by 1,1-diphenyl-2-picrvlhydrazyl (DPPH) method verifies the exsistence of radicals induced by plasma, which activize the immobilization reaction. Pure water contact angle on the membrane surface decreased with the increase of DMAEMA immobilization degree, which indicates an enhanced hydrophilicity for the modified membranes. The effects of immobilization degrees on pure water fluxes were also measured. It is shown that pure water fluxes first increased with immobilization degree and then decreased. Finally, permeation of bovine serum albumin (BSA) and lysozyme solution were measured to evaluate the antifouling property of the DMAEMA-modified membranes, from which it is shown that both hydrophilicity and electrostatic repulsion are beneficial for membrane antifouling.

  19. [Lipid composition in erythrocytic membranes of rats with various stress resistance during repeated immobilization].

    PubMed

    Tsygvintsev, A A; Bryndina, I G

    2011-01-01

    The dependence between variation of erythrocyte phospholipid composition and stress resistance was studied in chronic experiment on nonline male albino rats, previously differed by their behavior in the 'open field' test. A significant exhausting of membrane pool by the basic classes of phospholipids was registered under influence of 2 hours daily immobilization during 5, 10, 20, 30 days, however, their metabolism for resistant and predisposed to stress animals flows variously. PMID:21688664

  20. Nanoscale electron transport measurements of immobilized cytochrome P450 proteins

    NASA Astrophysics Data System (ADS)

    Bostick, Christopher D.; Flora, Darcy R.; Gannett, Peter M.; Tracy, Timothy S.; Lederman, David

    2015-04-01

    Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport (ETp) depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of ETp processes in the enzyme, in addition to occupying the active site.

  1. Nanoscale Electron Transport Measurements of Immobilized Cytochrome P450 Proteins

    PubMed Central

    Bostick, Christopher D.; Flora, Darcy R.; Gannett, Peter M.; Tracy, Timothy S.; Lederman, David

    2015-01-01

    Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site. PMID:25804257

  2. Specific covalent immobilization of proteins through dityrosine cross-links.

    PubMed

    Endrizzi, Betsy J; Huang, Gang; Kiser, Patrick F; Stewart, Russell J

    2006-12-19

    Dityrosine cross-links are widely observed in nature in structural proteins such as elastin and silk. Natural oxidative cross-linking between tyrosine residues is catalyzed by a diverse group of metalloenzymes. Dityrosine formation is also catalyzed in vitro by metal-peptide complexes such as Gly-Gly-His-Ni(II). On the basis of these observations, a system was developed to specifically and covalently surface immobilize proteins through dityrosine cross-links. Methacrylate monomers of the catalytic peptide Gly-Gly-His-Tyr-OH (GGHY) and the Ni(II)-chelating group nitrilotriacetic acid (NTA) were copolymerized with acrylamide into microbeads. Green fluorescent protein (GFP), as a model protein, was genetically tagged with a tyrosine-modified His6 peptide on its carboxy terminus. GFP-YGH6, specifically associated with the NTA-Ni(II) groups, was covalently coupled to the bead surface through dityrosine bond formation catalyzed by the colocalized GGHY-Ni(II) complex. After extensive washing with EDTA to disrupt metal coordination bonds, we observed that up to 75% of the initially bound GFP-YGH6 remained covalently bound to the bead while retaining its structure and activity. Dityrosine cross-linking was confirmed by quenching the reaction with free tyrosine. The method may find particular utility in the construction and optimization of protein microarrays. PMID:17154619

  3. Immobilization of imidazole moieties in polymer electrolyte composite membrane for elevated temperature fuel cells

    NASA Astrophysics Data System (ADS)

    Li, Ke; Zhou, Bei; Ye, Gongbo; Pan, Mu; Zhang, Haining

    2015-12-01

    Development of membrane electrolyte with reasonable proton conductivity at elevated temperature without external humidification is essential for practical applications of elevated temperature proton exchange membrane fuel cells. Herein, a novel polymer electrolyte composite membrane using imidazole as anhydrous proton carriers for elevated temperature fuel cells is investigated. The imidazole moieties are immobilized inside the Nafion/poly(tetrafluoroethylene) (PTFE) composite membrane through in situ formation of imidazole functionalized silica nanoparticles in Nafion dispersion. The thus-formed membrane exhibits strong Coulombic interaction between negatively charged sulfonic acid groups of Nafion and protonated imidazole moieties, leading to an anhydrous proton conductivity of 0.018 S cm-1 at 180 °C. With the introduction of PTFE matrix, the mechanical strength of the membrane is greatly improved. The peak power density of a single cell assembled from the hybrid membrane is observed to be 130 mW cm-2 under 350 mA cm-2 at 110 °C without external humidification and it remains stable for 20 h continuous operation. The obtained results demonstrate that the developed composite membranes could be utilized as promising membrane electrolytes for elevated temperature fuel cells.

  4. Removal of water contaminants by nanoscale zero-valent iron immobilized in PAN-based oxidized membrane

    NASA Astrophysics Data System (ADS)

    Liu, Chunyi; Li, Xiang; Ma, Bomou; Qin, Aiwen; He, Chunju

    2014-12-01

    The functionalizing nanoporous polyacrylonitrile-based oxidized membrane (PAN-OM) firmly immobilized with highly reactive nanoscale zero-valent iron (NZVI) are successfully prepared via an innovative in situ synthesis method. Due to the formation of ladder structure, the PAN-OM present excellent thermal and chemical stabilities as a new carrier for the in-situ growth of NZVI via firm chelation and reduction action, respectively, which prevent the aggregation and release of NZVI. The developed NZVI-immobilized membrane present effective decolorizing efficiency to both anionic methyl blue and cationic methylene blue with a pseudo-first-order decay and degrading efficiency to trichloroethylene (TCE). The regeneration and stability results show that NZVI-immobilized membrane system can be regenerated without obvious performance reduction, which remain the reactivity after half a year storage period. These results suggest that PAN-based oxidized membrane immobilized with NZVI exhibit significant potential for environmental applications.

  5. Circular dichroism spectroscopy of membrane proteins.

    PubMed

    Miles, A J; Wallace, B A

    2016-09-21

    Circular dichroism (CD) spectroscopy is a well-established technique for studying the secondary structures, dynamics, folding pathways, and interactions of soluble proteins, and is complementary to the high resolution but generally static structures produced by X-ray crystallography, NMR spectroscopy, and cryo electron microscopy. CD spectroscopy has special relevance for the study of membrane proteins, which are difficult to crystallise and largely ignored in structural genomics projects. However, the requirement for membrane proteins to be embedded in amphipathic environments such as membranes, lipid vesicles, detergent micelles, bicelles, oriented bilayers, or nanodiscs, in order for them to be soluble or dispersed in solution whilst maintaining their structure and function, necessitates the use of different experimental and analytical approaches than those employed for soluble proteins. This review discusses specialised methods for collecting and analysing membrane protein CD data, highlighting where protocols for soluble and membrane proteins diverge. PMID:27347568

  6. Phosphoinositide Control of Membrane Protein Function

    PubMed Central

    Logothetis, Diomedes E.; Petrou, Vasileios I.; Zhang, Miao; Mahajan, Rahul; Meng, Xuan-Yu; Adney, Scott K.; Cui, Meng; Baki, Lia

    2015-01-01

    Anionic phospholipids are critical constituents of the inner leaflet of the plasma membrane, ensuring appropriate membrane topology of transmembrane proteins. Additionally, in eukaryotes, the negatively charged phosphoinositides serve as key signals not only through their hydrolysis products but also through direct control of transmembrane protein function. Direct phosphoinositide control of the activity of ion channels and transporters has been the most convincing case of the critical importance of phospholipid-protein interactions in the functional control of membrane proteins. Furthermore, second messengers, such as [Ca2+]i, or posttranslational modifications, such as phosphorylation, can directly or allosterically fine-tune phospholipid-protein interactions and modulate activity. Recent advances in structure determination of membrane proteins have allowed investigators to obtain complexes of ion channels with phosphoinositides and to use computational and experimental approaches to probe the dynamic mechanisms by which lipid-protein interactions control active and inactive protein states. PMID:25293526

  7. Eggshell membrane: A natural substrate for immobilization and detection of DNA.

    PubMed

    Ray, Preetam Guha; Roy, Somenath

    2016-02-01

    Chemically modified eggshell membranes (ESM) have been explored as potentially novel platforms for immobilization of oligonucleotides and subsequent detection of target DNA. The fibrous network of the native ESM as well those functionalized with acetic acid or n-butyl acetate has been examined by field-emission scanning electron microscopy (FESEM). The formation of surface functional moieties has been confirmed by Fourier-transform infrared spectroscopy (FTIR). DNA molecules, with an end terminal -NH2 group (at 5' end) have been immobilized on the chemically modified ESM surface. The effect of surface modification on the DNA immobilization efficiency has been investigated using fluorescence microscopy and atomic force microscopy (AFM). The above studies concurrently suggest that functionalization of ESM with n-butyl acetate causes a better homogeneity of the DNA probes on the membrane surface. On-chip hybridization of the target DNA with the surface bound capture probes has been performed on the functionalized membranes. It is observed that n-butyl acetate modification of ESM pushes the limit of detection (LOD) of the DNA sensors by at least an order of magnitude compared to the other modification method. PMID:26652390

  8. Diffusion-limited attachment of nanoparticles to flexible membrane-immobilized receptors

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2016-04-01

    In biosystems, vesicles, virions, or metal particles of size ∼100 nm often diffuse in solution and interact with short (<10 nm) flexible receptors immobilized in a lipid membrane. The attachment kinetics of such nanoparticles can be limited by diffusion globally or locally. In the latter case, the calculation of the attachment rate is complicated by the dependence of the diffusion coefficient on the distance between a particle and membrane. The analysis, taking this factor into account, shows that the attachment rate constant is proportional to the receptor length and nearly independent of the particle radius.

  9. Effects of protein crowding on membrane systems.

    PubMed

    Guigas, Gernot; Weiss, Matthias

    2016-10-01

    Cellular membranes are typically decorated with a plethora of embedded and adsorbed macromolecules, e.g. proteins, that participate in numerous vital processes. With typical surface densities of 30,000 proteins per μm(2) cellular membranes are indeed crowded places that leave only few nanometers of private space for individual proteins. Here, we review recent advances in our understanding of protein crowding in membrane systems. We first give a brief overview on state-of-the-art approaches in experiment and simulation that are frequently used to study crowded membranes. After that, we review how crowding can affect diffusive transport of proteins and lipids in membrane systems. Next, we discuss lipid and protein sorting in crowded membrane systems, including effects like protein cluster formation, phase segregation, and lipid droplet formation. Subsequently, we highlight recent progress in uncovering crowding-induced conformational changes of membranes, e.g. membrane budding and vesicle formation. Finally, we give a short outlook on potential future developments in the field of crowded membrane systems. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26724385

  10. Tandem Facial Amphiphiles for Membrane Protein Stabilization

    PubMed Central

    Chae, Pil Seok; Gotfryd, Kamil; Pacyna, Jennifer; Miercke, Larry J. W.; Rasmussen, Søren G. F.; Robbins, Rebecca A.; Rana, Rohini R.; Loland, Claus J.; Kobilka, Brian; Stroud, Robert; Byrne, Bernadette; Gether, Ulrik; Gellman, Samuel H.

    2010-01-01

    We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles. PMID:21049926

  11. Protein Solvation in Membranes and at Water-Membrane Interfaces

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Chipot, Christophe; Wilson, Michael A.

    2002-01-01

    Different salvation properties of water and membranes mediate a host of biologically important processes, such as folding, insertion into a lipid bilayer, associations and functions of membrane proteins. These processes will be discussed in several examples involving synthetic and natural peptides. In particular, a mechanism by which a helical peptide becomes inserted into a model membrane will be described. Further, the molecular mechanism of recognition and association of protein helical segments in membranes will be discussed. These processes are crucial for proper functioning of a cell. A membrane-spanning domain of glycophorin A, which exists as a helical dimer, serves as the model system. For this system, the free energy of dissociation of the helices is being determined for both the wild type and a mutant, in which dimerization is disrupted.

  12. Crystallization of Membrane protein under Microgravity

    NASA Astrophysics Data System (ADS)

    Henning, C.; Frank, J.; Laubender, G.; Fromme, P.

    2002-01-01

    Proteins are biological molecules which catalyse all essential reactions of cells. The knowledge on the structure of these molecular machines is necessary for the understanding of their function. Many diseases are caused by defects of membrane proteins. In order to develop new medical therapies the construction principle of the proteins must be known. The main difficulty in the determination of the structure of these membrane protein complexes is the crystallisation. Membrane proteins are normally not soluble in water and have therefore to be solubilised from the membranes by use of detergents. The whole protein-detergent micelle must be crystallised to maintain the functional integrity of the protein complexes. These difficulties are the reasons for the fact that crystals of membrane proteins are difficult to grow and most of them are badly ordered, being not appropriate for X-ray structure analysis. The crystallisation of proteins under microgravity leads to the growth of better-ordered crystals by reduction of nucleation rate and the undisturbed growth of the hovering seeds by the absence of sedimentation and convection. The successful crystallistation of a membrane protein under microgravity has been performed during the space shuttle missions USML2 and STS95 in the Space Shuttle with Photosystem I as model protein. Photosystem I is a large membrane protein complex which catalyses one of the first and fundamental steps in oxygen photosynthesis. The crystals of Photosystem I, grown under microgravity were twenty times larger than all Photosystem I crystals which have been grown on earth. They were the basis for the determination of an improved X-ray structure of Photo- system I. These experiments opened the way for the structure enlightenment of more membrane proteins on the basis of microgravity experiments. On board of the International Space Station ideal conditions for the crystallisation of proteins under zero gravity are existing.

  13. Cell wall constrains lateral diffusion of plant plasma-membrane proteins

    PubMed Central

    Martinière, Alexandre; Lavagi, Irene; Nageswaran, Gayathri; Rolfe, Daniel J.; Maneta-Peyret, Lilly; Luu, Doan-Trung; Botchway, Stanley W.; Webb, Stephen E. D.; Mongrand, Sebastien; Maurel, Christophe; Martin-Fernandez, Marisa L.; Kleine-Vehn, Jürgen; Friml, Jirí; Moreau, Patrick; Runions, John

    2012-01-01

    A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein–protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction. PMID:22689944

  14. Protein immobilization and detection on laser processed polystyrene surfaces

    SciTech Connect

    Sarantopoulou, Evangelia; Kollia, Zoe; Palles, Dimitrios; Spyropoulos-Antonakakis, Nikolaos; Cefalas, Alkiviadis-Constantinos; Petrou, Panagiota S.; Kakabakos, Sotirios

    2011-09-15

    The bovine serum albumin (BSA)-polystyrene (PS) interface layer is laser photo activated at 157 nm for site selective multiple target-protein immobilization. The 5-15 nm photon induced interface layer has different chemical, wetting, and stiffness properties than the PS photon processed surface. The irradiated areas exhibit target-protein binding, followed by localized probe-target protein detection. The photon induced chemical modification of the BSA-PS interface layer is identified by: (1) Morphological, imaging, and analysis of surface parameters with atomic force microscopy, (2) spectroscopic shift (4 cm{sup -1}), of the amide I group and formation of new C=N, NH{sub 2}, C-O, C=O, and O-C=O groups following irradiation, identified with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and (3) the different hydrophilic/hydrophobic and force-distance response of the bare PS and BSA-PS surfaces. Near field edge diffraction (Fresnel) fluorescence imaging specifies the threshold photon energy and the fluence required to optically detect the protein binding on the photon induced BSA-PS interface layer. By approximating the Fresnel integrals with analytical functions, the threshold photon energy and the fluence are expressed as the sum of zero, first, and second order harmonic terms of two characteristic diffracted modes and they are specified to be 8.73x10{sup -9} Jand623 J m{sup -2}, respectively. Furthermore, a bioarray of three probe-target proteins is fabricated with 1.5 {mu}m spatial resolution using a 157 nm laser microstepper. The methodology eliminates the use of intermediate polymer layers between the blocking BSA protein and the PS substrate in bioarray fabrication.

  15. IFITM Proteins Restrict Viral Membrane Hemifusion

    PubMed Central

    Golfetto, Ottavia; Bungart, Brittani; Li, Minghua; Ding, Shilei; He, Yuxian; Liang, Chen; Lee, James C.; Gratton, Enrico; Cohen, Fredric S.; Liu, Shan-Lu

    2013-01-01

    The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous

  16. Membrane Protein Crystallization Using Laser Irradiation

    NASA Astrophysics Data System (ADS)

    Adachi, Hiroaki; Murakami, Satoshi; Niino, Ai; Matsumura, Hiroyoshi; Takano, Kazufumi; Inoue, Tsuyoshi; Mori, Yusuke; Yamaguchi, Akihito; Sasaki, Takatomo

    2004-10-01

    We demonstrate the crystallization of a membrane protein using femtosecond laser irradiation. This method, which we call the laser irradiated growth technique (LIGHT), is useful for producing AcrB crystals in a solution of low supersaturation range. LIGHT is characterized by reduced nucleation times. This feature is important for crystallizing membrane proteins because of their labile properties when solubilized as protein-detergent micelles. Using LIGHT, high-quality crystals of a membrane transporter protein, AcrB, were obtained. The resulting crystals were found to be of sufficiently high resolution for X-ray diffraction. The results reported here indicate that LIGHT is a powerful tool for membrane protein crystallization, as well as for the growth of soluble proteins.

  17. Genome-wide Membrane Protein Structure Prediction

    PubMed Central

    Piccoli, Stefano; Suku, Eda; Garonzi, Marianna; Giorgetti, Alejandro

    2013-01-01

    Transmembrane proteins allow cells to extensively communicate with the external world in a very accurate and specific way. They form principal nodes in several signaling pathways and attract large interest in therapeutic intervention, as the majority pharmaceutical compounds target membrane proteins. Thus, according to the current genome annotation methods, a detailed structural/functional characterization at the protein level of each of the elements codified in the genome is also required. The extreme difficulty in obtaining high-resolution three-dimensional structures, calls for computational approaches. Here we review to which extent the efforts made in the last few years, combining the structural characterization of membrane proteins with protein bioinformatics techniques, could help describing membrane proteins at a genome-wide scale. In particular we analyze the use of comparative modeling techniques as a way of overcoming the lack of high-resolution three-dimensional structures in the human membrane proteome. PMID:24403851

  18. Platelet adhesion and cellular interaction with poly(ethylene oxide) immobilized onto silicone rubber membrane surfaces.

    PubMed

    Hsiue, G H; Lee, S D; Chang, P C

    1996-01-01

    Cellular interaction and platelet adsorption were investigated on poly(ethylene oxide) (PEO) immobilized silicone rubber membrane (SR) which has polyacrylic acid grafts on the surfaces. Polyacrylic acid (PAA) had been introduced to the SR surface after Ar plasma treatment of SR surfaces to introduce peroxide groups. Surface characterizations were made using ATR-FTIR, ESCA, SEM, and contact angle measurements. Experimental results obtained by ESCA high resolution curve fitting spectra indicated that the amount of bisamino PEO of different molecular weights immobilized onto SR surfaces were similar, which showed that the influence of the length of molecular chains (-C-C-O-) on the reactivity of terminal amino group is negligible. The wettability of modified SR surfaces increased with an increase in PEO molecular weight. Biological studies such as corneal epithelial cell culture and blood platelet adhesion were performed to understand the biocompatibility of modified SR surfaces. Biological studies using corneal epithelial cells showed that cell migration, attachment and proliferation onto PEO-20000 immobilized SR surface were suppressed, whereas these biological activities on PEO-600 were enhanced. Another study on platelet adhesion revealed that many platelets attached to PEO-600 immobilized SR, while platelet deposition was rarely observed on SR grafted with PEO-3350. The effects of different PEO molecular chains on biological response were discussed. PMID:8836831

  19. Multipass Membrane Protein Structure Prediction Using Rosetta

    PubMed Central

    Yarov-Yarovoy, Vladimir; Schonbrun, Jack; Baker, David

    2006-01-01

    We describe the adaptation of the Rosetta de novo structure prediction method for prediction of helical transmembrane protein structures. The membrane environment is modeled by embedding the protein chain into a model membrane represented by parallel planes defining hydrophobic, interface, and polar membrane layers for each energy evaluation. The optimal embedding is determined by maximizing the exposure of surface hydrophobic residues within the membrane and minimizing hydrophobic exposure outside of the membrane. Protein conformations are built up using the Rosetta fragment assembly method and evaluated using a new membrane-specific version of the Rosetta low-resolution energy function in which residue–residue and residue–environment interactions are functions of the membrane layer in addition to amino acid identity, distance, and density. We find that lower energy and more native-like structures are achieved by sequential addition of helices to a growing chain, which may mimic some aspects of helical protein biogenesis after translocation, rather than folding the whole chain simultaneously as in the Rosetta soluble protein prediction method. In tests on 12 membrane proteins for which the structure is known, between 51 and 145 residues were predicted with root-mean-square deviation <4Å from the native structure. PMID:16372357

  20. Functional dynamics of cell surface membrane proteins

    NASA Astrophysics Data System (ADS)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  1. Inherently Tunable Electrostatic Assembly of Membrane Proteins

    SciTech Connect

    Liang, H.; Whited, G.; Nguyen, C.; Okerlund, A.; Stucky, G.D.

    2009-05-19

    Membrane proteins are a class of nanoscopic entities that control the matter, energy, and information transport across cellular boundaries. Electrostatic interactions are shown to direct the rapid co-assembly of proteorhodopsin (PR) and lipids into long-range crystalline arrays. The roles of inherent charge variations on lipid membranes and PR variants with different compositions are examined by tuning recombinant PR variants with different extramembrane domain sizes and charged amino acid substitutions, lipid membrane compositions, and lipid-to-PR stoichiometric ratios. Rational control of this predominantly electrostatic assembly for PR crystallization is demonstrated, and the same principles should be applicable to the assembly and crystallization of other integral membrane proteins.

  2. Protein-Free Cell Culture on an Artificial Substrate with Covalently Immobilized Insulin

    NASA Astrophysics Data System (ADS)

    Ito, Yoshihiro; Zheng, Ji; Imanishi, Yukio; Yonezawa, Kazuyoshi; Kasuga, Masato

    1996-04-01

    Insulin was immobilized on a surface-hydrolyzed poly(methyl methacrylate) film. Chinese hamster ovary cells overexpressing human insulin receptors were cultured on the film in the absence of serum or soluble proteins. Small amounts of immobilized insulin (1-10% of the required amount of free insulin) were sufficient to stimulate cell proliferation. In addition, the maximal mitogenic effect of immobilized insulin was greater than that of free insulin. Immobilized insulin activated the insulin receptor and down-stream signaling proteins, and this activation persisted for longer periods than that obtained with free insulin, probably explaining the greater mitogenic effect of the immobilized insulin. Finally the immobilized-insulin film was usable repeatedly without marked loss of activity.

  3. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

    Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

  4. Active Nuclear Import of Membrane Proteins Revisited.

    PubMed

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast. PMID:26473931

  5. Immobilized N-alkylated polyethylenimine avidly kills bacteria by rupturing cell membranes with no resistance developed.

    PubMed

    Milović, Nebojsa M; Wang, Jun; Lewis, Kim; Klibanov, Alexander M

    2005-06-20

    Several critical mechanistic and phenomenological aspects of the microbicidal surface coatings based on immobilized hydrophobic polycations, previously developed by us, are addressed. Using Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria, remarkable bactericidal action (up to a 10(9)-fold reduction in live bacteria count in the surface-exposed solution and a 100% inactivation of the surface-adhered bacteria) of an amino-glass slide covalently derivatized with N-hexyl,methyl-polyethylenimine (PEI) is found to be due to rupturing bacterial cell membranes by the polymeric chains. The bacteria fail to develop noticeable resistance to this lethal action over the course of many successive generations. Finally, the immobilized N-alkyl-PEI, while deadly to bacteria, is determined to be harmless to mammalian (monkey kidney) cells. PMID:15803464

  6. Helical Membrane Protein Conformations and their Environment

    PubMed Central

    Cross, Timothy A.; Murray, Dylan T.; Watts, Anthony

    2013-01-01

    Evidence that membrane proteins respond conformationally and functionally to their environment is gaining pace. Structural models, by necessity, have been characterized in preparations where the protein has been removed from its native environment. Different structural methods have used various membrane mimetics that have recently included lipid bilayers as a more native-like environment. Structural tools applied to lipid bilayer-embedded integral proteins are informing us about important generic characteristics of how membrane proteins respond to the lipid environment as compared with their response to other non-lipid environments. Here, we review the current status of the field, with specific reference to observations of some well-studied α-helical membrane proteins, as a starting point to aid the development of possible generic principals for model refinement. PMID:23996195

  7. Membrane protein expression in Lactococcus lactis.

    PubMed

    King, Martin S; Boes, Christoph; Kunji, Edmund R S

    2015-01-01

    The Gram-positive bacterium Lactococcus lactis has many properties that are ideal for the overproduction of membrane proteins in a functional form. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. The available promoter systems are strong and tightly regulated, allowing expression of toxic gene products in a controlled manner. Expressed membrane proteins are targeted exclusively to the cytoplasmic membrane, allowing the use of ionophores, ligands, and inhibitors to study activity of the membrane protein in whole cells. Constructed plasmids are stable and expression levels are highly reproducible. The relatively small genome size of the organism causes little redundancy, which facilitates complementation studies and allows for easier purification. The produced membrane proteins are often stable, as the organism has limited proteolytic capability, and they are readily solubilized from the membrane with mild detergents. Lactococci are multiple amino acid auxotrophs, allowing the incorporation of labels, such as selenomethionine. Among the few disadvantages are the low transformation frequency, AT-rich codon usage, and resistance to lysis by mechanical means, but these problems can be overcome fairly easily. We will describe in detail the protocols used to express membrane proteins in L. lactis, from cloning of the target gene to the isolation of membrane vesicles for the determination of expression levels. PMID:25857778

  8. Polyene antibiotic that inhibits membrane transport proteins

    PubMed Central

    te Welscher, Yvonne Maria; van Leeuwen, Martin Richard; de Kruijff, Ben; Dijksterhuis, Jan; Breukink, Eefjan

    2012-01-01

    The limited therapeutic arsenal and the increase in reports of fungal resistance to multiple antifungal agents have made fungal infections a major therapeutic challenge. The polyene antibiotics are the only group of antifungal antibiotics that directly target the plasma membrane via a specific interaction with the main fungal sterol, ergosterol, often resulting in membrane permeabilization. In contrast to other polyene antibiotics that form pores in the membrane, the mode of action of natamycin has remained obscure but is not related to membrane permeabilization. Here, we demonstrate that natamycin inhibits growth of yeasts and fungi via the immediate inhibition of amino acid and glucose transport across the plasma membrane. This is attributable to ergosterol-specific and reversible inhibition of membrane transport proteins. It is proposed that ergosterol-dependent inhibition of membrane proteins is a general mode of action of all the polyene antibiotics, of which some have been shown additionally to permeabilize the plasma membrane. Our results imply that sterol-protein interactions are fundamentally important for protein function even for those proteins that are not known to reside in sterol-rich domains. PMID:22733749

  9. Polyene antibiotic that inhibits membrane transport proteins.

    PubMed

    te Welscher, Yvonne Maria; van Leeuwen, Martin Richard; de Kruijff, Ben; Dijksterhuis, Jan; Breukink, Eefjan

    2012-07-10

    The limited therapeutic arsenal and the increase in reports of fungal resistance to multiple antifungal agents have made fungal infections a major therapeutic challenge. The polyene antibiotics are the only group of antifungal antibiotics that directly target the plasma membrane via a specific interaction with the main fungal sterol, ergosterol, often resulting in membrane permeabilization. In contrast to other polyene antibiotics that form pores in the membrane, the mode of action of natamycin has remained obscure but is not related to membrane permeabilization. Here, we demonstrate that natamycin inhibits growth of yeasts and fungi via the immediate inhibition of amino acid and glucose transport across the plasma membrane. This is attributable to ergosterol-specific and reversible inhibition of membrane transport proteins. It is proposed that ergosterol-dependent inhibition of membrane proteins is a general mode of action of all the polyene antibiotics, of which some have been shown additionally to permeabilize the plasma membrane. Our results imply that sterol-protein interactions are fundamentally important for protein function even for those proteins that are not known to reside in sterol-rich domains. PMID:22733749

  10. Biophysical EPR Studies Applied to Membrane Proteins

    PubMed Central

    Sahu, Indra D; Lorigan, Gary A

    2015-01-01

    Membrane proteins are very important in controlling bioenergetics, functional activity, and initializing signal pathways in a wide variety of complicated biological systems. They also represent approximately 50% of the potential drug targets. EPR spectroscopy is a very popular and powerful biophysical tool that is used to study the structural and dynamic properties of membrane proteins. In this article, a basic overview of the most commonly used EPR techniques and examples of recent applications to answer pertinent structural and dynamic related questions on membrane protein systems will be presented. PMID:26855825

  11. Immobilization of Firefly Luciferase on PVA-co-PE Nanofibers Membrane as Biosensor for Bioluminescent Detection of ATP.

    PubMed

    Wang, Wenwen; Zhao, Qinghua; Luo, Mengying; Li, Mufang; Wang, Dong; Wang, Yuedan; Liu, Qiongzhen

    2015-09-16

    The bioluminescent reaction catalyzed by firefly luciferase has become widely established as an outstanding analytical system for assay of adenosine triphosphate (ATP). When in solution, the luciferase is unstable and cannot be reused. The problem can be partially solved by immobilizing the luciferase on solid substrates. The poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibers membrane has abundant active hydroxyl groups on the surface. The PVA-co-PE nanofibers membrane was first activated by cyanuric chloride with triazinyl group. Then the activated PVA-co-PE nanofibers membrane was subsequently reacted with 1,3-propanediamine and biotin. The firefly luciferase was immobilized onto the surface of 1,3-propanediamine- and biotin-functionalized membranes. The surface chemical structure and morphologies of nanofibers membranes were characterized by FTIR-ATR spectra and SEM. The hydrophilicity of membranes was tested by water contact angle measurements. The detection of fluorescence intensity displayed that the firefly-luciferase-immobilized PVA-co-PE nanofibers membranes indicated high catalytic activity and efficiency. Especially, the firefly-luciferase-immobilized nanofiber membrane which was functionalized by biotin can be a promising candidate as biosensor for bioluminescent detection of ATP because of its high detection sensitivity. PMID:26275118

  12. Virus disinfection in water by biogenic silver immobilized in polyvinylidene fluoride membranes

    SciTech Connect

    Gusseme, B.D.; Fitts, J.; Hennebel, T.; Christiaens, E.; Saveyn, H.; Verbeken, K.; Boon, N.; Verstraete, W.

    2011-03-01

    The development of innovative water disinfection strategies is of utmost importance to prevent outbreaks of waterborne diseases related to poor treatment of (drinking) water. Recently, the association of silver nanoparticles with the bacterial cell surface of Lactobacillus fermentum (referred to as biogenic silver or bio-Ag{sup 0}) has been reported to exhibit antiviral properties. The microscale bacterial carrier matrix serves as a scaffold for Ag{sup 0} particles, preventing aggregation during encapsulation. In this study, bio-Ag{sup 0} was immobilized in different microporous PVDF membranes using two different pre-treatments of bio-Ag{sup 0} and the immersion-precipitation method. Inactivation of UZ1 bacteriophages using these membranes was successfully demonstrated and was most probably related to the slow release of Ag{sup +} from the membranes. At least a 3.4 log decrease of viruses was achieved by application of a membrane containing 2500 mg bio-Ag{sub powder}{sup 0} m{sup -2} in a submerged plate membrane reactor operated at a flux of 3.1 L m{sup -2} h{sup -1}. Upon startup, the silver concentration in the effluent initially increased to 271 {micro}g L{sup -1} but after filtration of 31 L m{sup -2}, the concentration approached the drinking water limit (= 100 {micro}g L{sup -1}). A virus decline of more than 3 log was achieved at a membrane flux of 75 L m{sup -2} h{sup -1}, showing the potential of this membrane technology for water disinfection on small scale. In biogenic silver, silver nanoparticles are attached to a bacterial carrier matrix. Bio-Ag{sup 0} was successfully immobilized in PVDF membranes using immersion-precipitation. The antiviral activity of this material was demonstrated in a plate membrane reactor. The antimicrobial mechanism was most probably related to the slow release of Ag{sup +} ions. The membranes can be applied for treatment of limited volumes of contaminated water.

  13. Towards Simulations of Outer Membrane Proteins in Lipopolysaccharide Membranes

    SciTech Connect

    Soares, Thereza A.; Straatsma, TP

    2007-12-26

    Biomolecular simulation derived properties of LPS membranes that impact the structural and internal dynamics of transmembrane proteins are shown to exhibit good agreement with available experimental data within the time scale simulated, chosen force field and simulation conditions. The molecular model used offers an accurate representation of the LPS layer, including the high asymmetry and low fluidity characteristics of outer membranes. This contribution describes the data intensive analysis of the large molecular time trajectories generated for these systems using massively parallel computing resources.

  14. Covalent immobilization of protein onto a functionalized hydrogenated diamond-like carbon substrate.

    PubMed

    Biswas, Hari Shankar; Datta, Jagannath; Chowdhury, D P; Reddy, A V R; Ghosh, Uday Chand; Srivastava, Arvind Kumar; Ray, Nihar Ranjan

    2010-11-16

    Hydrogenated diamond-like carbon (HDLC) has an atomically smooth surface that can be deposited on high-surface area substrata and functionalized with reactive chemical groups, providing an ideal substrate for protein immobilization. A synthetic sequence is described involving deposition and hydrogenation of DLC followed by chemical functionalization. These functional groups are reacted with amines on proteins causing covalent immobilization on contact. Raman measurements confirm the presence of these surface functional groups, and Fourier transform infrared spectroscopy (FTIR) confirms covalent protein immobilization. Atomic force microscopy (AFM) of immobilized proteins is reproducible because proteins do not move as a result of interactions with the AFM probe-tip, thus providing an advantage over mica substrata typically used in AFM studies of protein. HDLC offers many of the same technical advantages as oxidized graphene but also allows for coating large surface areas of biomaterials relevant to the fabrication of medical/biosensor devices. PMID:20949913

  15. Membrane proteins: is the future disc shaped?

    PubMed

    Lee, Sarah C; Pollock, Naomi L

    2016-08-15

    The use of styrene maleic acid lipid particles (SMALPs) for the purification of membrane proteins (MPs) is a rapidly developing technology. The amphiphilic copolymer of styrene and maleic acid (SMA) disrupts biological membranes and can extract membrane proteins in nanodiscs of approximately 10 nm diameter. These discs contain SMA, protein and membrane lipids. There is evidence that MPs in SMALPs retain their native structures and functions, in some cases with enhanced thermal stability. In addition, the method is compatible with biological buffers and a wide variety of biophysical and structural analysis techniques. The use of SMALPs to solubilize and stabilize MPs offers a new approach in our attempts to understand, and influence, the structure and function of MPs and biological membranes. In this review, we critically assess progress with this method, address some of the associated technical challenges, and discuss opportunities for exploiting SMA and SMALPs to expand our understanding of MP biology. PMID:27528746

  16. EH domain proteins regulate cardiac membrane protein targeting

    PubMed Central

    Gudmundsson, Hjalti; Hund, Thomas J.; Wright, Patrick J.; Kline, Crystal F.; Snyder, Jedidiah S.; Qian, Lan; Koval, Olha M.; Cunha, Shane R.; George, Manju; Rainey, Mark A.; Kashef, Farshid E.; Dun, Wen; Boyden, Penelope A.; Anderson, Mark E.; Band, Hamid; Mohler, Peter J.

    2010-01-01

    Rationale Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules. Membrane protein dynamics in health and disease are maintained by a complex ensemble of intracellular targeting, scaffolding, recycling, and degradation pathways. Surprisingly, despite decades of research linking dysfunction in membrane protein trafficking with human cardiovascular disease, essentially nothing is known regarding the molecular identity or function of these intracellular targeting pathways in excitable cardiomyocytes. Objective We sought to discover novel pathways for membrane protein targeting in primary cardiomyocytes. Methods and Results We report the initial characterization of a large family of membrane trafficking proteins in human heart. We employed a tissue-wide screen for novel ankyrin-associated trafficking proteins and identified four members of a unique Eps15 homology (EH) domain-containing protein family (EHD1, EHD2, EHD3, EHD4) that serve critical roles in endosome-based membrane protein targeting in other cell types. We show that EHD1-4 directly associate with ankyrin, provide the first information on the expression and localization of these molecules in primary cardiomyocytes, and demonstrate that EHD1-4 are co-expressed with ankyrin-B in the myocyte perinuclear region. Notably, the expression of multiple EHD proteins is increased in animal models lacking ankyrin-B, and EHD3-deficient cardiomyocytes display aberrant ankyrin-B localization and selective loss of Na/Ca exchanger expression and function. Finally, we report significant modulation of EHD expression following myocardial infarction, suggesting that these proteins may play a key role in regulating membrane excitability in normal and diseased heart. Conclusions Our findings identify and characterize a new class of cardiac trafficking proteins, define the first group of proteins associated with the ankyrin

  17. Detergents in Membrane Protein Purification and Crystallisation.

    PubMed

    Anandan, Anandhi; Vrielink, Alice

    2016-01-01

    Detergents play a significant role in structural and functional characterisation of integral membrane proteins (IMPs). IMPs reside in the biological membranes and exhibit a great variation in their structural and physical properties. For in vitro biophysical studies, structural and functional analyses, IMPs need to be extracted from the membrane lipid bilayer environment in which they are found and purified to homogeneity while maintaining a folded and functionally active state. Detergents are capable of successfully solubilising and extracting the IMPs from the membrane bilayers. A number of detergents with varying structure and physicochemical properties are commercially available and can be applied for this purpose. Nevertheless, it is important to choose a detergent that is not only able to extract the membrane protein but also provide an optimal environment while retaining the correct structural and physical properties of the protein molecule. Choosing the best detergent for this task can be made possible by understanding the physical and chemical properties of the different detergents and their interaction with the IMPs. In addition, understanding the mechanism of membrane solubilisation and protein extraction along with crystallisation requirements, if crystallographic studies are going to be undertaken, can help in choosing the best detergent for the purpose. This chapter aims to present the fundamental properties of detergents and highlight information relevant to IMP crystallisation. The first section of the chapter reviews the physicochemical properties of detergents and parameters essential for predicting their behaviour in solution. The second section covers the interaction of detergents with the biologic membranes and proteins followed by their role in membrane protein crystallisation. The last section will briefly cover the types of detergent and their properties focusing on custom designed detergents for membrane protein studies. PMID:27553232

  18. High-affinity immobilization of proteins using biotin- and GST-based coupling strategies.

    PubMed

    Hutsell, Stephanie Q; Kimple, Randall J; Siderovski, David P; Willard, Francis S; Kimple, Adam J

    2010-01-01

    Surface plasmon resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected across the sensor surface. This chapter focuses on high-affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction. PMID:20217614

  19. High affinity immobilization of proteins using biotin- and GST-based coupling strategies

    PubMed Central

    Hutsell, Stephanie Q.; Kimple, Randall J.; Siderovski, David P.; Willard, Francis S.; Kimple, Adam J.

    2011-01-01

    Surface Plasmon Resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected across the sensor surface. This chapter focuses on high affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction. PMID:20217614

  20. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  1. Detergent-Free Membrane Protein Purification.

    PubMed

    Rothnie, Alice J

    2016-01-01

    Membrane proteins are localized within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilizing or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilizing protein structure, is time consuming and expensive. Here we present a recently-developed method using a styrene-maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment. PMID:27485341

  2. Dielectrophoretic Sorting of Membrane Protein Nanocrystals

    PubMed Central

    Abdallah, Bahige G.; Chao, Tzu-Chiao; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

    2013-01-01

    Structure elucidation of large membrane protein complexes still comprises a considerable challenge yet is a key factor in drug development and disease combat. Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals, thus overcoming the experimental riddle faced in traditional crystallography methods. Here, we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis. We demonstrate the excellent sorting power of this new approach with numerical simulations of selected sub-micrometer beads in excellent agreement with experimental observations. Crystals from batch crystallization broths of the huge membrane protein complex photosystem I were sorted without further treatment, resulting in a high degree of monodispersity and crystallinity in the ~ 100 nm size range. Microfluidic integration, continuous sorting, and nanometer-sized crystal fractions make this method ideal for direct coupling to femtosecond nanocrystallography. PMID:24004002

  3. Membrane biotechnology, co-immobilization, and aqueous two-phase systems: alternatives in bioconversion of cellulose

    SciTech Connect

    Hahn-Haegerdal, B.; Andersson, E.; Lopez-Leiva, M.; Mattiasson, B.

    1981-01-01

    Three different techniques having complementary features have been applied to the bioconversion of cellulose to ethanol: (1) membrane biotechnology involving ultrafiltration and reverse osmosis allows conversion of particulate substrates with soluble biocatalysts, continuous removal of inhibitory products, and low-energy upgrading of dilute product streams; (2) co-immobilization of enzymes and microorganisms results in new metabolic combinations, allowing microbial conversion of nondigestible substrates, removal of inhibitory intermediates, and continuous operation; (3) aqueous two-phase systems are biocompatible and allow extractive bioconversions in that soluble biocatalysts and particulate substrates can be partitioned to one phase while products can be partitioned and upgraded in the other phase.

  4. Efficient preparation and analysis of membrane and membrane protein systems.

    PubMed

    Javanainen, Matti; Martinez-Seara, Hector

    2016-10-01

    Molecular dynamics (MD) simulations have become a highly important technique to consider lipid membrane systems, and quite often they provide considerable added value to laboratory experiments. Rapid development of both software and hardware has enabled the increase of time and size scales reachable by MD simulations to match those attainable by several accurate experimental techniques. However, until recently, the quality and maturity of software tools available for building membrane models for simulations as well as analyzing the results of these simulations have seriously lagged behind. Here, we discuss the recent developments of such tools from the end-users' point of view. In particular, we review the software that can be employed to build lipid bilayers and other related structures with or without embedded membrane proteins to be employed in MD simulations. Additionally, we provide a brief critical insight into force fields and MD packages commonly used for membrane and membrane protein simulations. Finally, we list analysis tools that can be used to study the properties of membrane and membrane protein systems. In all these points we comment on the respective compatibility of the covered tools. We also share our opinion on the current state of the available software. We briefly discuss the most commonly employed tools and platforms on which new software can be built. We conclude the review by providing a few ideas and guidelines on how the development of tools can be further boosted to catch up with the rapid pace at which the field of membrane simulation progresses. This includes improving the compatibility between software tools and promoting the openness of the codes on which these applications rely. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26947184

  5. NMR of Membrane Proteins: Beyond Crystals.

    PubMed

    Rajesh, Sundaresan; Overduin, Michael; Bonev, Boyan B

    2016-01-01

    Membrane proteins are essential for the flow of signals, nutrients and energy between cells and between compartments of the cell. Their mechanisms can only be fully understood once the precise structures, dynamics and interactions involved are defined at atomic resolution. Through advances in solution and solid state NMR spectroscopy, this information is now available, as demonstrated by recent studies of stable peripheral and transmembrane proteins. Here we highlight recent cases of G-protein coupled receptors, outer membrane proteins, such as VDAC, phosphoinositide sensors, such as the FAPP-1 pleckstrin homology domain, and enzymes including the metalloproteinase MMP-12. The studies highlighted have resulted in the determination of the 3D structures, dynamical properties and interaction surfaces for membrane-associated proteins using advanced isotope labelling strategies, solubilisation systems and NMR experiments designed for very high field magnets. Solid state NMR offers further insights into the structure and multimeric assembly of membrane proteins in lipid bilayers, as well as into interactions with ligands and targets. Remaining challenges for wider application of NMR to membrane structural biology include the need for overexpression and purification systems for the production of isotope-labelled proteins with fragile folds, and the availability of only a few expensive perdeuterated detergents.Step changes that may transform the field include polymers, such as styrene maleic acid, which obviate the need for detergent altogether, and allow direct high yield purification from cells or membranes. Broader demand for NMR may be facilitated by MODA software, which instantly predicts membrane interactive residues that can subsequently be validated by NMR. In addition, recent developments in dynamic nuclear polarization NMR instrumentation offer a remarkable sensitivity enhancement from low molarity samples and cell surfaces. These advances illustrate the current

  6. Surface recognition elements of membrane protein oligomerization.

    PubMed

    Rath, Arianna; Deber, Charles M

    2008-02-15

    Although certain membrane proteins are functional as monomeric polypeptides, others must assemble into oligomers to carry out their biological roles. High-resolution membrane protein structures provide a valuable resource for examining the sequence features that facilitate-or preclude-assembly of membrane protein monomers into multimeric structures. Here we have utilized a data set of 28 high-resolution alpha-helical membrane protein structures comprising 32 nonredundant polypeptides to address this issue. The lipid-exposed surfaces of membrane proteins that have reached their fully assembled and functional biological units have been compared with those of the individual subunits that build quaternary structures. Though the overall amino acid composition of each set of surfaces is similar, a key distinction-the distribution of small-xxx-small motifs-delineates subunits from membrane proteins that have reached a functioning oligomeric state. Quaternary structure formation may therefore be dictated by small-xxx-small motifs that are not satisfied by intrachain contacts. PMID:17729275

  7. Immobilization of proteins on glow discharge treated polymers

    NASA Astrophysics Data System (ADS)

    Kiaei, D.; Safranj, A.; Chen, J. P.; Johnston, A. B.; Zavala, F.; Deelder, A.; Castelino, J. B.; Markovic, V.; Hoffman, A. S.

    Certain glow discharge-treated surfaces have been shown to enhance retention of adsorbed proteins. On the basis of this phenomenon, we have investigated the possibility of immobilizing (a) albumin for developing thromboresistant and non-fouling surfaces, (b) antibodies for immuno-diagnostic assays and (c) enzymes for various biosensors and industrial bioprocesses. Albumin retention was highest on surfaces treated with tetrafluoroethylene (TFE) compared to untreated surfaces or other glow discharge treatments studied. Preadsorption of albumin on TFE-treated surfaces resulted in low fibrinogen adsorption and platelet adhesion. IgG retention was also highest on TFE-treated surfaces. The lower detection limits of both malaria antigen and circulating anodic antigen of the schistosomiasis worm were enhanced following glow discharge treatment of the assay plates with TFE. Both TFE and tetrachloroethylene (TCE) glow discharge treated surfaces showed high retention of adsorbed horseradish peroxidase (HRP). However, the retained specific activity of HRP after adsorption on TCE-treated surfaces was remarkably higher than on TFE-treated surfaces.

  8. Kinetic evaluation of nitrification performance in an immobilized cell membrane bioreactor.

    PubMed

    Güven, D; Ubay Çokgör, E; Sözen, S; Orhon, D

    2016-01-01

    High rate membrane bioreactor (MBR) systems operated at extremely low sludge ages (superfast membrane bioreactors (SFMBRs)) are inefficient to achieve nitrogen removal, due to insufficient retention time for nitrifiers. Moreover, frequent chemical cleaning is required due to high biomass flux. This study aims to satisfy the nitrification in SFMBRs by using sponge as carriers, leading to the extension of the residence time of microorganisms. In order to test the limits of nitrification, bioreactor was run under 52, 5 and 2 days of carrier residence time (CRT), with a hydraulic retention time of 6 h. Different degrees of nitrification were obtained for different CRTs. Sponge immobilized SFMBR operation with short CRT resulted in partial nitrification indicating selective dominancy of ammonia oxidizers. At higher CRT, simultaneous nitrification-denitrification was achieved when accompanying with oxygen limitation. Process kinetics was determined through evaluation of the results by a modeling study. Nitrifier partition in the reactor was also identified by model calibration. PMID:27332835

  9. Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium.

    PubMed

    Uchino, Yuichi; Shimmura, Shigeto; Miyashita, Hideyuki; Taguchi, Tetsushi; Kobayashi, Hisatoshi; Shimazaki, Jun; Tanaka, Junzo; Tsubota, Kazuo

    2007-04-01

    Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis. PMID:16924609

  10. Virus Disinfection in Water by Biogenic Silver Immobilized in Polyvinylidene Fluoride Membranes

    SciTech Connect

    B De Gusseme; T Hennebel; E Christiaens; H Saveyn; K Verbeken; J Fitts; N Boon; W Vertraete

    2011-12-31

    The development of innovative water disinfection strategies is of utmost importance to prevent outbreaks of waterborne diseases related to poor treatment of (drinking) water. Recently, the association of silver nanoparticles with the bacterial cell surface of Lactobacillus fermentum (referred to as biogenic silver or bio-Ag{sup 0}) has been reported to exhibit antiviral properties. The microscale bacterial carrier matrix serves as a scaffold for Ag{sup 0} particles, preventing aggregation during encapsulation. In this study, bio-Ag{sup 0} was immobilized in different microporous PVDF membranes using two different pre-treatments of bio-Ag{sup 0} and the immersion-precipitation method. Inactivation of UZ1 bacteriophages using these membranes was successfully demonstrated and was most probably related to the slow release of Ag{sup +} from the membranes. At least a 3.4 log decrease of viruses was achieved by application of a membrane containing 2500 mg bio-Ag{sup 0}{sub powder} m{sup -2} in a submerged plate membrane reactor operated at a flux of 3.1 L m{sup -2} h{sup -1}. Upon startup, the silver concentration in the effluent initially increased to 271 {mu}g L{sub -1} but after filtration of 31 L m{sup -2}, the concentration approached the drinking water limit (= 100 {mu}g L{sup -1}). A virus decline of more than 3 log was achieved at a membrane flux of 75 L m{sup -2} h{sup -1}, showing the potential of this membrane technology for water disinfection on small scale.

  11. Protein Stains to Detect Antigen on Membranes.

    PubMed

    Dsouza, Anil; Scofield, R Hal

    2015-01-01

    Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical. PMID:26139252

  12. Biophysical Characterization of Membrane Proteins in Nanodiscs

    PubMed Central

    Inagaki, Sayaka; Ghirlando, Rodolfo; Grisshammer, Reinhard

    2012-01-01

    Nanodiscs are self-assembled discoidal phospholipid bilayers surrounded and stabilized by membrane scaffold proteins (MSP), that have become a powerful and promising tool for the study of membrane proteins. Even though their reconstitution is highly regulated by the type of MSP and phospholipid input, a biophysical characterization leading to the determination of the stoichiometry of MSP, lipid and membrane protein is essential. This is important for biological studies, as the oligomeric state of membrane proteins often correlates with their functional activity. Typically combinations of several methods are applied using, for example, modified samples that incorporate fluorescent labels, along with procedures that result in nanodisc disassembly and lipid dissolution. To obtain a comprehensive understanding of the native properties of nanodiscs, modification-free analysis methods are required. In this work we provide a strategy, using a combination of dynamic light scattering and analytical ultracentrifugation, for the biophysical characterization of unmodified nanodiscs. In this manner we characterize the nanodisc preparation in terms of its overall polydispersity and characterize the hydrodynamically resolved nanodisc of interest in terms of its sedimentation coefficient, Stokes’ radius and overall protein and lipid stoichiometry. Functional and biological applications are also discussed for the study of the membrane protein embedded in nanodiscs under defined experimental conditions. PMID:23219517

  13. Two-Photon Fluorescence Anisotropy Imaging to Elucidate the Dynamics and the Stability of Immobilized Proteins.

    PubMed

    Orrego, Alejandro H; García, Carolina; Mancheño, José M; Guisán, Jose M; Lillo, M Pilar; López-Gallego, Fernando

    2016-01-28

    Time/spatial-resolved fluorescence determines anisotropy values of supported-fluorescent proteins through different immobilization chemistries, evidencing some of the molecular mechanisms that drive the stabilization of proteins at the interfaces with solid surfaces. Fluorescence anisotropy imaging provides a normalized protein mobility parameter that serves as a guide to study the effect of different immobilization parameters (length and flexibility of the spacer arm and multivalency of the protein-support interaction) on the final stability of the supported proteins. Proteins in a more constrained environment correspond to the most thermostable ones, as was shown by thermal inactivation studies. This work contributes to explain the experimental evidence found with conventional methods based on observable measurements; thus this advanced characterization technique provides reliable molecular information about the immobilized proteins with sub-micrometer spatial resolution. Such information has been very useful for fabricating highly stable heterogeneous biocatalysts with high interest in industrial developments. PMID:26716569

  14. Intrinsically disordered proteins drive membrane curvature

    PubMed Central

    Busch, David J.; Houser, Justin R.; Hayden, Carl C.; Sherman, Michael B.; Lafer, Eileen M.; Stachowiak, Jeanne C.

    2015-01-01

    Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures. PMID:26204806

  15. Intrinsically disordered proteins drive membrane curvature

    NASA Astrophysics Data System (ADS)

    Busch, David J.; Houser, Justin R.; Hayden, Carl C.; Sherman, Michael B.; Lafer, Eileen M.; Stachowiak, Jeanne C.

    2015-07-01

    Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.

  16. Class III viral membrane fusion proteins

    PubMed Central

    Backovic, Marija

    2010-01-01

    SUMMARY Accumulating structural studies of viral fusion glycoproteins have revealed unanticipated structural relationships between unrelated virus families and allowed the grouping of these membrane fusogens into three distinct classes. Here we review the newly identified group of class III viral fusion proteins, whose members include fusion proteins from rhabdoviruses, herpesviruses and baculoviruses. While clearly related in structure, the class III viral fusion proteins exhibit distinct structural features in their architectures as well as in their membrane-interacting fusion loops, which are likely related to their virus-specific differences in cellular entry. Further study of the similarities and differences in the class III viral fusion glycoproteins may provide greater insights into protein:membrane interactions that are key to promoting efficient bilayer fusion during virus entry. PMID:19356922

  17. Protein transfer to membranes upon shape deformation

    NASA Astrophysics Data System (ADS)

    Sagis, L. M. C.; Bijl, E.; Antono, L.; de Ruijter, N. C. A.; van Valenberg, H.

    2013-05-01

    Red blood cells, milk fat droplets, or liposomes all have interfaces consisting of lipid membranes. These particles show significant shape deformations as a result of flow. Here we show that these shape deformations can induce adsorption of proteins to the membrane. Red blood cell deformability is an important factor in several diseases involving obstructions of the microcirculatory system, and deformation induced protein adsorption will alter the rigidity of their membranes. Deformation induced protein transfer will also affect adsorption of cells onto implant surfaces, and the performance of liposome based controlled release systems. Quantitative models describing this phenomenon in biomaterials do not exist. Using a simple quantitative model, we provide new insight in this phenomenon. We present data that show convincingly that for cells or droplets with diameters upwards of a few micrometers, shape deformations induce adsorption of proteins at their interface even at moderate flow rates.

  18. Conformational flexibility of a model protein upon immobilization on self-assembled monolayers.

    PubMed

    Bigdeli, Saharnaz; Talasaz, AmirAli H; Ståhl, Patrik; Persson, Henrik H J; Ronaghi, Mostafa; Davis, Ronald W; Nemat-Gorgani, Mohsen

    2008-05-01

    The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD(+), some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed. PMID:18078298

  19. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    PubMed

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. PMID:26230624

  20. Helix insertion into bilayers and the evolution of membrane proteins

    PubMed Central

    2010-01-01

    Polytopic α-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic α-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of α-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins. Topics include: translocon-dependent membrane protein insertion, antibiotic peptides and proteins, in vitro insertion of membrane proteins, chaperone-mediated insertion of transmembrane helices, and C-terminal tail-anchored (TA) proteins. Analysis of the E. coli genome reveals several predicted C-terminal TA proteins that may be descendents of proteins involved in pre-cellular membrane protein insertion. Mechanisms of pre-translocon polytopic α-helical membrane protein insertion are discussed. PMID:20039094

  1. Protein separation using an electrically tunable membrane

    NASA Astrophysics Data System (ADS)

    Jou, Ining; Melnikov, Dmitriy; Gracheva, Maria

    Separation of small proteins by charge with a solid-state porous membrane requires control over the protein's movement. Semiconductor membrane has this ability due to the electrically tunable electric potential profile inside the nanopore. In this work we investigate the possibility to separate the solution of two similar sized proteins by charge. As an example, we consider two small globular proteins abundant in humans: insulin (negatively charged) and ubiquitin (neutral). We find that the localized electric field inside the pore either attracts or repels the charged protein to or from the pore wall which affects the delay time before a successful translocation of the protein through the nanopore. However, the motion of the uncharged ubiquitin is unaffected. The difference in the delay time (and hence the separation) can be further increased by the application of the electrolyte bias which induces an electroosmotic flow in the pore. NSF DMR and CBET Grant No. 1352218.

  2. Crystallization of Membrane Proteins by Vapor Diffusion

    PubMed Central

    Delmar, Jared A.; Bolla, Jani Reddy; Su, Chih-Chia; Yu, Edward W.

    2016-01-01

    X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization. PMID:25950974

  3. A fullerene C60-based ligand in a stationary phase for affine chromatography of membrane porphyrin-binding proteins

    NASA Astrophysics Data System (ADS)

    Amirshakhi, N.; Alyautdin, R. N.; Orlov, A. P.; Poloznikov, A. A.; Kuznetsov, D. A.

    2008-11-01

    A new affine chromatography technique is suggested for the purification of porphyrin-binding proteins (PBP) from mammal cell membranes. The procedure uses new fullerene-porphyrin ligands immobilized on agarose and bound to the polysaccharide matrix via the epoxycyclohexyl residue. A selective PBP stationary phase was used in a single-column chromatography run for the complete purification of a monomeric protein (17.6 kDa) from mitochondrial membranes of rat myocardium. This protein was characterized by high affinity for porphyrin-related structures. To separate it from other nonspecifically sorbed membrane proteins, synchronous linear pH and ionic strength gradients were used.

  4. Membrane protein structure from rotational diffusion☆

    PubMed Central

    Das, Bibhuti B.; Park, Sang Ho; Opella, Stanley J.

    2014-01-01

    The motional averaging of powder pattern line shapes is one of the most fundamental aspects of sold-state NMR. Since membrane proteins in liquid crystalline phospholipid bilayers undergo fast rotational diffusion, all of the signals reflect the angles of the principal axes of their dipole–dipole and chemical shift tensors with respect to the axis defined by the bilayer normal. The frequency span and sign of the axially symmetric powder patterns that result from motional averaging about a common axis provide sufficient structural restraints for the calculation of the three-dimensional structure of a membrane protein in a phospholipid bilayer environment. The method is referred to as rotationally aligned (RA) solid-state NMR and demonstrated with results on full-length, unmodified membrane proteins with one, two, and seven trans-membrane helices. RA solid-state NMR is complementary to other solid-state NMR methods, in particular oriented sample (OS) solid-state NMR of stationary, aligned samples. Structural distortions of membrane proteins from the truncations of terminal residues and other sequence modifications, and the use of detergent micelles instead of phospholipid bilayers have also been demonstrated. Thus, it is highly advantageous to determine the structures of unmodified membrane proteins in liquid crystalline phospholipid bilayers under physiological conditions. RA solid-state NMR provides a general method for obtaining accurate and precise structures of membrane proteins under near-native conditions. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. PMID:24747039

  5. Breaking the barriers in membrane protein crystallography.

    PubMed

    Kang, Hae Joo; Lee, Chiara; Drew, David

    2013-03-01

    As we appreciate the importance of stabilising membrane proteins, the barriers towards their structure determination are being broken down. This change in mindset comes hand-in-hand with more effort placed on developing methods focused at screening for membrane proteins which are naturally stable in detergent solution or improving those that are not so. In practice, however, it is not easy to decide the best strategy to monitor and improve detergent stability, requiring a decision-making process that can be even more difficult for those new to the field. In this review we outline the importance of membrane protein stability with discussions of the stabilisation strategies applied in context with the use of crystallisation scaffolds and the different types of crystallisation methods themselves. Where possible we also highlight areas that we think could push this field forward with emerging technologies, such as X-ray free electron lasers (X-feL), which could have a big impact on the membrane protein structural biology community. We hope this review will serve as a useful guide for those striving to solve structures of both pro- and eukaryotic membrane proteins. PMID:23291355

  6. Affinity purification of antibodies using immobilized FB domain of protein A.

    PubMed

    Solomon, B; Raviv, O; Leibman, E; Fleminger, G

    1992-04-24

    A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A. PMID:1517325

  7. Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports

    SciTech Connect

    Camarero, J A

    2007-02-21

    Immobilization of proteins onto surfaces is of great importance in numerous applications, including protein analysis, drug screening, and medical diagnostics, among others. The success of all these technologies relies on the immobilization technique employed to attach a protein to the corresponding surface. Non-specific physical adsorption or chemical cross-linking with appropriate surfaces results in the immobilization of the protein in random orientations. Site-specific covalent attachment, on the other hand, leads to molecules being arranged in a definite, orderly fashion and allows the use of spacers and linkers to help minimize steric hindrances between the protein and the surface. The present work reviews the latest chemical and biochemical developments for the site-specific covalent attachment of proteins onto solid supports.

  8. Curvature-mediated interactions between membrane proteins.

    PubMed Central

    Kim, K S; Neu, J; Oster, G

    1998-01-01

    Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions. PMID:9788923

  9. Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues.

    PubMed

    Coïc, Yves-Marie; Vincent, Michel; Gallay, Jacques; Baleux, Françoise; Mousson, Florence; Beswick, Veronica; Neumann, Jean-Michel; de Foresta, Béatrice

    2005-12-01

    Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H(+)-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H(+)-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185-97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (lambda(max)), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k(q)) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We

  10. Direct site-specific immobilization of protein A via aldehyde-hydrazide conjugation.

    PubMed

    Zang, Berlin; Ren, Jun; Xu, Li; Jia, Lingyun

    2016-01-01

    Immobilization of affinity ligands on supporting matrices is a key step for the preparation of affinity chromatography resins, and an efficient coupling strategy can significantly improve the validity and cost of the affinity system, especially for systems that employ expensive recombinant proteins or antibodies as affinity ligands. This study described a simple method for obtaining site-specific immobilization of protein A (the ligand) via aldehyde-hydrazide conjugation and its application in antibody purification via protein A chromatography. An aldehyde group was generated at the N-terminus of protein A in vivo by co-expressing a formylglycine-generating enzyme (FGE) and recombinant protein A containing a FGE recognizing sequence (aldehyde tag) in Escherichia coli. The resulting aldehyde allowed direct immobilization of protein A onto the hydrazide-modified agarose matrices under mild condition. We found that 100mM aniline was most effective for catalyzing the coupling reaction, and the recombinant protein A could be coupled with high selectivity, directly from a crude cell extract. The site-specific immobilized protein A showed good capacity for antibody purification. The specificity of the aldehyde-hydrazide reaction not only allowed site-specific immobilization of affinity ligands, but also improved the cost of the process by employing unpurified ligands, a method that might be of great use to industrial applications. PMID:26655104

  11. Highly efficient antibody immobilization with multimeric protein Gs coupled magnetic silica nanoparticles

    NASA Astrophysics Data System (ADS)

    Lee, J. H.; Choi, H. K.; Chang, J. H.

    2011-10-01

    This work reports the immobilization of monomeric, dimeric and trimer protein Gs onto silica magnetic nanoparticles for self-oriented antibody immobilization. To achieve this, we initially prepared the silica-coated magnetic nanoparticle having about 170 nm diameters. The surface of the silica coated magnetic nanoparticles was modified with 3- aminopropyl-trimethoxysilane (APTMS) to chemically link to multimeric protein Gs. The conjugation of amino groups on the SiO2-MNPs to cysteine tagged in multimeric protein Gs was performed using a sulfo-SMCC coupling procedure. The binding efficiencies of monomer, dimer and trimer were 77 %, 67 % and 55 % respectively. However, the efficiencies of antibody immobilization were 70 %, 83 % and 95 % for monomeric, dimeric and trimeric protein G, respectively. To prove the enhancement of accessibility by using multimeric protein G, FITC labeled goat-anti-mouse IgG was treated to mouse IgG immobilized magnetic silica nanoparticles through multimeric protein G. FITC labeled goat anti-mouse IgGs were more easily bound to mouse IgG immobilized by trimeric protein G than others. Finally protein G bound silica magnetic nanoparticles were utilized to develop highly sensitive immunoassay to detect hepatitis B antigen.

  12. Immobilization of two organometallic complexes into a single cage to construct protein-based microcompartments.

    PubMed

    Maity, Basudev; Fukumori, Kazuki; Abe, Satoshi; Ueno, Takafumi

    2016-04-01

    Natural protein-based microcompartments containing multiple enzymes promote cascade reactions within cells. We use the apo-ferritin protein cage to mimic such biocompartments by immobilizing two organometallic Ir and Pd complexes into the single protein cage. Precise locations of the metals and their accumulation mechanism were studied by X-ray crystallography. PMID:27021005

  13. Identifying the hub proteins from complicated membrane protein network systems.

    PubMed

    Shen, Yi-Zhen; Ding, Yong-Sheng; Gu, Quan; Chou, Kuo-Chen

    2010-05-01

    The so-called "hub proteins" are those proteins in a protein-protein interaction network system that have remarkably higher interaction relations (or degrees) than the others. Therefore, the information of hub proteins can provide very useful insights for selecting or prioritizing targets during drug development. In this paper, by combining the multi-agent-based method with the graphical spectrum analysis and immune-genetic algorithm, a novel simulator for identifying the hub proteins from membrane protein interaction networks is proposed. As a demonstration of using the simulator, two hub membrane proteins, YPL227C and YIL147C, were identified from a complicated network system consisting of 1500 membrane proteins. Meanwhile, along with the two identified hub proteins, their molecular functions, biological processes, and cellular components were also revealed. It is anticipated that the hub-protein-simulator may become a very useful tool for system biology and drug development, particularly in deciphering unknown protein functions, determining protein complexes, and in identifying the key targets from a complicated disease system. PMID:20507268

  14. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.

    PubMed

    Habicht, K-L; Singh, N S; Indig, F E; Wainer, I W; Moaddel, R; Shimmo, R

    2015-09-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006μM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  15. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  16. Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor.

    PubMed

    Giorno, L; Li, N; Drioli, E

    2003-12-20

    The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity. PMID:14595780

  17. Model-building codes for membrane proteins.

    SciTech Connect

    Shirley, David Noyes; Hunt, Thomas W.; Brown, W. Michael; Schoeniger, Joseph S.; Slepoy, Alexander; Sale, Kenneth L.; Young, Malin M.; Faulon, Jean-Loup Michel; Gray, Genetha Anne

    2005-01-01

    We have developed a novel approach to modeling the transmembrane spanning helical bundles of integral membrane proteins using only a sparse set of distance constraints, such as those derived from MS3-D, dipolar-EPR and FRET experiments. Algorithms have been written for searching the conformational space of membrane protein folds matching the set of distance constraints, which provides initial structures for local conformational searches. Local conformation search is achieved by optimizing these candidates against a custom penalty function that incorporates both measures derived from statistical analysis of solved membrane protein structures and distance constraints obtained from experiments. This results in refined helical bundles to which the interhelical loops and amino acid side-chains are added. Using a set of only 27 distance constraints extracted from the literature, our methods successfully recover the structure of dark-adapted rhodopsin to within 3.2 {angstrom} of the crystal structure.

  18. Transmembrane protein sorting driven by membrane curvature

    NASA Astrophysics Data System (ADS)

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-11-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  19. Transmembrane protein sorting driven by membrane curvature

    PubMed Central

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-01-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization. PMID:26522943

  20. Atomic-level analysis of membrane-protein structure.

    PubMed

    Hendrickson, Wayne A

    2016-06-01

    Membrane proteins are substantially more challenging than natively soluble proteins as subjects for structural analysis. Thus, membrane proteins are greatly underrepresented in structural databases. Recently, focused consortium efforts and advances in methodology for protein production, crystallographic analysis and cryo-EM analysis have accelerated the pace of atomic-level structure determination of membrane proteins. PMID:27273628

  1. Protein permeation through an electrically tunable membrane.

    PubMed

    Jou, Ining A; Melnikov, Dmitriy V; Gracheva, Maria E

    2016-05-20

    Protein filtration is important in many fields of science and technology such as medicine, biology, chemistry, and engineering. Recently, protein separation and filtering with nanoporous membranes has attracted interest due to the possibility of fast separation and high throughput volume. This, however, requires understanding of the protein's dynamics inside and in the vicinity of the nanopore. In this work, we utilize a Brownian dynamics approach to study the motion of the model protein insulin in the membrane-electrolyte electrostatic potential. We compare the results of the atomic model of the protein with the results of a coarse-grained and a single-bead model, and find that the coarse-grained representation of protein strikes the best balance between the accuracy of the results and the computational effort required. Contrary to common belief, we find that to adequately describe the protein, a single-bead model cannot be utilized without a significant effort to tabulate the simulation parameters. Similar to results for nanoparticle dynamics, our findings also indicate that the electric field and the electro-osmotic flow due to the applied membrane and electrolyte biases affect the capture and translocation of the biomolecule by either attracting or repelling it to or from the nanopore. Our computational model can also be applied to other types of proteins and separation conditions. PMID:27044064

  2. Outer membrane proteins of Methylococcus capsulatus (Bath).

    PubMed

    Fjellbirkeland, A; Kleivdal, H; Joergensen, C; Thestrup, H; Jensen, H B

    1997-08-01

    Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed by electron microscopy and by the content of beta-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes, this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl. PMID:9238104

  3. Major intrinsic proteins in biomimetic membranes.

    PubMed

    Nielsen, Claus Hélix

    2010-01-01

    Biological membranes define the structural and functional boundaries in living cells and their organelles. The integrity of the cell depends on its ability to separate inside from outside and yet at the same time allow massive transport of matter in and out the cell. Nature has elegantly met this challenge by developing membranes in the form of lipid bilayers in which specialized transport proteins are incorporated. This raises the question: is it possible to mimic biological membranes and create a membrane based sensor and/or separation device? In the development of a biomimetic sensor/separation technology, a unique class of membrane transport proteins is especially interesting-the major intrinsic proteins (MIPs). Generally, MIPs conduct water molecules and selected solutes in and out of the cell while preventing the passage of other solutes, a property critical for the conservation of the cells internal pH and salt concentration. Also known as water channels or aquaporins they are highly efficient membrane pore proteins some of which are capable of transporting water at very high rates up to 10(9) molecules per second. Some MIPs transport other small, uncharged solutes, such as glycerol and other permeants such as carbon dioxide, nitric oxide, ammonia, hydrogen peroxide and the metalloids antimonite, arsenite, silicic and boric acid depending on the effective restriction mechanism of the protein. The flux properties of MIPs thus lead to the question ifMIPs can be used in separation devices or as sensor devices based on, e.g., the selective permeation of metalloids. In principle a MIP based membrane sensor/separation device requires the supporting biomimetic matrix to be virtually impermeable to anything but water or the solute in question. In practice, however, a biomimetic support matrix will generally have finite permeabilities to both electrolytes and non-electrolytes. The feasibility of a biomimetic MIP device thus depends on the relative transport

  4. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

    PubMed Central

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J.

    2015-01-01

    The organization of proteins and lipids in the plasma membrane has been subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here, we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase, nor result in any enrichment of nanoscopic ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane. PMID:25897971

  5. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane.

    PubMed

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J

    2015-01-01

    The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane. PMID:25897971

  6. The Reticulon and Dp1/Yop1p Proteins Form Immobile Oligomers in the Tubular Endoplasmic Reticulum*S⃞

    PubMed Central

    Shibata, Yoko; Voss, Christiane; Rist, Julia M.; Hu, Junjie; Rapoport, Tom A.; Prinz, William A.; Voeltz, Gia K.

    2008-01-01

    We recently identified a class of membrane proteins, the reticulons and DP1/Yop1p, which shape the tubular endoplasmic reticulum (ER) in yeast and mammalian cells. These proteins are highly enriched in the tubular portions of the ER and virtually excluded from other regions. To understand how they promote tubule formation, we characterized their behavior in cellular membranes and addressed how their localization in the ER is determined. Using fluorescence recovery after photobleaching, we found that yeast Rtn1p and Yop1p are less mobile in the membrane than normal ER proteins. Sucrose gradient centrifugation and cross-linking analyses show that they form oligomers. Mutants of yeast Rtn1p, which no longer localize exclusively to the tubular ER or are even totally inactive in inducing ER tubules, are more mobile and oligomerize less extensively. The mammalian reticulons and DP1 are also relatively immobile and can form oligomers. The conserved reticulon homology domain that includes the two membrane-embedded segments is sufficient for the localization of the reticulons to the tubular ER, as well as for their diffusional immobility and oligomerization. Finally, ATP depletion in both yeast and mammalian cells further decreases the mobilities of the reticulons and DP1. We propose that oligomerization of the reticulons and DP1/Yop1p is important for both their localization to the tubular domains of the ER and for their ability to form tubules. PMID:18442980

  7. Preparation of a novel Zr(4+)-immobilized metal affinity membrane for selective adsorption of phosphoprotein.

    PubMed

    He, Maofang; Wang, Chaozhan; Wei, Yinmao

    2016-09-01

    In this study, a novel phosphate-Zr(4+) immobilized metal affinity membrane (IMAM) was prepared based on the surface initiated-atom transfer radical polymerization technique for the selective adsorption of phosphoprotein. The adsorption capacity and selectivity of the phosphate-Zr(4+) IMAM were evaluated by using the mixture of standard phosphoproteins (β-casein, ovalbumin) and nonphosphoproteins (bovine serum albumin and lysozyme) as model samples. The adsorption isotherms and competitive adsorption results demonstrated that the phosphate-Zr(4+) IMAM had higher binding capacity and selectivity for phosphoproteins over nonphosphoproteins. Moreover, the phosphate-Zr(4+) IMAM exhibited good re-usability and re-productivity. Finally, the phosphate-Zr(4+) IMAM was applied to separate phosphoprotein from real samples with high purity. Therefore, the as-prepared phosphate-Zr(4+) IMAM could be a promising affinity material for the efficient enrichment of phosphoprotein from complex bio-samples. PMID:27433983

  8. Development of 170 MHz Electrodeless Quartz-Crystal Microbalance Immunosensor with Nonspecifically Immobilized Receptor Proteins

    NASA Astrophysics Data System (ADS)

    Hirotsugu Ogi,; Hironao Nagai,; Yuji Fukunishi,; Taiji Yanagida,; Masahiko Hirao,; Masayoshi Nishiyama,

    2010-07-01

    Staphylococcus aureus protein A (SPA) shows high nonspecific binding affinity on a naked quartz surface, and it can be used as the receptor protein for detecting immunoglobulin G (IgG), the most important immunoglobulin. The immunosensor ability, however, significantly depends on the immobilization procedure. In this work, the effect of the nonspecific immobilization procedure on the sensor sensitivity is studied using a home-built electrodeless quartz-crystal microbalance (QCM) biosensor. The pure-shear vibration of a 9.7-μm-thick AT-cut quartz plate is excited and detected in liquids by the line antenna located outside the flow channel. SPA molecules are immobilized on the quartz surfaces, and human IgG is injected to monitor the binding reaction between SPA and IgG. This study reveals that a long (nearly 24 h) immersion procedure is required for immobilizing SPA to achieve the tight biding with the quartz surfaces.

  9. Development of 170 MHz Electrodeless Quartz-Crystal Microbalance Immunosensor with Nonspecifically Immobilized Receptor Proteins

    NASA Astrophysics Data System (ADS)

    Ogi, Hirotsugu; Nagai, Hironao; Fukunishi, Yuji; Yanagida, Taiji; Hirao, Masahiko; Nishiyama, Masayoshi

    2010-07-01

    Staphylococcus aureus protein A (SPA) shows high nonspecific binding affinity on a naked quartz surface, and it can be used as the receptor protein for detecting immunoglobulin G (IgG), the most important immunoglobulin. The immunosensor ability, however, significantly depends on the immobilization procedure. In this work, the effect of the nonspecific immobilization procedure on the sensor sensitivity is studied using a home-built electrodeless quartz-crystal microbalance (QCM) biosensor. The pure-shear vibration of a 9.7-µm-thick AT-cut quartz plate is excited and detected in liquids by the line antenna located outside the flow channel. SPA molecules are immobilized on the quartz surfaces, and human IgG is injected to monitor the binding reaction between SPA and IgG. This study reveals that a long (nearly 24 h) immersion procedure is required for immobilizing SPA to achieve the tight biding with the quartz surfaces.

  10. Glutaraldehyde activated eggshell membrane for immobilization of tyrosinase from Amorphophallus companulatus: application in construction of electrochemical biosensor for dopamine.

    PubMed

    Tembe, Sanket; Kubal, B S; Karve, Meena; D'Souza, S F

    2008-04-01

    Tyrosinase from a plant source Amorphophallus companulatus was immobilized on eggshell membrane using glutaraldehyde. Among the three different approaches used for immobilization, activation of eggshell membrane by glutaraldehyde followed by enzyme adsorption on activated support could stabilize the enzyme tyrosinase and was found to be effective. K(m) and V(max) values for dopamine hydrochloride calculated from Lineweaver-Burk plot were 0.67 mM and 0.08 mM min(-1), respectively. Studies on effect of pH showed retention of more than 90% activity over a pH range 5.0-6.5. Membrane bound enzyme exhibited consistent activity in the temperature range 20-45 degrees C. Shelf life of immobilized tyrosinase system was found to be more than 6 months when stored in phosphate buffer at 4 degrees C. An electrochemical biosensor for dopamine was developed by mounting the tyrosinase immobilized eggshell membrane on the surface of glassy carbon electrode. Dopamine concentrations were determined by the direct reduction of biocatalytically liberated quinone species at -0.19 V versus Ag/AgCl (3M KCl). Linearity was observed within the range of 50-250 microM with a detection limit of 25 microM. PMID:18358868

  11. Quantitative structure-retention relationships of flavonoids unraveled by immobilized artificial membrane chromatography.

    PubMed

    Santoro, Adriana Leandra; Carrilho, Emanuel; Lanças, Fernando Mauro; Montanari, Carlos Alberto

    2016-06-10

    The pharmacokinetic properties of flavonoids with differing degrees of lipophilicity were investigated using immobilized artificial membranes (IAMs) as the stationary phase in high performance liquid chromatography (HPLC). For each flavonoid compound, we investigated whether the type of column used affected the correlation between the retention factors and the calculated octanol/water partition (log Poct). Three-dimensional (3D) molecular descriptors were calculated from the molecular structure of each compound using i) VolSurf software, ii) the GRID method (computational procedure for determining energetically favorable binding sites in molecules of known structure using a probe for calculating the 3D molecular interaction fields, between the probe and the molecule), and iii) the relationship between partition and molecular structure, analyzed in terms of physicochemical descriptors. The VolSurf built-in Caco-2 model was used to estimate compound permeability. The extent to which the datasets obtained from different columns differ both from each other and from both the calculated log Poct and the predicted permeability in Caco-2 cells was examined by principal component analysis (PCA). The immobilized membrane partition coefficients (kIAM) were analyzed using molecular descriptors in partial least square regression (PLS) and a quantitative structure-retention relationship was generated for the chromatographic retention in the cholesterol column. The cholesterol column provided the best correlation with the permeability predicted by the Caco-2 cell model and a good fit model with great prediction power was obtained for its retention data (R(2)=0.96 and Q(2)=0.85 with four latent variables). PMID:26916828

  12. A Survey of Membrane Proteins in Human Serum

    PubMed Central

    Dung, Nguyen Tien; Van Chi, Phan

    2012-01-01

    Serum and membrane proteins are two of the most attractive targets for proteomic analysis. Previous membrane protein studies tend to focus on tissue sample, while membrane protein studies in serum are still limited. In this study, an analysis of membrane proteins in normal human serum was carried out. Nano-liquid chromatography-electrospray ionization mass spectrometry (NanoLC-ESI-MS/MS) and bioinformatics tools were used to identify membrane proteins. Two hundred and seventeen membrane proteins were detected in the human serum, of which 129 membrane proteins have at least one transmembrane domain (TMD). Further characterizations of identified membrane proteins including their subcellular distributions, molecular weights, post translational modifications, transmembrane domains and average of hydrophobicity, were also implemented. Our results showed the potential of membrane proteins in serum for diagnosis and treatment of diseases. PMID:25288886

  13. Facile and high-efficient immobilization of histidine-tagged multimeric protein G on magnetic nanoparticles

    PubMed Central

    2014-01-01

    This work reports the high-efficient and one-step immobilization of multimeric protein G on magnetic nanoparticles. The histidine-tagged (His-tag) recombinant multimeric protein G was overexpressed in Escherichia coli BL21 by the repeated linking of protein G monomers with a flexible linker. High-efficient immobilization on magnetic nanoparticles was demonstrated by two different preparation methods through the amino-silane and chloro-silane functionalization on silica-coated magnetic nanoparticles. Three kinds of multimeric protein G such as His-tag monomer, dimer, and trimer were tested for immobilization efficiency. For these tests, bicinchoninic acid (BCA) assay was employed to determine the amount of immobilized His-tag multimeric protein G. The result showed that the immobilization efficiency of the His-tag multimeric protein G of the monomer, dimer, and trimer was increased with the use of chloro-silane-functionalized magnetic nanoparticles in the range of 98% to 99%, rather than the use of amino-silane-functionalized magnetic nanoparticles in the range of 55% to 77%, respectively. PMID:25593554

  14. A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

    SciTech Connect

    Rosiere, T.K.; Marrs, J.A.; Bouck, G.B. )

    1990-04-01

    The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39.

  15. Anionic deep cavitands enable the adhesion of unmodified proteins at a membrane bilayer.

    PubMed

    Ghang, Yoo-Jin; Perez, Lizeth; Morgan, Melissa A; Si, Fang; Hamdy, Omar M; Beecher, Consuelo N; Larive, Cynthia K; Julian, Ryan R; Zhong, Wenwan; Cheng, Quan; Hooley, Richard J

    2014-12-28

    An anionic self-folding deep cavitand is capable of immobilizing unmodified proteins and enzymes at a supported lipid bilayer interface, providing a simple, soft bioreactive surface that allows enzymatic function under mild conditions. The adhesion is based on complementary charge interactions, and the hosts are capable of binding enzymes such as trypsin at the bilayer interface: the catalytic activity is retained upon adhesion, allowing selective reactions to be performed at the membrane surface. PMID:25366572

  16. Solution phase and membrane immobilized iron-based free radical reactions: Fundamentals and applications for water treatment

    NASA Astrophysics Data System (ADS)

    Lewis, Scott Romak

    Membrane-based separation processes have been used extensively for drinking water purification, wastewater treatment, and numerous other applications. Reactive membranes synthesized through functionalization of the membrane pores offer enhanced reactivity due to increased surface area at the polymer-solution interface and low diffusion limitations. Oxidative techniques utilizing free radicals have proven effective for both the destruction of toxic organics and non-environmental applications. Most previous work focuses on reactions in the homogeneous phase; however, the immobilization of reactants in membrane pores offers several advantages. The use of polyanions immobilized in a membrane or chelates in solution prevents ferric hydroxide precipitation at near-neutral pH, a common limitation of iron(Fe(II/III))-catalyzed hydrogen peroxide (H 2O2) decomposition. The objectives of this research are to develop a membrane-based platform for the generation of free radicals, degrade toxic organic compounds using this and similar solution-based reactions, degrade toxic organic compounds in droplet form, quantify hydroxyl radical production in these reactions, and develop kinetic models for both processes. In this study, a functionalized membrane containing poly(acrylic acid) (PAA) was used to immobilize iron ions and conduct free radical reactions by permeating H2O2 through the membrane. The membrane's responsive behavior to pH and divalent cations was investigated and modeled. The conversion of Fe(II) to Fe(III) in the membrane and its effect on the decomposition of hydrogen peroxide were monitored and used to develop kinetic models for predicting H2O2 decomposition in these systems. The rate of hydroxyl radical production, and hence contaminant degradation can be varied by changing the residence time, H2O2 concentration, and/or iron loading. Using these membrane-immobilized systems, successful removal of toxic organic compounds, such as pentachlorophenol (PCP), from water

  17. Nylon-Based Affinity Membranes: Impacts of Surface Modification on Protein Adsorption.

    PubMed

    Beeskow; Kroner; Anspach

    1997-12-15

    Nylon microfiltration membranes were activated with bisoxirane and formaldehyde at terminal amino groups and amide groups of the nylon polymer, respectively. Dextrans were covalently immobilized on these activated membranes to yield dextran-coated membrane matrices. Both procedures led to a significant reduction of hemoglobin adsorption; however, bisoxirane activation required additional cross-linking of dextran and a second dextran layer to yield comparable quality of dextran-coated membranes than formaldehyde activation. Formaldehyde activation was easiest and cheapest and resulted in membranes with highest dextran density and relatively lowest nonspecific hemoglobin adsorption. Dextrans of &Mmacr;w >/= 40,000 were required for bisoxirane-activated membranes, whereas dextrans of &Mmacr;w = 6000 were sufficient for formaldehyde-activated membranes. Both activation methods resulted in stable coatings at low and high pH; however, formaldehyde-activated membranes were unstable under strongly acidic conditions at pH < 3. Dextran coils were found responsible for the reduction of the hydraulic permeability but also for the high ligand densities obtained after immobilization of Cibacron Blue F3G-A (360 nmol/cm2) and iminodiacetic acid (400 nmol/cm2). The thermodynamics of protein adsorption on dye ligand affinity (DLA) membranes corresponded with chromatographic sorbents and dye ligand conjugates, with the dextran coating demonstrating similar structure than dextrans in solution. Protein adsorption took place in the extended coil structure of dextrans with binding capacities up to 730 µg/cm2 lysozyme on DLA membranes and 470 µg/cm2 concanavalin A on metal chelate affinity membranes. Copyright 1997 Academic Press. PMID:9792753

  18. Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

    PubMed

    Duquesne, Katia; Prima, Valérie; Sturgis, James N

    2016-01-01

    Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results. PMID:27485340

  19. Self diffusion of interacting membrane proteins.

    PubMed Central

    Abney, J R; Scalettar, B A; Owicki, J C

    1989-01-01

    A two-dimensional version of the generalized Smoluchowski equation is used to analyze the time (or distance) dependent self diffusion of interacting membrane proteins in concentrated membrane systems. This equation provides a well established starting point for descriptions of the diffusion of particles that interact through both direct and hydrodynamic forces; in this initial work only the effects of direct interactions are explicitly considered. Data describing diffusion in the presence of hard-core repulsions, soft repulsions, and soft repulsions with weak attractions are presented. The effect that interactions have on the self-diffusion coefficient of a real protein molecule from mouse liver gap junctions is also calculated. The results indicate that self diffusion is always inhibited by direct interactions; this observation is interpreted in terms of the caging that will exist at finite protein concentration. It is also noted that, over small distance scales, the diffusion coefficient is determined entirely by the very strong Brownian forces; therefore, as a function of displacement the self-diffusion coefficient decays (rapidly) from its value at infinite dilution to its steady-state interaction-averaged value. The steady-state self-diffusion coefficient describes motion over distance scales that range from approximately 10 nm to cellular dimensions and is the quantity measured in fluorescence recovery after photobleaching experiments. The short-ranged behavior of the diffusion coefficient is important on the interparticle-distance scale and may therefore influence the rate at which nearest-neighbor collisional processes take place. The hard-disk theoretical results presented here are in excellent agreement with lattice Monte-Carlo results obtained by other workers. The concentration dependence of experimentally measured diffusion coefficients of antibody-hapten complexes bound to the membrane surface is consistent with that predicted by the theory. The

  20. High Affinity Immobilization of Proteins Using the CrAsH/TC Tag.

    PubMed

    Schulte-Zweckel, Janine; Rosi, Federica; Sreenu, Domalapally; Schröder, Hendrik; Niemeyer, Christof M; Triola, Gemma

    2016-01-01

    Protein microarrays represent important tools for biomedical analysis. We have recently described the use of the biarsenical-tetracysteine (TC) tag for the preparation of protein microarrays. The unique feature of this tag enables the site-specific immobilization of TC-containing proteins on biarsenical-modified surfaces, resulting in a fluorescence enhancement that allows the direct quantification of the immobilized proteins. Moreover, the reversibility of the binding upon incubation with large quantities of thiols permits the detachment of the proteins from the surface, thereby enabling recovery of the substrate to extend the life time of the slide. Herein, we describe our recent results that further extend the applicability of the CrAsH/TC tag to the fabrication of biochips. With this aim, the immobilization of proteins on surfaces has been investigated using two different spacers and two TC tags, the minimal TC sequence (CCPGCC) and an optimized motif (FLNCCPGCCMEP). While the minimal peptide motif enables a rapid recycling of the slide, the optimized TC sequence reveals an increased affinity due to its greater resistance to displacement by thiols. Moreover, the developed methodology was applied to the immobilization of proteins via on-chip ligation of recombinant protein thioesters. PMID:27338319

  1. MemProtMD: Automated Insertion of Membrane Protein Structures into Explicit Lipid Membranes

    PubMed Central

    Stansfeld, Phillip J.; Goose, Joseph E.; Caffrey, Martin; Carpenter, Elisabeth P.; Parker, Joanne L.; Newstead, Simon; Sansom, Mark S.P.

    2015-01-01

    Summary There has been exponential growth in the number of membrane protein structures determined. Nevertheless, these structures are usually resolved in the absence of their lipid environment. Coarse-grained molecular dynamics (CGMD) simulations enable insertion of membrane proteins into explicit models of lipid bilayers. We have automated the CGMD methodology, enabling membrane protein structures to be identified upon their release into the PDB and embedded into a membrane. The simulations are analyzed for protein-lipid interactions, identifying lipid binding sites, and revealing local bilayer deformations plus molecular access pathways within the membrane. The coarse-grained models of membrane protein/bilayer complexes are transformed to atomistic resolution for further analysis and simulation. Using this automated simulation pipeline, we have analyzed a number of recently determined membrane protein structures to predict their locations within a membrane, their lipid/protein interactions, and the functional implications of an enhanced understanding of the local membrane environment of each protein. PMID:26073602

  2. Ultra-thin porous glass membranes--an innovative material for the immobilization of active species for optical chemosensors.

    PubMed

    Müller, R; Anders, N; Titus, J; Enke, D

    2013-03-30

    In addition to polymers, porous glasses can be used for the immobilization of indicators, chromoionophores or enzymes. Advantages of these materials include, among others, the photochemical and thermal stability. Porous glass membranes (CPG) based on phase-separated alkali borosilicate glasses with thicknesses of 250-300 μm and dimensions of approximately 9-13 mm² were used in this work. The average pore diameter was found to be between 12 and 112 nm. Initially, the membrane permeability for water was determined. Furthermore, the absorption spectra for the water-soaked membranes were recorded optically. CPG membranes which are pH-sensitive were prepared based on the covalent immobilization of thymol blue and a derivative of styryl acridine. In each case, the absorption spectra of the immobilized indicators are shown. The t90-times vary between 4 and 20 min and were determined for the thermodynamic equilibrium. The influence of the ionic strength on the characteristic curve is discussed and detailed results are given. After the storage time of about 900 days a pH-sensitivity for a CPG membrane styryl acridine derivative sample was still detectable. PMID:23598220

  3. Engineering Lipid Bilayer Membranes for Protein Studies

    PubMed Central

    Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton

    2013-01-01

    Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temperature, pH, ionic strength, adsorption behavior, conformational reorientation and surface density in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica, and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochemical functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiological conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biological conditions. This study summarizes the use of electrochemical impedance spectroscopy (EIS) data on artificial biological membranes that are closely matched with previously published biological systems using both black lipid membrane and patch clamp techniques. PMID:24185908

  4. Evaluation of Thermostabilities of Enzymes, Mediators and Immobilizing Membranes for Enzyme Sensors

    NASA Astrophysics Data System (ADS)

    Yamada, Yohei; Ohnishi, Yuki; Hayashi, Tetsuya; Isobe, Yoshifumi; Yabutani, Tomoki

    The stability of the constituents of electrochemical measurement, electron mediators, enzymes and enzyme-immobilizing membranes was evaluated under high temperature (maximum 75°C) by electrochemical analysis, UV-Vis spectrometry (UV-Vis) and UV circular dichroism (CD). As a result of stability evaluation of mediators at 75°C, electrochemical activity of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt (ABTS), potassium ferricyanide (K3[Fe(CN)6]) and ferrocenemethanol (FcOH) were not changed, but 2,6-dichloroindophenol (DCIP), p-benzoquinone (p-BQ), vitaminK3 (VK3) were greatly decreased. The stability of diaphorase from Bacillus stearothermophilus (DI) were compared between in-solution and in several types of membranes, Agarose H, Poly-L-lysine (PLL) and poly-ion-complex (PIC) by electrochemical analysis. In solution, activity and secondary structure of DI were changed at 65°C or higher. This tendency of activity was not much different in Agarose H but in PLL, the activity was almost kept until 70°C. It was suggested that DI was fixed on the electrodes in high concentration and the elimination of DI seldom arise in PLL from the magnitude of the current response and the results of prolonged stability evaluation.

  5. Biofield-effect protein-sensor: Plasma functionalization of polyaniline, protein immobilization, and sensing mechanism

    NASA Astrophysics Data System (ADS)

    Cho, Chae-Ryong; Lee, Hyun-Uk; Ahn, Kyun; Jeong, Se-Young; Choi, Jun-Hee; Kim, Jinwoo; Cho, Jiung

    2014-06-01

    We report the fabrication of a biofield-effect protein-sensor (BioFEP) based on atmospheric-pressure plasma (AP) treatment of a conducting polyaniline (PANI) film. Successive H2 and O2 AP (OHAP) treatment generated dominant hydrophilic -OH and O=CO- functional groups on the PANI film surface, which served as strong binding sites to immobilize bovine serum albumin (BSA) protein molecules. The output current changes of the BioFEP as a function of BSA concentration were obtained. The resistance of the OHAP surface could be sensitively increased from 2.5 × 108 Ω to 2.0 × 1012 Ω with increasing BSA concentrations in the range of 0.025-4 μg/ml. The results suggest that the method is a simple and cost-effective tool to determine the concentration of BSA by measuring electrical resistance.

  6. When physics takes over: BAR proteins and membrane curvature

    PubMed Central

    Simunovic, Mijo; Voth, Gregory A.; Callan-Jones, Andrew; Bassereau, Patricia

    2016-01-01

    Cell membranes become highly curved during membrane trafficking, cytokinesis, infection, immune response or cell motion. Bin/amphiphysin/Rvs (BAR) domain proteins with their intrinsically curved and anisotropic shape are involved in many of these processes, but with a large spectrum of modes of action. In vitro experiments and multiscale computer simulations have contributed in identifying a minimal set of physical parameters, namely protein density on the membrane, membrane tension, and membrane shape, that control how bound BAR domain proteins behave on the membrane. In this review, we summarize the multifaceted coupling of BAR proteins to membrane mechanics and propose a simple phase diagram that recapitulates the effects of these parameters. PMID:26519988

  7. SNARE proteins and ‘membrane rafts’

    PubMed Central

    Lang, Thorsten

    2007-01-01

    The original ‘lipid raft’ hypothesis proposed that lipid-platforms/rafts form in the exoplasmic plasmalemmal leaflet by tight clustering of sphingolipids and cholesterol. Their physical state, presumably similar to liquid-ordered phases in model membranes, would confer detergent resistance to rafts and enriched proteins therein. Based on this concept, detergent resistant membranes (DRMs) from solubilized cells were considered to reflect pre-existing ‘lipid rafts’ in live cells. To date, more than 200 proteins were found in DRMs including also members of the SNARE superfamily, which are small membrane proteins involved in intracellular fusion steps. Their raft association indicates that they are not uniformly distributed, and, indeed, microscopic studies revealed that SNAREs concentrate in submicrometre-sized, cholesterol-dependent clusters at which vesicles fuse. However, the idea that SNARE clusters are ‘lipid rafts’ was challenged, as they do not colocalize with raft markers, and SNAREs are excluded from liquid-ordered phases in model membranes. Independent from this disagreement, in recent years the solubilization criterion has been criticized for several reasons, calling for a more exact definition of rafts. At a recent consensus on a revised raft model, the term ‘lipid rafts’ was replaced by ‘membrane rafts’ that were defined as ‘small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes’. As a result, after dismissing the terms ‘detergent resistant’ and ‘liquid-ordered’, it now appears that SNARE clusters are bona fide ‘membrane rafts’. PMID:17478530

  8. Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

    PubMed

    Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

    2016-02-01

    A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. PMID:26522987

  9. Membrane protein structure determination by electron crystallography

    PubMed Central

    Ubarretxena-Belandia, Iban; Stokes, David L.

    2012-01-01

    During the past year, electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer. From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope. There have also been key developments in software, and in molecular replacement and phase extension methods designed to facilitate the process of structure determination. PMID:22572457

  10. Protein permeation through an electrically tunable membrane

    NASA Astrophysics Data System (ADS)

    Jou, Ining A.; Melnikov, Dmitriy V.; Gracheva, Maria E.

    2016-05-01

    Protein filtration is important in many fields of science and technology such as medicine, biology, chemistry, and engineering. Recently, protein separation and filtering with nanoporous membranes has attracted interest due to the possibility of fast separation and high throughput volume. This, however, requires understanding of the protein’s dynamics inside and in the vicinity of the nanopore. In this work, we utilize a Brownian dynamics approach to study the motion of the model protein insulin in the membrane–electrolyte electrostatic potential. We compare the results of the atomic model of the protein with the results of a coarse-grained and a single-bead model, and find that the coarse-grained representation of protein strikes the best balance between the accuracy of the results and the computational effort required. Contrary to common belief, we find that to adequately describe the protein, a single-bead model cannot be utilized without a significant effort to tabulate the simulation parameters. Similar to results for nanoparticle dynamics, our findings also indicate that the electric field and the electro-osmotic flow due to the applied membrane and electrolyte biases affect the capture and translocation of the biomolecule by either attracting or repelling it to or from the nanopore. Our computational model can also be applied to other types of proteins and separation conditions.

  11. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling. PMID:26152529

  12. Immobilization-stabilization of proteins on nanofibrillated cellulose derivatives and their bioactive film formation.

    PubMed

    Arola, Suvi; Tammelin, Tekla; Setälä, Harri; Tullila, Antti; Linder, Markus B

    2012-03-12

    In a number of different applications for enzymes and specific binding proteins a key technology is the immobilization of these proteins to different types of supports. In this work we describe a concept for protein immobilization that is based on nanofibrillated cellulose (NFC). NFC is a form of cellulose where fibers have been disintegrated into fibrils that are only a few nanometers in diameter and have a very large aspect ratio. Proteins were conjugated through three different strategies using amine, epoxy, and carboxylic acid functionalized NFC. The conjugation chemistries were chosen according to the reactive groups on the NFC derivatives; epoxy amination, heterobifunctional modification of amino groups, and EDC/s-NHS activation of carboxylic acid groups. The conjugation reactions were performed in solution and immobilization was performed by spin coating the protein-NCF conjugates. The structure of NFC was shown to be advantageous for both protein performance and stability. The use of NFC allows all covalent chemistry to be performed in solution, while the immobilization is achieved by a simple spin coating or spreading of the protein-NFC conjugates on a support. This allows more scalable methods and better control of conditions compared to the traditional methods that depend on surface reactions. PMID:22248303

  13. Preparation of λN-GST fusion protein for affinity immobilization of RNA.

    PubMed

    Di Tomasso, Geneviève; Lampron, Philipe; Omichinski, James G; Legault, Pascale

    2012-01-01

    Affinity purification of in vitro transcribed RNA is becoming an attractive alternative to purification using standard denaturing gel electrophoresis. Affinity purification is particularly advantageous because it can be performed in a few hours under non-denaturing conditions. However, the performance of affinity purification methods can vary tremendously depending on the RNA immobilization matrix. It was previously shown that RNA immobilization via an optimized λN-GST fusion protein bound to glutathione-Sepharose resin allows affinity purification of RNA with very high purity and yield. This Chapter outlines the experimental procedure employed to prepare the λN-GST fusion protein used for RNA immobilization in successful affinity purifications of RNA. It describes the details of protein expression and purification as well as routine quality control analyses. PMID:23065558

  14. Statistical thermodynamic analysis of peptide and protein insertion into lipid membranes.

    PubMed Central

    Ben-Shaul, A; Ben-Tal, N; Honig, B

    1996-01-01

    A statistical thermodynamic approach is used to analyze the various contributions to the free energy change associated with the insertion of proteins and protein fragments into lipid bilayers. The partition coefficient that determines the equilibrium distribution of proteins between the membrane and the solution is expressed as the ratio between the partition functions of the protein in the two phases. It is shown that when all of the relevant degrees of freedom (i.e., those that change their character upon insertion into the membrane) can be treated classically, the partition coefficient is fully determined by the ratio of the configurational integrals and thus does not involve any mass-dependent factors, a conclusion that is also valid for related processes such as protein adsorption on a membrane surface or substrate binding to proteins. The partition coefficient, and hence the transfer free energy, depend only on the potential energy of the protein in the membrane. Expressing this potential as a sum of a "static" term, corresponding to the equilibrium (minimal free energy) configuration of the protein in the membrane, and a "dynamical" term representing fluctuations around the equilibrium configuration, we show that the static term contains the "solvation" and "lipid perturbation" contributions to the transfer free energy. The dynamical term is responsible for the "immobilization" free energy, reflecting the loss of translational and rotational entropy of the protein upon incorporation into the membrane. Based on a recent molecular theory of lipid-protein interactions, the lipid perturbation and immobilization contributions are then expressed in terms of the elastic deformation free energy resulting from the perturbation of the lipid environment by the foreign (protein) inclusion. The model is formulated for cylindrically shaped proteins, and numerical estimates are given for the insertion of an alpha-helical peptide into a lipid bilayer. The immobilization

  15. Golgi protein FAPP2 tubulates membranes

    PubMed Central

    Cao, Xinwang; Coskun, Ünal; Rössle, Manfred; Buschhorn, Sabine B.; Grzybek, Michal; Dafforn, Timothy R.; Lenoir, Marc; Overduin, Michael; Simons, Kai

    2009-01-01

    The Golgi-associated four-phosphate adaptor protein 2 (FAPP2) has been shown to possess transfer activity for glucosylceramide both in vitro and in cells. We have previously shown that FAPP2 is involved in apical transport from the Golgi complex in epithelial MDCK cells. In this paper we assign an unknown activity for the protein as well as providing structural insight into protein assembly and a low-resolution envelope structure. By applying analytical ultracentrifugation and small-angle x-ray scattering, we show that FAPP2 is a dimeric protein in solution, having a curved shape 30 nm in length. The purified FAPP2 protein has the capability to form tubules from membrane sheets in vitro. This activity is dependent on the phosphoinositide-binding activity of the PH domain of FAPP2. These data suggest that FAPP2 functions directly in the formation of apical carriers in the trans-Golgi network. PMID:19940249

  16. Exploiting Microbeams for Membrane Protein Structure Determination.

    PubMed

    Warren, Anna J; Axford, Danny; Paterson, Neil G; Owen, Robin L

    2016-01-01

    A reproducible, and sample independent means of predictably obtaining large, well-ordered crystals has proven elusive in macromolecular crystallography. In the structure determination pipeline, crystallisation often proves to be a rate-limiting step, and the process of obtaining even small or badly ordered crystals can prove time-consuming and laborious. This is particularly true in the field of membrane protein crystallography and this is reflected in the limited number of unique membrane protein structures deposited in the protein data bank (less than 650 by June 2016 - http://blanco.biomol.uci.edu/mpstruc ). Over recent years the requirement for, and time and cost associated with obtaining, large crystals has been partially alleviated through the development of beamline instrumentation allowing data collection, and structure solution, from ever-smaller crystals. Advances in several areas have led to a step change in what might be considered achievable during a synchrotron trip over the last decade. This chapter will briefly review the current status of the field, the tools available to ease data collection and processing, and give some examples of exploitation of these for membrane protein microfocus macromolecular crystallography. PMID:27553238

  17. Development of continuous microwave-assisted protein digestion with immobilized enzyme.

    PubMed

    Chen, Zhengyi; Li, Yongle; Lin, Shuhai; Wei, Meiping; Du, Fuyou; Ruan, Guihua

    2014-03-01

    In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC-MS(n). Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS-PAGE and LC-MS(n). Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC-MS(n) profiles of the hydrolysate was compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research. PMID:24530398

  18. Co-Immobilization of Proteins and DNA Origami Nanoplates to Produce High-Contrast Biomolecular Nanoarrays.

    PubMed

    Hager, Roland; Burns, Jonathan R; Grydlik, Martyna J; Halilovic, Alma; Haselgrübler, Thomas; Schäffler, Friedrich; Howorka, Stefan

    2016-06-01

    The biofunctionalization of nanopatterned surfaces with DNA origami nanostructures is an important topic in nanobiotechnology. An unexplored challenge is, however, to co-immobilize proteins with DNA origami at pre-determined substrate sites in high contrast relative to the nontarget areas. The immobilization should, in addition, preferably be achieved on a transparent substrate to allow ultrasensitive optical detection. If successful, specific co-binding would be a step towards stoichiometrically defined arrays with few to individual protein molecules per site. Here, we successfully immobilize with high specificity positively charged avidin proteins and negatively charged DNA origami nanoplates on 100 nm-wide carbon nanoislands while suppressing undesired adsorption to surrounding nontarget areas. The arrays on glass slides achieve unprecedented selectivity factors of up to 4000 and allow ultrasensitive fluorescence read-out. The co-immobilization onto the nanoislands leads to layered biomolecular architectures, which are functional because bound DNA origami influences the number of capturing sites on the nanopatches for other proteins. The novel hybrid DNA origami-protein nanoarrays allow the fabrication of versatile research platforms for applications in biosensing, biophysics, and cell biology, and, in addition, represent an important step towards single-molecule protein arrays. PMID:27062557

  19. Purification of proteins containing zinc finger domains using Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Voráčková, Irena; Suchanová, Šárka; Ulbrich, Pavel; Diehl, William E.; Ruml, Tomáš

    2011-01-01

    Heterologous proteins are frequently purified by Immobilized Metal Ion Affinity Chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e. CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state. PMID:21600288

  20. Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

    PubMed Central

    Witte, Martin D.; Wu, Tongfei; Guimaraes, Carla P.; Theile, Christopher S.; Blom, Annet E.M.; Ingram, Jessica R.; Li, Zeyang; Kundrat, Lenka; Goldberg, Shalom D.; Ploegh, Hidde L.

    2016-01-01

    Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bioorthogonal reactive groups and affinity handles. This protocol describes immobilization of sortase A on a solid support (sepharose beads). Immobilization of sortase A simplifies downstream purification of a protein of interest after labeling of its N- or C- terminus. Small batch and larger scale continuous flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca2+-independent variant of sortase A with increased catalytic activity. This heptamutant variant of sortase A (7M) was generated by combining previously published mutations and this immobilized enzyme can used for the modification of calcium-senstive substrates or in instances where low temperatures are needed. Preparation of immobilized sortase A takes 1–2 days. Batch reactions take 3–12 hours and flow reactions proceed at 0.5 mL per hour, depending on the geometry of the reactor used. PMID:25719269

  1. Outer membrane proteins of pathogenic spirochetes

    PubMed Central

    Cullen, Paul A.; Haake, David A.; Adler, Ben

    2009-01-01

    Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis. PMID:15449605

  2. Serial Millisecond Crystallography of Membrane Proteins.

    PubMed

    Jaeger, Kathrin; Dworkowski, Florian; Nogly, Przemyslaw; Milne, Christopher; Wang, Meitian; Standfuss, Joerg

    2016-01-01

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a powerful method to determine high-resolution structures of pharmaceutically relevant membrane proteins. Recently, the technology has been adapted to carry out serial millisecond crystallography (SMX) at synchrotron sources, where beamtime is more abundant. In an injector-based approach, crystals grown in lipidic cubic phase (LCP) or embedded in viscous medium are delivered directly into the unattenuated beam of a microfocus beamline. Pilot experiments show the application of microjet-based SMX for solving the structure of a membrane protein and compatibility of the method with de novo phasing. Planned synchrotron upgrades, faster detectors and software developments will go hand-in-hand with developments at free-electron lasers to provide a powerful methodology for solving structures from microcrystals at room temperature, ligand screening or crystal optimization for time-resolved studies with minimal or no radiation damage. PMID:27553240

  3. An all-aqueous route to polymer brush-modified membranes with remarkable permeabilites and protein capture rates

    PubMed Central

    Anuraj, Nishotha; Bhattacharjee, Somnath; Geiger, James H.; Baker, Gregory L.; Bruening, Merlin L.

    2011-01-01

    Microporous membranes are attractive for protein purification because convection rapidly brings proteins to binding sites. However, the low binding capacity of such membranes limits their applications. This work reports a rapid, aqueous procedure to create highly permeable, polymer brush-modified membranes that bind large amounts of protein. The synthetic method includes a 10-min adsorption of a macroinitiator in a hydroxylated nylon membrane and a subsequent 5-min aqueous atom transfer radical polymerization of 2-(methacryloyloxy)ethyl succinate from the immobilized initiator to form poly(acid) brushes. This procedure likely leads to more swollen, less dense brushes than polymerization from silane initiators, and thus requires less polymer to achieve the same binding capacity. The hydraulic permeability of the poly(acid) membranes is 4-fold higher than that of similar membranes prepared by growing brushes from immobilized silane initiators. These brush-containing nylon membranes bind 120 mg/cm3 of lysozyme using solution residence times as short as 35 ms, and when functionalized with nitrilotriacetate (NTA)-Ni2+ complexes, they capture 85 mg/cm3 of histidine6-tagged (His-tagged) Ubiquitin. Additionally the NTA-Ni2+-functionalized membranes isolate His-tagged myo-inositol-1-phosphate synthase directly from cell extracts and show >90% recovery of His-tagged proteins. PMID:22287817

  4. Binding contribution between synaptic vesicle membrane and plasma membrane proteins in neurons: an AFM study.

    PubMed

    Sritharan, K C; Quinn, A S; Taatjes, D J; Jena, B P

    1998-01-01

    The final step in the exocytotic process is the docking and fusion of membrane-bound secretory vesicles at the cell plasma membrane. This docking and fusion is brought about by several participating vesicle membrane, plasma membrane and soluble cytosolic proteins. A clear understanding of the interactions between these participating proteins giving rise to vesicle docking and fusion is essential. In this study, the binding force profiles between synaptic vesicle membrane and plasma membrane proteins have been examined for the first time using the atomic force microscope. Binding force contributions of a synaptic vesicle membrane protein VAMP1, and the plasma membrane proteins SNAP-25 and syntaxin, are also implicated from these studies. Our study suggests that these three proteins are the major, if not the only contributors to the interactive binding force that exist between the two membranes. PMID:10452835

  5. Lipidic phase membrane protein serial femtosecond crystallography

    PubMed Central

    Johansson, Linda C; Arnlund, David; White, Thomas A; Katona, Gergely; DePonte, Daniel P; Weierstall, Uwe; Doak, R Bruce; Shoeman, Robert L; Lomb, Lukas; Malmerberg, Erik; Davidsson, Jan; Nass, Karol; Liang, Mengning; Andreasson, Jakob; Aquila, Andrew; Bajt, Sasa; Barthelmess, Miriam; Barty, Anton; Bogan, Michael J; Bostedt, Christoph; Bozek, John D; Caleman, Carl; Coffee, Ryan; Coppola, Nicola; Ekeberg, Tomas; Epp, Sascha W; Erk, Benjamin; Fleckenstein, Holger; Foucar, Lutz; Graafsma, Heinz; Gumprecht, Lars; Hajdu, Janos; Hampton, Christina Y; Hartmann, Robert; Hartmann, Andreas; Hauser, Günter; Hirsemann, Helmut; Holl, Peter; Hunter, Mark S; Kassemeyer, Stephan; Kimmel, Nils; Kirian, Richard A; Maia, Filipe R N C; Marchesini, Stefano; Martin, Andrew V; Reich, Christian; Rolles, Daniel; Rudek, Benedikt; Rudenko, Artem; Schlichting, Ilme; Schulz, Joachim; Seibert, M Marvin; Sierra, Raymond G; Soltau, Heike; Starodub, Dmitri; Stellato, Francesco; Stern, Stephan; Strüder, Lothar; Timneanu, Nicusor; Ullrich, Joachim; Wahlgren, Weixiao Y; Wang, Xiaoyu; Weidenspointner, Georg; Wunderer, Cornelia; Fromme, Petra; Chapman, Henry N; Spence, John C H; Neutze, Richard

    2012-01-01

    X-ray free electron laser (X-feL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-feL beam using a sponge phase micro-jet. PMID:22286383

  6. Theoretical analysis of protein organization in lipid membranes.

    PubMed

    Gil, T; Ipsen, J H; Mouritsen, O G; Sabra, M C; Sperotto, M M; Zuckermann, M J

    1998-11-10

    The fundamental physical principles of the lateral organization of trans-membrane proteins and peptides as well as peripheral membrane proteins and enzymes are considered from the point of view of the lipid-bilayer membrane, its structure, dynamics, and cooperative phenomena. Based on a variety of theoretical considerations and model calculations, the nature of lipid-protein interactions is considered both for a single protein and an assembly of proteins that can lead to aggregation and protein crystallization in the plane of the membrane. Phenomena discussed include lipid sorting and selectivity at protein surfaces, protein-lipid phase equilibria, lipid-mediated protein-protein interactions, wetting and capillary condensation as means of protein organization, mechanisms of two-dimensional protein crystallization, as well as non-equilibrium organization of active proteins in membranes. The theoretical findings are compared with a variety of experimental data. PMID:9804966

  7. Immobilization of catalase on electrospun PVA/PA6-Cu(II) nanofibrous membrane for the development of efficient and reusable enzyme membrane reactor.

    PubMed

    Feng, Quan; Zhao, Yong; Wei, Anfang; Li, Changlong; Wei, Qufu; Fong, Hao

    2014-09-01

    In this study, a mat/membrane consisting of overlaid PVA/PA6-Cu(II) composite nanofibers was prepared via the electrospinning technique followed by coordination/chelation with Cu(II) ions; an enzyme of catalase (CAT) was then immobilized onto the PVA/PA6-Cu(II) nanofibrous membrane. The amount of immobilized catalase reached a high value of 64 ± 4.6 mg/g, while the kinetic parameters (Vmax and Km) of enzyme were 3774 μmol/mg·min and 41.13 mM, respectively. Furthermore, the thermal stability and storage stability of immobilized catalase were improved significantly. Thereafter, a plug-flow type of immobilized enzyme membrane reactor (IEMR) was assembled from the PVA/PA6-Cu(II)-CAT membrane. With the increase of operational pressure from 0.02 to 0.2 MPa, the flux value of IEMR increased from 0.20 ± 0.02 to 0.76 ± 0.04 L/m(2)·min, whereas the conversion ratio of H2O2 decreased slightly from 92 ± 2.5% to 87 ± 2.1%. After 5 repeating cycles, the production capacity of IEMR was merely decreased from 0.144 ± 0.006 to 0.102 ± 0.004 mol/m(2)·min. These results indicated that the assembled IEMR possessed high productivity and excellent reusability, suggesting that the IEMR based on electrospun PVA/PA6-Cu(II) nanofibrous membrane might have great potential for various applications, particularly those related to environmental protection. PMID:25093534

  8. Direct dye binding--a quantitative assay for solid-phase immobilized protein.

    PubMed

    Bonde, M; Pontoppidan, H; Pepper, D S

    1992-01-01

    A direct dye-binding procedure was established for the quantification of protein after its immobilization on a solid phase, using IgG and BSA as model proteins. The assay, which in the range 0-5 mg protein/ml gel correlates well with indirect protein determination by A280 as well as determination of protein hydrolyzed from the gel, is based on a modified Bradford dye-binding assay. As the protein coupled to the gel binds the dye, a decrease in A465 of the supernatant is measured. Three solid supports commonly used for protein immobilization (Sepharose, Sephadex, Sephacryl) were found to be compatible with the dye-binding assay while nonspecific dye binding was found to HEMA gels. Protein was coupled to Sephacryl S-1000 using three different activation methods (aldehyde, hydrazine, and adipic acid dihydrazide). Artifactual dye-binding was not observed using any of the three different "linkers." The assay is easily carried out and represents a useful tool, e.g., when optimizing procedures for protein immobilization. PMID:1595895

  9. Synergistic effects of amine and protein modified epoxy-support on immobilized lipase activity.

    PubMed

    Cui, Caixia; Tao, Yifeng; Ge, Chunling; Zhen, Yueju; Chen, Biqiang; Tan, Tianwei

    2015-09-01

    We have developed an improved and effective method to immobilize Yarrowia lipolytica lipase Lip2 (YLIP2) on an epoxy poly-(glycidylmethacrylate-triallyisocyanurate-ethyleneglycoldimethacrylate) (PGMA-TAIC-EGDMA) support structure with or without amine or/and protein modifications. Our results show that there is an increase in the activity of the immobilized lipase on n-butylamine (BA) modified support (420U/g support) and the biocompatible gelatin modified support (600U/g support) when compared to the support without modification (240U/g support). To further study the influences of BA and gelatin modification on the activity of the immobilized lipase, gelatin and BA were concurrently used to decorate the support structure. Lipase immobilized on 2% BA/gelatin (1:1) modified support obtained the highest activity (1180U/g support), which was five-fold higher than that on a native support structure. These results suggest that the activity of a support-immobilized lipase depends on the support surface properties and a moderate support surface micro-environment was crucial for elevated activity. Collectively, these data show that a combined gelatin and BA modification regulates the support surface more suitable for immobilizing YLIP2. PMID:26073154

  10. Membrane tension controls the assembly of curvature-generating proteins

    PubMed Central

    Simunovic, Mijo; Voth, Gregory A.

    2015-01-01

    Proteins containing a Bin/Amphiphysin/Rvs (BAR) domain regulate membrane curvature in the cell. Recent simulations have revealed that BAR proteins assemble into linear aggregates, strongly affecting membrane curvature and its in-plane stress profile. Here, we explore the opposite question: do mechanical properties of the membrane impact protein association? By using coarse-grained molecular dynamics simulations, we show that increased surface tension significantly impacts the dynamics of protein assembly. While tensionless membranes promote a rapid formation of long-living linear aggregates of N-BAR proteins, increase in tension alters the geometry of protein association. At high tension, protein interactions are strongly inhibited. Increasing surface density of proteins leads to a wider range of protein association geometries, promoting the formation of meshes, which can be broken apart with membrane tension. Our work indicates that surface tension may play a key role in recruiting proteins to membrane-remodelling sites in the cell. PMID:26008710

  11. Crystallizing Membrane Proteins Using Lipidic Mesophases

    PubMed Central

    Caffrey, Martin; Cherezov, Vadim

    2009-01-01

    A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human engineered β2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour. PMID:19390528

  12. Effect of oral creatine supplementation on human muscle GLUT4 protein content after immobilization.

    PubMed

    Op 't Eijnde, B; Ursø, B; Richter, E A; Greenhaff, P L; Hespel, P

    2001-01-01

    The purpose of this study was to investigate the effect of oral creatine supplementation on muscle GLUT4 protein content and total creatine and glycogen content during muscle disuse and subsequent training. A double-blind placebo-controlled trial was performed with 22 young healthy volunteers. The right leg of each subject was immobilized using a cast for 2 weeks, after which subjects participated in a 10-week heavy resistance training program involving the knee-extensor muscles (three sessions per week). Half of the subjects received creatine monohydrate supplements (20 g daily during the immobilization period and 15 and 5 g daily during the first 3 and the last 7 weeks of rehabilitation training, respectively), whereas the other 11 subjects ingested placebo (maltodextrine). Muscle GLUT4 protein content and glycogen and total creatine concentrations were assayed in needle biopsy samples from the vastus lateralis muscle before and after immobilization and after 3 and 10 weeks of training. Immobilization decreased GLUT4 in the placebo group (-20%, P < 0.05), but not in the creatine group (+9% NS). Glycogen and total creatine were unchanged in both groups during the immobilization period. In the placebo group, during training, GLUT4 was normalized, and glycogen and total creatine were stable. Conversely, in the creatine group, GLUT4 increased by approximately 40% (P < 0.05) during rehabilitation. Muscle glycogen and total creatine levels were higher in the creatine group after 3 weeks of rehabilitation (P < 0.05), but not after 10 weeks of rehabilitation. We concluded that 1) oral creatine supplementation offsets the decline in muscle GLUT4 protein content that occurs during immobilization, and 2) oral creatine supplementation increases GLUT4 protein content during subsequent rehabilitation training in healthy subjects. PMID:11147785

  13. Encapsulation and immobilization of papain in electrospun nanofibrous membranes of PVA cross-linked with glutaraldehyde vapor.

    PubMed

    Moreno-Cortez, Iván E; Romero-García, Jorge; González-González, Virgilio; García-Gutierrez, Domingo I; Garza-Navarro, Marco A; Cruz-Silva, Rodolfo

    2015-01-01

    In this paper, papain enzyme (E.C. 3.4.22.2, 1.6 U/mg) was successfully immobilized in poly(vinyl alcohol) (PVA) nanofibers prepared by electrospinning. The morphology of the electrospun nanofibers was characterized by scanning electron microscopy (SEM) and the diameter distribution was in the range of 80 to 170 nm. The presence of the enzyme within the PVA nanofibers was confirmed by infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDXS) analyses. The maximum catalytic activity was reached when the enzyme loading was 13%. The immobilization of papain in the nanofiber membrane was achieved by chemical crosslinking with a glutaraldehyde vapor treatment (GAvt). The catalytic activity of the immobilized papain was 88% with respect to the free enzyme. The crosslinking time by GAvt to immobilize the enzyme onto the nanofiber mat was 24h, and the enzyme retained its catalytic activity after six cycles. The crosslinked samples maintained 40% of their initial activity after being stored for 14 days. PVA electrospun nanofibers are excellent matrices for the immobilization of enzymes due to their high surface area and their nanoporous structure. PMID:25953572

  14. The potential of immobilized artificial membrane chromatography to predict human oral absorption.

    PubMed

    Tsopelas, Fotios; Vallianatou, Theodosia; Tsantili-Kakoulidou, Anna

    2016-01-01

    The potential of immobilized artificial membrane (IAM) chromatography to estimate human oral absorption (%HOA) was investigated. For this purpose, retention indices on IAM stationary phases reported previously by our group or measured by other authors under similar conditions were used to model %HOA data, compiled from literature sources. Considering the pH gradient in gastrointestinal tract, the highest logkw(IAM) values were considered, obtained either at pH7.4 or 5.5, defined as logkw(IAM)(best). Non linear models were established upon introduction of additional parameters and after exclusion of drugs which are substrates either to efflux or uptake transporters. The best model included Abraham's hydrogen-bond acidity parameter, molecular weight as well as the positively and negatively charged molecular fractions. For reasons of comparison between IAM chromatography and traditional lipophilicity, corresponding models were derived by replacing IAM retention factors with octanol-water distribution coefficients (logD). An overexpression of electrostatic interactions with phosphate anions was observed in the case of IAM retention as expressed by the negative contribution of the positively charged fraction F(+). The same parameter is statistically significant also in the logD model, but with a positive sign, indicating the attraction of basic drugs in the negatively charged inner membrane. To validate the obtained models a blind test set of 22 structurally diverse drugs was used, whose logkw(IAM)(best) values were determined and analyzed in the present study under similar conditions. IAM retention factors were further compared with MDCK cell lines permeability data taken from literature for a set of validation drugs. The overexpression of electrostatic interactions with phosphate anions on IAM surface was also evident in respect to MDCK permeability. In contrast to the clear classification between drugs with high and poor (or intermediate) absorption provided by MDCK

  15. Co-immobilization of glucose oxidase and hexokinase on silicate hybrid sol-gel membrane for glucose and ATP detections.

    PubMed

    Liu, Songqin; Sun, Yueming

    2007-01-15

    The co-immobilization of glucose oxidase (GOD) and hexokinase/glucose-6-phosphate dehydrogenase (HEX) in the silica hybrid sol-gel film for development of amperometric biosensors was investigated. The silica hybrid film fabricated by hydrolysis of the mixture of tetraethyl orthosilicate and 3-(trimethoxysiyl)propyl methacrylate possessed a three-dimension vesicle structure and good uniformity and conformability, and was ready for enzyme immobilization. The electrochemical and spectroscopic measurements showed that the silica hybrid sol-gel provided excellent matrice for the enzyme immobilization and that the immobilized enzyme retained its bioactivity effectively. The immobilized GOD could catalyze the oxidation of glucose, which could be used to determine glucose at +1.0 V without help of any mediator. The competition between GOD and HEX for the substrate glucose involving ATP as a co-substrate led to a decrease of the glucose response, which allowed us to develop an ATP sensor with a good stability. The fabricated silica hybrid sol-gel matrice offered a stage for further study of immobilization and electrochemistry of proteins. PMID:16687247

  16. A novel route for immobilization of proteins to silica particles incorporating silaffin domains.

    PubMed

    Nam, Dong Hyun; Won, Keehoon; Kim, Yong Hwan; Sang, Byung In

    2009-01-01

    In the diatom Cylindrotheca fusiformis, modified peptides called silaffin polypeptides are responsible for silica deposition in vivo at ambient conditions. Recently, it was discovered that the synthetic R5 peptide, the repeat unit of silaffin polypeptide without post-translational modification, was capable of precipitating silica in vitro and at ambient conditions. Herein, chimeric proteins were generated by incorporating synthetic silaffin R5 peptides and related unmodified silaffin domains (R1-R7) from Cylindrotheca fusiformis onto green fluorescent protein (GFP) by recombinant DNA technology and their ability to cause silicification was also examined. GFP chimeric proteins showed silicification at very low concentrations (600-700 microg/mL) when compared with adding excess amounts of R5 peptides (10 mg/mL) as previously reported. Sensitive to pH conditions, only the GFP-R1 chimera showed silicification activity at pH 8.0. The protein immobilization efficiencies of these chimeras were unexpectedly high ranging from 75 to 85%, with the R1 silaffin-protein construct showing excellent immobilization efficiency and a constant molar ratio of silica to protein ranging from 250 to 350 over a wide pH range. The average silica particle sizes had a tendency to decrease as pH increased to basic conditions. This study demonstrated the production of nanoscale immobilized protein, fabricated via silaffin-fused proteins. PMID:19774662

  17. Quantitative time-resolved measurement of membrane protein-ligand interactions using microcantilever array sensors

    NASA Astrophysics Data System (ADS)

    Braun, Thomas; Ghatkesar, Murali Krishna; Backmann, Natalija; Grange, Wilfried; Boulanger, Pascale; Letellier, Lucienne; Lang, Hans-Peter; Bietsch, Alex; Gerber, Christoph; Hegner, Martin

    2009-03-01

    Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor-the FhuA receptor of Escherichia coli-is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein-ligand interactions in a micro-array format.

  18. Nramp defines a family of membrane proteins.

    PubMed Central

    Cellier, M; Privé, G; Belouchi, A; Kwan, T; Rodrigues, V; Chia, W; Gros, P

    1995-01-01

    Nramp (natural resistance-associated macrophage protein) is a newly identified family of integral membrane proteins whose biochemical function is unknown. We report on the identification of Nramp homologs from the fly Drosophila melanogaster, the plant Oryza sativa, and the yeast Saccharomyces cerevisiae. Optimal alignment of protein sequences required insertion of very few gaps and revealed remarkable sequence identity of 28% (yeast), 40% (plant), and 55% (fly) with the mammalian proteins (46%, 58%, and 73% similarity), as well as a common predicted transmembrane topology. This family is defined by a highly conserved hydrophobic core encoding 10 transmembrane segments. Other features of this hydrophobic core include several invariant charged residues, helical periodicity of sequence conservation suggesting conserved and nonconserved faces for several transmembrane helices, a consensus transport signature on the intracytoplasmic face of the membrane, and structural determinants previously described in ion channels. These characteristics suggest that the Nramp polypeptides form part of a group of transporters or channels that act on as yet unidentified substrates. Images Fig. 1 PMID:7479731

  19. From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

    PubMed Central

    Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

    2013-01-01

    Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

  20. From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

    PubMed

    Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

    2013-01-01

    Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

  1. Amphipathic agents for membrane protein study.

    PubMed

    Sadaf, Aiman; Cho, Kyung Ho; Byrne, Bernadette; Chae, Pil Seok

    2015-01-01

    Membrane proteins (MPs) are insoluble in aqueous media as a result of incompatibility between the hydrophilic property of the solvent molecules and the hydrophobic nature of MP surfaces, normally associated with lipid membranes. Amphipathic compounds are necessary for extraction of these macromolecules from the native membranes and their maintenance in solution. The amphipathic agents surround the hydrophobic segments of MPs, thus serving as a membrane mimetic system. Of the available amphipathic agents, detergents are most widely used for MP manipulation. However, MPs encapsulated by conventional detergent micelles have a tendency to undergo structural degradation, hampering MP advance, and necessitating the development of novel detergents with enhanced efficacy for MP study. In this chapter, we will introduce both conventional and novel classes of detergents and discuss about the chemical structures, design principles, and efficacies of these compounds for MP solubilization and stabilization. The behaviors of those agents toward MP crystallization will be a primary topic in our discussion. This discussion highlights the common features of popular conventional/novel detergents essential for successful MP structural study. The conclusions reached by this discussion would not only enable MP scientists to rationally select a set of detergent candidates among a large number of detergents but also provide detergent inventors with useful guidelines in designing novel amphipathic systems. PMID:25950960

  2. Reconstitution of the membrane protein OmpF into biomimetic block copolymer-phospholipid hybrid membranes.

    PubMed

    Bieligmeyer, Matthias; Artukovic, Franjo; Nussberger, Stephan; Hirth, Thomas; Schiestel, Thomas; Müller, Michaela

    2016-01-01

    Structure and function of many transmembrane proteins are affected by their environment. In this respect, reconstitution of a membrane protein into a biomimetic polymer membrane can alter its function. To overcome this problem we used membranes formed by poly(1,4-isoprene-block-ethylene oxide) block copolymers blended with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. By reconstituting the outer membrane protein OmpF from Escherichia coli into these membranes, we demonstrate functionality of this protein in biomimetic lipopolymer membranes, independent of the molecular weight of the block copolymers. At low voltages, the channel conductance of OmpF in 1 M KCl was around 2.3 nS. In line with these experiments, integration of OmpF was also revealed by impedance spectroscopy. Our results indicate that blending synthetic polymer membranes with phospholipids allows for the reconstitution of transmembrane proteins under preservation of protein function, independent of the membrane thickness. PMID:27547605

  3. Reconstitution of the membrane protein OmpF into biomimetic block copolymer–phospholipid hybrid membranes

    PubMed Central

    Bieligmeyer, Matthias; Artukovic, Franjo; Hirth, Thomas; Schiestel, Thomas

    2016-01-01

    Summary Structure and function of many transmembrane proteins are affected by their environment. In this respect, reconstitution of a membrane protein into a biomimetic polymer membrane can alter its function. To overcome this problem we used membranes formed by poly(1,4-isoprene-block-ethylene oxide) block copolymers blended with 1,2-diphytanoyl-sn-glycero-3-phosphocholine. By reconstituting the outer membrane protein OmpF from Escherichia coli into these membranes, we demonstrate functionality of this protein in biomimetic lipopolymer membranes, independent of the molecular weight of the block copolymers. At low voltages, the channel conductance of OmpF in 1 M KCl was around 2.3 nS. In line with these experiments, integration of OmpF was also revealed by impedance spectroscopy. Our results indicate that blending synthetic polymer membranes with phospholipids allows for the reconstitution of transmembrane proteins under preservation of protein function, independent of the membrane thickness. PMID:27547605

  4. Membrane curvature and its generation by BAR proteins

    PubMed Central

    Mim, Carsten; Unger, Vinzenz M

    2012-01-01

    Membranes are flexible barriers that surround the cell and its compartments. To execute vital functions such as locomotion or receptor turnover, cells need to control the shapes of their membranes. In part, this control is achieved through membrane-bending proteins, such as the bin/amphiphysin/rvs domain (BAR) proteins. Many open questions remain about the mechanisms by which membrane-bending proteins function. Addressing this shortfall, recent structures of BAR protein:membrane complexes support existing mechanistic models, but also produced novel insights into how BAR-domain proteins sense, stabilize and generate curvature. Here we review these recent findings, focusing on how BAR proteins interact with the membrane, and how the resulting scaffold structures might aid the recruitment of other proteins to the sites where membranes are bent. PMID:23058040

  5. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    NASA Astrophysics Data System (ADS)

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-01

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

  6. Membrane protein assembly: genetic, evolutionary and medical perspectives.

    PubMed

    Manoil, C; Traxler, B

    1995-01-01

    Lipid bilayers are delicate structures that are easily disrupted by a variety of amphipathic molecules. Yet the viability of a cell requires the continued assembly of large amphipathic proteins within its membranes without damage. The need to minimize bilayer disruption may account for a number of fundamental features of membrane protein assembly. These include the use of redundant sequence information to establish the topologies and folded structures of membrane proteins, and the existence of efficient mechanisms to rid cells of misassembled proteins. Most missense mutations that inactivate a membrane protein probably do so by altering the folding of the membrane-inserted structure rather than by rearranging the topology or by changing key residues involved directly in function. Such misfolded membrane proteins may be toxic to cells if they escape cellular safeguards. This toxicity may underlie some human degenerative diseases due to mutant membrane proteins. PMID:8825471

  7. Polydopamine Grafted Porous Graphene as Biocompatible Nanoreactor for Efficient Identification of Membrane Proteins.

    PubMed

    Fang, Xiaoni; Zhao, Jingjing; Zhang, Kun; Yang, Pengyuan; Qiao, Liang; Liu, Baohong

    2016-03-16

    Functional nanomaterials, used as nanoreactors, have shown great advantages in a variety of applications in biomedical fields. Herein, we designed a novel nanoreactor system toward the application in membrane proteomics by using polydopamine-coated nanoporous graphene foams (NGFs-PD) prepared by a facile in situ oxidative polymerization. Taking advantage of the unique 3-D structure and surface functionalization, NGFs-PD can quickly adsorb a large amount of hydrophobic membrane proteins dissolved in sodium dodecyl sulfonate (SDS)/methanol and hydrophilic trypsin in aqueous solution, and then confine the proteolysis in the nanoscale domains to fasten the reaction rate. Therefore, the current nanoreactor system combines the multifunctions of highly efficient solubilization, immobilization, and proteolysis of membrane proteins. With the nanoreactor, digestion of standard membrane proteins can be finished in 10 min. 893 membrane proteins were identified from human glioma cells (U251). All these superiorities indicate that the biocompatible NGFs-PD nanoreactor system is of great promise to facilitate high-throughput membrane proteomic analysis. PMID:26913964

  8. Chemoselective Immobilization of Proteins by Microcontact Printing and Bioorthogonal Click Reactions

    PubMed Central

    Tolstyka, Zachary P.; Richardson, Wade; Bat, Erhan; Stevens, Caitlin J.; Parra, Dayanara P.; Dozier, Jonathan K.; Distefano, Mark D.; Dunn, Bruce; Maynard, Heather D.

    2014-01-01

    Herein, a combination of microcontact printing of functionalized alkanethiols and site-specific modification of proteins is utilized to chemoselectively immobilize proteins onto gold surfaces either by oxime or copper catalyzed alkyne-azide click chemistry. Two molecules capable of click reactions, an aminooxy-functionalized alkanethiol and an azide-functionalized alkanethiol, were synthesized, and self-assembled monolayer (SAM) formation on gold was confirmed by IR spectroscopy. The alkanethiols were then individually patterned onto gold surfaces by microcontact printing. Site-specifically modified proteins, horse heart myoglobin (HHMb) containing an N-terminal α-oxoamide and a red-fluorescent protein (mCherry-CVIA) with a C-terminal alkyne, respectively were immobilized by incubation onto the stamped functionalized alkanethiol patterns. Pattern formation was confirmed by fluorescence microscopy. PMID:24166802

  9. A saposin-lipoprotein nanoparticle system for membrane proteins.

    PubMed

    Frauenfeld, Jens; Löving, Robin; Armache, Jean-Paul; Sonnen, Andreas F-P; Guettou, Fatma; Moberg, Per; Zhu, Lin; Jegerschöld, Caroline; Flayhan, Ali; Briggs, John A G; Garoff, Henrik; Löw, Christian; Cheng, Yifan; Nordlund, Pär

    2016-04-01

    A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptTSo2 by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state. PMID:26950744

  10. Large-scale proteomic analysis of membrane proteins

    SciTech Connect

    Ahram, Mamoun; Springer, David L.

    2004-10-01

    Proteomic analysis of membrane proteins is promising in identification of novel candidates as drug targets and/or disease biomarkers. Despite notable technological developments, obstacles related to extraction and solubilization of membrane proteins are frequently encountered. A critical discussion of the different preparative methods of membrane proteins is offered in relation to downstream proteomic applications, mainly gel-based analyses and mass spectrometry. Unknown proteins are often identified by high-throughput profiling of membrane proteins. In search for novel membrane proteins, analysis of protein sequences using computational tools is performed to predict for the presence of transmembrane domains. Here, we also present these bioinformatic tools with the human proteome as a case study. Along with technological innovations, advancements in the areas of sample preparation and computational prediction of membrane proteins will lead to exciting discoveries.

  11. Membrane shape instabilities induced by BAR domain proteins

    NASA Astrophysics Data System (ADS)

    Baumgart, Tobias

    2014-03-01

    Membrane curvature has developed into a forefront of membrane biophysics. Numerous proteins involved in membrane curvature sensing and membrane curvature generation have recently been discovered, including proteins containing the crescent-shaped BAR domain as membrane binding and shaping module. Accordingly, the structure determination of these proteins and their multimeric complexes is increasingly well-understood. Substantially less understood, however, are thermodynamic and kinetic aspects and the detailed mechanisms of how these proteins interact with membranes in a curvature-dependent manner. New experimental approaches need to be combined with established techniques to be able to fill in these missing details. Here we use model membrane systems in combination with a variety of biophysical techniques to characterize mechanistic aspects of BAR domain protein function. This includes a characterization of membrane curvature sensing and membrane generation. We also establish kinetic and thermodynamic aspects of BAR protein dimerization in solution, and investigate kinetic aspects of membrane binding. We present two new approaches to investigate membrane shape instabilities and demonstrate that membrane shape instabilities can be controlled by protein binding and lateral membrane tension. This work is supported through NIH grant GM-097552 and NSF grant CBET-1053857.

  12. Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems.

    PubMed

    Cheng, Chi-Yuan; Han, Songi

    2013-01-01

    Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments. PMID:23331309

  13. Dynamic Nuclear Polarization Methods in Solids and Solutions to Explore Membrane Proteins and Membrane Systems

    NASA Astrophysics Data System (ADS)

    Cheng, Chi-Yuan; Han, Songi

    2013-04-01

    Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

  14. Mass Spectrometry of Membrane Proteins: A Focus on Aquaporins

    PubMed Central

    Schey, Kevin L.; Grey, Angus C.; Nicklay, Joshua J.

    2015-01-01

    Membrane proteins are abundant, critically important biomolecules that conduct essential functions in all cells and are the targets of a significant number of therapeutic drugs. However, the analysis of their expression, modification, protein–protein interactions, and structure by mass spectrometry has lagged behind similar studies of soluble proteins. Here we review the limitations to analysis of integral membrane and membrane-associated proteins and highlight advances in sample preparation and mass spectrometry methods that have led to the successful analysis of this protein class. Advances in the analysis of membrane protein posttranslational modification, protein–protein interaction, protein structure, and tissue distributions by imaging mass spectrometry are discussed. Furthermore, we focus our discussion on the application of mass spectrometry for the analysis of aquaporins as a prototypical integral membrane protein and how advances in analytical methods have revealed new biological insights into the structure and function of this family of proteins. PMID:23394619

  15. Evaluation of protein immobilization capacity on various carbon nanotube embedded hydrogel biomaterials.

    PubMed

    Derkus, Burak; Emregul, Kaan Cebesoy; Emregul, Emel

    2015-11-01

    This study investigates effective immobilization of proteins, an important procedure in many fields of bioengineering and medicine, using various biomaterials. Gelatin, alginate and chitosan were chosen as polymeric carriers, and applied in both their composites and nanocomposite forms in combination with carbon nanotubes (CNTs). The prepared nano/composite structures were characterized using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TG) and contact angle analysis (CA). Electrochemical impedance spectroscopy analysis revealed gelatin composites in general to exhibit better immobilization performance relative to the native gelatin which can be attributed to enhanced film morphologies of the composite structures. Moreover, superior immobilization efficiencies were obtained with the addition of carbon nanotubes, due to their conducting and surface enhancement features, especially in the gelatin-chitosan structures due to the presence of structural active groups. PMID:26249574

  16. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    PubMed Central

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-01

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes. PMID:25635869

  17. A Prediction Model for Membrane Proteins Using Moments Based Features.

    PubMed

    Butt, Ahmad Hassan; Khan, Sher Afzal; Jamil, Hamza; Rasool, Nouman; Khan, Yaser Daanial

    2016-01-01

    The most expedient unit of the human body is its cell. Encapsulated within the cell are many infinitesimal entities and molecules which are protected by a cell membrane. The proteins that are associated with this lipid based bilayer cell membrane are known as membrane proteins and are considered to play a significant role. These membrane proteins exhibit their effect in cellular activities inside and outside of the cell. According to the scientists in pharmaceutical organizations, these membrane proteins perform key task in drug interactions. In this study, a technique is presented that is based on various computationally intelligent methods used for the prediction of membrane protein without the experimental use of mass spectrometry. Statistical moments were used to extract features and furthermore a Multilayer Neural Network was trained using backpropagation for the prediction of membrane proteins. Results show that the proposed technique performs better than existing methodologies. PMID:26966690

  18. A Prediction Model for Membrane Proteins Using Moments Based Features

    PubMed Central

    Butt, Ahmad Hassan; Khan, Sher Afzal; Jamil, Hamza; Rasool, Nouman; Khan, Yaser Daanial

    2016-01-01

    The most expedient unit of the human body is its cell. Encapsulated within the cell are many infinitesimal entities and molecules which are protected by a cell membrane. The proteins that are associated with this lipid based bilayer cell membrane are known as membrane proteins and are considered to play a significant role. These membrane proteins exhibit their effect in cellular activities inside and outside of the cell. According to the scientists in pharmaceutical organizations, these membrane proteins perform key task in drug interactions. In this study, a technique is presented that is based on various computationally intelligent methods used for the prediction of membrane protein without the experimental use of mass spectrometry. Statistical moments were used to extract features and furthermore a Multilayer Neural Network was trained using backpropagation for the prediction of membrane proteins. Results show that the proposed technique performs better than existing methodologies. PMID:26966690

  19. “Versatile toolset” for DNA or protein immobilization: Toward a single-step chemistry

    NASA Astrophysics Data System (ADS)

    Berthelot, Thomas; Garcia, Alexandre; Le, Xuan Tuan; El Morsli, Jenna; Jégou, Pascale; Palacin, Serge; Viel, Pascal

    2011-02-01

    Covalent immobilization of non-modified biological materials as proteins or nucleic acids has been performed through a single and soft method. Based on diazonium salt chemistry, this protocol leads to an ultrathin grafted film, on metallic or polymer materials, which can eventually be used as a self-adhesive primer for immobilizing biological materials from aqueous solutions through a simple dipping step. Moreover, this self-adhesive primer may be patterned by cheap and easy methods as ink or UV masking. Biological models as low molecular weight DNA from salmon sperm and glucose oxidase (GOD) were covalently immobilized by this soft procedure. In order to evaluate the consequences of this non-specific covalent immobilization method on biological activity, enzymatic activity of GOD was monitored by electrochemical detection of hydrogen peroxide (H2O2). We thus demonstrate that such a self-adhesive primer represents a new and alternative process offering a versatile toolset for immobilizing biological material for biosensor development on conductive and non-conductive materials.

  20. Immobilization of Bone Morphogenetic Protein on DOPA- or Dopamine-Treated Titanium Surfaces to Enhance Osseointegration

    PubMed Central

    Kang, Jeonghwa; Tada, Seiichi; Kitajima, Takashi; Son, Tae Il; Aigaki, Toshiro; Ito, Yoshihiro

    2013-01-01

    Titanium was treated with 3,4-dihydroxy-L-phenylalanine (DOPA) or dopamine to immobilize bone morphogenetic protein-2 (BMP2), a biomolecule. DOPA and dopamine solutions turned into suspensions, and precipitates were produced at high pH. Both treatments produced a brown surface on titanium that was thicker at high pH than low pH. Dopamine produced a thicker layer than DOPA. The hydrophobicity of the surfaces increased after treatment with dopamine independent of pH. Furthermore, there were more amino groups in the layers formed at pH 8.5 than pH 4.5 in both treatments. Dopamine treatment produced more amino groups in the layer than DOPA. BMP2 was immobilized on the treated surfaces via a coupling reaction using carbodiimide. More BMP2 was immobilized on surfaces treated at pH 8.5 than pH 4.5 in both treatments. The immobilized BMP induced specific signal transduction and alkali phosphatase, a differentiation marker. Thus, the present study demonstrates that titanium treated with DOPA or dopamine can become bioactive via the surface immobilization of BMP2, which induces specific signal transduction. PMID:24459666

  1. Role of membrane contact sites in protein import into mitochondria

    PubMed Central

    Horvath, Susanne E; Rampelt, Heike; Oeljeklaus, Silke; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus

    2015-01-01

    Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture. PMID:25514890

  2. Dynamic membrane protein topological switching upon changes in phospholipid environment

    PubMed Central

    Vitrac, Heidi; MacLean, David M.; Jayaraman, Vasanthi; Bogdanov, Mikhail; Dowhan, William

    2015-01-01

    A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids. PMID:26512118

  3. Protein crystals on phase-separating model membranes

    NASA Astrophysics Data System (ADS)

    Manley, Suliana; Horton, Margaret; Leszczynski, Szymon; Gast, Alice

    2006-03-01

    We study the interplay between the crystallization of proteins tethered to membranes and separation within the membranes of giant unilamellar vesicles (GUVs) composed of DOPC, sphingomyelin (SM), and cholesterol. These model membranes phase separate into coexisting liquid domains below a miscibility transition temperature. This phase separation captures some aspects of the formation of lipid rafts in cell membranes and demonstrates the influence of membrane composition on raft formation. Real cell membranes have a much more complicated structure. There are additional physical constraints present in cell membranes, such as their attachment to the cytoskeleton and the presence of membrane bound proteins. The self-association of membrane proteins can influence the membrane phase behavior. We begin to investigate these effects on model tethered protein- loaded membranes by incorporating a small amount of biotin-X- DPPE into our GUVs. The biotinylated lipid partitions into a cholesterol-poor phase; thus, streptavidin binds preferentially to one of the membrane phases. As streptavidin assembles to form crystalline domains, it restricts the membrane mobility. We examine the effect of this protein association on lipid phase separation, as well as the effect of the lipid phase separation on the crystallization of the tethered proteins.

  4. Membrane Protein Structure and Dynamics from NMR Spectroscopy

    NASA Astrophysics Data System (ADS)

    Hong, Mei; Zhang, Yuan; Hu, Fanghao

    2012-05-01

    We review the current state of membrane protein structure determination using solid-state nuclear magnetic resonance (NMR) spectroscopy. Multidimensional magic-angle-spinning correlation NMR combined with oriented-sample experiments has made it possible to measure a full panel of structural constraints of membrane proteins directly in lipid bilayers. These constraints include torsion angles, interatomic distances, oligomeric structure, protein dynamics, ligand structure and dynamics, and protein orientation and depth of insertion in the lipid bilayer. Using solid-state NMR, researchers have studied potassium channels, proton channels, Ca2+ pumps, G protein-coupled receptors, bacterial outer membrane proteins, and viral fusion proteins to elucidate their mechanisms of action. Many of these membrane proteins have also been investigated in detergent micelles using solution NMR. Comparison of the solid-state and solution NMR structures provides important insights into the effects of the solubilizing environment on membrane protein structure and dynamics.

  5. Structure Determination of Membrane Proteins by Nuclear Magnetic Resonance Spectroscopy

    NASA Astrophysics Data System (ADS)

    Opella, Stanley J.

    2013-06-01

    Many biological membranes consist of 50% or more (by weight) membrane proteins, which constitute approximately one-third of all proteins expressed in biological organisms. Helical membrane proteins function as receptors, enzymes, and transporters, among other unique cellular roles. Additionally, most drugs have membrane proteins as their receptors, notably the superfamily of G protein-coupled receptors with seven transmembrane helices. Determining the structures of membrane proteins is a daunting task because of the effects of the membrane environment; specifically, it has been difficult to combine biologically compatible environments with the requirements for the established methods of structure determination. There is strong motivation to determine the structures in their native phospholipid bilayer environment so that perturbations from nonnatural lipids and phases do not have to be taken into account. At present, the only method that can work with proteins in liquid crystalline phospholipid bilayers is solid-state NMR spectroscopy.

  6. Marginally hydrophobic transmembrane α-helices shaping membrane protein folding

    PubMed Central

    De Marothy, Minttu T; Elofsson, Arne

    2015-01-01

    Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices—implying a dynamic relationship between membrane proteins and their environment. PMID:25970811

  7. Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells

    PubMed Central

    Weghuber, Julian; Aichinger, Michael C.; Brameshuber, Mario; Wieser, Stefan; Ruprecht, Verena; Plochberger, Birgit; Madl, Josef; Horner, Andreas; Reipert, Siegfried; Lohner, Karl; Henics, Tamás; Schütz, Gerhard J.

    2011-01-01

    Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides—in particular a CAMP with Lysine–Leucine–Lysine repeats (termed KLK)—affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents. PMID:21718688

  8. Prediction of oral absorption in humans by experimental immobilized artificial membrane chromatography indices and physicochemical descriptors.

    PubMed

    Kotecha, Jignesh; Shah, Shailesh; Rathod, Ishwarsinh; Subbaiah, Gunta

    2008-08-01

    The purpose of the present study was to examine the human oral absorption (HOA) predictability of the experimentally determined immobilized artificial membrane (IAM) chromatography capacity factor (log k'IAM) in conjunction with physicochemical descriptors. Transcellular permeation was modeled based on determination of log k'IAM considering pH partition hypothesis, and the independent variables were polar surface area (PSA) and molecular weight (MW). The correlation between log k'IAM determined at different pH and n-octanol/water partition coefficient (log P) and contribution of polarity (PSA) and size (MW) in the transcellular permeation model were the extension to the previous work. A data set of 37 compounds with partition coefficient values taken from the literature was employed to show importance of ionic interaction in oral absorption prediction. The highest log k'IAM value among screened pH 4.5, 5.5, 6.5 and 7.4 (log k'IAM4.5-7.4) in conjunction with PSA predicted HOA with coefficient of determination (CD) of 0.9001 compare to log k'IAM4.5-7.4 alone with CD of 0.8454 after excluding bretylium from the set of 28 structurally diverse drugs for known reason. PSA helped to avoid over estimation of HOA for amiloride, famotidine and furosemide. The model was tested for its applicability in drug development program and found to predict oral absorption using physically meaningful and structurally related properties making them relatively straightforward for a medicinal chemist to interpret. PMID:18524510

  9. Designing Mimics of Membrane Active Proteins

    PubMed Central

    Sgolastra, Federica; deRonde, Brittany M.; Sarapas, Joel M.; Som, Abhigyan; Tew, Gregory N.

    2014-01-01

    CONSPECTUS As a semi-permeable barrier that controls the flux of biomolecules in and out the cell, the plasma membrane is critical in cell function and survival. Many proteins interact with the plasma membrane and modulate its physiology. Within this large landscape of membrane-active molecules, researchers have focused significant attention on two specific classes of peptides, antimicrobial peptides (AMPs) and cell penetrating peptides (CPPs) because of their unique properties. In this account, we describe our efforts over the last decade to build and understand synthetic mimics of antimicrobial peptides (SMAMPs). These endeavors represent one specific example of a much larger effort to understand how synthetic molecules interact with and manipulate the plasma membrane. Using both defined molecular weight oligomers and easier to produce, but heterogeneous, polymers, it has been possible to generate scaffolds with biological potency superior to the natural analogs. In one case, a compound has progressed through a phase II clinical trial for pan)staph infections. Modern biophysical assays highlighted the interplay between the synthetic scaffold and lipid composition leading to negative Gaussian curvature, a requirement for both pore formation and endosomal escape. The complexity of this interplay between lipids, bilayer components, and the scaffolds remains to be better resolved, but significant new insight has been provided. It is worthwhile to consider the various aspects of permeation and how these are related to ‘pore formation.’ More recently, our efforts have expanded toward protein transduction domains, or cell penetrating peptide, mimics. The combination of unique molecular scaffolds and guanidinium) rich side chains has produced an array of polymers with robust transduction (and delivery) activity. Being a new area, the fundamental interactions between these new scaffolds and the plasma membrane are just beginning to be understood. Negative Gaussian

  10. Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

    PubMed Central

    MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

    2008-01-01

    Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

  11. Diffusing proteins on a fluctuating membrane: Analytical theory and simulations

    NASA Astrophysics Data System (ADS)

    Reister-Gottfried, Ellen; Leitenberger, Stefan M.; Seifert, Udo

    2010-03-01

    Using analytical calculations and computer simulations, we consider both the lateral diffusion of a membrane protein and the fluctuation spectrum of the membrane in which the protein is embedded. The membrane protein interacts with the membrane shape through its spontaneous curvature and bending rigidity. The lateral motion of the protein may be viewed as diffusion in an effective potential, hence, the effective mobility is always reduced compared to the case of free diffusion. Using a rigorous path-integral approach, we derive an analytical expression for the effective diffusion coefficient for small ratios of temperature and bending rigidity, which is the biologically relevant limit. Simulations show very good quantitative agreement with our analytical result. The analysis of the correlation functions contributing to the diffusion coefficient shows that the correlations between the stochastic force of the protein and the response in the membrane shape are responsible for the reduction. Our quantitative analysis of the membrane height correlation spectrum shows an influence of the protein-membrane interaction causing a distinctly altered wave-vector dependence compared to a free membrane. Furthermore, the time correlations exhibit the two relevant time scales of the system: that of membrane fluctuations and that of lateral protein diffusion with the latter typically much longer than the former. We argue that the analysis of the long-time decay of membrane height correlations can thus provide a new means to determine the effective diffusion coefficient of proteins in the membrane.

  12. Integrated system for extraction, purification, and digestion of membrane proteins.

    PubMed

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

    2016-05-01

    An integrated system was developed for directly processing living cells into peptides of membrane proteins. Living cells were directly injected into the system and cracked in a capillary column by ultrasonic treatment. Owing to hydrophilicity for broken pieces of the cell membrane, the obtained membranes were retained in a well-designed bi-filter. While cytoplasm proteins were eluted from the bi-filter, the membranes were dissolved and protein released by flushing 4 % SDS buffer through the bi-filter. The membrane proteins were subsequently transferred into a micro-reactor and covalently bound in the reactor for purification and digestion. As the system greatly simplified the whole pretreatment processes and minimized both sample loss and contamination, it could be used to analyze the membrane proteome samples of thousand-cell-scales with acceptable reliability and stability. We totally identified 1348 proteins from 5000 HepG2 cells, 615 of which were annotated as membrane proteins. In contrast, with conventional method, only 233 membrane proteins were identified. It is adequately demonstrated that the integrated system shows promising practicability for the membrane proteome analysis of small amount of cells. Graphical Abstract The legend of online abstract figure is (a) schematic illustration of membrane proteins extraction, purification and digestion from living cells; (b) diagrammatic sketch of the automatic integrated membrane proteome analysis system. PMID:26922343

  13. Polypyrrole-supported membrane proteins for bio-inspired ion channels.

    PubMed

    Pérez-Madrigal, Maria M; del Valle, Luis J; Armelin, Elaine; Michaux, Catherine; Roussel, Guillaume; Perpète, Eric A; Alemán, Carlos

    2015-01-28

    Biomedical platforms constructed by immobilizing membrane proteins in matrixes made of synthetic organic polymers is a challenge because the structure and function of these proteins are affected by environmental conditions. In this work, an operative composite that regulates the diffusion of alkali ions has been prepared by functionalizing a supporting matrix made of poly(N-methylpyrrole) (PNMPy) with a β-barrel membrane protein (Omp2a) that forms channels and pores. The protein has been unequivocally identified in the composite, and its structure has been shown to remain unaltered. The PNMPy-Omp2a platform fulfills properties typically associated with functional bio-interfaces with biomedical applications (e.g., biocompatibility, biodegrabadility, and hydrophilicity). The functionality of the immobilized protein has been examined by studying the passive ion transport response in the presence of electrolytic solutions with Na(+) and K(+) concentrations close to those found in blood. Although the behavior of PNMPy and PNMPy-Omp2a is very similar for solutions with very low concentration, the resistance of the latter decreases drastically when the concentration of ions increases to ∼100 mM. This reduction reflects an enhanced ion exchange between the biocomposite and the electrolytic medium, which is not observed in PNMPy, evidencing that PNMPy-Omp2a is particularly well suited to prepare bioinspired channels and smart biosensors. PMID:25585165

  14. Extracellular Protease Digestion to Evaluate Membrane Protein Cell Surface Localization

    PubMed Central

    Besingi, Richard N.; Clark, Patricia L.

    2016-01-01

    Membrane proteins play crucial roles in signaling and as anchors for cell surface display. Proper secretion of a membrane protein can be evaluated by its susceptibility to digestion by an extracellular protease, but this requires a crucial control to confirm membrane integrity during digestion. This protocol describes how to use this approach to determine how efficiently a protein is secreted to the outer surface of Gram-negative bacteria. Its success relies upon careful selection of an appropriate intracellular reporter protein that will remain undigested if the membrane barrier remains intact, but is rapidly digested when cells are lysed prior to evaluation. Reporter proteins that are resistant to proteases (e.g. maltose-binding protein) do not return accurate results; in contrast, proteins that are more readily digested (e.g. SurA) serve as more sensitive reporters of membrane integrity, yielding more accurate measurements of membrane protein localization. Similar considerations apply when evaluating membrane protein localization in other contexts, including eukaryotic cells and organelle membranes. Evaluating membrane protein localization using this approach requires only standard biochemistry laboratory equipment for cell lysis, gel electrophoresis and western blotting. After expression of the protein of interest, this procedure can be completed in 4 h. PMID:26584447

  15. Fluctuating hydrodynamics of multicomponent membranes with embedded proteins

    SciTech Connect

    Camley, Brian A.; Brown, Frank L. H.

    2014-08-21

    A simulation method for the dynamics of inhomogeneous lipid bilayer membranes is presented. The membrane is treated using stochastic Saffman-Delbrück hydrodynamics, coupled to a phase-field description of lipid composition and discrete membrane proteins. Multiple applications are considered to validate and parameterize the model. The dynamics of membrane composition fluctuations above the critical point and phase separation dynamics below the critical point are studied in some detail, including the effects of adding proteins to the mixture.

  16. Immobilization of proteins on agarose beads, monitored in real time by bead injection spectroscopy

    PubMed Central

    Ruzicka*, Jaromir; Carroll, Andrea D.; Lähdesmäki, Ilkka

    2006-01-01

    Summary This work introduces a novel tool for the examination and optimization of protein immobilization protocols, by measuring the rate and yield of coupling reactions, as they take place on the surface of agarose beads in a well-stirred microreactor. The power of the Bead Injection Spectroscopy (BIS) technique is demonstrated on examples of amino coupling reactions for albumin, ovalbumin, lysozyme, human IgG, ribonuclease A and cytochrome C, using commercially available Aminolink® agarose beads. It was found, surprisingly, that currently recommended protocols for reductive amination can be shortened from several hours to several minutes, and that, contrary to literature data, the yield of coupling is dependent on pH and the isoelectric point of the protein. In addition, leakage of immobilized ligands can be measured by direct spectroscopic interrogation of captured beads in situ. The methodology presented in this work documents that BIS is a useful tool for quality control of agarose-based chromatographic supports, as well as for the optimization of a wide variety of immobilization chemistries, as used for synthesis of chromatographic supports, immobilization of enzymes, and derivatization of biosensing surfaces. PMID:16802025

  17. Design of affinity tags for one-step protein purification from immobilized zinc columns

    SciTech Connect

    Pasquinelli, R.S.; Shepherd, R.E.; Koepsel, R.R.; Zhao, A.; Ataai, M.M.

    2000-02-01

    Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to e superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. for example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper the authors have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

  18. Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces.

    PubMed

    Wylie, Benjamin J; Dzikovski, Boris G; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H; McDermott, Ann E

    2015-04-01

    We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces. PMID:25828256

  19. Preparation of a Cu(II)-PVA/PA6 Composite Nanofibrous Membrane for Enzyme Immobilization

    PubMed Central

    Feng, Quan; Tang, Bin; Wei, Qufu; Hou, Dayin; Bi, Songmei; Wei, Anfang

    2012-01-01

    PVA/PA6 composite nanofibers were formed by electrospinning. Cu(II)-PVA/PA6 metal chelated nanofibers, prepared by the reaction between PVA/PA6 composite nanofibers and Cu2+ solution, were used as the support for catalase immobilization. The result of the experiments showed that PVA/PA6 composite nanofibers had an excellent chelation capacity for Cu2+ ions, and the structures of nanofibers were stable during the reaction with Cu2+ solution. The adsorption of Cu(II) onto PVA/PA6 composite nanofibers was studied by the Langmuir isothermal adsorption model. The maximum amount of coordinated Cu(II) (qm) was 3.731 mmol/g (dry fiber), and the binding constant (Kl) was 0.0593 L/mmol. Kinetic parameters were analyzed for both immobilized and free catalases. The value of Vmax (3774 μmol/mg·min) for the immobilized catalases was smaller than that of the free catalases (4878 μmol/mg·min), while the Km for the immobilized catalases was larger. The immobilized catalases showed better resistance to pH and temperature than that of free form, and the storage stabilities, reusability of immobilized catalases were significantly improved. The half-lives of free and immobilized catalases were 8 days and 24 days, respectively. PMID:23202922

  20. Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

    PubMed

    Bogdanov, Mikhail; Heacock, Philip N; Dowhan, William

    2010-01-01

    A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system. PMID:20419405

  1. Design of an optically stable pH sensor based on immobilization of Giemsa on triacetylcellulose membrane.

    PubMed

    Khodadoust, Saeid; Kouri, Narges Cham; Talebiyanpoor, Mohammad Sharif; Deris, Jamile; Pebdani, Arezou Amiri

    2015-12-01

    In this work a simple, inexpensive, and sensitive optical sensor based on triacetylcellulose membrane as solid support was developed by using immobilization of Giemsa indicator for pH measurement. In this method, the influence variables on the membrane performance including pH concentration of indicator, response time, ionic strength, and reversibility were investigated. At optimum values of all variables the response of optical pH sensor is linear in the pH range of 3.0-12.0. This optical sensor was produced through simultaneous binding of the Giemsa on the activated triacetylcellulose membrane which responded to the pH changes in a broader linear range within less than 2.0 min and suitable reproducibility (RSD<5%). Stability results showed that this sensor was stable after 6 months of storage in the water/ethanol (50:50, v/v) solution without any measurable divergence in response properties (less than 5% RSD). PMID:26354268

  2. Structure Determination of Membrane Proteins by Nuclear Magnetic Resonance Spectroscopy

    PubMed Central

    Opella, Stanley J.

    2014-01-01

    Many biological membranes consist of 50% or more (by weight) membrane proteins, which constitute approximately one-third of all proteins expressed in biological organisms. Helical membrane proteins function as receptors, enzymes, and transporters, among other unique cellular roles. Additionally, most drugs have membrane proteins as their receptors, notably the superfamily of G protein–coupled receptors with seven transmembrane helices. Determining the structures of membrane proteins is a daunting task because of the effects of the membrane environment; specifically, it has been difficult to combine biologically compatible environments with the requirements for the established methods of structure determination. There is strong motivation to determine the structures in their native phospholipid bilayer environment so that perturbations from nonnatural lipids and phases do not have to be taken into account. At present, the only method that can work with proteins in liquid crystalline phospholipid bilayers is solid-state NMR spectroscopy. PMID:23577669

  3. Membrane-Mediated Interaction between Strongly Anisotropic Protein Scaffolds

    PubMed Central

    Schweitzer, Yonatan; Kozlov, Michael M.

    2015-01-01

    Specialized proteins serve as scaffolds sculpting strongly curved membranes of intracellular organelles. Effective membrane shaping requires segregation of these proteins into domains and is, therefore, critically dependent on the protein-protein interaction. Interactions mediated by membrane elastic deformations have been extensively analyzed within approximations of large inter-protein distances, small extents of the protein-mediated membrane bending and small deviations of the protein shapes from isotropic spherical segments. At the same time, important classes of the realistic membrane-shaping proteins have strongly elongated shapes with large and highly anisotropic curvature. Here we investigated, computationally, the membrane mediated interaction between proteins or protein oligomers representing membrane scaffolds with strongly anisotropic curvature, and addressed, quantitatively, a specific case of the scaffold geometrical parameters characterizing BAR domains, which are crucial for membrane shaping in endocytosis. In addition to the previously analyzed contributions to the interaction, we considered a repulsive force stemming from the entropy of the scaffold orientation. We computed this interaction to be of the same order of magnitude as the well-known attractive force related to the entropy of membrane undulations. We demonstrated the scaffold shape anisotropy to cause a mutual aligning of the scaffolds and to generate a strong attractive interaction bringing the scaffolds close to each other to equilibrium distances much smaller than the scaffold size. We computed the energy of interaction between scaffolds of a realistic geometry to constitute tens of kBT, which guarantees a robust segregation of the scaffolds into domains. PMID:25710602

  4. A novel lipoprotein nanoparticle system for membrane proteins

    PubMed Central

    Frauenfeld, Jens; Löving, Robin; Armache, Jean-Paul; Sonnen, Andreas; Guettou, Fatma; Moberg, Per; Zhu, Lin; Jegerschöld, Caroline; Flayhan, Ali; Briggs, John A.G.; Garoff, Henrik; Löw, Christian; Cheng, Yifan; Nordlund, Pär

    2016-01-01

    Membrane proteins are of outstanding importance in biology, drug discovery and vaccination. A common limiting factor in research and applications involving membrane proteins is the ability to solubilize and stabilize membrane proteins. Although detergents represent the major means for solubilizing membrane proteins, they are often associated with protein instability and poor applicability in structural and biophysical studies. Here, we present a novel lipoprotein nanoparticle system that allows for the reconstitution of membrane proteins into a lipid environment that is stabilized by a scaffold of Saposin proteins. We showcase the applicability of the method on two purified membrane protein complexes as well as the direct solubilization and nanoparticle-incorporation of a viral membrane protein complex from the virus membrane. We also demonstrate that this lipid nanoparticle methodology facilitates high-resolution structural studies of membrane proteins in a lipid environment by single-particle electron cryo-microscopy (cryo-EM) and allows for the stabilization of the HIV-envelope glycoprotein in a functional state. PMID:26950744

  5. Mechanism study of dual-frequency ultrasound assisted enzymolysis on rapeseed protein by immobilized Alcalase.

    PubMed

    Wang, Bei; Meng, Tingting; Ma, Haile; Zhang, Yanyan; Li, Yunliang; Jin, Jian; Ye, Xiaofei

    2016-09-01

    The mechanism of ultrasound field promoting enzymolysis efficiency is difficult to study, because the reaction system mixes with enzymes, proteins and hydrolysates. Immobilized enzyme is a good option that can be used to investigate the mechanism by separating enzymes out from the system after enzymolysis. The objective of this study was by using immobilized Alcalase to investigate the effects and mechanisms of the promotion of dual-frequency ultrasound (DFU) assisted-enzymolysis on rapeseed protein. Based on single factor experiments, response surface methodology model with three factors - hydrolysis time, power density and solid-liquid ratio at three levels was utilized to optimize the degree of hydrolysis (DH). Circular dichroism (CD) was used to analyze the secondary structure change of the protein, scanning electron microscopy (SEM) was used to analyze the surface microstructure change of the enzyme. The results showed that with DFU assisted-enzymolysis, the DH increased by 74.38% at the optimal levels for power density 57W/L, solid-liquid ratio 5.3g/L and enzymolysis time 76min. After DFU assisted-enzymolysis, the yield of soluble solids content, including protein, peptides and total sugar in hydrolysate increased by 64.61%, 40.88% and 23.60%, respectively. CD analysis showed that after DFU assisted-enzymolysis, the number of α-helix and random coil decreased by 10.7% and 4.5%, β-chain increased by 2.4%. SEM showed that the degree of surface roughness of immobilized Alcalase increased. The above results indicated that the improvement of hydrolysis by DFU assisted-enzymolysis was achieved by enhancing the solid solubility, changing the molecular structure of protein and increased the surface area of immobilized enzyme. PMID:27150775

  6. Smad2/3 Proteins Are Required for Immobilization-induced Skeletal Muscle Atrophy.

    PubMed

    Tando, Toshimi; Hirayama, Akiyoshi; Furukawa, Mitsuru; Sato, Yuiko; Kobayashi, Tami; Funayama, Atsushi; Kanaji, Arihiko; Hao, Wu; Watanabe, Ryuichi; Morita, Mayu; Oike, Takatsugu; Miyamoto, Kana; Soga, Tomoyoshi; Nomura, Masatoshi; Yoshimura, Akihiko; Tomita, Masaru; Matsumoto, Morio; Nakamura, Masaya; Toyama, Yoshiaki; Miyamoto, Takeshi

    2016-06-01

    Skeletal muscle atrophy promotes muscle weakness, limiting activities of daily living. However, mechanisms underlying atrophy remain unclear. Here, we show that skeletal muscle immobilization elevates Smad2/3 protein but not mRNA levels in muscle, promoting atrophy. Furthermore, we demonstrate that myostatin, which negatively regulates muscle hypertrophy, is dispensable for denervation-induced muscle atrophy and Smad2/3 protein accumulation. Moreover, muscle-specific Smad2/3-deficient mice exhibited significant resistance to denervation-induced muscle atrophy. In addition, expression of the atrogenes Atrogin-1 and MuRF1, which underlie muscle atrophy, did not increase in muscles of Smad2/3-deficient mice following denervation. We also demonstrate that serum starvation promotes Smad2/3 protein accumulation in C2C12 myogenic cells, an in vitro muscle atrophy model, an effect inhibited by IGF1 treatment. In vivo, we observed IGF1 receptor deactivation in immobilized muscle, even in the presence of normal levels of circulating IGF1. Denervation-induced muscle atrophy was accompanied by reduced glucose intake and elevated levels of branched-chain amino acids, effects that were Smad2/3-dependent. Thus, muscle immobilization attenuates IGF1 signals at the receptor rather than the ligand level, leading to Smad2/3 protein accumulation, muscle atrophy, and accompanying metabolic changes. PMID:27129272

  7. Protein-Induced Modulation of Chloroplast Membrane Morphology

    PubMed Central

    Machettira, Anu B.; Groß, Lucia E.; Tillmann, Bodo; Weis, Benjamin L.; Englich, Gisela; Sommer, Maik S.; Königer, Martina; Schleiff, Enrico

    2012-01-01

    Organelles are surrounded by membranes with a distinct lipid and protein composition. While it is well established that lipids affect protein functioning and vice versa, it has been only recently suggested that elevated membrane protein concentrations may affect the shape and organization of membranes. We therefore analyzed the effects of high chloroplast envelope protein concentrations on membrane structures using an in vivo approach with protoplasts. Transient expression of outer envelope proteins or protein domains such as CHUP1-TM–GFP, outer envelope protein of 7 kDa–GFP, or outer envelope protein of 24 kDa–GFP at high levels led to the formation of punctate, circular, and tubular membrane protrusions. Expression of inner membrane proteins such as translocase of inner chloroplast membrane 20, isoform II (Tic20-II)–GFP led to membrane protrusions including invaginations. Using increasing amounts of DNA for transfection, we could show that the frequency, size, and intensity of these protrusions increased with protein concentration. The membrane deformations were absent after cycloheximide treatment. Co-expression of CHUP1-TM–Cherry and Tic20-II–GFP led to membrane protrusions of various shapes and sizes including some stromule-like structures, for which several functions have been proposed. Interestingly, some structures seemed to contain both proteins, while others seem to contain one protein exclusively, indicating that outer and inner envelope dynamics might be regulated independently. While it was more difficult to investigate the effects of high expression levels of membrane proteins on mitochondrial membrane shapes using confocal imaging, it was striking that the expression of the outer membrane protein Tom20 led to more elongate mitochondria. We discuss that the effect of protein concentrations on membrane structure is possibly caused by an imbalance in the lipid to protein ratio and may be involved in a signaling pathway regulating membrane

  8. An Integrated Framework Advancing Membrane Protein Modeling and Design

    PubMed Central

    Weitzner, Brian D.; Duran, Amanda M.; Tilley, Drew C.; Elazar, Assaf; Gray, Jeffrey J.

    2015-01-01

    Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design. PMID:26325167

  9. An Integrated Framework Advancing Membrane Protein Modeling and Design.

    PubMed

    Alford, Rebecca F; Koehler Leman, Julia; Weitzner, Brian D; Duran, Amanda M; Tilley, Drew C; Elazar, Assaf; Gray, Jeffrey J

    2015-09-01

    Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design. PMID:26325167

  10. Sampling the membrane: function of rhomboid-family proteins.

    PubMed

    Lemberg, Marius K

    2013-05-01

    Rhomboids constitute a conserved protein superfamily that specifically binds membrane proteins and directs them into various different cellular pathways ranging from regulated secretion to endoplasmic reticulum (ER)-associated degradation (ERAD). Rhomboid proteases are known to release protein domains from membranes by a cut in their membrane anchor, whereas an emerging new class of rhomboid-family proteins lacks key catalytic residues and is not proteolytically active. Recent work has shown that these rhomboid pseudoproteases, including iRhoms and derlins, bind membrane proteins to regulate their fate, but the underlying molecular mechanism is not known. This review summarizes recent advances in the molecular understanding of rhomboid-family proteins and discusses common principles in how they recognize and bind proteins in the plane of the membrane. PMID:23369641

  11. A topological and conformational stability alphabet for multipass membrane proteins.

    PubMed

    Feng, Xiang; Barth, Patrick

    2016-03-01

    Multipass membrane proteins perform critical signal transduction and transport across membranes. How transmembrane helix (TMH) sequences encode the topology and conformational flexibility regulating these functions remains poorly understood. Here we describe a comprehensive analysis of the sequence-structure relationships at multiple interacting TMHs from all membrane proteins with structures in the Protein Data Bank (PDB). We found that membrane proteins can be deconstructed in interacting TMH trimer units, which mostly fold into six distinct structural classes of topologies and conformations. Each class is enriched in recurrent sequence motifs from functionally unrelated proteins, revealing unforeseen consensus and evolutionary conserved networks of stabilizing interhelical contacts. Interacting TMHs' topology and local protein conformational flexibility were remarkably well predicted in a blinded fashion from the identified binding-hotspot motifs. Our results reveal universal sequence-structure principles governing the complex anatomy and plasticity of multipass membrane proteins that may guide de novo structure prediction, design, and studies of folding and dynamics. PMID:26780406

  12. Intrinsic membrane association of Drosophila cysteine string proteins.

    PubMed

    Mastrogiacomo, A; Kohan, S A; Whitelegge, J P; Gundersen, C B

    1998-09-25

    Cysteine string proteins (csps) are highly conserved constituents of vertebrate and invertebrate secretory organelles. Biochemical and immunoprecipitation experiments implied that vertebrate csps were integral membrane proteins that were tethered to the outer leaflet of secretory vesicles via the fatty acyl residues of their extensively acylated cysteine string. Independently, work of others suggested that Drosophila csps were peripheral membrane proteins that were anchored to membranes by a mechanism that was independent of the cysteine string and its fatty acyl residues. We extended these investigation and found first that sodium carbonate treatment partially stripped both csps and the integral membrane protein, synaptotagmin, from Drosophila membranes. Concomitantly, carbonate released fatty acids into the medium, arguing that it has a mild, solubilizing effect on these membranes. Second, we observed that Drosophila csps behaved like integral membrane proteins in Triton X-114 partitioning experiments. Third, we found that when membrane-bound csps were deacylated, they remained membrane bound. Moreover, it appeared that hydrophobic interactions were necessary for this persistent membrane association of csps. Thus, neither reducing conditions, urea, nor chaotropic agents displaced deacylated csps from membranes. Only detergents were effective in solubilizing deacylated csps. Finally, by virtue of the inaccessibility of deacylated csps to thiol alkylation by the membrane-impermeant alkylating reagent, iodoacetic acid, we inferred that it was the cysteine string domain that mediated the membrane association of deacylated csps. Thus, we conclude that under physiological conditions csps are integral membrane proteins of secretory organelles, and that the cysteine string domain plays a vital role in the membrane association of these proteins. PMID:9771899

  13. The Use of Detergents to Purify Membrane Proteins.

    PubMed

    Orwick-Rydmark, Marcella; Arnold, Thomas; Linke, Dirk

    2016-01-01

    Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, biomolecular NMR, or electron microscopy. A brief summary of alternative membrane protein solubilizing and stabilizing systems is also included. Protocols in this unit include the isolation and solubilization of biological membranes and phase separation; support protocols for detergent removal, detergent exchange, and the determination of critical micelle concentration using different methods are also included. PMID:27038269

  14. Effects of porogen and cross-linking agents on improved properties of silica-supported macroporous chitosan membranes for enzyme immobilization.

    PubMed

    Yang, Wen-Yi; Thirumavalavan, Munusamy; Lee, Jiunn-Fwu

    2015-04-01

    A series of silica-supported macroporous chitosan membranes (CM15, CM20, and CM25) was prepared by varying the ratio of 70-230-μm-sized silica particles. These synthesized membranes were further cross-linked using different cross-linking agents for covalent immobilization of biological macromolecules especially enzymes and in this study, Bovine serum albumin and laccase. Effects of silica particle and cross-linking agents on their flow rates, surface properties, and chemical and biological properties were explored. Pore size of as-synthesized membranes was 0.1192, 0.1268, and 0.1623 μm, respectively, for CM15, CM20, and CM25. The effect of various parameters such as temperature and pH on the relative activity of both free and immobilized enzymes was studied in details. The relative enzyme activity upon immobilization was greatly enhanced several folds of its original activity. The stability of enzymes over a range of temperature and pH was significantly improved by immobilization. The optimum temperature and pH were determined to be 50 °C and pH 3, respectively, for both the free and the immobilized enzymes. The immobilized enzyme possessed good operational stability and reusability properties that support its potentiality for practical applications. Among three membranes, CM25 is confirmed to be efficient candidate due to its improved characteristics. PMID:25432857

  15. Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in Meat by Using Phages Immobilized on Modified Cellulose Membranes

    PubMed Central

    Anany, H.; Chen, W.; Pelton, R.; Griffiths, M. W.

    2011-01-01

    The ability of phages to specifically interact with and lyse their host bacteria makes them ideal antibacterial agents. The range of applications of bacteriophage can be extended by their immobilization on inert surfaces. A novel method for the oriented immobilization of bacteriophage has been developed. The method was based on charge differences between the bacteriophage head, which exhibits an overall net negative charge, and the tail fibers, which possess an overall net positive charge. Hence, the head would be more likely to attach to positively charged surfaces, leaving the tails free to capture and lyse bacteria. Cellulose membranes modified so that they had a positive surface charge were used as the support for phage immobilization. It was established that the number of infective phages immobilized on the positively charged cellulose membranes was significantly higher than that on unmodified membranes. Cocktails of phages active against Listeria or Escherichia coli immobilized on these membranes were shown to effectively control the growth of L. monocytogenes and E. coli O157:H7 in ready-to-eat and raw meat, respectively, under different storage temperatures and packaging conditions. The phage storage stability was investigated to further extend their industrial applications. It was shown that lyophilization can be used as a phage-drying method to maintain their infectivity on the newly developed bioactive materials. In conclusion, utilizing the charge difference between phage heads and tails provided a simple technique for oriented immobilization applicable to a wide range of phages and allowed the retention of infectivity. PMID:21803890

  16. Bilayer-thickness-mediated interactions between integral membrane proteins.

    PubMed

    Kahraman, Osman; Koch, Peter D; Klug, William S; Haselwandter, Christoph A

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  17. Bilayer-thickness-mediated interactions between integral membrane proteins

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Koch, Peter D.; Klug, William S.; Haselwandter, Christoph A.

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  18. Tetra Detector Analysis of Membrane Proteins

    PubMed Central

    Robbins, Rebecca A.; Stroud, Robert M.

    2014-01-01

    Well-characterized membrane protein detergent complexes (PDC) that are pure, homogenous and stable with minimized excess detergent micelles are essential for functional assays and crystallization studies. Procedural steps to measure the mass, size, shape, homogeneity and molecular composition of PDCs and their host detergent micelle using size exclusion chromatography (SEC) with a Viscotek tetra detector array (TDA; absorbance, refractive index, light scattering and viscosity detectors) are presented. The value of starting with a quality PDC sample, the precision and accuracy of the results, and the use of a digital bench top refractometer are emphasized. An alternate and simplified purification and characterization approach using SEC with dual absorbance and refractive index detectors to optimize detergent and lipid concentration while measuring the PDC homogeneity are also described. Applications relative to purification and characterization goals are illustrated as well. PMID:25081744

  19. Detergent-Specific Membrane Protein Crystallization Screens

    NASA Technical Reports Server (NTRS)

    Wiener, Michael

    2007-01-01

    A suite of reagents has been developed for three-dimensional crystallization of integral membranes present in solution as protein-detergent complexes (PDCs). The compositions of these reagents have been determined in part by proximity to the phase boundaries (lower consolute boundaries) of the detergents present in the PDCs. The acquisition of some of the requisite phase-boundary data and the preliminary design of several of the detergent- specific screens was supported by a NASA contract. At the time of expiration of the contract, a partial set of preliminary screens had been developed. This work has since been extended under non-NASA sponsorship, leading to near completion of a set of 20 to 30 different and unique detergent- specific 96-condition screens.

  20. Succinimidyl Ester Surface Chemistry: Implications of the Competition between Aminolysis and Hydrolysis on Covalent Protein Immobilization

    PubMed Central

    2015-01-01

    N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins. PMID:25317495

  1. Towards Co-Evolution of Membrane Proteins and Metabolism

    NASA Astrophysics Data System (ADS)

    Wilson, Michael A.; Wei, Chenyu; Pohorille, Andrew

    2014-12-01

    Primordial metabolism co-evolved with the earliest membrane peptides to produce more environmentally fit progeny. Here, we map a continuous, evolutionary path that connects nascent biochemistry with simple, membrane-bound oligopeptides, ion channels and, further, membrane proteins capable of energy transduction and utilization of energy for active transport.

  2. Guided reconstitution of membrane protein fragments.

    PubMed

    Cohen, Leah S; Arshava, Boris; Kauffman, Sarah; Mathew, Elizabeth; Fracchiolla, Katrina E; Ding, Fa-Xiang; Dumont, Mark E; Becker, Jeffrey M; Naider, Fred

    2014-01-01

    Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs. Short peptides representing the N-terminal (NT) and C-terminal (CT) regions of the first extracellular loop (EL1) of Ste2p, the Saccharomyces cerevisiae alpha-factor mating receptor, were activated using these reagents and the efficiencies of activation and rates of heterodimerization were analyzed. Activation of NT peptides with DTNP and 4-PDS resulted in about 60% yield, but heterodimerization was rapid and nearly quantitative. Double transmembrane domain protein fragments were biosynthesized and used in Guided Reconstitution reactions. A 102-residue fragment, 2TM-tail [Ste2p(G31-I120C)], was heterodimerized with CT-EL1-tail(DTNP) at pH 4.6 with a yield of ∼75%. A 132-residue fragment, 2TMlong-tail [Ste2p(M1-I120C)], was expressed in both unlabeled and (15)N-labeled forms and used with a peptide comprising the third transmembrane domain, to generate a 180-residue segmentally labeled 3TM protein that was found to be segmentally labeled using [(15)N,(1)H]-HSQC analysis. Our data indicate that the Guided Reconstitution method would be applicable to the segmental labeling of a membrane protein with 3 transmembrane domains and may prove useful in the preparation of an intact reconstituted GPCR for use in biophysical analysis and structure determination. PMID:23897574

  3. Expression strategies for structural studies of eukaryotic membrane proteins.

    PubMed

    Lyons, Joseph A; Shahsavar, Azadeh; Paulsen, Peter Aasted; Pedersen, Bjørn Panyella; Nissen, Poul

    2016-06-01

    Integral membrane proteins in eukaryotes are central to various cellular processes and key targets in structural biology, biotechnology and drug development. However, the number of available structures for eukaryotic membrane protein belies their physiological importance. Recently, the number of available eukaryotic membrane protein structures has been steadily increasing due to the development of novel strategies in construct design, expression and structure determination. Here, we examine the major expression systems exploited for eukaryotic membrane proteins. Additionally we strive to tabulate and describe the recent expression strategies in eukaryotic membrane protein structural biology. We find that a majority of targets have been expressed in advanced host systems and modified from their wild-type form with distinct focus on conformation and thermostabilisation. However, strategies for native protein purification should also be considered where possible, particularly in light of the recent advances in single particle cryo electron microscopy. PMID:27362979

  4. Anomalous diffusion of proteins in sheared lipid membranes.

    PubMed

    Khoshnood, Atefeh; Jalali, Mir Abbas

    2013-09-01

    We use coarse grained molecular dynamics simulations to investigate diffusion properties of sheared lipid membranes with embedded transmembrane proteins. In membranes without proteins, we find normal in-plane diffusion of lipids in all flow conditions. Protein embedded membranes behave quite differently: by imposing a simple shear flow and sliding the monolayers of the membrane over each other, the motion of protein clusters becomes strongly superdiffusive in the shear direction. In such a circumstance, the subdiffusion regime is predominant perpendicular to the flow. We show that superdiffusion is a result of accelerated chaotic motions of protein-lipid complexes within the membrane voids, which are generated by hydrophobic mismatch or the transport of lipids by proteins. PMID:24125292

  5. Size-dependent protein segregation at membrane interfaces

    NASA Astrophysics Data System (ADS)

    Schmid, Eva M.; Bakalar, Matthew H.; Choudhuri, Kaushik; Weichsel, Julian; Ann, Hyoung Sook; Geissler, Phillip L.; Dustin, Michael L.; Fletcher, Daniel A.

    2016-07-01

    Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane proteins whose organization is critical for intracellular signalling. To isolate the role of membrane protein size in pattern formation, we reconstituted model membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between membrane proteins can drastically alter their organization at membrane interfaces, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally driven membrane height fluctuations that transiently limit access to the interface. This sensitive and highly effective means of physically segregating proteins has implications for cell-cell contacts such as T-cell immunological synapses (for example, CD45 exclusion) and epithelial cell junctions (for example, E-cadherin enrichment), as well as for protein sorting at intracellular contact points between membrane-bound organelles.

  6. Membrane protein structures without crystals, by single particle electron cryomicroscopy

    PubMed Central

    Vinothkumar, Kutti R

    2015-01-01

    It is an exciting period in membrane protein structural biology with a number of medically important protein structures determined at a rapid pace. However, two major hurdles still remain in the structural biology of membrane proteins. One is the inability to obtain large amounts of protein for crystallization and the other is the failure to get well-diffracting crystals. With single particle electron cryomicroscopy, both these problems can be overcome and high-resolution structures of membrane proteins and other labile protein complexes can be obtained with very little protein and without the need for crystals. In this review, I highlight recent advances in electron microscopy, detectors and software, which have allowed determination of medium to high-resolution structures of membrane proteins and complexes that have been difficult to study by other structural biological techniques. PMID:26435463

  7. In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF.

    PubMed

    Moldes, Cristina; García, Pedro; García, José L; Prieto, María A

    2004-06-01

    A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-beta-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme. PMID:15184113

  8. Energy-coupled outer membrane transport proteins and regulatory proteins.

    PubMed

    Braun, Volkmar; Endriss, Franziska

    2007-06-01

    FhuA and FecA are two examples of energy-coupled outer membrane import proteins of gram-negative bacteria. FhuA transports iron complexed by the siderophore ferrichrome and serves as a receptor for phages, a toxic bacterial peptide, and a toxic protein. FecA transports diferric dicitrate and regulates transcription of an operon encoding five ferric citrate (Fec) transport genes. Properties of FhuA mutants selected according to the FhuA crystal structure are described. FhuA mutants in the TonB box, the hatch, and the beta-barrel are rather robust. TonB box mutants in FhuA FecA, FepA, Cir, and BtuB are compared; some mutations are suppressed by mutations in TonB. Mutant studies have not revealed a ferrichrome diffusion pathway, and tolerance to mutations in the region linking the TonB box to the hatch does not disclose a mechanism for how energy transfer from the cytoplasmic membrane to FhuA changes the conformation of FhuA such that bound substrates are released, the pore is opened, and substrates enter the periplasm, or how surface loops change their conformation such that TonB-dependent phages bind irreversibly and release their DNA into the cells. The FhuA and FecA crystal structures do not disclose the mechanism of these proteins, but they provide important information for specific functional studies. FecA is also a regulatory protein that transduces a signal from the cell surface into the cytoplasm. The interacting subdomains of the proteins in the FecA --> FecR --> FecI --> RNA polymerase signal transduction pathway resulting in fecABCDE transcription have been determined. Energy-coupled transporters transport not only iron and vitamin B12, but also other substrates of very low abundance such as sugars across the outer membrane; transcription regulation of the transport genes may occur similarly to that of the Fec transport genes. PMID:17370038

  9. Membrane interaction of retroviral Gag proteins

    PubMed Central

    Dick, Robert A.; Vogt, Volker M.

    2014-01-01

    Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses – MA, CA, and NC – provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV) appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding. PMID:24808894

  10. Integrated reactive ion etching to pattern cross-linked hydrophilic polymer structures for protein immobilization

    NASA Astrophysics Data System (ADS)

    Bhatnagar, Parijat; Strickland, Aaron D.; Kim, Il; Malliaras, George G.; Batt, Carl A.

    2007-04-01

    Patterning of cross-linked hydrophilic polymer features using reactive ion etching (RIE) capable of covalently immobilizing proteins has been achieved. Projection photolithography was used to pattern photoresist to create micromolds. Vapor phase molecular self-assembly of polymerizable monolayer in molds allowed covalent binding of hydrogel on surface during free-radical polymerization. Excess hydrogel blanket film was consumed with oxygen RIE resulting into hydrogel pattern of 1μm size aligned to prefabricated silicon oxide structures. Proteins were finally coupled through their primary amine groups selectively to acid functionalized hydrogel features through stable amide linkages using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysulfosuccinimide.

  11. The derivatization of oxidized polysaccharides for protein immobilization and affinity chromatography.

    PubMed

    Junowicz, E; Charm, S E

    1976-03-25

    The present report describes the preparation of modified polysaccharides matrices useful for the synthesis of affinity adsorbents and immobilized proteins. Hydrazido-matrices were synthesized by condensing an excess of the bifunctional reagent, adipic acid dihydrazide, with periodate oxidized cellulose paper, Sephadex, or Sepharose matrices. Ribonucleotide dialdehyde cofactors, glyceraldehyde 3-phosphate, pyridoxal 5'-phosphate and oxidized DNAase B were separately bound to the hydrazido-polymers. Azido-matrices obtained by modification of the hydrazido-derivatives were coupled to specific amino ligands such as amino acids and proteins. Several adsorbents were prepared and used as models for affinity chromatography. PMID:1260016

  12. Single molecule techniques for the study of membrane proteins.

    PubMed

    García-Sáez, Ana J; Schwille, Petra

    2007-08-01

    Single molecule techniques promise novel information about the properties and behavior of individual particles, thus enabling access to molecular heterogeneities in biological systems. Their recent developments to accommodate membrane studies have significantly deepened the understanding of membrane proteins. In this short review, we will describe the basics of the three most common single-molecule techniques used on membrane proteins: fluorescence correlation spectroscopy, single particle tracking, and atomic force microscopy. We will discuss the most relevant findings made during the recent years and their contribution to the membrane protein field. PMID:17497147

  13. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

  14. Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins.

    PubMed

    Forato, Florian; Liu, Hao; Benoit, Roland; Fayon, Franck; Charlier, Cathy; Fateh, Amina; Defontaine, Alain; Tellier, Charles; Talham, Daniel R; Queffélec, Clémence; Bujoli, Bruno

    2016-06-01

    Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl3 as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr(4+), afforded better immobilization of the peptides and proteins and efficient capture of their targets. PMID:27166821

  15. Conductimetric biosensor for the detection of uric Acid by immobilization uricase on nata de coco membrane-pt electrode.

    PubMed

    Mulyasuryani, Ani; Srihardiastutie, Arie

    2011-01-01

    A conductimetric enzyme biosensor for uric acid detection has been developed. The uricase, as enzyme, is isolated from Candida utilis and immobilized on a nata de coco membrane-Pt electrode. The biosensor demonstrates a linear response to urate over the concentration range 1-6 ppm and has good selectivity properties. The response is affected by the membrane thickness and pH change in the range 7.5-9.5. The response time is three minutes in aqueous solutions and in human serum samples. Application of the biosensor to the determination of uric acid in human serum gave results that compared favourably with those obtained by medical laboratory. The operational stability of the biosensor was not less than three days and the relative error is smaller than 10%. PMID:21792276

  16. Network pattern of residue packing in helical membrane proteins and its application in membrane protein structure prediction.

    PubMed

    Pabuwal, Vagmita; Li, Zhijun

    2008-01-01

    De novo protein structure prediction plays an important role in studies of helical membrane proteins as well as structure-based drug design efforts. Developing an accurate scoring function for protein structure discrimination and validation remains a current challenge. Network approaches based on overall network patterns of residue packing have proven useful in soluble protein structure discrimination. It is thus of interest to apply similar approaches to the studies of residue packing in membrane proteins. In this work, we first carried out such analysis on a set of diverse, non-redundant and high-resolution membrane protein structures. Next, we applied the same approach to three test sets. The first set includes nine structures of membrane proteins with the resolution worse than 2.5 A; the other two sets include a total of 101 G-protein coupled receptor models, constructed using either de novo or homology modeling techniques. Results of analyses indicate the two criteria derived from studying high-resolution membrane protein structures are good indicators of a high-quality native fold and the approach is very effective for discriminating native membrane protein folds from less-native ones. These findings should be of help for the investigation of the fundamental problem of membrane protein structure prediction. PMID:18178566

  17. The Hydrophobic Insertion Mechanism of Membrane Curvature Generation by Proteins

    PubMed Central

    Campelo, Felix; McMahon, Harvey T.; Kozlov, Michael M.

    2008-01-01

    A wide spectrum of intracellular processes is dependent on the ability of cells to dynamically regulate membrane shape. Membrane bending by proteins is necessary for the generation of intracellular transport carriers and for the maintenance of otherwise intrinsically unstable regions of high membrane curvature in cell organelles. Understanding the mechanisms by which proteins curve membranes is therefore of primary importance. Here we suggest, for the first time to our knowledge, a quantitative mechanism of lipid membrane bending by hydrophobic or amphipathic rodlike inclusions which simulate amphipathic α-helices—structures shown to sculpt membranes. Considering the lipid monolayer matrix as an anisotropic elastic material, we compute the intramembrane stresses and strains generated by the embedded inclusions, determine the resulting membrane shapes, and the accumulated elastic energy. We characterize the ability of an inclusion to bend membranes by an effective spontaneous curvature, and show that shallow rodlike inclusions are more effective in membrane shaping than are lipids having a high propensity for curvature. Our computations provide experimentally testable predictions on the protein amounts needed to generate intracellular membrane shapes for various insertion depths and membrane thicknesses. We also predict that the ability of N-BAR domains to produce membrane tubules in vivo can be ascribed solely to insertion of their amphipathic helices. PMID:18515373

  18. A comprehensive strategy to identify stoichiometric membrane protein interactomes

    PubMed Central

    Gokhale, Avanti; Perez-Cornejo, Patricia; Duran, Charity; Hartzell, H. Criss; Faundez, Victor

    2012-01-01

    There are numerous experimental approaches to identify the interaction networks of soluble proteins, but strategies for the identification of membrane protein interactomes remain limited. We discuss in detail the logic of an experimental design that led us to identify the interactome of a membrane protein of complex membrane topology, the calcium activated chloride channel Anoctamin 1/Tmem16a (Ano1). We used covalent chemical stabilizers of protein-protein interactions combined with magnetic bead immuno-affinity chromatography, quantitative SILAC mass-spectrometry and in silico network construction. This strategy led us to define a putative Ano1 interactome from which we selected key components for functional testing. We propose a combination of procedures to narrow down candidate proteins interacting with a membrane protein of interest for further functional studies. PMID:23676845

  19. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  20. Negative Ions Enhance Survival of Membrane Protein Complexes.

    PubMed

    Liko, Idlir; Hopper, Jonathan T S; Allison, Timothy M; Benesch, Justin L P; Robinson, Carol V

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein. Graphical Abstract ᅟ. PMID:27106602

  1. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-04-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  2. Sonochemical synthesis of (3-aminopropyl)triethoxysilane-modified monodispersed silica nanoparticles for protein immobilization

    SciTech Connect

    Shen, Shou-Cang; Ng, Wai Kiong; Chia, Leonard; Dong, Yuan-Cai; Tan, Reginald B.H.

    2011-10-15

    Graphical abstract: 3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by rapid sonochemical co-condensation to achieve high capability for protein immobilization. Highlights: {yields} Amino-modified monodispersed silica nanoparticles were synthesized by rapid co-condensation. {yields} Strong positive charge was created by aminopropyl-modification. {yields} Capability for immobilization of negatively charged protein was enhanced. {yields} Electrostatic interaction between proteins and surface contributed to the enhanced adsorption. -- Abstract: 3-Aminopropyltriethoxysilane modified monodispersed silica nanoparticles were synthesized by a rapid sonochemical co-condensation synthesis procedure. The chemical nature of surface organic modifier on the obtained modified silica nanoparticle was characterized by {sup 13}C and {sup 29}Si MAS Nuclear Magnetic Resonance (NMR) spectroscopies, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA)- differential scanning calorimetry (DSC). Due to the strengthened positive surface charge of the silica nanoparticles by the modification with aminopropyl groups, the capability for bovine serum albumin (BSA) adsorption was significantly increased as compared with bare silica nanoparticles. 80 mg/g BSA was adsorbed on modified silica nanoparticles, whereas only 20 mg/g BSA could be loaded on pure silica nanoparticles. The enhanced positive surface charge repelled proteins with net positive charge and the modified silica nanoparticles exhibited negligible adsorption of lysozyme, thus a selective adsorption of proteins could be achieved.

  3. 4 °C preparation of ferrite nanoparticles having protein molecules immobilized on their surfaces

    NASA Astrophysics Data System (ADS)

    Nishimura, K.; Hasegawa, M.; Ogura, Y.; Nishi, T.; Kataoka, K.; Handa, H.; Abe, M.

    2002-05-01

    Trypsin, a proteolytic enzyme or a protein, was immobilized onto the surfaces of ferrite (a Fe3O4-γFe2O3 mixed solution) fine particles, ˜8 nm in size, during the process in which the particles were synthesized from an aqueous solution. The process was performed in the open air at a temperature as low as 4 °C and on near-neutral condition of pH⩽9, which is compatible with most of the bioactive molecules as well as trypsin. Therefore this technique is advantageous for preparing magnetite particles having biomolecules immobilized on their surfaces, which will be used for biomedical applications utilizing magnetic separation technique.

  4. Immobilization of Aspergillus oryzae  β-Galactosidase on Cellulose Acetate-Polymethylmethacrylate Membrane and Its Application in Hydrolysis of Lactose from Milk and Whey

    PubMed Central

    Ansari, Shakeel Ahmed; Satar, Rukhsana; Kashif Zaidi, Syed; Ahmad, Abrar

    2014-01-01

    The present study demonstrates the immobilization of Aspergillus oryzae β-galactosidase on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane and its application in hydrolyzing lactose in dairy industries. The effect of physical and chemical denaturants like pH, temperature, product inhibition by galactose, storage stability, and reuse number of the enzyme immobilized on CA-PMMA membrane has been investigated. Lactose was hydrolyzed from milk and whey in batch reactors at 50°C by free and immobilized β-galactosidase (IβG). Optimum pH for the free and immobilized enzyme was found to be the same, that is, 4.5. However, IβG retained greater fractions of catalytic activity at lower and higher pH ranges. The temperature optimum for the immobilized enzyme was increased by 10°C. Moreover, Michaelis-Menten constant was increased for IβG as compared to the native one while maximum reaction rate was reduced for the immobilized enzyme. The preserved activity of free and immobilized enzyme was found to be 45% and 83%, respectively, after five weeks of storage at 4°C. Reusability of IβG was observed to be 86% even after fifth repeated use, thereby signifying its application in lactose hydrolysis (as shown in lab-scale batch reactors) in various dairy products including milk and whey. PMID:27350979

  5. Composition fluctuations, correlated response, and protein solvation in membranes

    NASA Astrophysics Data System (ADS)

    McConnell, Harden

    2010-05-01

    Membrane composition fluctuations are deduced from the deuterium NMR relaxation data of S. L. Veatch et al. [Proc. Natl. Acad. Sci. U.S.A. 104, 17650 (2007)]. A theoretical model for these fluctuations is used to determine the parameters of a correlation function. A fluctuation-response relation is then derived to infer the response of a lipid bilayer membrane to perturbations, such as the presence of a protein. The energy of the correlated response is shown to decrease as a bilayer miscibility critical point is approached from higher temperatures. Near the critical temperature the low energy of the composition response facilitates the lipid solvation of membrane proteins and minimizes lipid-mediated nonspecific protein-protein interactions. This facilitated lipid solvation of membrane proteins may be the basis of reports that at the growth temperature, the lipids of animal cell membranes have compositions such that they are within ˜10° of a miscibility critical point.

  6. Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications.

    PubMed

    Hiruma-Shimizu, Kazumi; Shimizu, Hiroki; Thompson, Gary S; Kalverda, Arnout P; Patching, Simon G

    2015-01-01

    Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR. PMID:26906947

  7. X-ray Diffraction from Membrane Protein Nanocrystals

    PubMed Central

    Hunter, M.S.; DePonte, D.P.; Shapiro, D.A.; Kirian, R.A.; Wang, X.; Starodub, D.; Marchesini, S.; Weierstall, U.; Doak, R.B.; Spence, J.C.H.; Fromme, P.

    2011-01-01

    Membrane proteins constitute >30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is <300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 μm. The results demonstrate that there are membrane protein crystals that contain <100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain <200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells. PMID:21190672

  8. Protein quality control at the inner nuclear membrane

    PubMed Central

    Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J.; Le Dez, Gaëlle; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D.; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwenaël; Ljungdahl, Per O.; Knop, Michael

    2015-01-01

    The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by ER-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc72,3. However, little is known regarding protein quality control at the INM. Here we describe a protein degradation pathway at the INM mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi34. We report that the As complex functions together with the ubiquitin conjugating enzymes Ubc6andUbc7to degrade soluble and integral membrane proteins. Genetic evidence suggest that the Asi ubiquitin ligase defines a pathway distinct from but complementary to ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer (tFT)5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquity ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalised integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. PMID:25519137

  9. Translation Levels Control Multi-Spanning Membrane Protein Expression

    PubMed Central

    Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.

    2012-01-01

    Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane. PMID:22563408

  10. Highly-sensitive organophosphorus pesticide biosensors based on CdTe quantum dots and bi-enzyme immobilized eggshell membranes.

    PubMed

    Xue, Gao; Yue, Zhao; Bing, Zhang; Yiwei, Tang; Xiuying, Liu; Jianrong, Li

    2016-02-01

    An optical biosensing method using CdTe quantum dots (QDs) and bi-enzyme-immobilized eggshell membranes for the determination of organophosphorus pesticides (OPs) has been developed. Increasing amounts of OPs led to a decrease of the enzymatic activity and thus a decrease in the production of hydrogen peroxide (H2O2), which can quench the fluorescence of the CdTe QDs. Under the optimum conditions, there was a good linear relationship between the enzyme inhibition percentage and the logarithm of paraoxon or parathion concentration in the range of 1.0 × 10(-11)-1.0 × 10(-6) mol L(-1). The detection limit (S/N = 3) of the proposed biosensors were as low as 4.30 × 10(-12) mol L(-1) for paraoxon and 2.47 × 10(-12) mol L(-1) for parathion. The bi-enzyme-immobilized eggshell membrane demonstrated a long shelf-life of at least 2 months and the results showed good repeatability. The proposed method was successfully applied to the determination of the OPs in real fruit samples with satisfactory results. PMID:26688862

  11. Immobilization of immunoglobulin-G-binding domain of Protein A on a gold surface modified with biotin ligase.

    PubMed

    Miyao, Hiroki; Ikeda, Yusuke; Shiraishi, Arata; Kawakami, Yuji; Sueda, Shinji

    2015-09-01

    Protein A from Staphylococcus aureus specifically binds to the Fc region of immunoglobulin G (IgG) and is widely used as a scaffold for the immobilization of IgG antibodies on solid supports. It is known that the oriented immobilization of Protein A on solid supports enhances its antibody-binding capability in comparison with immobilization in a random manner. In the current work, we developed a novel method for the oriented immobilization of the IgG-binding domain of Protein A based on the biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property in that the enzyme, biotin protein ligase (BPL), forms a stable complex with its biotinylated substrate protein, biotin carboxyl carrier protein (BCCP). Here, BCCP was fused to the IgG-binding domain of Protein A, and the resulting fusion protein was immobilized on the BPL-modified gold surface of the sensor chip for quartz crystal microbalance through complexation between BCCP and BPL. The layer of the IgG-binding domain prepared in this way successfully captured the antibody, and the captured antibody retained high antigen-binding capability. PMID:25998102

  12. An integrated, peptide-based approach to site-specific protein immobilization for detection of biomolecular interactions.

    PubMed

    Kruis, Ilmar C; Löwik, Dennis W P M; Boelens, Wilbert C; van Hest, Jan C M; Pruijn, Ger J M

    2016-09-21

    We have developed an integrated solution for the site-specific immobilization of proteins on a biosensor surface, which may be widely applicable for high throughput analytical purposes. The gold surface of a biosensor was coated with an anti-fouling layer of zwitterionic peptide molecules from which leucine zipper peptides protrude. Proteins of interest, the autoantigenic proteins La and U1A, were immobilized via a simple incubation procedure by using the complementary leucine zipper sequence as a genetically fused binding tag. This tag forms a strong coiled-coil interaction that is stable during multiple consecutive measurements and under common regeneration conditions. Visualization of the immobilized proteins of interest via antibody binding with multiplex surface plasmon resonance imaging demonstrated 2.5 times higher binding responses than when these proteins were randomly attached to the surface via the commonly applied activated ester-mediated coupling. The proteins could also be immobilized in a leucine zipper-dependent manner directly from complex mixtures like bacterial lysates, eliminating the need for laborious purification steps. This method allows the production of uniform functional protein arrays by control over immobilized protein orientation and geometry and is compatible with high-throughput procedures. PMID:27328408

  13. Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

    PubMed Central

    Bohnert, Maria; Wenz, Lena-Sophie; Zerbes, Ralf M.; Horvath, Susanne E.; Stroud, David A.; von der Malsburg, Karina; Müller, Judith M.; Oeljeklaus, Silke; Perschil, Inge; Warscheid, Bettina; Chacinska, Agnieszka; Veenhuis, Marten; van der Klei, Ida J.; Daum, Günther; Wiedemann, Nils; Becker, Thomas; Pfanner, Nikolaus; van der Laan, Martin

    2012-01-01

    Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins. PMID:22918945

  14. Curvature Forces in Membrane Lipid-Protein Interactions

    NASA Astrophysics Data System (ADS)

    Brown, Michael F.

    2012-02-01

    Membrane protein conformational changes, folding, and stability may all involve elastic deformation of the bilayer. Non-specific properties of the bilayer play a significant role in modulating protein conformational energetics. A flexible-surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. The FSM describes elastic coupling of membrane lipids to integral membrane proteins. Curvature and hydrophobic matching to the lipid bilayer entails a stress field that explains membrane protein stability. Rhodopsin provides an important example, where solid-state NMR and FTIR spectroscopy characterize the energy landscape of the dynamically activated receptor. Time-resolved UV-visible and FTIR spectroscopic studies show how membrane lipids affect the metarhodopsin equilibrium due to non-specific material properties. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress on rhodopsin function are all explained by the new biomembrane model. By contrast, the older fluid-mosaic model fails to account for such effects on membrane protein activity. According to the FSM proteins are regulated by membrane lipids whose spontaneous curvature most closely matches the activated state within the lipid membrane.

  15. Prediction of lipid-binding regions in cytoplasmic and extracellular loops of membrane proteins as exemplified by protein translocation membrane proteins.

    PubMed

    Keller, Rob C A

    2013-01-01

    The presence of possible lipid-binding regions in the cytoplasmic or extracellular loops of membrane proteins with an emphasis on protein translocation membrane proteins was investigated in this study using bioinformatics. Recent developments in approaches recognizing lipid-binding regions in proteins were found to be promising. In this study a total bioinformatics approach specialized in identifying lipid-binding helical regions in proteins was explored. Two features of the protein translocation membrane proteins, the position of the transmembrane regions and the identification of additional lipid-binding regions, were analyzed. A number of well-studied protein translocation membrane protein structures were checked in order to demonstrate the predictive value of the bioinformatics approach. Furthermore, the results demonstrated that lipid-binding regions in the cytoplasmic and extracellular loops in protein translocation membrane proteins can be predicted, and it is proposed that the interaction of these regions with phospholipids is important for proper functioning during protein translocation. PMID:22961045

  16. Membrane-Protein Crystallography and Potentiality for Drug Design

    NASA Astrophysics Data System (ADS)

    Yamashita, Atsuko

    Structure-based drug design for membrane proteins is far behind that for soluble proteins due to difficulty in crystallographic structure determination, despite the fact that about 60% of FDA-approved drugs target membrane proteins located at the cell surface. Stable homologs for a membrane protein of interest, such as prokaryotic neurotransmitter transporter homolog LeuT, might enable cooperative analyses by crystallography and functional assays, provide useful information for functional mechanisms, and thus serve as important probes for drug design based on mechanisms as well as structures.

  17. BPROMPT: A consensus server for membrane protein prediction.

    PubMed

    Taylor, Paul D; Attwood, Teresa K; Flower, Darren R

    2003-07-01

    Protein structure prediction is a cornerstone of bioinformatics research. Membrane proteins require their own prediction methods due to their intrinsically different composition. A variety of tools exist for topology prediction of membrane proteins, many of them available on the Internet. The server described in this paper, BPROMPT (Bayesian PRediction Of Membrane Protein Topology), uses a Bayesian Belief Network to combine the results of other prediction methods, providing a more accurate consensus prediction. Topology predictions with accuracies of 70% for prokaryotes and 53% for eukaryotes were achieved. BPROMPT can be accessed at http://www.jenner.ac.uk/BPROMPT. PMID:12824397

  18. The Protein 4.1 family: hub proteins in animals for organizing membrane proteins.

    PubMed

    Baines, Anthony J; Lu, Hui-Chun; Bennett, Pauline M

    2014-02-01

    Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and

  19. Refractive index matching to develop transparent polyaphrons: Characterization of immobilized proteins.

    PubMed

    Ward, Keeran; Stuckey, David C

    2016-06-01

    Refractive index matching was used to create optically transparent polyaphrons to enable proteins adsorbed to the aphron surface to be characterized. Due to the significant light scattering created by polyaphrons, refractive index matching allowed for representative circular dichroism (CD) spectra and acceptable structural characterization. The method utilized n-hexane as the solvent phase, a mixture of glycerol and phosphate buffer (30% [w/v]) as the aqueous phase, and the non-ionic surfactants, Laureth-4 and Kolliphor P-188. Deconvolution of CD spectra revealed that the immobilized protein adapted its native conformation, showing that the adsorbed protein interacted only with the bound water layer ("soapy shell") of the aphron. Isothermal calorimetry further demonstrated that non-ionic surfactant interactions were virtually non-existent, even at the high concentrations used (5% [w/v]), proving that non-ionic surfactants can preserve protein conformation. PMID:26952359

  20. A Usual G-Protein-Coupled Receptor in Unusual Membranes.

    PubMed

    Chawla, Udeep; Jiang, Yunjiang; Zheng, Wan; Kuang, Liangju; Perera, Suchithranga M D C; Pitman, Michael C; Brown, Michael F; Liang, Hongjun

    2016-01-11

    G-protein-coupled receptors (GPCRs) are the largest family of membrane-bound receptors and constitute about 50% of all known drug targets. They offer great potential for membrane protein nanotechnologies. We report here a charge-interaction-directed reconstitution mechanism that induces spontaneous insertion of bovine rhodopsin, the eukaryotic GPCR, into both lipid- and polymer-based artificial membranes. We reveal a new allosteric mode of rhodopsin activation incurred by the non-biological membranes: the cationic membrane drives a transition from the inactive MI to the activated MII state in the absence of high [H(+)] or negative spontaneous curvature. We attribute this activation to the attractive charge interaction between the membrane surface and the deprotonated Glu134 residue of the rhodopsin-conserved ERY sequence motif that helps break the cytoplasmic "ionic lock". This study unveils a novel design concept of non-biological membranes to reconstitute and harness GPCR functions in synthetic systems. PMID:26633591

  1. Overcoming bottlenecks in the membrane protein structural biology pipeline.

    PubMed

    Hardy, David; Bill, Roslyn M; Jawhari, Anass; Rothnie, Alice J

    2016-06-15

    Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future. PMID:27284049

  2. Immobilization of soluble protein complexes in MAS solid-state NMR: Sedimentation versus viscosity.

    PubMed

    Sarkar, Riddhiman; Mainz, Andi; Busi, Baptiste; Barbet-Massin, Emeline; Kranz, Maximilian; Hofmann, Thomas; Reif, Bernd

    2016-01-01

    In recent years, MAS solid-state NMR has emerged as a technique for the investigation of soluble protein complexes. It was found that high molecular weight complexes do not need to be crystallized in order to obtain an immobilized sample for solid-state NMR investigations. Sedimentation induced by sample rotation impairs rotational diffusion of proteins and enables efficient dipolar coupling based cross polarization transfers. In addition, viscosity contributes to the immobilization of the molecules in the sample. Natural Deep Eutectic Solvents (NADES) have very high viscosities, and can replace water in living organisms. We observe a considerable amount of cross polarization transfers for NADES solvents, even though their molecular weight is too low to yield significant sedimentation. We discuss how viscosity and sedimentation both affect the quality of the obtained experimental spectra. The FROSTY/sedNMR approach holds the potential to study large protein complexes, which are otherwise not amenable for a structural characterization using NMR. We show that using this method, backbone assignments of the symmetric proteasome activator complex (1.1MDa), and high quality correlation spectra of non-symmetric protein complexes such as the prokaryotic ribosome 50S large subunit binding to trigger factor (1.4MDa) are obtained. PMID:27017576

  3. Multifunctional Electrospun Nanofibers Incorporated with an Anti-infection Drug and Immobilized with Proteins

    NASA Astrophysics Data System (ADS)

    Zhou, Shufei

    Electrospinning has been used to fabricate ultrafine fibers with sizes ranging from nano to micrometers. Nanofibers electrospun from biocompatible and biodegradable polymers have been extensively investigated for their potential applications in wound healing and tissue regeneration. These nanofiber materials can be modified to incorporate bioactive molecules, such as antibacterial agents that provide infection control, or functional proteins which promote cell proliferation and tissue reconstruction. Despite the numerous studies on the development and design of nanofibers for biomedical applications, there has been little research on multifunctional nanofibers that are incorporated with both antibacterial drug(s) and bioactive proteins. The objective of the current study is, therefore, to develop nanofibers that are functionalized by several bioactive molecules. In this study, electrospinning was utilized to fabricate nanofibers from biodegradable polymers PLLA (Poly-L-lactide) and the copolymer PLLA-PEG (Polyethylene glycol)-NH2.A water soluble antibiotic drug, Tetracycline Hydrochloride (TCH), was incorporated into the electrospun nanofibers via emulsion electrospinning. The TCH-loaded nanofibers were surface modified to produce functional groups that can be further conjugated with a model protein, Bovine Serum Albumin (BSA).Drug releasing profiles of the medicated nanofibers were monitored and their antimicrobial properties were evaluated. Proteins (BSAs) immobilized on the fiber surface were verified by ATR-FTIR. The number of immobilized BSAs was determined using a UV-Vis spectrophotometer. The results of the study suggested that this multifunctional nanofibrous material could be a promising material for wound dressing or scaffolds for tissue engineering.

  4. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

    PubMed

    Ford, Nicole R; Hecht, Karen A; Hu, DeHong; Orr, Galya; Xiong, Yijia; Squier, Thomas C; Rorrer, Gregory L; Roesijadi, Guritno

    2016-03-18

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms. PMID:26746113

  5. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Melén, Karin; Blomberg, Anders; von Heijne, Gunnar

    2006-07-01

    Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing 600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na+/K+ homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies. caffeine | paraquat | salt tolerance | yeast

  6. Membrane proteins of Mycoplasma bovis and their role in pathogenesis.

    PubMed

    Adamu, James Y; Wawegama, Nadeeka K; Browning, Glenn F; Markham, Philip F

    2013-10-01

    Mycoplasma membrane proteins influence cell shape, cell division, motility and adhesion to host cells, and are thought to be integrally involved in the pathogenesis of mycoplasmoses. Many of the membrane proteins predicted from mycoplasma genome sequences remain hypothetical, as their presence in cellular protein preparations is yet to be established experimentally. Recent genome sequences of several strains of Mycoplasma bovis have provided further insight into the potential role of the membrane proteins of this pathogen in colonisation and infection. This review highlights recent advances in knowledge about the influence of M. bovis membrane proteins on the pathogenesis of infection with this species and identifies future research directions for enhancing our understanding of the role of these proteins. PMID:23810376

  7. Glycosomal membrane proteins and lipids from Leishmania mexicana.

    PubMed

    Quiñones, Wilfredo; Cáceres, Ana J; Ruiz, Maria Tibisay; Concepción, Juan Luis

    2015-04-01

    Constituents of the glycosomal membrane from Leishmania mexicana should play a critical role in the coordination of metabolic processes occurring in the cytosol and those compartmentalized within glycosomes. We have made an inventory of glycosomal membrane-associated proteins using approaches specific for enriching both integral and peripheral membrane proteins. Surprisingly, 70% of the proteins were recovered in the hydrophobic fraction of membranes solubilized with Triton X-114, while 20% were present in the soluble fraction obtained upon treatment with Na2CO3. 14 major polypeptides, ranging in molecular weight from 65 to 16 kDa, were found to be associated with the membrane, nine of them behaving as integral membrane proteins. Assessment of their topology in the membrane indicated that the polypeptides of 56, 50, 46 and 32 kDa have no domains exposed to the cytosol. The 50 kDa protein is the most abundant one of the glycosomal membrane, where it is peripherically located at the matrix face. The major phospholipids of glycosomal membranes are phosphatidyl-ethanolamine, phosphatidyl-choline and phosphatidyl-serine, with smaller proportions of sphingomyelin and phosphatidyl-inositol. The sterols found were of 5-dehydroepisterol, ergosta-5,7,24(24(1))-trien-3β-ol, and also their precursors, consistent with the notion that these organelles are involved in de novo biosynthesis of sterols in trypanosomatids. PMID:25499533

  8. Concentrating membrane proteins using asymmetric traps and AC electric fields.

    PubMed

    Cheetham, Matthew R; Bramble, Jonathan P; McMillan, Duncan G G; Krzeminski, Lukasz; Han, Xiaojun; Johnson, Benjamin R G; Bushby, Richard J; Olmsted, Peter D; Jeuken, Lars J C; Marritt, Sophie J; Butt, Julea N; Evans, Stephen D

    2011-05-01

    Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol.2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery 2006, 5, 993). However, the structure-function relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structure-function determination of these species (Poo et al. Nature 1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a "nested trap" and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins. PMID:21476549

  9. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography

    PubMed Central

    Weierstall, Uwe; James, Daniel; Wang, Chong; White, Thomas A.; Wang, Dingjie; Liu, Wei; Spence, John C.H.; Doak, R. Bruce; Nelson, Garrett; Fromme, Petra; Fromme, Raimund; Grotjohann, Ingo; Kupitz, Christopher; Zatsepin, Nadia A.; Liu, Haiguang; Basu, Shibom; Wacker, Daniel; Han, Gye Won; Katritch, Vsevolod; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Klinker, Markus; Gati, Cornelius; Shoeman, Robert L.; Barty, Anton; Chapman, Henry N.; Kirian, Richard A.; Beyerlein, Kenneth R.; Stevens, Raymond C.; Li, Dianfan; Shah, Syed T.A.; Howe, Nicole; Caffrey, Martin; Cherezov, Vadim

    2014-01-01

    Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously-renewed source of material for serial femtosecond crystallography. Data collected from sub-10 μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor. PMID:24525480

  10. Methods for Mapping of Interaction Networks Involving Membrane Proteins

    SciTech Connect

    Hooker, Brian S.; Bigelow, Diana J.; Lin, Chiann Tso

    2007-11-23

    Numerous approaches have been taken to study protein interactions, such as tagged protein complex isolation followed by mass spectrometry, yeast two-hybrid methods, fluorescence resonance energy transfer, surface plasmon resonance, site-directed mutagenesis, and crystallography. Membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is not suitable for the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.

  11. Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Shang, E S; Foley, D M; Blanco, D R; Champion, C I; Mirzabekov, T; Sokolov, Y; Kagan, B L; Miller, J N; Lovett, M A

    1995-01-01

    We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease. Images PMID:7593626

  12. The Origin and Early Evolution of Membrane Proteins

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Schweighofer, Karl; Wilson, Michael A.

    2005-01-01

    Membrane proteins mediate functions that are essential to all cells. These functions include transport of ions, nutrients and waste products across cell walls, capture of energy and its transduction into the form usable in chemical reactions, transmission of environmental signals to the interior of the cell, cellular growth and cell volume regulation. In the absence of membrane proteins, ancestors of cell (protocells), would have had only very limited capabilities to communicate with their environment. Thus, it is not surprising that membrane proteins are quite common even in simplest prokaryotic cells. Considering that contemporary membrane channels are large and complex, both structurally and functionally, a question arises how their presumably much simpler ancestors could have emerged, perform functions and diversify in early protobiological evolution. Remarkably, despite their overall complexity, structural motifs in membrane proteins are quite simple, with a-helices being most common. This suggests that these proteins might have evolved from simple building blocks. To explain how these blocks could have organized into functional structures, we performed large-scale, accurate computer simulations of folding peptides at a water-membrane interface, their insertion into the membrane, self-assembly into higher-order structures and function. The results of these simulations, combined with analysis of structural and functional experimental data led to the first integrated view of the origin and early evolution of membrane proteins.

  13. Towards fuel cell membranes with improved lifetime: Aquivion® Perfluorosulfonic Acid membranes containing immobilized radical scavengers

    NASA Astrophysics Data System (ADS)

    D'Urso, C.; Oldani, C.; Baglio, V.; Merlo, L.; Aricò, A. S.

    2014-12-01

    A facile synthesis, based on a wet impregnation technique and a thermal treatment, of a novel silica-supported cerium-oxide-based radical scavenger bearing sulfonic acid functionalities is presented. This material is loaded as a filler in ePTFE reinforced membranes (called R79-02S) prepared starting from Aquivion® Perfluoro-Sulfonic Acid (PFSA) dispersions. The aim is to mitigate the peroxy radicals attack to the polymeric membrane under fuel cell operating conditions. These membranes show much longer (7 times more) life-time in Accelerated Stress Tests (AST) and reduced fluoride release (about one half) in Fenton's tests than the radical scavenger-free membrane without any loss in electrochemical performance. Scavenger-free Aquivion® PFSA-based membrane durability is about 200 h in AST whereas the same membrane containing the newly developed radical scavenger exceeds 1400 h. These results confirm the stability of the modified membranes and the excellent activity of the composite scavenger in mitigating the polymer electrolyte degradation.

  14. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization

    PubMed Central

    Hansen, Debra T.; Robida, Mark D.; Craciunescu, Felicia M.; Loskutov, Andrey V.; Dörner, Katerina; Rodenberry, John-Charles; Wang, Xiao; Olson, Tien L.; Patel, Hetal; Fromme, Petra; Sykes, Kathryn F.

    2016-01-01

    Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins. PMID:26908053

  15. Improved 1H amide resonance line narrowing in oriented sample solid-state NMR of membrane proteins in phospholipid bilayers

    NASA Astrophysics Data System (ADS)

    Lu, George J.; Park, Sang Ho; Opella, Stanley J.

    2012-07-01

    We demonstrate 1H amide resonance line widths <300 Hz in 1H/15N heteronuclear correlation (HETCOR) spectra of membrane proteins in aligned phospholipid bilayers. This represents a substantial improvement over typically observed line widths of ˜1 kHz. Furthermore, in a proton detected local field (PDLF) version of the experiment that measures heteronuclear dipolar couplings, line widths <130 Hz are observed. This dramatic line narrowing of 1H amide resonances enables many more individual signals to be resolved and assigned from uniformly 15N labeled membrane proteins in phospholipid bilayers under physiological conditions of temperature and pH. Finding that the decrease in line widths occurs only for membrane proteins that undergo fast rotational diffusion around the bilayer normal, but not immobile molecules, such as peptide single crystals, identifies a potential new direction for pulse sequence development that includes overall molecular dynamics in their design.

  16. Heterotypic and homotypic associations between ezrin and moesin, two putative membrane-cytoskeletal linking proteins.

    PubMed Central

    Gary, R; Bretscher, A

    1993-01-01

    Ezrin and moesin are components of actin-rich cell surface structures that are thought to function as membrane-cytoskeletal linking proteins. Here we show that a stable complex of ezrin and moesin can be isolated from cultured cells by immunoprecipitation with specific antibodies. The capacity of these two proteins to interact directly was confirmed with a blot-overlay procedure in which biotin-tagged proteins in solution were incubated with immobilized binding partners. In addition to the heterotypic association of ezrin and moesin, homotypic binding of ezrin to ezrin and of moesin to moesin was also demonstrated in vitro. These results suggest mechanisms by which ezrin and moesin might participate in dynamic aspects of cortical cytoskeletal structure. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8248180

  17. MALDI Tissue Profiling of Integral Membrane Proteins from Ocular Tissues

    PubMed Central

    Thibault, Danielle B.; Gillam, Christopher J.; Grey, Angus C.; Han, Jun; Schey, Kevin L.

    2008-01-01

    MALDI tissue profiling and imaging have become valuable tools for rapid, direct analysis of tissues to investigate spatial distributions of proteins, potentially leading to an enhanced understanding of the molecular basis of disease. Sample preparation methods developed to date for these techniques produce protein expression profiles from predominantly hydrophilic, soluble proteins. The ability to obtain information about the spatial distribution of integral membrane proteins is critical to more fully understand their role in physiological processes, including transport, adhesion, and signaling. In this communication, a sample preparation method for direct tissue profiling of integral membrane proteins is presented. Spatially resolved profiles for the abundant lens membrane proteins aquaporin 0 (AQP0) and MP20, and the retinal membrane protein opsin, were obtained using this method. MALDI tissue profiling results were validated by analysis of dissected tissue prepared by traditional membrane protein processing methods. Furthermore, direct tissue profiling of lens membrane proteins revealed aged related post-translational modifications, as well as a novel modification that had not been detected using conventional tissue homogenization methods. PMID:18396059

  18. Dynamic Nuclear Polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces

    PubMed Central

    Wylie, Benjamin J; Dzikovski, Boris G.; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H.; McDermott, Ann E.

    2016-01-01

    We demonstrate that dynamic nuclear polarization (DNP) of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of 6-fold for the dimeric protein. The enhancement affect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces. PMID:25828256

  19. The amino-terminal structure of human fragile X mental retardation protein obtained using precipitant-immobilized imprinted polymers

    NASA Astrophysics Data System (ADS)

    Hu, Yufeng; Chen, Zhenhang; Fu, Yanjun; He, Qingzhong; Jiang, Lun; Zheng, Jiangge; Gao, Yina; Mei, Pinchao; Chen, Zhongzhou; Ren, Xueqin

    2015-03-01

    Flexibility is an intrinsic property of proteins and essential for their biological functions. However, because of structural flexibility, obtaining high-quality crystals of proteins with heterogeneous conformations remain challenging. Here, we show a novel approach to immobilize traditional precipitants onto molecularly imprinted polymers (MIPs) to facilitate protein crystallization, especially for flexible proteins. By applying this method, high-quality crystals of the flexible N-terminus of human fragile X mental retardation protein are obtained, whose absence causes the most common inherited mental retardation. A novel KH domain and an intermolecular disulfide bond are discovered, and several types of dimers are found in solution, thus providing insights into the function of this protein. Furthermore, the precipitant-immobilized MIPs (piMIPs) successfully facilitate flexible protein crystal formation for five model proteins with increased diffraction resolution. This highlights the potential of piMIPs for the crystallization of flexible proteins.

  20. Thermodynamics of protein driven self assembly in membranes

    NASA Astrophysics Data System (ADS)

    Natesan, Ramakrishnan; Tourdot, Richard; Bradley, Ryan; Radhakrishnan, Ravi

    2013-03-01

    Recent experimental evidences strongly point to the role of proteins and other membrane binding macromolecules in reshaping biological membranes, at length scales of the molecule and the structure enclosed by the membrane. In this work, we investigate the interplay between the membrane curvature induced at the molecular scale, mainly due to peripheral membrane proteins, and the resulting membrane morphologies, of varying complexity, observed at the mesoscale. The biological membrane, in our approach, is represented by a dynamically triangulated surface while the proteins are modeled as curvature fields on the membrane, which can either be isotropic or anisotropic. Thermal undulations in the membrane and cooperativity in the curvature field, due to the stabilization of a nematic phase, drives the membrane into conformations that resembles those in experiments in vivo and vitro. The stability of these structures are examined by two approaches to compute the free energy of the system: (i) Widom insertion technique to compute excess chemical potentials and (ii) thermodynamic integration using the Kirkwood coupling parameter to compute absolute free energies. Building on these methods, we propose a hybrid scheeme that couples both the approaches for computing free energies.

  1. Membrane Protein Production in the Yeast, S. cerevisiae.

    PubMed

    Cartwright, Stephanie P; Mikaliunaite, Lina; Bill, Roslyn M

    2016-01-01

    The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca(2+)-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes. PMID:27485327

  2. Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli.

    PubMed

    Velkov, Tony; Jones, Alun; Lim, Maria L R

    2008-01-01

    A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni(2+)-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor. PMID:18800304

  3. Immobilization of multivalent glycoprobes on gold surfaces for sensing proteins and macrophages.

    PubMed

    Gade, Madhuri; Khandelwal, Puneet; Sangabathuni, Sivakoti; Bavireddi, Harikrishna; Murthy, Raghavendra Vasudeva; Poddar, Pankaj; Kikkeri, Raghavendra

    2016-04-01

    The multivalent display of carbohydrates on the cell surface provides cooperative binding to improve the specific biological events. In addition to multivalency, the spatial arrangement and orientation of sugars with respect to external stimuli also trigger carbohydrate-protein interactions. Herein, we report a non-covalent host-guest strategy to immobilize heptavalent glyco-β-cyclodextrin on gold-coated glass slides to study multivalent carbohydrate-protein interactions. We have found that the localization of sugar entities on surfaces using β-cyclodextrin (β-CD) chemistry increased the avidity of carbohydrate-protein and carbohydrate-macrophage interactions compared to monovalent-β-CD sugar coated surfaces. This platform is expected to be a promising tool to amplify the avidity of sugar-mediated interactions on surfaces and contribute to the development of next generation bio-medical products. PMID:26934683

  4. Membrane proteins of dense lysosomes from Chinese hamster ovary cells

    SciTech Connect

    Chance, S.C.

    1987-01-01

    In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

  5. The electrical interplay between proteins and lipids in membranes.

    PubMed

    Richens, Joanna L; Lane, Jordan S; Bramble, Jonathan P; O'Shea, Paul

    2015-09-01

    All molecular interactions that are relevant to cellular and molecular structures are electrical in nature but manifest in a rich variety of forms that each has its own range and influences on the net effect of how molecular species interact. This article outlines how electrical interactions between the protein and lipid membrane components underlie many of the activities of membrane function. Particular emphasis is placed on spatially localised behaviour in membranes involving modulation of protein activity and microdomain structure. The interactions between membrane lipids and membrane proteins together with their role within cell biology represent an enormous body of work. Broad conclusions are not easy given the complexities of the various systems and even consensus with model membrane systems containing two or three lipid types is difficult. By defining two types of broad lipid-protein interaction, respectively Type I as specific and Type II as more non-specific and focussing on the electrical interactions mostly in the extra-membrane regions it is possible to assemble broad rules or a consensus of the dominant features of the interplay between these two fundamentally important classes of membrane component. This article is part of a special issue entitled: Lipid-protein interactions. PMID:25817548

  6. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    PubMed

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane. PMID:26621472

  7. Laccase immobilization on bacterial nanocellulose membranes: Antimicrobial, kinetic and stability properties.

    PubMed

    Sampaio, Liliana M P; Padrão, Jorge; Faria, Jorge; Silva, João P; Silva, Carla J; Dourado, Fernando; Zille, Andrea

    2016-07-10

    This work studied the physical immobilization of a commercial laccase on bacterial nanocellulose (BNC) aiming to identify the laccase antibacterial properties suitable for wound dressings. Physico-chemical analysis demonstrates that the BNC structure is manly formed by pure crystalline Iα cellulose. The pH optimum and activation energy of free laccase depends on the substrate employed corresponding to pH 6, 7, 3 and 57, 22, 48kJmol(-1) for 2,6-dimethylphenol (DMP), catechol and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), respectively. The Michaelis-Menten constant (Km) value for the immobilized laccase (0.77mM) was found to be almost double of that of the free enzyme (0.42mM). However, the specific activities of immobilized and free laccase are similar suggesting that the cage-like structure of BNC allows entrapped laccase to maintain some flexibility and favour substrate accessibility. The results clearly show the antimicrobial effect of laccase in Gram-positive (92%) and Gram-negative (26%) bacteria and cytotoxicity acceptable for wound dressing applications. PMID:27106145

  8. Structures and Mechanisms of Viral Membrane Fusion Proteins

    PubMed Central

    White, Judith M.; Delos, Sue E.; Brecher, Matthew; Schornberg, Kathryn

    2009-01-01

    Recent work has identified three distinct classes of viral membrane fusion proteins based on structural criteria. In addition, there are at least four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. Viral fusion proteins also contain different types of fusion peptides and vary in their reliance on accessory proteins. These differing features combine to yield a rich diversity of fusion proteins. Yet despite this staggering diversity, all characterized viral fusion proteins convert from a fusion-competent state (dimers or trimers, depending on the class) to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide, attached to the target membrane, and the transmembrane domain, attached to the viral membrane, into close proximity thereby facilitating the union of viral and target membranes. During these conformational conversions, the fusion proteins induce membranes to progress through stages of close apposition, hemifusion, and then the formation of small, and finally large, fusion pores. Clearly, highly divergent proteins have converged on the same overall strategy to mediate fusion, an essential step in the life cycle of every enveloped virus. PMID:18568847

  9. Membranes Do Not Tell Proteins How To Fold.

    PubMed

    Popot, Jean-Luc; Engelman, Donald M

    2016-01-12

    Which properties of the membrane environment are essential for the folding and oligomerization of transmembrane proteins? Because the lipids that surround membrane proteins in situ spontaneously organize into bilayers, it may seem intuitive that interactions with the bilayer provide both hydrophobic and topological constraints that help the protein to achieve a stable and functional three-dimensional structure. However, one may wonder whether folding is actually driven by the membrane environment or whether the folded state just reflects an adaptation of integral proteins to the medium in which they function. Also, apart from the overall transmembrane orientation, might the asymmetry inherent in biosynthesis processes cause proteins to fold to out-of-equilibrium, metastable topologies? Which of the features of a bilayer are essential for membrane protein folding, and which are not? To which extent do translocons dictate transmembrane topologies? Recent data show that many membrane proteins fold and oligomerize very efficiently in media that bear little similarity to a membrane, casting doubt on the essentiality of many bilayer constraints. In the following discussion, we argue that some of the features of bilayers may contribute to protein folding, stability and regulation, but they are not required for the basic three-dimensional structure to be achieved. This idea, if correct, would imply that evolution has steered membrane proteins toward an accommodation to biosynthetic pathways and a good fit into their environment, but that their folding is not driven by the latter or dictated by insertion apparatuses. In other words, the three-dimensional structure of membrane proteins is essentially determined by intramolecular interactions and not by bilayer constraints and insertion pathways. Implications are discussed. PMID:26649989

  10. TMBETA-NET: discrimination and prediction of membrane spanning beta-strands in outer membrane proteins.

    PubMed

    Gromiha, M Michael; Ahmad, Shandar; Suwa, Makiko

    2005-07-01

    We have developed a web-server, TMBETA-NET for discriminating outer membrane proteins and predicting their membrane spanning beta-strand segments. The amino acid compositions of globular and outer membrane proteins have been systematically analyzed and a statistical method has been proposed for discriminating outer membrane proteins. The prediction of membrane spanning segments is mainly based on feed forward neural network and refined with beta-strand length. Our program takes the amino acid sequence as input and displays the type of the protein along with membrane-spanning beta-strand segments as a stretch of highlighted amino acid residues. Further, the probability of residues to be in transmembrane beta-strand has been provided with a coloring scheme. We observed that outer membrane proteins were discriminated with an accuracy of 89% and their membrane spanning beta-strand segments at an accuracy of 73% just from amino acid sequence information. The prediction server is available at http://psfs.cbrc.jp/tmbeta-net/. PMID:15980447

  11. Protein-driven membrane stresses in fusion and fission

    PubMed Central

    Kozlov, Michael M.; McMahon, Harvey T.; Chernomordik, Leonid V.

    2013-01-01

    Cellular membranes undergo continuous remodeling. Exocytosis and endocytosis, mitochondrial fusion and fission, entry of enveloped viruses into host cellsand release of the newly assembled virions, cell-to-cell fusion and cell division, and budding and fusion of transport carriers all proceed via topologically similar, but oppositely ordered, membrane rearrangements. The biophysical similarities and differences between membrane fusion and fission become more evident if we disregard the accompanying biological processes and consider only remodeling of the lipid bilayer. The forces that determine the bilayer propensity to undergo fusion or fission come from proteins and inmost cases from membrane-bound proteins. In this review, we consider the mechanistic principles underlying the fusion and fission reactions and discuss the current hypotheses on how specific proteins act in the two types of membrane remodeling. PMID:20638285

  12. Association of Influenza Virus Proteins with Membrane Rafts

    PubMed Central

    Veit, Michael; Thaa, Bastian

    2011-01-01

    Assembly and budding of influenza virus proceeds in the viral budozone, a domain in the plasma membrane with characteristics of cholesterol/sphingolipid-rich membrane rafts. The viral transmembrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) are intrinsically targeted to these domains, while M2 is seemingly targeted to the edge of the budozone. Virus assembly is orchestrated by the matrix protein M1, binding to all viral components and the membrane. Budding progresses by protein- and lipid-mediated membrane bending and particle scission probably mediated by M2. Here, we summarize the experimental evidence for this model with emphasis on the raft-targeting features of HA, NA, and M2 and review the functional importance of raft domains for viral protein transport, assembly and budding, environmental stability, and membrane fusion. PMID:22312341

  13. Membrane protein biosensing with plasmonic nanopore arrays and pore-spanning lipid membranes

    PubMed Central

    Im, Hyungsoon; Wittenberg, Nathan J.; Lesuffleur, Antoine; Lindquist, Nathan C.; Oh, Sang-Hyun

    2010-01-01

    Integration of solid-state biosensors and lipid bilayer membranes is important for membrane protein research and drug discovery. In these sensors, it is critical that the solid-state sensing material does not have adverse effects on the conformation or functionality of membrane-bound molecules. In this work, pore-spanning lipid membranes are formed over an array of periodic nanopores in free-standing gold films for surface plasmon resonance (SPR) kinetic binding assays. The ability to perform kinetic assays with a transmembrane protein is demonstrated with α-hemolysin (α-HL). The incorporation of α-HL into the membrane followed by specific antibody binding (anti-α-HL) red-shifts the plasmon resonance of the gold nanopore array, which is optically monitored in real time. Subsequent fluorescence imaging reveals that the antibodies primarily bind in nanopore regions, indicating that α-HL incorporation preferentially occurs into areas of pore-spanning lipid membranes. PMID:21218136

  14. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates

    PubMed Central

    Ju, Jung Won; Kim, Ho-Cheol; Shin, Hyun-Il; Kim, Yu Jung; Kim, Dong-Myung

    2015-01-01

    Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA. PMID:26599101

  15. Architecture and Function of Mechanosensitive Membrane Protein Lattices

    PubMed Central

    Kahraman, Osman; Koch, Peter D.; Klug, William S.; Haselwandter, Christoph A.

    2016-01-01

    Experiments have revealed that membrane proteins can form two-dimensional clusters with regular translational and orientational protein arrangements, which may allow cells to modulate protein function. However, the physical mechanisms yielding supramolecular organization and collective function of membrane proteins remain largely unknown. Here we show that bilayer-mediated elastic interactions between membrane proteins can yield regular and distinctive lattice architectures of protein clusters, and may provide a link between lattice architecture and lattice function. Using the mechanosensitive channel of large conductance (MscL) as a model system, we obtain relations between the shape of MscL and the supramolecular architecture of MscL lattices. We predict that the tetrameric and pentameric MscL symmetries observed in previous structural studies yield distinct lattice architectures of MscL clusters and that, in turn, these distinct MscL lattice architectures yield distinct lattice activation barriers. Our results suggest general physical mechanisms linking protein symmetry, the lattice architecture of membrane protein clusters, and the collective function of membrane protein lattices. PMID:26771082

  16. Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

    PubMed Central

    Choi, Ryan; Kelley, Angela; Leibly, David; Nakazawa Hewitt, Stephen; Napuli, Alberto; Van Voorhis, Wesley

    2011-01-01

    The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure. PMID:21904040

  17. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.

    PubMed

    Baker, Lindsay A; Daniëls, Mark; van der Cruijsen, Elwin A W; Folkers, Gert E; Baldus, Marc

    2015-06-01

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR. PMID:25956570

  18. Direct and reversible immobilization and microcontact printing of functional proteins on glass using a genetically appended silica-binding tag.

    PubMed

    Coyle, Brandon L; Baneyx, François

    2016-05-19

    Fusion of disulfide-constrained or linear versions of the Car9 dodecapeptide to model fluorescent proteins support their on-contact and oriented immobilization onto unmodified glass. Bound proteins can be released and the surface regenerated by incubation with l-lysine. This noncovalent chemistry enables rapid and reversibe microcontact printing of tagged proteins and speeds up the production of bicontinuous protein patterns. PMID:27157272

  19. Formation of oxygen from water, photosensitized by (bpy)/sub 2/Ru(bpy(C/sub 17/)/sub 2/)/sup 2+/, immobilized in a lipid bilayer membrane

    SciTech Connect

    Knerel'man, E.I.; Shafirovich, V.Ya.

    1988-04-01

    A system has been constructed, composed of (bpy)/sub 2/Ru(bpy(C/sub 17/)/sub 2/)/sup 2+/, as photosensitizer, immobilized in a lipid bilayer membrane, K/sub 2/S/sub 2/O/sub 8/ as electron acceptor, in the surrounding outer solution, and a cobalt catalyst attached to the membrane. Irradiation of this system with visible light leads to the formation of oxygen from water, in quantum yields ranging from 20-40%.

  20. Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading.

    PubMed

    Chen, Bo; Pernodet, Nadine; Rafailovich, Miriam H; Bakhtina, Asya; Gross, Richard A

    2008-12-01

    A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity. PMID:18991420

  1. Isolation and identification of Enterococcus faecalis membrane proteins using membrane shaving, 1D SDS/PAGE, and mass spectrometry.

    PubMed

    Cathro, Peter; McCarthy, Peter; Hoffmann, Peter; Zilm, Peter

    2016-06-01

    Enterococcus faecalis is a significant nosocomial pathogen, which is able to survive in diverse environments and resist killing with antimicrobial therapies. The expression of cell membrane proteins play an important role in how bacteria respond to environmental stress. As such, the capacity to identify and study membrane protein expression is critical to our understanding of how specific proteins influence bacterial survival. Here, we describe a combined approach to identify membrane proteins of E. faecalis ATCC V583 using membranes fractionated by either 1D SDS/PAGE or membrane shaving, coupled with LC-ESI mass spectrometry. We identified 222 membrane-associated proteins, which represent approximately 24% of the predicted membrane-associated proteome: 170 were isolated using 1D SDS/PAGE and 68 with membrane shaving, with 36 proteins being common to both the techniques. Of the proteins identified by membrane shaving, 97% were membrane-associated with the majority being integral membrane proteins (89%). Most of the proteins identified with known physiology are involved with transportation across the membrane. The combined 1D SDS/PAGE and membrane shaving approach has produced the greatest number of membrane proteins identified from E. faecalis to date. These protocols will aid future researchers investigating changes in the membrane proteome of E. faecalis by improving our understanding of how E. faecalis adapts and responds to its environment. PMID:27419061

  2. Mixing and Matching Detergents for Membrane Protein NMR Structure Determination

    SciTech Connect

    Columbus, Linda; Lipfert, Jan; Jambunathan, Kalyani; Fox, Daniel A.; Sim, Adelene Y.L.; Doniach, Sebastian; Lesley, Scott A.

    2009-10-21

    One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

  3. A highly selective optode for determination of Hg (II) by a modified immobilization of indigo carmine on a triacetylcellulose membrane

    NASA Astrophysics Data System (ADS)

    Tavallali, Hossein; Shaabanpur, Elham; Vahdati, Parvin

    2012-04-01

    A new mercury optical sensor was designed with indigo carmine (IC) as a dye indicator. The water-soluble indicator was lipophilized in the form of an ion-pair with N-cetyl pyridinium chloride (CPC) and dissolved in methanol (70 °C), then immobilized on a triacetylcellulose membrane. This optode exhibits a linear range of 24.0-468.0 μM of the Hg (II) ion concentration with detection limit of 7.2 μM at 669.5 nm. Response time was within 8-10 min, depending on the Hg (II) ion concentration. The sensor could readily be regenerated with a hydrochloric acid solution (0.01 M) in a reversible manner and its response was reproducible (RSD = 3.2%). The method was applied to the determination of mercury content of a variety of samples which gave satisfactory results.

  4. Growth of silicone-immobilized bacteria on polycarbonate membrane filters, a technique to study microcolony formation under anaerobic conditions.

    PubMed Central

    Højberg, O; Binnerup, S J; Sørensen, J

    1997-01-01

    A technique was developed to study microcolony formation by silicone-immobilized bacteria on polycarbonate membrane filters under anaerobic conditions. A sudden shift to anaerobiosis was obtained by submerging the filters in medium which was depleted for oxygen by a pure culture of bacteria. The technique was used to demonstrate that preinduction of nitrate reductase under low-oxygen conditions was necessary for nonfermenting, nitrate-respiring bacteria, e.g., Pseudomonas spp., to cope with a sudden lack of oxygen. In contrast, nitrate-respiring, fermenting bacteria, e.g., Bacillus and Escherichia spp., formed microcolonies under anaerobic conditions with or without the presence of nitrate and irrespective of aerobic or anaerobic preculture conditions. PMID:9212439

  5. A highly selective optode for determination of Hg (II) by a modified immobilization of indigo carmine on a triacetylcellulose membrane.

    PubMed

    Tavallali, Hossein; Shaabanpur, Elham; Vahdati, Parvin

    2012-04-01

    A new mercury optical sensor was designed with indigo carmine (IC) as a dye indicator. The water-soluble indicator was lipophilized in the form of an ion-pair with N-cetyl pyridinium chloride (CPC) and dissolved in methanol (70 °C), then immobilized on a triacetylcellulose membrane. This optode exhibits a linear range of 24.0-468.0 μM of the Hg (II) ion concentration with detection limit of 7.2 μM at 669.5 nm. Response time was within 8-10 min, depending on the Hg (II) ion concentration. The sensor could readily be regenerated with a hydrochloric acid solution (0.01 M) in a reversible manner and its response was reproducible (RSD=3.2%). The method was applied to the determination of mercury content of a variety of samples which gave satisfactory results. PMID:22277622

  6. Decoding signals for membrane protein assembly using alkaline phosphatase fusions.

    PubMed Central

    McGovern, K; Ehrmann, M; Beckwith, J

    1991-01-01

    We have used genetic methods to investigate the role of the different domains of a bacterial cytoplasmic membrane protein, MalF, in determining its topology. This was done by analyzing the effects of MalF topology of deleting various domains of the protein using MalF-alkaline phosphatase fusion proteins. Our results show that the cytoplasmic domains of the protein are the pre-eminent topogenic signals. These domains contain information that determines their cytoplasmic location and, thus, the orientation of the membrane spanning segments surrounding them. Periplasmic domains do not appear to have equivalent information specifying their location and membrane spanning segments do not contain information defining their orientation in the membrane. The strength of cytoplasmic domains as topogenic signals varies, correlated with the density of positively charged amino acids within them. Images PMID:1915262

  7. Organization and dynamics of SNARE proteins in the presynaptic membrane

    PubMed Central

    Milovanovic, Dragomir; Jahn, Reinhard

    2015-01-01

    Our view of the lateral organization of lipids and proteins in the plasma membrane has evolved substantially in the last few decades. It is widely accepted that many, if not all, plasma membrane proteins and lipids are organized in specific domains. These domains vary widely in size, composition, and stability, and they represent platforms governing diverse cell functions. The presynaptic plasma membrane is a well-studied example of a membrane which undergoes rearrangements, especially during exo- and endocytosis. Many proteins and lipids involved in presynaptic function are known, and major efforts have been made to understand their spatial organization and dynamics. Here, we focus on the mechanisms underlying the organization of SNAREs, the key proteins of the fusion machinery, in distinct domains, and we discuss the functional significance of these clusters. PMID:25852575

  8. Preparation and characterization of Pd/Fe bimetallic nanoparticles immobilized on Al2O3/PVDF membrane: Parameter optimization and dechlorination of dichloroacetic acid.

    PubMed

    Zhang, Lijuan; Meng, Zhaohong; Zang, Shuying

    2015-05-01

    Using a liquid-solid phase inversion method, a hybrid matrix poly(vinylidene fluoride) (PVDF) membrane was prepared with alumina (Al2O3) nanoparticle addition. Pd/Fe nanoparticles (NPs) were successfully immobilized on the Al2O3/PVDF membrane, which was characterized by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The micrographs showed that the Pd/Fe NPs were dispersed homogeneously. Several important experimental parameters were optimized, including the mechanical properties, contact angle and surface area of Al2O3/PVDF composite membranes with different Al2O3 contents. At the same time, the ferrous ion concentration and the effect of hydrophilization were studied. The results showed that the modified Al2O3/PVDF membrane functioned well as a support. The Al2O3/PVDF membrane with immobilized Pd/Fe NPs exhibited high efficiency in terms of dichloroacetic acid (DCAA) dechlorination. Additionally, a reaction pathway for DCAA dechlorination by Pd/Fe NPs immobilized on the Al2O3/PVDF membrane system was proposed. PMID:25968273

  9. Immobilization of tris(2 pyridyl) methylamine in a PVC-Membrane Sensor and Characterization of the Membrane Properties

    PubMed Central

    2012-01-01

    Background Due to the increasing industrial use of titanium compounds, its determination is the subject of considerable efforts. The ionophore or membrane active recognition is the most important component of any polymeric membrane sensor. The sensor’s response depends on the ionophore and bonding between the ionophore and the target ion. Ionophores with molecule-sized dimensions containing cavities or semi-cavities can surround the target ion. The bond between the ionophore and target ion gives different selectivity and sensitivity toward the other ions. Therefore, ionophores with different binding strengths can be used in the sensor. Results In the present work, poly (vinyl chloride) (PVC) based membrane incorporating tris (2 pyridyl) methylamine (tpm) as an ionophore has been prepared and explored as a titanium(III) selective sensor. Conclusions The strengths of the ion–ionophore (Ti(OH)2+-tpm) interactions and the role of ionophore on membrane were tested by various techniques such as elemental analysis, UV–vis, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and powder X-ray diffraction (XRD). All data approved the successful incorporation of organic group via covalent bond. PMID:22564322

  10. Membrane attack complex (MAC)-mediated damage to spermatozoa: protection of the cells by the presence on their membranes of MAC inhibitory proteins.

    PubMed Central

    Rooney, I A; Davies, A; Morgan, B P

    1992-01-01

    Although antibody and complement are known to cause immobilization and killing of spermatozoa in vitro the components of the complement system mediating these effects remain undefined. Here we have examined the effects of the membrane attack complex (MAC) on spermatozoa and demonstrate that spermatotoxic effects are dependent on assembly of the complete MAC. We subsequently examined the presence and functional significance of the complement regulatory proteins decay accelerating factor (DAF), MAC-inhibiting protein (MIP) and CD59 antigen on spermatozoa. Both DAF and CD59 antigen were present on the membranes of these cells. Neutralization of CD59 antigen with specific antibodies increased the susceptibility of the cells to MAC-mediated damage, suggesting a role for this molecule in the protection of spermatozoa from complement-mediated damage in the female reproductive tract. Images Figure 2 Figure 4 PMID:1374057

  11. Transport proteins of the plant plasma membrane

    NASA Technical Reports Server (NTRS)

    Assmann, S. M.; Haubrick, L. L.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

  12. Brain phosphoproteome obtained by a FASP-based method reveals plasma membrane protein topology.

    PubMed

    Wiśniewski, Jacek R; Nagaraj, Nagarjuna; Zougman, Alexandre; Gnad, Florian; Mann, Matthias

    2010-06-01

    Taking advantage of the recently developed Filter Assisted Sample Preparation (FASP) method for sample preparation, we performed an in-depth analysis of phosphorylation sites in mouse brain. To maximize the number of detected phosphorylation sites, we fractionated proteins by size exclusion chromatography (SEC) or separated tryptic peptides on an anion exchanger (SAX) prior or after the TiO(2)-based phosphopeptide enrichment, respectively. SEC allowed analysis of minute tissue samples (1 mg total protein), and resulted in identification of more than 4000 sites in a single experiment, comprising eight fractions. SAX in a pipet tip format offered a convenient and rapid way to fractionate phosphopeptides and mapped more than 5000 sites in a single six fraction experiment. To enrich peptides containing phosphotyrosine residues, we describe a filter aided antibody capturing and elution (FACE) method that requires only the uncoupled instead of resin-immobilized capture reagent. In total, we identified 12,035 phosphorylation sites on 4579 brain proteins of which 8446 are novel. Gene Ontology annotation reveals that 23% of identified sites are located on plasma membrane proteins, including a large number of ion channels and transporters. Together with the glycosylation sites from a recent large-scale study, they can confirm or correct predicted membrane topologies of these proteins, as we show for the examples calcium channels and glutamate receptors. PMID:20415495

  13. Immobilized metal ion affinity partitioning, a method combining metal-protein interaction and partitioning of proteins in aqueous two-phase systems.

    PubMed

    Birkenmeier, G; Vijayalakshmi, M A; Stigbrand, T; Kopperschläger, G

    1991-02-22

    Immobilized metal ions were used for the affinity extraction of proteins in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran or PEG and salt. Soluble chelating polymers were prepared by covalent attachment of metal-chelating groups to PEG. The effect on the partitioning of proteins of such chelating PEG derivatives coordinated with different metal ions is demonstrated. The proteins studied were alpha 2-macroglobulin, tissue plasminogen activator, superoxide dismutase and monoclonal antibodies. The results indicate that immobilized metal ion affinity partitioning provides excellent potential for the extraction of proteins. PMID:1710621

  14. NMR Structures of Membrane Proteins in Phospholipid Bilayers

    PubMed Central

    Radoicic, Jasmina; Lu, George J.; Opella, Stanley J.

    2014-01-01

    Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins; especially those accomplished with the proteins in phospholipid bilayer environments where they function. PMID:25032938

  15. Comprehensive Proteomic Analysis of Membrane Proteins in Toxoplasma gondii*

    PubMed Central

    Che, Fa-Yun; Madrid-Aliste, Carlos; Burd, Berta; Zhang, Hongshan; Nieves, Edward; Kim, Kami; Fiser, Andras; Angeletti, Ruth Hogue; Weiss, Louis M.

    2011-01-01

    Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is an important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: one-dimensional gel liquid chromatography-tandem MS analysis (one-dimensional gel electrophoresis LC-MS/MS), biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer “sandwich” gel electrophoresis with multidimensional protein identification technology. A total of 2241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T. gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially suitable targets for the development of therapeutic strategies. PMID:20935347

  16. Specific antibody immobilization with biotin-poly(L-lysine)-g-poly(ethylene glycol) and protein A on microfluidic chips.

    PubMed

    Wen, Xiufang; He, Hongyan; Lee, L James

    2009-10-31

    Highly efficient antibody immobilization is crucial for conducting high-performance immunoassays such as enzyme-linked immunosorbent assay (ELISA) in microarray and microfluidic biochips. In this study, a biotin-poly(L-lysine)-g-poly(ethylene glycol) (biotin-PLL-g-PEG) and protein A-based technique was developed to immobilize antibody on the surface of poly(methyl methacrylate) (PMMA) microchannels. First, PMMA surface was activated by oxygen plasma, followed by poly(acrylic acid) (PAA) grafting to add functional carboxyl group for subsequent binding. After the biotin-PLL-g-PEG molecules reacted with carboxyl groups through the electrostatic interactions, biotinylated protein A was immobilized on the surface through a linking molecule, neutravidin. To evaluate the applicability of this novel immobilization strategy, human interferon-gamma (IFN-gamma) was used as a model protein. Since protein A could better control the immobilization orientation, and the combination of biotin-PLL-g-PEG and PLL-g-PEG could adjust the conformation of antibodies, antigen capture efficiency and detection signals were significantly improved on the microchips by using this strategy. The optimal grafting conditions were also experimentally determined: the biotin grafting ratio of 0.189 in the PLL-g-PEG molecule and the mixture ratio of 85% (biotin-PLL-g-PEG to PLL-g-PEG). This surface modification can be applied for targeted drug delivery, biosensor and other immunoassay applications. PMID:19647744

  17. Polyether sulfone/hydroxyapatite mixed matrix membranes for protein purification

    NASA Astrophysics Data System (ADS)

    Sun, Junfen; Wu, Lishun

    2014-07-01

    This work proposes a novel approach for protein purification from solution using mixed matrix membranes (MMMs) comprising of hydroxyapatite (HAP) inside polyether sulfone (PES) matrix. The influence of HAP particle loading on membrane morphology is studied. The MMMs are further characterized concerning permeability and adsorption capacity. The MMMs show purification of protein via both diffusion as well as adsorption, and show the potential of using MMMs for improvements in protein purification techniques. The bovine serum albumin (BSA) was used as a model protein. The properties and structures of MMMs prepared by immersion phase separation process were characterized by pure water flux, BSA adsorption and scanning electron microscopy (SEM).

  18. Spectroscopic study of 3-Hydroxyflavone - protein interaction in lipidic bi-layers immobilized on silver nanoparticles.

    PubMed

    Voicescu, Mariana; Ionescu, Sorana; Nistor, Cristina L

    2017-01-01

    The interaction of 3-Hydroxyflavone with serum proteins (BSA and HSA) in lecithin lipidic bi-layers (PC) immobilized on silver nanoparticles (SNPs), was studied by fluorescence and Raman spectroscopy. BSA secondary structure was quantified with a deconvolution algorithm, showing a decrease in α-helix structure when lipids were added to the solution. The effect of temperature on the rate of the excited-state intra-molecular proton transfer and on the dual fluorescence emission of 3-HF in the HSA/PC/SNPs systems was discussed. Evaluation of the antioxidant activity of 3-HF in HSA/PC/SNPs systems was also studied. The antioxidant activity of 3-HF decreased in the presence of SNPs. The results are discussed with relevance to the secondary structure of proteins and of the 3-HF based nano-systems to a topical formulation useful in the oxidative stress process. PMID:27380623

  19. Immobilized WNT Proteins Act as a Stem Cell Niche for Tissue Engineering.

    PubMed

    Lowndes, Molly; Rotherham, Michael; Price, Joshua C; El Haj, Alicia J; Habib, Shukry J

    2016-07-12

    The timing, location, and level of WNT signaling are highly regulated during embryonic development and for the maintenance of adult tissues. Consequently the ability to provide a defined and directed source of WNT proteins is crucial to fully understand its role in tissue development and to mimic its activity in vitro. Here we describe a one-step immobilization technique to covalently bind WNT3A proteins as a basal surface with easy storage and long-lasting activity. We show that this platform is able to maintain adult and embryonic stem cells while also being adaptable for 3D systems. Therefore, this platform could be used for recapitulating specific stem cell niches with the goal of improving tissue engineering. PMID:27411105

  20. Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

    PubMed Central

    2015-01-01

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

  1. Exceptional overproduction of a functional human membrane protein.

    PubMed

    Nyblom, Maria; Oberg, Fredrik; Lindkvist-Petersson, Karin; Hallgren, Karin; Findlay, Heather; Wikström, Jennie; Karlsson, Anders; Hansson, Orjan; Booth, Paula J; Bill, Roslyn M; Neutze, Richard; Hedfalk, Kristina

    2007-11-01

    Eukaryotic--especially human--membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization. PMID:17869538

  2. Phytochemicals perturb membranes and promiscuously alter protein function.

    PubMed

    Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

    2014-08-15

    A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

  3. In Situ Quantification of Protein Binding to the Plasma Membrane

    PubMed Central

    Smith, Elizabeth M.; Hennen, Jared; Chen, Yan; Mueller, Joachim D.

    2015-01-01

    This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus. PMID:26039166

  4. Clipping or Extracting: Two Ways to Membrane Protein Degradation.

    PubMed

    Avci, Dönem; Lemberg, Marius K

    2015-10-01

    Protein degradation is a fundamentally important process that allows cells to recognize and remove damaged protein species and to regulate protein abundance according to functional need. A fundamental challenge is to understand how membrane proteins are recognized and removed from cellular organelles. While most of our understanding of this mechanism comes from studies on p97/Cdc48-mediated protein dislocation along the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, recent studies have revealed intramembrane proteolysis to be an additional mechanism that can extract transmembrane segments. Here, we review these two principles in membrane protein degradation and discuss how intramembrane proteolysis, which introduces an irreversible step in protein dislocation, is used to drive regulated protein turnover. PMID:26410407

  5. Electrospun regenerated cellulose nanofibrous membranes surface-grafted with polymer chains/brushes via the atom transfer radical polymerization method for catalase immobilization.

    PubMed

    Feng, Quan; Hou, Dayin; Zhao, Yong; Xu, Tao; Menkhaus, Todd J; Fong, Hao

    2014-12-10

    In this study, an electrospun regenerated cellulose (RC) nanofibrous membrane with fiber diameters of ∼200-400 nm was prepared first; subsequently, 2-hydroxyethyl methacrylate (HEMA), 2-dimethylaminoethyl methacrylate (DMAEMA), and acrylic acid (AA) were selected as the monomers for surface grafting of polymer chains/brushes via the atom transfer radical polymerization (ATRP) method. Thereafter, four nanofibrous membranes (i.e., RC, RC-poly(HEMA), RC-poly(DMAEMA), and RC-poly(AA)) were explored as innovative supports for immobilization of an enzyme of bovine liver catalase (CAT). The amount/capacity, activity, stability, and reusability of immobilized catalase were evaluated, and the kinetic parameters (Vmax and Km) for immobilized and free catalase were determined. The results indicated that the respective amounts/capacities of immobilized catalase on RC-poly(HEMA) and RC-poly(DMAEMA) nanofibrous membranes reached 78 ± 3.5 and 67 ± 2.7 mg g(-1), which were considerably higher than the previously reported values. Meanwhile, compared to that of free CAT (i.e., 18 days), the half-life periods of RC-CAT, RC-poly(HEMA)-CAT, RC-poly(DMAEMA)-CAT, and RC-poly(AA)-CAT were 49, 58, 56, and 60 days, respectively, indicating that the storage stability of immobilized catalase was also significantly improved. Furthermore, the immobilized catalase exhibited substantially higher resistance to temperature variation (tested from 5 to 70 °C) and lower degree of sensitivity to pH value (tested from 4.0 and 10.0) than the free catalase. In particular, according to the kinetic parameters of Vmax and Km, the nanofibrous membranes of RC-poly(HEMA) (i.e., 5102 μmol mg(-1) min(-1) and 44.89 mM) and RC-poly(DMAEMA) (i.e., 4651 μmol mg(-1) min(-1) and 46.98 mM) had the most satisfactory biocompatibility with immobilized catalase. It was therefore concluded that the electrospun RC nanofibrous membranes surface-grafted with 3-dimensional nanolayers of polymer chains/brushes would be

  6. Active membrane transport and receptor proteins from bacteria.

    PubMed

    Saidijam, M; Bettaney, K E; Szakonyi, G; Psakis, G; Shibayama, K; Suzuki, S; Clough, J L; Blessie, V; Abu-Bakr, A; Baumberg, S; Meuller, J; Hoyle, C K; Palmer, S L; Butaye, P; Walravens, K; Patching, S G; O'reilly, J; Rutherford, N G; Bill, R M; Roper, D I; Phillips-Jones, M K; Henderson, P J F

    2005-08-01

    A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides. PMID:16042616

  7. Domain formation in membranes caused by lipid wetting of protein.

    PubMed

    Akimov, Sergey A; Frolov, Vladimir A J; Kuzmin, Peter I; Zimmerberg, Joshua; Chizmadzhev, Yuri A; Cohen, Fredric S

    2008-05-01

    Formation of rafts and other domains in cell membranes is considered as wetting of proteins by lipids. The membrane is modeled as a continuous elastic medium. Thermodynamic functions of the lipid films that wet proteins are calculated using a mean-field theory of liquid crystals as adapted to biomembranes. This approach yields the conditions necessary for a macroscopic wetting film to form; its thickness could also be determined. It is shown that films of macroscopic thicknesses form around large (tens nanometers in diameter) lipid-protein aggregates; only thin adsorption films form around single proteins or small complexes. The means by which wetting films can facilitate the merger of these aggregates is considered. It is shown that a wetting film prevents a protein from leaving an aggregate. Using experimentally derived values of elastic moduli and spontaneous curvatures as well as height mismatch between aggregates and bulk membrane, we obtained numerical results, which can be compared with the experimental data. PMID:18643096

  8. Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

    2010-03-01

    The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

  9. The functions of tryptophan residues in membrane proteins

    SciTech Connect

    Schiffer, M.; Chang, C.H.; Stevens, F.J.

    1994-08-01

    Membrane proteins in general have a significantly higher Trp content than do soluble proteins. This is especially true for the M and L subunits of the photosynthetic reaction center from purple bacteria. The Trp residues are located mostly in the segments that connect the transmembrane helices. Further, they are concentrated at the periplasmic side of the complex. Within the protein subunits, many form hydrogen bonds with carbonyl oxygens of the main chain, thereby stabilizing the protein. On the surface of the molecule, they are correctly positioned to form hydrogen bonds with the lipid head groups while their hydrophobic rings are immersed in the lipid part of the bilayer. We suggest that Trp residues are involved in the translocation of protein through the membrane and that following translocation, Trp residues serve as anchors on the periplasmic side of the membrane.

  10. Mechanisms of integral membrane protein insertion and folding

    PubMed Central

    2014-01-01

    The biogenesis, folding, and structure of α-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, and the transport of solutes and signals across membranes. Nearly all MPs require translocons—often referred to as protein-conducting channels—for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of membrane protein insertion and the folding of polytopic membrane proteins in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure, it is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion. PMID:25277655

  11. Predictive energy landscapes for folding membrane protein assemblies

    NASA Astrophysics Data System (ADS)

    Truong, Ha H.; Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.

    2015-12-01

    We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na+-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

  12. Membrane protein properties revealed through data-rich electrostatics calculations

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.; Grabe, Michael

    2015-01-01

    SUMMARY The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem including: full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane induced pKa shifts, calculation of non-polar energies, and command-line scripting for large scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane potentially revealing interesting functional information. PMID:26118532

  13. Expression of two membrane fusion proteins, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, in choroid plexus epithelium.

    PubMed

    Chung, I; Burkart, A; Szmydynger-Chodobska, J; Dodd, K A; Trimble, W S; Miller, K V; Shim, M; Chodobski, A

    2003-01-01

    In addition to being the major site of cerebrospinal fluid formation, the choroid plexus epithelium emerges as an important source of polypeptides in the brain. Physiologically regulated release of some polypeptides synthesized by the choroid plexus has been shown. The molecular mechanisms underlying this polypeptide secretion have not been characterized, however. In the present study, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, two membrane fusion proteins playing a critical role in exocytosis in neurons and endocrine cells, were found to be expressed in the choroid plexus epithelium. It was also shown that in choroidal epithelium, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein stably interact. Two members of the vesicle-associated membrane protein family, vesicle-associated membrane protein-1 and vesicle-associated membrane protein-2, were expressed in the rat choroid plexus at the messenger RNA and protein level. However, their newly discovered isoforms, vesicle-associated membrane protein-1b and vesicle-associated membrane protein-2b, produced by alternative RNA splicing, were not detected in choroidal tissue. Immunohistochemistry demonstrated that vesicle-associated membrane protein is confined to the cytoplasm of choroidal epithelium, whereas synaptosome-associated protein of 25 kDa is associated with plasma membranes, albeit with a varied cellular distribution among species studied. Specifically, in the rat choroid plexus, synaptosome-associated protein of 25 kDa was localized to the basolateral membrane domain of choroidal epithelium and was expressed in small groups of cells. In comparison, in ovine and human choroidal tissues, apical staining for synaptosome-associated protein of 25 kDa was found in the majority of epithelial cells. These species-related differences in cellular synaptosome-associated protein of 25 kDa distribution suggested that the synaptosome-associated protein of

  14. The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice

    PubMed Central

    You, Jae-Sung; Anderson, Garrett B.; Dooley, Matthew S.; Hornberger, Troy A.

    2015-01-01

    ABSTRACT The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate

  15. The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice.

    PubMed

    You, Jae-Sung; Anderson, Garrett B; Dooley, Matthew S; Hornberger, Troy A

    2015-09-01

    The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate that the

  16. Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

    PubMed Central

    Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M.; Climent, Víctor; Sanz, Jesús M.

    2014-01-01

    We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. PMID:24498237

  17. Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.

    PubMed

    Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M; Climent, Víctor; Sanz, Jesús M

    2014-01-01

    We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. PMID:24498237

  18. Setting up and running molecular dynamics simulations of membrane proteins.

    PubMed

    Kandt, Christian; Ash, Walter L; Tieleman, D Peter

    2007-04-01

    Molecular dynamics simulations have become a popular and powerful technique to study lipids and membrane proteins. We present some general questions and issues that should be considered prior to embarking on molecular dynamics simulation studies of membrane proteins and review common simulation methods. We suggest a practical approach to setting up and running simulations of membrane proteins, and introduce two new (related) methods to embed a protein in a lipid bilayer. Both methods rely on placing lipids and the protein(s) on a widely spaced grid and then 'shrinking' the grid until the bilayer with the protein has the desired density, with lipids neatly packed around the protein. When starting from a grid based on a single lipid structure, or several potentially different lipid structures (method 1), the bilayer will start well-packed but requires more equilibration. When starting from a pre-equilibrated bilayer, either pure or mixed, most of the structure of the bilayer stays intact, reducing equilibration time (method 2). The main advantages of these methods are that they minimize equilibration time and can be almost completely automated, nearly eliminating one time consuming step in MD simulations of membrane proteins. PMID:17367719

  19. Disrupting Microtubules Network Immobilizes Amoeboid Chemotactic Receptor in the Plasma Membrane

    PubMed Central

    de Keijzer, S.; Galloway, J.; Harms, G.S.; Devreotes, P.N.; Iglesias, P.A.

    2011-01-01

    Signaling cascades are initiated in the plasma membrane via activation of one molecule by another. The interaction depends on the mutual availability of the molecules to each other and this is determined by their localization and lateral diffusion in the cell membrane. The cytoskeleton plays a very important role in this process by enhancing or restricting the possibility of the signaling partners to meet in the plasma membrane. In this study we explored the mode of diffusion of the cAMP receptor, cAR1, in the plasma membrane of Dictyostelium discoideum cells and how this is regulated by the cytoskeleton. Single-particle tracking of fluorescently labeled cAR1 using total internal reflection microscopy showed that 70% of the cAR1 molecules were mobile. These receptors showed directed motion and we demonstrate that this is not because of tracking along the actin cytoskeleton. Instead, destabilization of the microtubules abolished cAR1 mobility in the plasma membrane and this was confirmed by fluorescence recovery after photobleaching. As a result of microtubule stabilization, one of the first downstream signaling events, the jump of the PH domain of CRAC, was decreased. These results suggest a role for microtubules in cAR1 dynamics and in the ability of cAR1 molecules to interact with their signaling partners. PMID:21334306

  20. Genomic analysis of membrane protein families: abundance and conserved motifs

    PubMed Central

    Liu, Yang; Engelman, Donald M; Gerstein, Mark

    2002-01-01

    Background Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities. Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well-characterized membrane protein families in 26 recently sequenced genomes. To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families. Results Analysis of the occurrence and composition of these families revealed several interesting trends. The number of assigned membrane protein domains has an approximately linear relationship to the total number of open reading frames (ORFs) in 26 genomes studied. Caenorhabditis elegans is an apparent outlier, because of its high representation of seven-span transmembrane (7-TM) chemoreceptor families. In all genomes, including that of C. elegans, the number of distinct membrane protein families has a logarithmic relation to the number of ORFs. Glycine, proline, and tyrosine locations tend to be conserved in transmembrane regions within families, whereas isoleucine, valine, and methionine locations are relatively mutable. Analysis of motifs in putative transmembrane helices reveals that GxxxG and GxxxxxxG (which can be written GG4 and GG7, respectively; see Materials and methods) are among the most prevalent. This was noted in earlier studies; we now find these motifs are particularly well conserved in families, however, especially those corresponding to transporters, symporters, and channels. Conclusions We carried out a genome-wide analysis on patterns of the classified polytopic membrane protein families and analyzed the distribution of conserved amino acids and motifs in the transmembrane helix regions in these families. PMID:12372142

  1. Ubiquilins Chaperone and Triage Mitochondrial Membrane Proteins for Degradation.

    PubMed

    Itakura, Eisuke; Zavodszky, Eszter; Shao, Sichen; Wohlever, Matthew L; Keenan, Robert J; Hegde, Ramanujan S

    2016-07-01

    We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions. PMID:27345149

  2. Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins

    PubMed Central

    Sanz-Hernández, Máximo; Vostrikov, Vitaly V.; Veglia, Gianluigi; De Simone, Alfonso

    2016-01-01

    The structural dynamics governing collective motions in oligomeric membrane proteins play key roles in vital biomolecular processes at cellular membranes. In this study, we present a structural refinement approach that combines solid-state NMR experiments and molecular simulations to accurately describe concerted conformational transitions identifying the overall structural, dynamical, and topological states of oligomeric membrane proteins. The accuracy of the structural ensembles generated with this method is shown to reach the statistical error limit, and is further demonstrated by correctly reproducing orthogonal NMR data. We demonstrate the accuracy of this approach by characterising the pentameric state of phospholamban, a key player in the regulation of calcium uptake in the sarcoplasmic reticulum, and by probing its dynamical activation upon phosphorylation. Our results underline the importance of using an ensemble approach to characterise the conformational transitions that are often responsible for the biological function of oligomeric membrane protein states. PMID:26975211

  3. Protein Analysis by Ambient Ionization Mass Spectrometry Using Trypsin-Immobilized Organosiloxane Polymer Surfaces.

    PubMed

    Dulay, Maria T; Eberlin, Livia S; Zare, Richard N

    2015-12-15

    In the growing field of proteomic research, rapid and simple protein analysis is a crucial component of protein identification. We report the use of immobilized trypsin on hybrid organic-inorganic organosiloxane (T-OSX) polymers for the on-surface, in situ digestion of four model proteins: melittin, cytochrome c, myoglobin, and bovine serum albumin. Tryptic digestion products were sampled, detected, and identified using desorption electrospray ionization mass spectrometry (DESI-MS) and nanoDESI-MS. These novel, reusable T-OSX arrays on glass slides allow for protein digestion in methanol:water solvents (1:1, v/v) and analysis directly from the same polymer surface without the need for sample preparation, high temperature, and pH conditions typically required for in-solution trypsin digestions. Digestion reactions were conducted with 2 μL protein sample droplets (0.35 mM) at incubation temperatures of 4, 25, 37, and 65 °C and digestion reaction times between 2 and 24 h. Sequence coverages were dependent on the hydrophobicity of the OSX polymer support and varied by temperature and digestion time. Under the best conditions, the sequence coverages, determined by DESI-MS, were 100% for melittin, 100% for cytochrome c, 90% for myoglobin, and 65% for bovine serum albumin. PMID:26567450

  4. Membrane bending by protein crowding is affected by protein lateral confinement.

    PubMed

    Derganc, Jure; Čopič, Alenka

    2016-06-01

    Crowding of asymmetrically-distributed membrane proteins has been recently recognized as an important factor in remodeling of biological membranes, for example during transport vesicle formation. In this paper, we theoretically analyze the effect of protein crowding on membrane bending and examine its dependence on protein size, shape, transmembrane asymmetry and lateral confinement. We consider three scenarios of protein lateral organization, which are highly relevant for cellular membranes in general: freely diffusing membrane proteins without lateral confinement, the presence of a diffusion barrier and interactions with a vesicular coat. We show that protein crowding affects vesicle formation even if the proteins are distributed symmetrically across the membrane and that this effect depends significantly on lateral confinement. The largest crowding effect is predicted for the proteins that are confined to the forming vesicle by a diffusion barrier. We calculate the bending properties of a crowded membrane and find that its spontaneous curvature depends primarily on the degree of transmembrane asymmetry, and its effective bending modulus on the type of lateral confinement. Using the example of COPII vesicle formation from the endoplasmic reticulum, we analyze the energetic cost of vesicle formation. The results provide a novel insight into the effects of lateral and transmembrane organization of membrane proteins, and can guide data interpretation and future experimental approaches. PMID:26969088

  5. Structural adaptations of proteins to different biological membranes

    PubMed Central

    Pogozheva, Irina D.; Tristram-Nagle, Stephanie; Mosberg, Henry I.; Lomize, Andrei L.

    2013-01-01

    To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and β) and the dipolarity/polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, β-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30-31 Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic plasma membranes (23.9, 28.6 and 33.5 Å, respectively). Protein and lipid polarity parameters abruptly change in the lipid carbonyl zone that matches the calculated hydrophobic boundaries. Maxima of positively charged protein groups correspond to the location of lipid phosphates at 20-22 Å distances from the membrane center. Locations of Tyr atoms coincide with hydrophobic boundaries, while distributions maxima of Trp rings are shifted by 3-4 Å toward the membrane center. Distributions of Trp atoms indicate the presence of two 5-8 Å-wide midpolar regions with intermediate π* values within the hydrocarbon core, whose size and symmetry depend on the lipid composition of membrane leaflets. Midpolar regions are especially asymmetric in outer bacterial membranes and cell membranes of mesophilic but not hyperthermophilic archaebacteria, indicating the larger width of the central nonpolar region in the later case. In artificial lipid bilayers, midpolar regions are observed up to the level of acyl chain double bonds. PMID:23811361

  6. Quality control of nonstop membrane proteins at the ER membrane and in the cytosol.

    PubMed

    Arakawa, Shunsuke; Yunoki, Kaori; Izawa, Toshiaki; Tamura, Yasushi; Nishikawa, Shuh-Ichi; Endo, Toshiya

    2016-01-01

    Since messenger RNAs without a stop codon (nonstop mRNAs) for organelle-targeted proteins and their translation products (nonstop proteins) generate clogged translocon channels as well as stalled ribosomes, cells have mechanisms to degrade nonstop mRNAs and nonstop proteins and to clear the translocons (e.g. the Sec61 complex) by release of nonstop proteins into the organellar lumen. Here we followed the fate of nonstop endoplasmic reticulum (ER) membrane proteins with different membrane topologies in yeast to evaluate the importance of the Ltn1-dependent cytosolic degradation and the Dom34-dependent release of the nonstop membrane proteins. Ltn1-dependent degradation differed for membrane proteins with different topologies and its failure did not affect ER protein import or cell growth. On the other hand, failure in the Dom34-dependent release of the nascent polypeptide from the ribosome led to the block of the Sec61 channel and resultant inhibition of other protein import into the ER caused cell growth defects. Therefore, the nascent chain release from the translation apparatus is more instrumental in clearance of the clogged ER translocon channel and thus maintenance of normal cellular functions. PMID:27481473

  7. Quality control of nonstop membrane proteins at the ER membrane and in the cytosol

    PubMed Central

    Arakawa, Shunsuke; Yunoki, Kaori; Izawa, Toshiaki; Tamura, Yasushi; Nishikawa, Shuh-ichi; Endo, Toshiya

    2016-01-01

    Since messenger RNAs without a stop codon (nonstop mRNAs) for organelle-targeted proteins and their translation products (nonstop proteins) generate clogged translocon channels as well as stalled ribosomes, cells have mechanisms to degrade nonstop mRNAs and nonstop proteins and to clear the translocons (e.g. the Sec61 complex) by release of nonstop proteins into the organellar lumen. Here we followed the fate of nonstop endoplasmic reticulum (ER) membrane proteins with different membrane topologies in yeast to evaluate the importance of the Ltn1-dependent cytosolic degradation and the Dom34-dependent release of the nonstop membrane proteins. Ltn1-dependent degradation differed for membrane proteins with different topologies and its failure did not affect ER protein import or cell growth. On the other hand, failure in the Dom34-dependent release of the nascent polypeptide from the ribosome led to the block of the Sec61 channel and resultant inhibition of other protein import into the ER caused cell growth defects. Therefore, the nascent chain release from the translation apparatus is more instrumental in clearance of the clogged ER translocon channel and thus maintenance of normal cellular functions. PMID:27481473

  8. Plasma Surface Modification for Immobilization of Bone Morphogenic Protein-2 on Polycaprolactone Scaffolds

    NASA Astrophysics Data System (ADS)

    Kim, Byung Hoon; Myung, Sung Woon; Jung, Sang Chul; Ko, Yeong Mu

    2013-11-01

    The immobilization of recombinant human bone formation protein-2 (rhBMP-2) on polycaprolactone (PCL) scaffolds was performed by plasma polymerization. RhBMP-2, which induces osteoblast differentiation in various cell types, is a growth factor that plays an important role in bone formation and repair. The surface of the PCL scaffold was functionalized with the carboxyl groups of plasma-polymerized acrylic acid (PPAA) thin films. Plasma polymerization was carried out at a discharge power of 60 W at an acrylic acid flow rate of 7 sccm for 5 min. The PPAA thin film exhibited moderate hydrophilic properties and possessed a high density of carboxyl groups. Carboxyl groups and rhBMP-2 on the PCL scaffolds surface were identified by attenuated total reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The alkaline phosphatase activity assay showed that the rhBMP-2 immobilized PCL scaffold increased the level of MG-63 cell differentiation. Plasma surface modification for the preparation of biomaterials, such as biofunctionalized polymer scaffolds, can be used for the binding of bioactive molecules in tissue engineering.

  9. Pulsed ESR dipolar spectroscopy for distance measurements in immobilized spin labeled proteins in liquid solution

    PubMed Central

    Yang, Zhongyu; Liu, Yangping; Borbat, Peter; Zweier, Jay L.; Freed, Jack H.; Hubbell, Wayne L.

    2012-01-01

    Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer- range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron-electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized. PMID:22676043

  10. Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

    PubMed Central

    Meyer, Virginia; Swanson, Michael A.; Clouston, Laura J.; Boratyński, Przemysław J.; Stein, Richard A.; Mchaourab, Hassane S.; Rajca, Andrzej; Eaton, Sandra S.; Eaton, Gareth R.

    2015-01-01

    Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label. PMID:25762332

  11. MreB-Dependent Organization of the E. coli Cytoplasmic Membrane Controls Membrane Protein Diffusion.

    PubMed

    Oswald, Felix; Varadarajan, Aravindan; Lill, Holger; Peterman, Erwin J G; Bollen, Yves J M

    2016-03-01

    The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs. PMID:26958890

  12. Lipopolysaccharide Membranes and Membrane Proteins of Pseudomonas aeruginosa Studied by Computer Simulation

    SciTech Connect

    Straatsma, TP

    2006-12-01

    Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium with high metabolic versatility and an exceptional ability to adapt to a wide range of ecological environments, including soil, marches, coastal habitats, plant and animal tissues. Gram-negative microbes are characterized by the asymmetric lipopolysaccharide outer membrane, the study of which is important for a number of applications. The adhesion to mineral surfaces plays a central role in characterizing their contribution to the fate of contaminants in complex environmental systems by effecting microbial transport through soils, respiration redox chemistry, and ion mobility. Another important application stems from the fact that it is also a major opportunistic human pathogen that can result in life-threatening infections in many immunocompromised patients, such as lung infections in children with cystic fibrosis, bacteraemia in burn victims, urinary-tract infections in catheterized patients, hospital-acquired pneumonia in patients on respirators, infections in cancer patients receiving chemotherapy, and keratitis and corneal ulcers in users of extended-wear soft contact lenses. The inherent resistance against antibiotics which has been linked with the specific interactions in the outer membrane of P. aeruginosa makes these infections difficult to treat. Developments in simulation methodologies as well as computer hardware have enabled the molecular simulation of biological systems of increasing size and with increasing accuracy, providing detail that is difficult or impossible to obtain experimentally. Computer simulation studies contribute to our understanding of the behavior of proteins, protein-protein and protein-DNA complexes. In recent years, a number of research groups have made significant progress in applying these methods to the study of biological membranes. However, these applications have been focused exclusively on lipid bilayer membranes and on membrane proteins in lipid

  13. [Multiparticle computer simulation of protein interactions in the photosynthetic membrane].

    PubMed

    Riznichenko, G Iu; Kovalenko, I B; Abaturova, A M; D'iakonova, A N; Kniazeva, O S; Ustinin, D M; Khrushchev, S S; Rubin, A B

    2011-01-01

    The basic principles of the design of direct multiparticle models and the results of multiparticle computer simulation of electron transfer by mobile protein carriers in the photosynthetic membrane of a chloroplast thylakoid are presented. The reactions of complex formation of the protein plastocyanin with the protein cytochrome f and the pigment-protein complex of photosystem I, as well as of the protein ferredoxin with the protein FNR and photosystem 1 are considered. The role of diffusion and electrostatic interactions is discussed, and the effect of the shape of the reaction volume and ionic strength on the rate of electron transport are discussed. PMID:22117434

  14. Encapsulated membrane proteins: A simplified system for molecular simulation.

    PubMed

    Lee, Sarah C; Khalid, Syma; Pollock, Naomi L; Knowles, Tim J; Edler, Karen; Rothnie, Alice J; R T Thomas, Owen; Dafforn, Timothy R

    2016-10-01

    Over the past 50years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modelling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex. An area where both challenges are present is that of membranes containing membrane proteins. In this review we analyse a new practical approach to membrane protein study that offers a potential new route to high resolution structures and the possibility to simplify simulations. These new approaches collectively recognise that preservation of the interaction between the membrane protein and the lipid bilayer is often essential to maintain structure and function. The new methods preserve these interactions by producing nano-scale disc shaped particles that include bilayer and the chosen protein. Currently two approaches lead in this area: the MSP system that relies on peptides to stabilise the discs, and SMALPs where an amphipathic styrene maleic acid copolymer is used. Both methods greatly enable protein production and hence have the potential to accelerate atomic resolution structure determination as well as providing a simplified format for simulations of membrane protein dynamics. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26946242

  15. Swimbladder membrane protein of an abyssal fish, Coryphaenoides acrolepis.

    PubMed

    Mosholder, R S; Josephson, R V; Phleger, C F

    1979-01-01

    Protein components of the membranous foamy tissue collected from the swimbladder of Coryphaenoides acrolepis, a continental slope/deep sea grenadier fish, were partially fractionated and characterized by procedures used successfully for erythrocyte membrane proteins. Methods involving pH and ionic strength adjustment in the presence of EDTA and beta-mercaptoethanol resulted in some protein fractionation but no distinct separation or isolation of membrane proteins. Gel filtration by Sephadex G-100 and Sepharose 2B in the presence of dodecyl sulfate partially fractionated protein species between 18,000 and 150,000 molecular weight (as confirmed by dodecyl sulfate polyacrylamide gel electrophoresis). Low molecular weight proteins were resolvable into a few diffuse and streaky bands by dodecyl sulfate and chloral hydrate polyacrylamide gel electrophoresis, the former giving superior reso-ution. A major fraction of large molecular weight protein (greater than or equal to 40 X 10(6)) was not resolved by any method. A possible explanation for these unusual findings is that decompression due to rapid ascent of the fish from deep ocean caused irreversible alteration of swimbladder membrane proteins. PMID:504363

  16. [Elution of urinary proteins preserved on nitrocellulose membrane with heating].

    PubMed

    Qin, Weiwei; Gao, Youhe

    2015-09-01

    The preservation of urinary proteins on a membrane plays a vital role in biomarker research, and the efficient elution of proteins preserved on nitrocellulose membrane (NC membrane) determines the application of this method. During the heating elution procedure, we raised the temperature to reduce the intense vortexing time, and kept gentle rotating while precipitation to prevent nitrocellulose reformation. We also used SDS-PAGE and LC-MS/MS to analyze the urinary proteins prepared by heating elution procedure, intense vortexing elution procedure and acetone precipitation method. There was no degradation of proteins prepared by heating elution procedure. Compared with proteins prepared by heating elution method and acetone precipitation method, the overlapping rates of the proteins was almost the same (92.6% versus 96.8%) and the ratios of CV values (< 20%) of the proteins were both high (85.2% and 94.4%). The heating elution procedure achieved good technical reproducibility, and was much simpler and more efficient than the previous one. It can facilitate the application of the preservation of urinary proteins on membrane. PMID:26955716

  17. Catalytic properties of endoxylanase fusion proteins from Neocallimastix frontalis and effect of immobilization onto metal-chelate matrix.

    PubMed

    Mesta, Laurent; Heyraud, Alain; Joseleau, Jean Paul; Coulet, Pierre R

    2003-03-20

    The production of hybrid enzymes with novel properties and the research for new methods for enzyme immobilization in bioreactors are of major interest in biotechnology. We report here the second part of a study concerning the improvement of the properties of the endoxylanase XYN3A4 from the anaerobic fungi Neocallimastix frontalis. The effects of gene fusion and immobilization on metal-chelate matrix are also compared for the reference enzymes XYN3, XYN3A, XYN4 used for the construction of the fusion protein XYN3A4. The influence of the metal ion in the immobilization process was first investigated and best immobilization yields were obtained with the Cu(II) ion whereas best coupling efficiencies were reached with the Ni(II) ion. It was also observed that XYN3, XYN3A and XYN34 had a lower rate of hydrolysis when immobilized on Ni(II)-IDA and more difficulties to accomodate small substrates than the soluble enzymes. Nevertheless, a major difference was noted during the hydrolysis of birchwood xylan and it appears that the reaction using the immobilized XYN3A4 chimeric enzyme leads to the accumulation of a specific product. PMID:12615394

  18. Ethanol production by Saccharomyces cerevisiae immobilized in hollow-fiber membrane bioreactors

    SciTech Connect

    Inloes, D.S.; Taylor, D.P.; Cohen, S.N.; Michaels, A.S.; Robertson, C.S.

    1983-07-01

    Saccharomyces cerevisiae ATCC 4126 was grown within the macroporous matrix of asymmetric-walled polysulfone hollow-fiber membranes and on the exterior surfaces of isotropic-walled polypropylene hollow-fiber membranes. Nutrients were supplied and products were removed by single-pass perfusion of the fiber lumens. Growth of yeast cells within the macrovoids of the asymmetric-walled membranes attained densities of greater than 10 to the power of 10 cells per ml and in some regions accounted for nearly 100% of the available macrovoid volume, forming a tissue-like mass. A radial distribution of cell packing existed across the fiber wall, indicating an inadequate glucose supply to cells located beyond 100 mum from the lumen surface. By comparison, yeast cell growth on the exterior surfaces of the isotropic-walled membranes resulted in an average density of 3.5 x 10 to the power of 9 viable cells per ml. Ethanol production by reactors containing isotropic polypropylene fibers reached a maximum value of 26 g/liter-h based on the total reactor volume. Reactor performance depended on the fiber packing density and on the glucose medium flow rate and was limited by low nutrient and product transport rates. The inhibition of ethanol production and the reduction in fermentation efficiency arose primarily from the accumulation of CO/sub 2/ gas within the sealed reactor shell space. (Refs. 37).

  19. Development of a novel pH sensor based upon Janus Green B immobilized on triacetyl cellulose membrane: Experimental design and optimization

    NASA Astrophysics Data System (ADS)

    Chamkouri, Narges; Niazi, Ali; Zare-Shahabadi, Vali

    2016-03-01

    A novel pH optical sensor was prepared by immobilizing an azo dye called Janus Green B on the triacetylcellulose membrane. Condition of the dye solution used in the immobilization step, including concentration of the dye, pH, and duration were considered and optimized using the Box-Behnken design. The proposed sensor showed good behavior and precision (RSD < 5%) in the pH range of 2.0-10.0. Advantages of this optical sensor include on-line applicability, no leakage, long-term stability (more than 6 months), fast response time (less than 1 min), high selectivity and sensitivity as well as good reversibility and reproducibility.

  20. Novel Approaches to Immobilized Heteropoly Acid Systems for High Temperature, Low Relative Humidity Polymer-Type Membranes - Final Report

    SciTech Connect

    Herring, Andrew M; Horan, James L; Aieta, Niccolo V; Sachdeva, Sonny; Kuo, Mei-Chen; Ren, Hui; Lingutla, Anitha; Emery, Michael; Haugen, Gregory M; Yandrasits, Michael A; Sharma, Neeraj; Coggio, William D; Hamrock, Steven J; Frey, Matthew H

    2012-05-20

    Original research was carried out at the CSM and the 3M Company from March 2007 through September 2011. The research was aimed at developing new to the world proton electrolyte materials for use in hydrogen fuel cells, in particular with high proton conductivity under hot and dry conditions (>100mS/cm at 120°C and 50%RH). Broadly stated, the research at 3M and between 3M and CSM that led to new materials took place in two phases: In the first phase, hydrocarbon membranes that could be formed by photopolymerization of monomer mixtures were developed for the purpose of determining the technical feasibility of achieving the program's Go/No-Go decision conductivity target of >100mS/cm at 120°C and 50%RH. In the second phase, attempts were made to extend the achieved conductivity level to fluorinated material systems with the expectation that durability and stability would be improved (over the hydrocarbon material). Highlights included: Multiple lots of an HPA-immobilized photocurable terpolymer derived from di-vinyl-silicotungstic acid (85%), n-butyl acrylate, and hexanediol diacrylate were prepared at 3M and characterized at 3M to exhibit an initial conductivity of 107mS/cm at 120°C and 47%RH (PolyPOM85v) using a Bekktech LLC sample fixture and TestEquity oven. Later independent testing by Bekktech LLC, using a different preheating protocol, on the same material, yielded a conductivity value of approximately 20mS/cm at 120°C and 50%RH. The difference in measured values is likely to have been the result of an instability of properties for the material or a difference in the measurement method. A dispersed catalyst fuel cell was fabricated and tested using a 150¼m thick HPA-based photocurable membrane (above, PolyPOM75v), exhibiting a current density of greater than 300mA/cm2 at 0.5V (H2/Air 800/1800sccm 70°C/75%RH ambient outlet pressure). Multiple lots of a co-polymer based on poly-trifluorovinylether (TFVE) derived HPA were synthesized and fabricated into

  1. Amyloid protein unfolding and insertion kinetics on neuronal membrane mimics

    NASA Astrophysics Data System (ADS)

    Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan

    2010-03-01

    Atomistic details of beta-amyloid (Aβ ) protein unfolding and lipid interaction kinetics mediated by the neuronal membrane surface are important for developing new therapeutic strategies to prevent and cure Alzheimer's disease. Using all-atom MD simulations, we explored the early unfolding and insertion kinetics of 40 and 42 residue long Aβ in binary lipid mixtures with and without cholesterol that mimic the cholesterol-depleted and cholesterol-enriched lipid nanodomains of neurons. The protein conformational transition kinetics was evaluated from the secondary structure profile versus simulation time plot. The extent of membrane disruption was examined by the calculated order parameters of lipid acyl chains and cholesterol fused rings as well as the density profiles of water and lipid headgroups at defined regions across the lipid bilayer from our simulations. Our results revealed that both the cholesterol content and the length of the protein affect the protein-insertion and membrane stability in our model lipid bilayer systems.

  2. VAMP-1: a synaptic vesicle-associated integral membrane protein.

    PubMed

    Trimble, W S; Cowan, D M; Scheller, R H

    1988-06-01

    Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters. PMID:3380805

  3. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    PubMed Central

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  4. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    PubMed

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  5. Supramolecular Chemistry And Self-assembly Special Feature: Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands

    NASA Astrophysics Data System (ADS)

    Hodneland, Christian D.; Lee, Young-Sam; Min, Dal-Hee; Mrksich, Milan

    2002-04-01

    This paper describes a method for the selective and covalent immobilization of proteins to surfaces with control over the density and orientation of the protein. The strategy is based on binding of the serine esterase cutinase to a self-assembled monolayer presenting a phosphonate ligand and the subsequent displacement reaction that covalently binds the ligand to the enzyme active site. Surface plasmon resonance (SPR) spectroscopy showed that cutinase binds irreversibly to a monolayer presenting the capture ligand at a density of 1% mixed among tri(ethylene glycol) groups. The covalent immobilization is specific for cutinase, and the glycol-terminated monolayer effectively prevents unwanted nonspecific adsorption of proteins. To demonstrate that the method could be used to immobilize proteins of interest, a cutinase-calmodulin fusion protein was constructed and immobilized to the monolayer. SPR showed that calcineurin selectively associated with the immobilized calmodulin. This capture ligand immobilization method combines the advantages that the immobilization reaction is highly selective for the intended protein, the tether is covalent and, hence, stable, and the method avoids the need for synthetic modification and rigorous purification of proteins before immobilization. These characteristics make the method well suited to a range of applications and, in particular, for constructing protein microarrays.

  6. Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia.

    PubMed Central

    Kropinski, A M; Parr, T R; Angus, B L; Hancock, R E; Ghiorse, W C; Greenberg, E P

    1987-01-01

    The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl. Images PMID:3025168

  7. Quality control of integral membrane proteins by assembly-dependent membrane integration.

    PubMed

    Feige, Matthias J; Hendershot, Linda M

    2013-08-01

    Cell-surface multiprotein complexes are synthesized in the endoplasmic reticulum (ER), where they undergo cotranslational membrane integration and assembly. The quality control mechanisms that oversee these processes remain poorly understood. We show that less hydrophobic transmembrane (TM) regions derived from several single-pass TM proteins can enter the ER lumen completely. Once mislocalized, they are recognized by the Hsp70 chaperone BiP. In a detailed analysis for one of these proteins, the αβT cell receptor (αβTCR), we show that unassembled ER-lumenal subunits are rapidly degraded, whereas specific subunit interactions en route to the native receptor promote membrane integration of the less hydrophobic TM segments, thereby stabilizing the protein. For the TCR α chain, both complete ER import and subunit assembly depend on the same pivotal residue in its TM region. Thus, membrane integration linked to protein assembly allows cellular quality control of membrane proteins and connects the lumenal ER chaperone machinery to membrane protein biogenesis. PMID:23932713

  8. Viral fusion protein transmembrane domain adopts β-strand structure to facilitate membrane topological changes for virus–cell fusion

    PubMed Central

    Yao, Hongwei; Lee, Michelle W.; Waring, Alan J.; Wong, Gerard C. L.; Hong, Mei

    2015-01-01

    The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive α-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magic-angle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly α-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the β-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. 31P NMR spectra and small-angle X-ray scattering (SAXS) data show that this β-strand–rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. 1H-31P 2D correlation spectra and 2H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the β-strand as the fusogenic conformation. PMID:26283363

  9. Single-Molecule Microscopy Reveals Plasma Membrane Microdomains Created by Protein-Protein Networks that Exclude or Trap Signaling Molecules in T Cells

    PubMed Central

    Douglass, Adam D.; Vale, Ronald D.

    2010-01-01

    Summary Membrane subdomains have been implicated in T cell signaling, although their properties and mechanisms of formation remain controversial. Here, we have used single-molecule and scanning confocal imaging to characterize the behavior of GFP-tagged signaling proteins in Jurkat T cells. We show that the coreceptor CD2, the adaptor protein LAT, and tyrosine kinase Lck cocluster in discrete microdomains in the plasma membrane of signaling T cells. These microdomains require protein-protein interactions mediated through phosphorylation of LAT and are not maintained by interactions with actin or lipid rafts. Using a two color imaging approach that allows tracking of single molecules relative to the CD2/LAT/Lck clusters, we demonstrate that these microdomains exclude and limit the free diffusion of molecules in the membrane but also can trap and immobilize specific proteins. Our data suggest that diffusional trapping through protein-protein interactions creates microdomains that concentrate or exclude cell surface proteins to facilitate T cell signaling. PMID:15960980

  10. Macroporous polyacrylamide-based monolithic column with immobilized pH gradient for protein analysis.

    PubMed

    Zhu, Guijie; Yuan, Huiming; Zhao, Peng; Zhang, Lihua; Liang, Zhen; Zhang, Weibing; Zhang, Yukui

    2006-09-01

    Monolithic materials were prepared in capillaries by in situ polymerization of acrylamide, glycidyl methacrylate, and N,N'-methylenebisacrylamide in the presence of 1,4-butanediol, dodecanol, and DMSO as porogens. With Ampholine attached to the surface of the porous monolith via epoxide groups, a monolithic-IPG (M-IPG) was formed and showed good mechanical and chemical stability. With such a column immobilized by Ampholine 3.5-10, IEF-MIX 3.6-9.3 was separated and good linearity was obtained. The CIEF behavior of M-IPG was evinced by comparing the current with that in the open tubular capillary. In addition, the protein mixtures excreted from lung cancer cells of rats were analyzed with such a new M-IPG column. PMID:16915568

  11. Quartz crystal microbalance (QCM): useful for developing procedures for immobilization of proteins on solid surfaces.

    PubMed

    Sha, Xue; Sun, Chengjun; Xu, Xiaohe; Alexander, Laura; Loll, Patrick J; Penn, Lynn S

    2012-12-01

    We demonstrate the combined use of liquid and air measurements with the quartz crystal microbalance (QCM) for quantitative analysis of multistep reaction procedures leading to immobilized proteins on solid surfaces. Reactions are conducted on the surfaces of QCM sensor crystals and are quantified by measurements of resonant frequency of the crystals before and after each reaction step. When reactions are conducted in the flow cell of the QCM in the presence of solvent, measurement of resonant frequency can be made in situ (liquid measurement). When reactions cannot be conducted in the flow cell because of temperatures or solvents not tolerated by the cell, frequency can be measured after evaporation of solvent (air measurement). Each reaction step can be analyzed by either liquid or air measurement so that the whole multistep procedure is addressed, no matter how diverse the chemical nature of the steps. We conducted identical multistep procedures on two different starting surfaces, gold and silica, and found comparable results. PMID:23121645

  12. Lipids assist the membrane insertion of a BAM-independent outer membrane protein

    PubMed Central

    Huysmans, Gerard H. M.; Guilvout, Ingrid; Chami, Mohamed; Nickerson, Nicholas N.; Pugsley, Anthony P.

    2015-01-01

    Like several other large, multimeric bacterial outer membrane proteins (OMPs), the assembly of the Klebsiella oxytoca OMP PulD does not rely on the universally conserved β-barrel assembly machinery (BAM) that catalyses outer membrane insertion. The only other factor known to interact with PulD prior to or during outer membrane targeting and assembly is the cognate chaperone PulS. Here, in vitro translation-transcription coupled PulD folding demonstrated that PulS does not act during the membrane insertion of PulD, and engineered in vivo site-specific cross-linking between PulD and PulS showed that PulS binding does not prevent membrane insertion. In vitro folding kinetics revealed that PulD is atypical compared to BAM-dependent OMPs by inserting more rapidly into membranes containing E. coli phospholipids than into membranes containing lecithin. PulD folding was fast in diC14:0-phosphatidylethanolamine liposomes but not diC14:0-phosphatidylglycerol liposomes, and in diC18:1-phosphatidylcholine liposomes but not in diC14:1-phosphatidylcholine liposomes. These results suggest that PulD efficiently exploits the membrane composition to complete final steps in insertion and explain how PulD can assemble independently of any protein-assembly machinery. Lipid-assisted assembly in this manner might apply to other large OMPs whose assembly is BAM-independent. PMID:26463896

  13. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    PubMed

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  14. A new window into the molecular physiology of membrane proteins

    PubMed Central

    Landreh, Michael; Robinson, Carol V

    2015-01-01

    Integral membrane proteins comprise ∼25% of the human proteome. Yet, our understanding of their molecular physiology is still in its infancy. This can be attributed to two factors: the experimental challenges that arise from the difficult chemical nature of membrane proteins, and the unclear relationship between their activity and their native environment. New approaches are therefore required to address these challenges. Recent developments in mass spectrometry have shown that it is possible to study membrane proteins in a solvent-free environment and provide detailed insights into complex interactions, ligand binding and folding processes. Interestingly, not only detergent micelles but also lipid bilayer nanodiscs or bicelles can serve as a means for the gentle desolvation of membrane proteins in the gas phase. In this manner, as well as by direct addition of lipids, it is possible to study the effects of different membrane components on the structure and function of the protein components allowing us to add functional data to the least accessible part of the proteome. PMID:25630257

  15. A new window into the molecular physiology of membrane proteins.

    PubMed

    Landreh, Michael; Robinson, Carol V

    2015-01-15

    Integral membrane proteins comprise ∼25% of the human proteome. Yet, our understanding of their molecular physiology is still in its infancy. This can be attributed to two factors: the experimental challenges that arise from the difficult chemical nature of membrane proteins, and the unclear relationship between their activity and their native environment. New approaches are therefore required to address these challenges. Recent developments in mass spectrometry have shown that it is possible to study membrane proteins in a solvent-free environment and provide detailed insights into complex interactions, ligand binding and folding processes. Interestingly, not only detergent micelles but also lipid bilayer nanodiscs or bicelles can serve as a means for the gentle desolvation of membrane proteins in the gas phase. In this manner, as well as by direct addition of lipids, it is possible to study the effects of different membrane components on the structure and function of the protein components allowing us to add functional data to the least accessible part of the proteome. PMID:25630257

  16. Palmitoylation of POTE family proteins for plasma membrane targeting

    SciTech Connect

    Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

    2007-11-23

    The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane.

  17. Photolabeling of brain membrane proteins by lysergic acid diethylamide.

    PubMed

    Mahon, A C; Hartig, P R

    1982-04-01

    3H-Lysergic acid diethylamide (3H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a subpopulation of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. 3H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays. PMID:7087658

  18. Photolabeling of brain membrane proteins by lysergic acid diethylamide

    SciTech Connect

    Mahon, A.C.; Hartig, P.R.

    1982-04-05

    /sup 3/H-Lysergic acid diethylamide (/sup 3/H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a sub-population of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. /sup 3/H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays.

  19. Reconstructing protein remodeled membranes in molecular detail from mesoscopic models

    PubMed Central

    Lyman, Edward; Cui, Haosheng; Voth, Gregory A.

    2014-01-01

    We present a method for “inverse coarse graining,” rebuilding a higher resolution model from a lower resolution one, in order to rebuild protein coats for remodeled membranes of complex topology. The specific case of membrane remodeling by N-BAR domain containing proteins is considered here, although the overall method is general and thus applicable to other membrane remodeling phenomena. Our approach begins with a previously developed, discretized mesoscopic continuum membrane model (EM2) which has been shown to capture the reticulated membrane topologies often observed for N-BAR/liposome systems by electron microscopy (EM). The information in the EM2 model — directions of the local curvatures and a low resolution sample of the membrane surface — is then used to construct a coarse-grained (CG) system with one site per lipid and 26 sites per protein. We demonstrate the approach on pieces of EM2 structures with three different topologies that have been observed by EM: A tubule, a “Y” junction, and a torus. We show that the approach leads to structures that are stable under subsequent constant temperature CG simulation, and end by considering the future application of the methodology as a hybrid approach that combines experimental information with computer modeling. PMID:21503332

  20. Glycan Moieties as Bait to Fish Plasma Membrane Proteins.

    PubMed

    Fang, Fei; Zhao, Qun; Sui, Zhigang; Liang, Yu; Jiang, Hao; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-05-17

    Plasma membrane proteome analysis is of significance for screening candidate biomarkers and drug targets. However, due to their low abundance and lack of specific groups that can enable their capture, the plasma membrane proteins (PMPs) are under-represented. On the basis of the fact that PMPs are embedded in or anchored to the phospholipid bilayer of the plasma