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Sample records for protein surfaces reveals

  1. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    PubMed

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters. PMID:26371748

  2. An optimized isolation of biotinylated cell surface proteins reveals novel players in cancer metastasis

    PubMed Central

    Karhemo, Piia-Riitta; Ravela, Suvi; Laakso, Marko; Ritamo, Ilja; Tatti, Olga; Mäkinen, Selina; Goodison, Steve; Stenman, Ulf-Håkan; Hölttä, Erkki; Hautaniemi, Sampsa; Valmu, Leena; Lehti, Kaisa; Laakkonen, Pirjo

    2012-01-01

    Details of metastasis, the deadliest aspect of cancer, are unclear. Cell surface proteins play central roles in adhesive contacts between the tumor cell and the stroma during metastasis. We optimized a fast, small-scale isolation of biotinylated cell surface proteins to reveal novel metastasis-associated players froman isogenic pair of human MDA-MB-435 cancer cells with opposite metastatic phenotypes. Isolated proteins were trypsin digested and analyzed using LC–MS/MS followed by quantitation with the Progenesis LC–MS software. Sixteen proteins displayed over twofold expression differences between the metastatic and non-metastatic cells. Interestingly, overexpression of most of them (14/16) in the metastatic cells indicates a gain of novel surface protein profile as compared to the non-metastatic one. All five validated, differentially expressed proteins showed higher expression in the metastatic cells in culture, and four of these were further validated in vivo. Moreover, we analyzed the expression of two of the identified proteins, CD109 and ITGA6 in 3-dimensional cultures of six melanoma cell lines. Both proteins marked the surface of cells derived from melanoma metastasis over cells derived from primary melanoma. These unbiased identification and validation of both known and novel metastasis-associated proteins indicate a reliable approach for the identification of differentially expressed surface proteins. PMID:22813880

  3. Weak protein-protein interactions revealed by immiscible filtration assisted by surface tension.

    PubMed

    Berry, Scott M; Chin, Emily N; Jackson, Shawn S; Strotman, Lindsay N; Goel, Mohit; Thompson, Nancy E; Alexander, Caroline M; Miyamoto, Shigeki; Burgess, Richard R; Beebe, David J

    2014-02-15

    Biological mechanisms are often mediated by transient interactions between multiple proteins. The isolation of intact protein complexes is essential to understanding biochemical processes and an important prerequisite for identifying new drug targets and biomarkers. However, low-affinity interactions are often difficult to detect. Here, we use a newly described method called immiscible filtration assisted by surface tension (IFAST) to isolate proteins under defined binding conditions. This method, which gives a near-instantaneous isolation, enables significantly higher recovery of transient complexes compared to current wash-based protocols, which require reequilibration at each of several wash steps, resulting in protein loss. The method moves proteins, or protein complexes, captured on a solid phase through one or more immiscible-phase barriers that efficiently exclude the passage of nonspecific material in a single operation. We use a previously described polyol-responsive monoclonal antibody to investigate the potential of this new method to study protein binding. In addition, difficult-to-isolate complexes involving the biologically and clinically important Wnt signaling pathway were isolated. We anticipate that this simple, rapid method to isolate intact, transient complexes will enable the discoveries of new signaling pathways, biomarkers, and drug targets. PMID:24215910

  4. Weak protein-protein interactions revealed by immiscible filtration assisted by surface tension (IFAST)

    PubMed Central

    Berry, Scott M.; Chin, Emily N.; Jackson, Shawn S.; Strotman, Lindsay N.; Goel, Mohit; Thompson, Nancy E.; Alexander, Caroline M.; Miyamoto, Shigeki; Burgess, Richard R.; Beebe, David J.

    2013-01-01

    Biological mechanisms are often mediated by transient interactions between multiple proteins. The isolation of intact protein complexes is essential to understanding biochemical processes and an important prerequisite for identifying new drug targets and biomarkers. However, low-affinity interactions are often difficult to detect. Here, we use a newly described method called immiscible filtration assisted by surface tension (IFAST) to isolate proteins under defined binding conditions. This method, that gives a near-instantaneous isolation, enables significantly higher recovery of transient complexes as compared to current wash-based protocols, which require re-equilibration at each of several wash steps, resulting in protein loss. The method moves proteins, or protein complexes, captured on a solid phase through one or more immiscible phase barriers that efficiently exclude the passage of non-specific material in a single operation. We use a previously described polyol-responsive monoclonal antibody (PR-mAb) to investigate the potential of this new method to study protein-binding. In addition, difficult-to-isolate complexes involving the biologically and clinically important Wnt signaling pathway were isolated. We anticipate that this simple, rapid method to isolate intact, transient complexes will enable the discoveries of new signaling pathways, biomarkers, and drug targets. PMID:24215910

  5. Local Geometry and Evolutionary Conservation of Protein Surfaces Reveal the Multiple Recognition Patches in Protein-Protein Interactions

    PubMed Central

    Laine, Elodie; Carbone, Alessandra

    2015-01-01

    Protein-protein interactions (PPIs) are essential to all biological processes and they represent increasingly important therapeutic targets. Here, we present a new method for accurately predicting protein-protein interfaces, understanding their properties, origins and binding to multiple partners. Contrary to machine learning approaches, our method combines in a rational and very straightforward way three sequence- and structure-based descriptors of protein residues: evolutionary conservation, physico-chemical properties and local geometry. The implemented strategy yields very precise predictions for a wide range of protein-protein interfaces and discriminates them from small-molecule binding sites. Beyond its predictive power, the approach permits to dissect interaction surfaces and unravel their complexity. We show how the analysis of the predicted patches can foster new strategies for PPIs modulation and interaction surface redesign. The approach is implemented in JET2, an automated tool based on the Joint Evolutionary Trees (JET) method for sequence-based protein interface prediction. JET2 is freely available at www.lcqb.upmc.fr/JET2. PMID:26690684

  6. An analysis of surface proteomics results reveals novel candidates for intracellular/surface moonlighting proteins in bacteria.

    PubMed

    Wang, Wangfei; Jeffery, Constance J

    2016-04-26

    Proteins expressed on the bacterial cell surface play important roles in infection and virulence and can be targets for vaccine development or used as biomarkers. Surprisingly, an increasing number of surface proteins are being found to be identical to intracellular enzymes and chaperones, and a few dozen intracellular/surface moonlighting proteins have been found that have different functions inside the cell and on the cell surface. The results of twenty-two published bacterial surface proteomics studies were analyzed using bioinformatics tools to consider how many additional intracellular proteins are also found on the cell surface. More than 1000 out of the 3619 proteins observed on the cell surface lack the transmembrane alpha-helices or transmembrane beta-barrels found in integral membrane proteins and also lack the signal peptides found in proteins secreted through the Sec pathway. Many of the proteins found on the cell surface are intracellular chaperones or enzymes involved in central metabolic pathways, including some that have previously been shown to have a moonlighting function on the cell surface in at least one species, such as Hsp60/GroEL, DnaK, glyceraldehyde 3-phosphate dehydrogenase, enolase, and fructose 1,6-bisphosphate aldolase. The results of the proteomics studies suggest they could also be moonlighting on the surface of many other species. Hundreds of other intracellular proteins are also found on the cell surface, although a second function on the surface has not yet been demonstrated, for example, glutamine synthetase, gamma-glutamyl phosphate reductase, and cysteine desulfurase. The presence of intracellular proteins on the cell surface is more common than previously expected and suggests that many additional proteins might be candidates for being intracellular/surface moonlighting proteins. PMID:26938107

  7. Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns.

    PubMed

    Arcinas, Arthur; Yen, Ten-Yang; Kebebew, Electron; Macher, Bruce A

    2009-08-01

    Cell surface proteins have been shown to be effective therapeutic targets. In addition, shed forms of these proteins and secreted proteins can serve as biomarkers for diseases, including cancer. Thus, identification of cell surface and secreted proteins has been a prime area of interest in the proteomics field. Most cell surface and secreted proteins are known to be glycosylated, and therefore, a proteomics strategy targeting these proteins was applied to obtain proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using two-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), Hurthle cell carcinoma (XTC-1), and anaplastic thyroid cancer (ARO and DRO-1) were evaluated. An average of 150 glycoproteins were identified per cell line, of which more than 57% are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g., CD44, galectin 3 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in the more well-differentiated thyroid cancer cell lines (follicular, Hurthle cell and papillary), or by cell lines derived from undifferentiated tumors that are uniformly fatal forms of thyroid cancer (i.e., anaplastic). On the basis of the results obtained, a

  8. Structure Function Studies of Vaccinia Virus Host Range Protein K1 Reveal a Novel Functional Surface for Ankyrin Repeat Proteins

    SciTech Connect

    Li, Yongchao; Meng, Xiangzhi; Xiang, Yan; Deng, Junpeng

    2010-06-15

    Poxvirus host tropism at the cellular level is regulated by virus-encoded host range proteins acting downstream of virus entry. The functioning mechanisms of most host range proteins are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand interaction through a concave surface. We report here the crystal structure of one of the ANK repeat-containing host range proteins, the vaccinia virus K1 protein. The structure, at a resolution of 2.3 {angstrom}, showed that K1 consists entirely of ANK repeats, including seven complete ones and two incomplete ones, one each at the N and C terminus. Interestingly, Phe82 and Ser83, which were previously shown to be critical for K1's function, are solvent exposed and located on a convex surface, opposite the consensus ANK interaction surface. The importance of this convex surface was further supported by our additional mutagenesis studies. We found that K1's host range function was negatively affected by substitution of either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83. Altogether, our data showed that K1 residues on a continuous convex ANK repeat surface are critical for the host range function, suggesting that K1 functions through ligand interaction and does so with a novel ANK interaction surface.

  9. Rapid cell-surface prion protein conversion revealed using a novel cell system

    PubMed Central

    Goold, R.; Rabbanian, S.; Sutton, L.; Andre, R.; Arora, P.; Moonga, J.; Clarke, A.R.; Schiavo, G.; Jat, P.; Collinge, J.; Tabrizi, S.J.

    2011-01-01

    Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrPC). Here we develop a unique cell system in which epitope-tagged PrPC is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrPC, when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrPSc). Using this epitope-tagged PrPSc, we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion. PMID:21505437

  10. The identification and functional characterization of WxL proteins from Enterococcus faecium reveal surface proteins involved in extracellular matrix interactions.

    PubMed

    Galloway-Peña, Jessica R; Liang, Xiaowen; Singh, Kavindra V; Yadav, Puja; Chang, Chungyu; La Rosa, Sabina Leanti; Shelburne, Samuel; Ton-That, Hung; Höök, Magnus; Murray, Barbara E

    2015-03-01

    The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species. PMID:25512313

  11. PSC: protein surface classification

    PubMed Central

    Tseng, Yan Yuan; Li, Wen-Hsiung

    2012-01-01

    We recently proposed to classify proteins by their functional surfaces. Using the structural attributes of functional surfaces, we inferred the pairwise relationships of proteins and constructed an expandable database of protein surface classification (PSC). As the functional surface(s) of a protein is the local region where the protein performs its function, our classification may reflect the functional relationships among proteins. Currently, PSC contains a library of 1974 surface types that include 25 857 functional surfaces identified from 24 170 bound structures. The search tool in PSC empowers users to explore related surfaces that share similar local structures and core functions. Each functional surface is characterized by structural attributes, which are geometric, physicochemical or evolutionary features. The attributes have been normalized as descriptors and integrated to produce a profile for each functional surface in PSC. In addition, binding ligands are recorded for comparisons among homologs. PSC allows users to exploit related binding surfaces to reveal the changes in functionally important residues on homologs that have led to functional divergence during evolution. The substitutions at the key residues of a spatial pattern may determine the functional evolution of a protein. In PSC (http://pocket.uchicago.edu/psc/), a pool of changes in residues on similar functional surfaces is provided. PMID:22669905

  12. Structure Function Studies of Vaccinia Virus Host Range Protein K1 Reveal a Novel Functional Surface for Ankyrin Repeat Proteins▿

    PubMed Central

    Li, Yongchao; Meng, Xiangzhi; Xiang, Yan; Deng, Junpeng

    2010-01-01

    Poxvirus host tropism at the cellular level is regulated by virus-encoded host range proteins acting downstream of virus entry. The functioning mechanisms of most host range proteins are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand interaction through a concave surface. We report here the crystal structure of one of the ANK repeat-containing host range proteins, the vaccinia virus K1 protein. The structure, at a resolution of 2.3 Å, showed that K1 consists entirely of ANK repeats, including seven complete ones and two incomplete ones, one each at the N and C terminus. Interestingly, Phe82 and Ser83, which were previously shown to be critical for K1's function, are solvent exposed and located on a convex surface, opposite the consensus ANK interaction surface. The importance of this convex surface was further supported by our additional mutagenesis studies. We found that K1's host range function was negatively affected by substitution of either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83. Altogether, our data showed that K1 residues on a continuous convex ANK repeat surface are critical for the host range function, suggesting that K1 functions through ligand interaction and does so with a novel ANK interaction surface. PMID:20089642

  13. The Plasmodium vivax Merozoite Surface Protein 3β Sequence Reveals Contrasting Parasite Populations in Southern and Northwestern Thailand

    PubMed Central

    Kuamsab, Napaporn; Sattabongkot, Jetsumon; Sirichaisinthop, Jeeraphat; Jongwutiwes, Somchai; Cui, Liwang

    2014-01-01

    Background Malaria control efforts have a significant impact on the epidemiology and parasite population dynamics. In countries aiming for malaria elimination, malaria transmission may be restricted to limited transmission hot spots, where parasite populations may be isolated from each other and experience different selection forces. Here we aim to examine the Plasmodium vivax population divergence in geographically isolated transmission zones in Thailand. Methodology We employed the P. vivax merozoite surface protein 3β (PvMSP3β) as a molecular marker for characterizing P. vivax populations based on the extensive diversity of this gene in Southeast Asian parasite populations. To examine two parasite populations with different transmission levels in Thailand, we obtained 45 P. vivax isolates from Tak Province, northwestern Thailand, where the annual parasite incidence (API) was more than 2%, and 28 isolates from Yala and Narathiwat Provinces, southern Thailand, where the API was less than 0.02%. We sequenced the PvMSP3β gene and examined its genetic diversity and molecular evolution between the parasite populations. Principal Findings Of 58 isolates containing single PvMSP3β alleles, 31 sequence types were identified. The overall haplotype diversity was 0.77±0.06 and nucleotide diversity 0.0877±0.0054. The northwestern vivax malaria population exhibited extensive haplotype diversity (HD) of PvMSP3β (HD = 1.0). In contrast, the southern parasite population displayed a single PvMSP3β allele (HD = 0), suggesting a clonal population expansion. This result revealed that the extent of allelic diversity in P. vivax populations in Thailand varies among endemic areas. Conclusion Malaria parasite populations in a given region may vary significantly in genetic diversity, which may be the result of control and influenced by the magnitude of malaria transmission intensity. This is an issue that should be taken into account for the implementation of P. vivax

  14. Atomic force microscopy reveals a dual collagen-binding activity for the staphylococcal surface protein SdrF.

    PubMed

    Herman-Bausier, Philippe; Dufrêne, Yves F

    2016-02-01

    Staphylococcus epidermidis causes nosocomial infections by colonizing and forming biofilms on indwelling medical devices. This process involves specific interactions between cell wall-anchored (CWA) proteins and host proteins adsorbed onto the biomaterial. Here, we have explored the molecular forces by which the S. epidermidis CWA protein serine-aspartate repeat protein F (SdrF) binds to type I collagen, by means of advanced atomic force microscopy (AFM) techniques. Using single-cell force spectroscopy, we found that SdrF mediates bacterial adhesion to collagen-coated substrates through both weak and strong bonds. Single-molecule force spectroscopy demonstrated that these bonds involve the A and B regions of SdrF, thus revealing that the protein is capable of dual ligand-binding activity. Both weak and strong bonds showed high dissociation rates, indicating they are much less stable than those formed by the well-characterized 'dock, lock and latch' mechanism. Collectively, our results show that CWA proteins can bind to ligands by novel mechanisms. We anticipate that AFM will greatly contribute to the identification of novel binding partners and binding mechanisms in staphylococcal CWA proteins. PMID:26481199

  15. Surface Mediated Protein Disaggregation

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Kumar, Sanat K.

    2014-03-01

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

  16. Histophilus somni Surface Proteins.

    PubMed

    Corbeil, Lynette B

    2016-01-01

    The pathogen surface is usually the first site of interaction with the host. Histophilus somni was earlier thought to only have an outer membrane on its surface. Now it is known that the surface is composed of many virulence factors, including outer membrane proteins, lipooligosaccharide or endotoxin, a fibrillar network, and an exopolysaccharide. Outer membrane blebs, endotoxin, the fibrillar network, and the exopolysaccharide are also shed from the surface. This review will focus on the surface proteins of this pathogen that may colonize the mucosal surface of ruminants as a commensal or may cause pneumonia, septicemia, myocarditis, thrombotic meningoencephalitis, arthritis, and/or abortion. The major outer membrane protein has been well studied. Since its size and epitopes vary from strain to strain, it may be useful for typing strains. Iron-regulated OMPs have also received much attention because of their role in iron uptake for in vivo growth of H. somni. Other OMPs may be protective, based on passive immunization with monospecific antibodies and active immunization experiments. The surface and shed fibrillar network has been shown to be an immunoglobulin-binding protein in that it binds bovine IgG2 by the Fc portion. Two repeat domains (DR1 and DR2) have cytotoxic Fic motifs. Vaccine studies with recombinant DR2 are promising. Studies of the bacterial genome as well as comparison of surface proteins of different strains from the various H. somni syndromes and carrier states will be discussed and have provided much insight into pathogenesis and protection. PMID:26728061

  17. Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril.

    PubMed Central

    Weerkamp, A H; Handley, P S; Baars, A; Slot, J W

    1986-01-01

    The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease

  18. Surface Relaxation in Protein Crystals

    NASA Technical Reports Server (NTRS)

    Boutet, S.; Robinson, I. K.; Hu, Z. W.; Thomas, B. R.; Chernov, A. A.

    2002-01-01

    Surface X-ray diffraction measurements were performed on (111) growth faces of crystals of the Cellular iron-storage protein horse spleen ferritin. Crystal Trunkation Rods (CTR) were measured. A fit of the measured profile of the CTR revealed a surface roughness of 48 +/- 4.5 A and a top layer spacing contraction of 3.9 +/- 1.5%. In addition to the peak from the CTR, the rocking curves of the crystals displayed unexpected extra peaks. Multiple-scattering is demonstrated to account for them. Future applications of the method could allow the exploration of hydration effects on the growth of protein crystals.

  19. Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy

    PubMed Central

    Correa, Bruna R.; Bettoni, Fabiana; Koyama, Fernanda C.; Navarro, Fabio C.P.; Perez, Rodrigo O.; Mariadason, John; Sieber, Oliver M.; Strausberg, Robert L.; Simpson, Andrew J.G.; Jardim, Denis L.F.; Reis, Luiz Fernando L.; Parmigiani, Raphael B.; Galante, Pedro A.F.; Camargo, Anamaria A.

    2014-01-01

    We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients. PMID:25193853

  20. Heterogeneous surfaces to repel proteins.

    PubMed

    Shen, Lei; Zhu, Jintao

    2016-02-01

    The nonspecific adsorption of proteins is usually undesirable on solid surfaces as it induces adverse responses, such as platelet adhesion on medical devices, negative signals of biosensors and contamination blockage of filtration membranes. Thus, an important scheme in material science is to design and fabricate protein-repulsive surfaces. Early approaches in this field focused on homogeneous surfaces comprised of single type functionality. Yet, recent researches have demonstrated that surfaces with heterogeneities (chemistry and topography) show promising performance against protein adsorption. In this review, we will summarize the recent achievements and discuss the new perspectives in the research of developing and characterizing heterogeneous surfaces to repel proteins. The protein repulsion mechanisms of different heterogeneous surfaces will also be discussed in details, followed by the perspective and challenge of this emerging field. PMID:26691416

  1. 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.

  2. cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences

    SciTech Connect

    Stubbs, J.D.; Bui, A. San Francisco State Univ., CA ); Lekutis, C.; Singer, K.L.; Srinivasan, U.; Parry, G. ); Yuzuki, D. )

    1990-11-01

    A 2.1-kilobase cDNA coding for a surface protein of mammary epithelial cells has been isolated from a mouse mammary gland {lambda}gt11 cDNA library. Sequence analysis of this cDNA reveals an open reading frame of 1,389 base pairs that defines a protein with a molecular mass of 51.5 dKa. Structural analysis of the predicted sequence identifies two putative functional domains of the protein: (i) an N-terminal cysteine-rich region that is similar to epidermal growth factor-like domains of Drosophila Notch-1 protein and (ii) a large segment of the sequence that exhibited 54.5% identify with C-terminal domains of human coagulation factors VIII and V. These similarities in structure are used to predict the possible functions of the protein and its means of interaction with the cell surface. mRNA expression was detectable in mammary tissue from nonpregnant animals but was maximal in the lactating gland. In cultured cells, mRNA levels also correlated with the degree of cellular differentiation.

  3. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    SciTech Connect

    Feng, Yingang; Song, Xiaxia; Lin, Jinzhong; Xuan, Jinsong; Cui, Qiu; Wang, Jinfeng

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  4. Multifunctional proteins revealed by overlapping clustering in protein interaction network

    PubMed Central

    Chapple, Charles E.; Guénoche, Alain; Brun, Christine

    2012-01-01

    Motivation: Multifunctional proteins perform several functions. They are expected to interact specifically with distinct sets of partners, simultaneously or not, depending on the function performed. Current graph clustering methods usually allow a protein to belong to only one cluster, therefore impeding a realistic assignment of multifunctional proteins to clusters. Results: Here, we present Overlapping Cluster Generator (OCG), a novel clustering method which decomposes a network into overlapping clusters and which is, therefore, capable of correct assignment of multifunctional proteins. The principle of OCG is to cover the graph with initial overlapping classes that are iteratively fused into a hierarchy according to an extension of Newman's modularity function. By applying OCG to a human protein–protein interaction network, we show that multifunctional proteins are revealed at the intersection of clusters and demonstrate that the method outperforms other existing methods on simulated graphs and PPI networks. Availability: This software can be downloaded from http://tagc.univ-mrs.fr/welcome/spip.php?rubrique197 Contact: brun@tagc.univ-mrs.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22080466

  5. Silicon-wafer-surface damage revealed by surface photovoltage measurements

    NASA Astrophysics Data System (ADS)

    Goodman, Alvin M.

    1982-11-01

    Anomalous results of surface photovoltage (SPV) measurements on Si wafers are shown to be associated with a damaged region beneath the illuminated surface of the wafer being measured. The anomaly is a concave-upward curvature of the I0(α-1) plot with an r2 value, derived from linear regression analysis, less than the normally observed minimum value (˜0.98). Removal of the damaged region by an appropriate etching procedure allows subsequent SPV measurements whose results are substantially free of the previously observed anomaly. The qualitative character of the anomaly can be reproduced by a simple theoretical model in which only one effect of the damage is considered; this effect is a diminished quantum efficiency for hole-electron pair generation by photon absorption in the damaged region. The results suggest the use of SPV measurements as a test procedure for revealing the presence of surface damage in Si wafers.

  6. Genetic structure of Plasmodium vivax using the merozoite surface protein 1 icb5-6 fragment reveals new hybrid haplotypes in southern Mexico

    PubMed Central

    2014-01-01

    Background Plasmodium vivax is a protozoan parasite with an extensive worldwide distribution, being highly prevalent in Asia as well as in Mesoamerica and South America. In southern Mexico, P. vivax transmission has been endemic and recent studies suggest that these parasites have unique biological and genetic features. The msp1 gene has shown high rate of nucleotide substitutions, deletions, insertions, and its mosaic structure reveals frequent events of recombination, maybe between highly divergent parasite isolates. Methods The nucleotide sequence variation in the polymorphic icb5-6 fragment of the msp1 gene of Mexican and worldwide isolates was analysed. To understand how genotype diversity arises, disperses and persists in Mexico, the genetic structure and genealogical relationships of local isolates were examined. To identify new sequence hybrids and their evolutionary relationships with other P. vivax isolates circulating worldwide two haplotype networks were constructed questioning that two portions of the icb5-6 have different evolutionary history. Results Twelve new msp1 icb5-6 haplotypes of P. vivax from Mexico were identified. These nucleotide sequences show mosaic structure comprising three partially conserved and two variable subfragments and resulted into five different sequence types. The variable subfragment sV1 has undergone recombination events and resulted in hybrid sequences and the haplotype network allocated the Mexican haplotypes to three lineages, corresponding to the Sal I and Belem types, and other more divergent group. In contrast, the network from icb5-6 fragment but not sV1 revealed that the Mexican haplotypes belong to two separate lineages, none of which are closely related to Sal I or Belem sequences. Conclusions These results suggest that the new hybrid haplotypes from southern Mexico were the result of at least three different recombination events. These rearrangements likely resulted from the recombination between haplotypes of

  7. Whole genome sequencing of phage resistant Bacillus anthracis mutants reveals an essential role for cell surface anchoring protein CsaB in phage AP50c adsorption

    PubMed Central

    2012-01-01

    Background Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix. Methods Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection. Results Using whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains. Conclusions Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in

  8. Protein Structures Revealed at Record Pace

    ScienceCinema

    Greg Hura

    2010-01-08

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  9. Protein Structures Revealed at Record Pace

    ScienceCinema

    Hura, Greg

    2013-05-29

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  10. Protein Structures Revealed at Record Pace

    SciTech Connect

    Hura, Greg

    2009-01-01

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  11. Protein Structures Revealed at Record Pace

    SciTech Connect

    Greg Hura

    2009-07-09

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  12. Revealing protein dynamics by photobleaching techniques.

    PubMed

    van Drogen, Frank; Peter, Matthias

    2004-01-01

    Green fluorescent proteins (GFPs) are widely used tools to visualize proteins and study their intracellular distribution. One feature of working with GFP variants, photobleaching, has recently been combined with an older technique known as fluorescence recovery after photobleaching (FRAP) to study protein kinetics in vivo. During photobleaching, fluorochromes get destroyed irreversibly by repeated excitation with an intensive light source. When the photobleaching is applied to a restricted area or structure, recovery of fluorescence will be the result of active or passive diffusion from fluorescent molecules from unbleached surrounding areas. Fluorescence loss in photobleaching (FLIP) is a variant of FRAP where an area is bleached, and loss of fluorescence in surrounding areas is observed. FLIP can be used to study the dynamics of different pools of a protein or can show how a protein diffuses, or is transported through a cell or cellular structure. Here, we discuss these photobleaching fluorescent imaging techniques, illustrated with examples of these techniques applied to proteins of the Saccharomyces cerevisiae pheromone response MAPK pathway. PMID:15173624

  13. Optical tweezers reveal how proteins alter replication

    NASA Astrophysics Data System (ADS)

    Chaurasiya, Kathy

    Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

  14. Nanoscopy Reveals Surface-Metallic Black Phosphorus

    NASA Astrophysics Data System (ADS)

    Abate, Yohannes

    Nanolayer and two-dimensional (2D) materials............. 1 such as graphene... 2,3 , boron nitride... 1,4 , transition metal dichalcogenides... 1 , 5 - 8 (TMDCs), and black phosphorus (BP)... 1 , 9 - 13 have intriguing fundamental physical properties and bear promise of important applications in electronics and optics... 9 , 14 , 15 . Of them, BP... 11 , 12 , 16 is a novel layered material that has been theoretically predicted... 10 to acquire plasmonic behavior for frequencies below ~0.4 eV when highly doped. The electronic properties of BP are unique due to its anisotropic structure . Advantages of BP as a material for nanoelectronics and nanooptics are due to the fact that, in contrast to metals, the free carrier density in it can be dynamically controlled by chemical or electrostatic gating, which has been demonstrated by its use in field-effect transistors.... 9 , 14 , 15 Despite all the interest that BP attracts, near-field and plasmonic properties of BP have not yet been investigated experimentally. Here we report the first observation of nanoscopic near-field properties of BP. We have discovered near-field patterns of outside bright fringes and high surface polarizability of nanofilm BP consistent with its surface-metallic, plasmonic behavior at mid-infrared (mid-IR) frequencies below critical frequency ωm ~ 1176 cm -1 . This has allowed us to estimate plasma frequency ωp ~ 0 . 4 eV, carrier density n ~ 1 . 1 × 1011 nm-1 and the thickness of the surface metallic layer of ~ 1 nm . We have also observed similar behavior in other nanolayer semiconductors such as TMDC MoS 2 and topological insulator Bi 2 Te 3 but not in insulators such as boron nitride. This new phenomenon is attributed to surface band-bending and charging of the semiconductor nanofilms. The surface plasmonic behavior has been found for 10-40 nm BP thickness but absent for 4 nm BP thickness. This discovery opens up a new field of research and potential applications in nanoelectronics

  15. Dawn at Ceres reveals an ammoniated surface

    NASA Astrophysics Data System (ADS)

    Pieters, Carle M.; De Sanctis, Maria Cristina; Ammannito, Eleonora; Raponi, Andrea; Combe, Jean-Philippe; McCord, Thomas B.; McSween, Harry; McFadden, Lucy A.; Marchi, Simone; Capaccioni, Fabrizio; Capria, Maria Teresa; Carrozzo, Giacomo; Ciarniello, Mauro; Longobardo, Andrea; Fonte, Sergio; Formisano, Michelangelo; Frigeri, Alessandro; Giardino, Marco; Magni, Gianfranco; Palomba, Enersto; Tosi, Federico; Turrini, Diego; Zambon, Francesca; Jaumann, Ralf; Feldman, William; Prettyman, Thomas; Toplis, Michael; Raymond, Carol A.; Russell, Christopher T.

    2015-11-01

    The Visible and Infrared Mapping Spectrometer (VIR) on board the Dawn spacecraft has observed Ceres’ surface and acquired spectra (0.5 to 5 µm) since January 2015. Here we report the average Ceres spectrum, including the important spectral range (2.6-2.9 µm) previously precluded from (telescopic) measurements due to telluric atmospheric absorptions. The VIR data confirm that the surface is very dark with an average albedo of 0.090 ±0.006 at 0.55 µm, consistent with Hubble Space Telescope data (Li et al., Icarus, 2006) and contains no prominent absorption features in the visible and near-Infrared at wavelengths less than 2.5 µm. Ceres’ average spectrum, however, is characterized by a prominent diagnostic absorption band at 2.7 µm along with weaker absorption bands observed between 3.05-3.1, 3.3-3.4 and 3.9-4 µm. We modeled the new VIR spectra of Ceres with various ices, meteorites, silicates, carbonates, and hydrates using Hapke theory. Results of the spectral modeling indicate that extensive water ice is not present in spectra representing the typical surface acquired to date at relatively low spatial resolution (<11 km/pixel). The best fit is obtained with a mixture of ammoniated phyllosilicates mixed with other clays, Mg-carbonates, serpentine, and a strongly absorbing material, such as magnetite (De Sanctis et al., Nature, 2015, in review). The presence of ammonia-bearing materials in the crust across much of the surface has implications for the origin of Ceres and its internal structure and evolution. At the time of this presentation the Dawn spacecraft will have also completed its high altitude mapping orbit to look for anticipated small-scale mineralogy variations across this remarkable dwarf planet.Acknowledgements: VIR is funded by the Italian Space Agency-ASI and was developed under the leadership of INAF, Rome-Italy. The instrument was built by Selex-Galileo, Florence-Italy. The analyses are supported by ASI, NASA, and the German Space Agency

  16. Synthetic protein interactions reveal a functional map of the cell

    PubMed Central

    Berry, Lisa K; Ólafsson, Guðjón; Ledesma-Fernández, Elena; Thorpe, Peter H

    2016-01-01

    To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001 PMID:27098839

  17. Glycobiology of surface layer proteins.

    PubMed

    Schäffer, C; Messner, P

    2001-07-01

    Over the last two decades, a significant change of perception has taken place regarding prokaryotic glycoproteins. For many years, protein glycosylation was assumed to be limited to eukaryotes; but now, a wealth of information on structure, function, biosynthesis and molecular biology of prokaryotic glycoproteins has accumulated, with surface layer (S-layer) glycoproteins being one of the best studied examples. With the designation of Archaea as a second prokaryotic domain of life, the occurrence of glycosylated S-layer proteins had been considered a taxonomic criterion for differentiation between Bacteria and Archaea. Extensive structural investigations, however, have demonstrated that S-layer glycoproteins are present in both domains. Among Gram-positive bacteria, S-layer glycoproteins have been identified only in bacilli. In Gram-negative organisms, their presence is still not fully investigated; presently, there is no indication for their existence in this class of bacteria. Extensive biochemical studies of the S-layer glycoprotein from Halobacterium halobium have, at least in part, unravelled the glycosylation pathway in Archaea; molecular biological analyses of these pathways have not been performed, so far. Significant observations concern the occurrence of unusual linkage regions both in archaeal and bacterial S-layer glycoproteins. Regarding S-layer glycoproteins of bacteria, first genetic data have shed some light into the molecular organization of the glycosylation machinery in this domain. In addition to basic S-layer glycoprotein research, the biotechnological application potential of these molecules has been explored. With the development of straightforward molecular biological methods, fascinating possibilities for the expression of prokaryotic glycoproteins will become available. S-layer glycoprotein research has opened up opportunities for the production of recombinant glycosylation enzymes and tailor-made S-layer glycoproteins in large quantities

  18. Mosaic nature of the wolbachia surface protein.

    PubMed

    Baldo, Laura; Lo, Nathan; Werren, John H

    2005-08-01

    Lateral gene transfer and recombination play important roles in the evolution of many parasitic bacteria. Here we investigate intragenic recombination in Wolbachia bacteria, considered among the most abundant intracellular bacteria on earth. We conduct a detailed analysis of the patterns of variation and recombination within the Wolbachia surface protein, utilizing an extensive set of published and new sequences from five main supergroups of Wolbachia. Analysis of nucleotide and amino acid sequence variations confirms four hypervariable regions (HVRs), separated by regions under strong conservation. Comparison of shared polymorphisms reveals a complex mosaic structure of the gene, characterized by a clear intragenic recombining of segments among several distinct strains, whose major recombination effect is shuffling of a relatively conserved set of amino acid motifs within each of the four HVRs. Exchanges occurred both within and between the arthropod supergroups. Analyses based on phylogenetic methods and a specific recombination detection program (MAXCHI) significantly support this complex partitioning of the gene, indicating a chimeric origin of wsp. Although wsp has been widely used to define macro- and microtaxonomy among Wolbachia strains, these results clearly show that it is not suitable for this purpose. The role of wsp in bacterium-host interactions is currently unknown, but results presented here indicate that exchanges of HVR motifs are favored by natural selection. Identifying host proteins that interact with wsp variants should help reveal how these widespread bacterial parasites affect and evolve in response to the cellular environments of their invertebrate hosts. PMID:16030235

  19. Metal complexes as "protein surface mimetics".

    PubMed

    Hewitt, Sarah H; Wilson, Andrew J

    2016-07-28

    A key challenge in chemical biology is to identify small molecule regulators for every single protein. However, protein surfaces are notoriously difficult to recognise with synthetic molecules, often having large flat surfaces that are poorly matched to traditional small molecules. In the surface mimetic approach, a supramolecular scaffold is used to project recognition groups in such a manner as to make multivalent non-covalent contacts over a large area of protein surface. Metal based supramolecular scaffolds offer unique advantages over conventional organic molecules for protein binding, including greater stereochemical and geometrical diversity conferred through the metal centre and the potential for direct assessment of binding properties and even visualisation in cells without recourse to further functionalisation. This feature article will highlight the current state of the art in protein surface recognition using metal complexes as surface mimetics. PMID:27353704

  20. Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily.

    PubMed

    Yeung, Ho; Squire, Christopher J; Yosaatmadja, Yuliana; Panjikar, Santosh; López, Gemma; Molina, Antonio; Baker, Edward N; Harris, Paul W R; Brimble, Margaret A

    2016-07-01

    Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63-residue member of this family, snakin-1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin-1, determined by a novel combination of racemic protein crystallization and radiation-damage-induced phasing (RIP), is reported. Racemic crystals of snakin-1 and quasi-racemic crystals incorporating an unnatural 4-iodophenylalanine residue were prepared from chemically synthesized d- and l-proteins. Breakage of the C-I bonds in the quasi-racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces. PMID:27145301

  1. Characterizing the statistical properties of protein surfaces

    NASA Astrophysics Data System (ADS)

    Bak, Ji Hyun; Bitbol, Anne-Florence; Bialek, William

    Proteins and their interactions form the body of the signaling transduction pathway in many living systems. In order to ensure the accuracy as well as the specificity of signaling, it is crucial that proteins recognize their correct interaction partners. How difficult, then, is it for a protein to discriminate its correct interaction partner(s) from the possibly large set of other proteins it may encounter in the cell? An important ingredient of recognition is shape complementarity. The ensemble of protein shapes should be constrained by the need for maintaining functional interactions while avoiding spurious ones. To address this aspect of protein recognition, we consider the ensemble of proteins in terms of the shapes of their surfaces. We take into account the high-resolution structures of E.coli non-DNA-binding cytoplasmic proteins, retrieved from the Protein Data Bank. We aim to characterize the statistical properties of the protein surfaces at two levels: First, we study the intrinsic dimensionality at the level of the ensemble of the surface objects. Second, at the level of the individual surfaces, we determine the scale of shape variation. We further discuss how the dimensionality of the shape space is linked to the statistical properties of individual protein surfaces. Jhb and WB acknowledge support from National Science Foundation Grants PHY-1305525 and PHY-1521553. AFB acknowledges support from the Human Frontier Science Program.

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

    PubMed

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

    2016-04-21

    The addition of three N-terminal histidines to β-lactamase inhibitor protein was shown experimentally to increase its binding potency to an Au(111) surface substantially but the binding mechanism was not resolved. Here, we propose a complete adsorption mechanism for this fusion protein by means of a multi-scale simulation approach and free energy calculations. We find that adsorption is a three-step process: (i) recognition of the surface predominantly by the histidine fusion peptide and formation of an encounter complex facilitated by a reduced dielectric screening of water in the interfacial region, (ii) adsorption of the protein on the surface and adoption of a specific binding orientation, and (iii) adaptation of the protein structure on the metal surface accompanied by induced fit. We anticipate that the mechanistic features of protein adsorption to an Au(111) surface revealed here can be extended to other inorganic surfaces and proteins and will therefore aid the design of specific protein-surface interactions. PMID:27021898

  3. Protein Binding for Detection of Small Changes on Nanoparticle Surface

    PubMed Central

    Zeng, Shang; Huang, Yu-ming M.; Chang, Chia-en A.; Zhong, Wenwan

    2014-01-01

    Protein adsorption on nanoparticles is closely associated with the physicochemical properties of particles, in particular, their surface property. We synthesized two batches of polyacrylic acid-coated nanoparticles under almost identical conditions except for heating duration and found differences in the head-group structure of the polyacrylic acid. The structure change was confirmed by NMR and MS. The two batches of particles had varied binding affinities to a selected group of proteins. Computational work confirmed that the head group of the polymer on the surface of a nanoparticle could directly interact with a protein, and small structural changes in the head group were sufficient to result in a significant difference in the free energy of binding. Our results demonstrate that protein adsorption is so sensitive to the surface property of particles that it can reveal even small variations in the structure of a nanoparticle surface ligand, and should be useful for quick assessment of nanoparticle properties. PMID:24482794

  4. Osmolyte perturbation reveals conformational equilibria in spin-labeled proteins

    PubMed Central

    López, Carlos J; Fleissner, Mark R; Guo, Zhefeng; Kusnetzow, Ana K; Hubbell, Wayne L

    2009-01-01

    Recent evidence suggests that proteins at equilibrium can exist in a manifold of conformational substates, and that these substates play important roles in protein function. Therefore, there is great interest in identifying regions in proteins that are in conformational exchange. Electron paramagnetic resonance spectra of spin-labeled proteins containing the nitroxide side chain (R1) often consist of two (or more) components that may arise from slow exchange between conformational substates (lifetimes > 100 ns). However, crystal structures of proteins containing R1 have shown that multicomponent spectra can also arise from equilibria between rotamers of the side chain itself. In this report, it is shown that these scenarios can be distinguished by the response of the system to solvent perturbation with stabilizing osmolytes such as sucrose. Thus, site-directed spin labeling (SDSL) emerges as a new tool to explore slow conformational exchange in proteins of arbitrary size, including membrane proteins in a native-like environment. Moreover, equilibrium between substates with even modest differences in conformation is revealed, and the simplicity of the method makes it suitable for facile screening of multiple proteins. Together with previously developed strategies for monitoring picosecond to millisecond backbone dynamics, the results presented here expand the timescale over which SDSL can be used to explore protein flexibility. PMID:19585559

  5. Protein-RNA networks revealed through covalent RNA marks

    PubMed Central

    Lapointe, Christopher P.; Wilinski, Daniel; Saunders, Harriet A. J.; Wickens, Marvin

    2015-01-01

    Protein-RNA networks are ubiquitous and central in biological control. We present an approach, termed “RNA Tagging,” that identifies protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or crosslinking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA using high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The method revealed that while RNA-binding proteins productively bind specific RNAs to control their function, they also “sample” RNAs without exerting a regulatory effect. We exploited the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. The RNA Tagging approach is well-suited to detect and analyze protein-RNA networks in vivo. PMID:26524240

  6. Osmolyte perturbation reveals conformational equilibria in spin-labeled proteins.

    PubMed

    López, Carlos J; Fleissner, Mark R; Guo, Zhefeng; Kusnetzow, Ana K; Hubbell, Wayne L

    2009-08-01

    Recent evidence suggests that proteins at equilibrium can exist in a manifold of conformational substates, and that these substates play important roles in protein function. Therefore, there is great interest in identifying regions in proteins that are in conformational exchange. Electron paramagnetic resonance spectra of spin-labeled proteins containing the nitroxide side chain (R1) often consist of two (or more) components that may arise from slow exchange between conformational substates (lifetimes > 100 ns). However, crystal structures of proteins containing R1 have shown that multicomponent spectra can also arise from equilibria between rotamers of the side chain itself. In this report, it is shown that these scenarios can be distinguished by the response of the system to solvent perturbation with stabilizing osmolytes such as sucrose. Thus, site-directed spin labeling (SDSL) emerges as a new tool to explore slow conformational exchange in proteins of arbitrary size, including membrane proteins in a native-like environment. Moreover, equilibrium between substates with even modest differences in conformation is revealed, and the simplicity of the method makes it suitable for facile screening of multiple proteins. Together with previously developed strategies for monitoring picosecond to millisecond backbone dynamics, the results presented here expand the timescale over which SDSL can be used to explore protein flexibility. PMID:19585559

  7. Surface waves of Min-proteins

    NASA Astrophysics Data System (ADS)

    Fischer-Friedrich, Elisabeth; Nguyen van yen, Romain; Kruse, Karsten

    2007-03-01

    In the bacterium Escherichia coli, the Min-proteins show pronounced pole-to-pole oscillations. They are functional for suppressing cell division at the cell ends, leaving the center as the only possible site for division. Analyzing different models of Min-protein dynamics in a bacterial geometry, we find waves on the cytoplasmic membrane. Interestingly, the surface wave solutions of different models belong to different symmetry classes. We suggest that experiments on Min-protein surface waves in vitro are helpful in distinguishing between different classes of models of Min-protein dynamics.

  8. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

    PubMed

    Shankaran, Mahalakshmi; King, Chelsea L; Angel, Thomas E; Holmes, William E; Li, Kelvin W; Colangelo, Marc; Price, John C; Turner, Scott M; Bell, Christopher; Hamilton, Karyn L; Miller, Benjamin F; Hellerstein, Marc K

    2016-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  9. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

    PubMed Central

    Shankaran, Mahalakshmi; King, Chelsea L.; Angel, Thomas E.; Holmes, William E.; Li, Kelvin W.; Colangelo, Marc; Price, John C.; Turner, Scott M.; Bell, Christopher; Hamilton, Karyn L.; Miller, Benjamin F.; Hellerstein, Marc K.

    2015-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  10. Characterizing the statistical properties of protein surfaces

    NASA Astrophysics Data System (ADS)

    Bak, Ji Hyun; Bitbol, Anne-Florence; Bialek, William

    2015-03-01

    Proteins and their interactions form the body of the signaling transduction pathway in many living systems. In order to ensure the accuracy as well as the specificity of signaling, it is crucial that proteins recognize their correct interaction partners. How difficult, then, is it for a protein to discriminate its correct interaction partner(s) from the possibly large set of other proteins it may encounter in the cell? An important ingredient of recognition is shape complementarity. The ensemble of protein shapes should be constrained by the need for maintaining functional interactions while avoiding spurious ones. To address this aspect of protein recognition, we consider the ensemble of proteins in terms of their three-dimensional shapes, more precisely in terms of their solvent-excluded surfaces. We take into account all high-resolution structures from E.coli non-DNA-binding cytoplasmic proteins that can be retrieved from the Protein Data Bank. We aim to characterize the statistical properties of the ensemble of protein surfaces, including the dimensionality of the space of surfaces.

  11. Protein Folding and Misfolding on Surfaces

    PubMed Central

    Stefani, Massimo

    2008-01-01

    Protein folding, misfolding and aggregation, as well as the way misfolded and aggregated proteins affects cell viability are emerging as key themes in molecular and structural biology and in molecular medicine. Recent advances in the knowledge of the biophysical basis of protein folding have led to propose the energy landscape theory which provides a consistent framework to better understand how a protein folds rapidly and efficiently to the compact, biologically active structure. The increased knowledge on protein folding has highlighted its strict relation to protein misfolding and aggregation, either process being in close competition with the other, both relying on the same physicochemical basis. The theory has also provided information to better understand the structural and environmental factors affecting protein folding resulting in protein misfolding and aggregation into ordered or disordered polymeric assemblies. Among these, particular importance is given to the effects of surfaces. The latter, in some cases make possible rapid and efficient protein folding but most often recruit proteins/peptides increasing their local concentration thus favouring misfolding and accelerating the rate of nucleation. It is also emerging that surfaces can modify the path of protein misfolding and aggregation generating oligomers and polymers structurally different from those arising in the bulk solution and endowed with different physical properties and cytotoxicities. PMID:19330090

  12. Surface and tribological properties of seed proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aqueous solutions of oat and lupin proteins were investigated for their surface, interfacial, friction and wear properties. The investigated oat proteins included those that were also chemically modified using a variety of methods (acetylation, succinylation, x-linking) and combinations of methods....

  13. Surface protein of a Streptococcus agalactiae isolate.

    PubMed Central

    de Cueninck, B J

    1979-01-01

    A Streptococcus agalactiae isolate of bovine origin was cultured in broth; log-phase cells were washed and radioiodinated and subsequently extracted at low pH in the presence of a nonionic detergent. A protein antigen was purified from concentrated extract by ultracentrifugation, gel filtration, and ion-exchange chromatography. The molecular weight of the protein was estimated at 31,800. The agglutinogenic character of the protein indicated its localization at the cell surface. Images PMID:381197

  14. The surface modification of stainless steel and the correlation between the surface properties and protein adsorption.

    PubMed

    Kang, Chan-Koo; Lee, Yoon-Sik

    2007-07-01

    Protein adsorption on a biomaterial surface is of great importance as it usually induces unfavorable biological cascades, with the result that much surface modification research has had to be performed in an effort to prevent this. In this study, we developed surface modification methods for stainless steel, which is a representative metal for biomedical device. The stainless steels were first smoothened to different extents by electropolishing, in order to obtain a rough or smooth surface. On these two kinds of substrates, we introduced epoxide groups to the metal surface by silanization with 3-glycidoxypropyltrimethoxysilane (GPTS). Then, various polymers such as poly(ethylene glycol) (PEG), poly(tetrahydrofuran glycol) (PTG), poly(propylene glycol) (PPG) and poly(dimethylsiloxane) (PDMS) were grafted on the silanized stainless steels. Each surface modification step was confirmed by various analytical methods. Contact angle measurement revealed that the surface hydrophilicity was controllable by polymer grafting. Root-mean-square (RMS) data of atomic force microscopy showed that surface roughness was dramatically changed by electropolishing. Based on these results, the correlation between surface properties and protein adsorption was investigated. In the protein adsorption study, we observed that all of the polymer-grafted stainless steels exhibited lower protein adsorption, when compared with bare stainless steel. Moreover, a hydrophilic and smooth surface was found to be the best of choice for decreasing the protein adsorption. PMID:17277988

  15. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein

    SciTech Connect

    Dooley, J.S.G.; Trust, T.J.

    1988-02-01

    The surface protein composition of members of a serogroup of Aeromonas hydrophila was examined. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed a 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase /sup 125/I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size required for A. hydrophila S-layer anchoring. The native S layer was permeable to /sup 125/I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behavior characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein.

  16. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins.

    PubMed

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina; Cruz-Zaragoza, Luis Daniel; Garcia, Esther; Clausen, Mathias P; Schliebs, Wolfgang; Erdmann, Ralf; Eggeling, Christian

    2016-08-12

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future. PMID:27311714

  17. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins*

    PubMed Central

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina; Cruz-Zaragoza, Luis Daniel; Garcia, Esther; Clausen, Mathias P.; Schliebs, Wolfgang; Erdmann, Ralf; Eggeling, Christian

    2016-01-01

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future. PMID:27311714

  18. Silk protein aggregation kinetics revealed by Rheo-IR.

    PubMed

    Boulet-Audet, Maxime; Terry, Ann E; Vollrath, Fritz; Holland, Chris

    2014-02-01

    The remarkable mechanical properties of silk fibres stem from a multi-scale hierarchical structure created when an aqueous protein "melt" is converted to an insoluble solid via flow. To directly relate a silk protein's structure and function in response to flow, we present the first application of a Rheo-IR platform, which couples cone and plate rheology with attenuated total reflectance infrared spectroscopy. This technique provides a new window into silk processing by linking shear thinning to an increase in molecular alignment, with shear thickening affecting changes in the silk protein's secondary structure. Additionally, compared to other static characterization methods for silk, Rheo-IR proved particularly useful at revealing the intrinsic difference between natural (native) and reconstituted silk feedstocks. Hence Rheo-IR offers important novel insights into natural silk processing. This has intrinsic academic merit, but it might also be useful when designing reconstituted silk analogues alongside other polymeric systems, whether natural or synthetic. PMID:24200713

  19. Effect of the interplay between protein and surface on the properties of adsorbed protein layers.

    PubMed

    Ouberai, Myriam M; Xu, Kairuo; Welland, Mark E

    2014-08-01

    Although protein adsorption to surface is a common phenomenon, investigation of the process is challenging due to the complexity of the interplay between external factors, protein and surface properties. Therefore experimental approaches have to measure the properties of adsorbed protein layers with high accuracy in order to achieve a comprehensive description of the process. To this end, we used a combination of two biosensing techniques, dual polarization interferometry and quartz crystal microbalance with dissipation. From this, we are able to extract surface coverage values, layer structural parameters, water content and viscoelastic properties to examine the properties of protein layers formed at the liquid/solid interface. Layer parameters were examined upon adsorption of proteins of varying size and structural properties, on surfaces with opposite polarity. We show that "soft" proteins such as unfolded α-synuclein and high molecular weight albumin are highly influenced by the surface polarity, as they form a highly diffuse and hydrated layer on the hydrophilic silica surface as opposed to the denser, less hydrated layer formed on a hydrophobic methylated surface. These layer properties are a result of different orientations and packing of the proteins. By contrast, lysozyme is barely influenced by the surface polarity due to its intrinsic structural stability. Interestingly, we show that for a similar molecular weight, the unfolded α-synuclein forms a layer with the highest percentage of solvation not related to surface coverage but resulting from the highest water content trapped within the protein. Together, these data reveal a trend in layer properties highlighting the importance of the interplay between protein and surface for the design of biomaterials. PMID:24780165

  20. 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

  1. Rhabdovirus Matrix Protein Structures Reveal a Novel Mode of Self-Association

    PubMed Central

    Graham, Stephen C.; Verma, Anil; Gholami, Alireza; Talbi, Chiraz; Owens, Raymond J.; Stuart, David I.; Grimes, Jonathan M.; Bourhy, Hervé

    2008-01-01

    The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins. PMID:19112510

  2. Adhesion of mussel foot proteins to different substrate surfaces

    PubMed Central

    Lu, Qingye; Danner, Eric; Waite, J. Herbert; Israelachvili, Jacob N.; Zeng, Hongbo; Hwang, Dong Soo

    2013-01-01

    Mussel foot proteins (mfps) have been investigated as a source of inspiration for the design of underwater coatings and adhesives. Recent analysis of various mfps by a surface forces apparatus (SFA) revealed that mfp-1 functions as a coating, whereas mfp-3 and mfp-5 resemble adhesive primers on mica surfaces. To further refine and elaborate the surface properties of mfps, the force–distance profiles of the interactions between thin mfp (i.e. mfp-1, mfp-3 or mfp-5) films and four different surface chemistries, namely mica, silicon dioxide, polymethylmethacrylate and polystyrene, were measured by an SFA. The results indicate that the adhesion was exquisitely dependent on the mfp tested, the substrate surface chemistry and the contact time. Such studies are essential for understanding the adhesive versatility of mfps and related/similar adhesion proteins, and for translating this versatility into a new generation of coatings and (including in vivo) adhesive materials. PMID:23173195

  3. Designed protein reveals structural determinants of extreme kinetic stability

    PubMed Central

    Broom, Aron; Ma, S. Martha; Xia, Ke; Rafalia, Hitesh; Trainor, Kyle; Colón, Wilfredo; Gosavi, Shachi; Meiering, Elizabeth M.

    2015-01-01

    The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge. PMID:26554002

  4. A Mass Spectrometric-Derived Cell Surface Protein Atlas

    PubMed Central

    Bausch-Fluck, Damaris; Hofmann, Andreas; Bock, Thomas; Frei, Andreas P.; Cerciello, Ferdinando; Jacobs, Andrea; Moest, Hansjoerg; Omasits, Ulrich; Gundry, Rebekah L.; Yoon, Charles; Schiess, Ralph; Schmidt, Alexander; Mirkowska, Paulina; Härtlová, Anetta; Van Eyk, Jennifer E.; Bourquin, Jean-Pierre; Aebersold, Ruedi; Boheler, Kenneth R.; Zandstra, Peter; Wollscheid, Bernd

    2015-01-01

    Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments. PMID:25894527

  5. Function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations

    SciTech Connect

    Nutt, David; Smith, Jeremy C

    2008-10-01

    Atomistic molecular dynamics simulations are used to investigate the mechanism by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, binds to ice. Comparison of structural and dynamic properties of the water around the three faces of the triangular prism-shaped protein in aqueous solution reveals that at low temperature the water structure is ordered and the dynamics slowed down around the ice-binding face of the protein, with a disordering effect observed around the other two faces. These results suggest a dual role for the solvation water around the protein. The preconfigured solvation shell around the ice-binding face is involved in the initial recognition and binding of the antifreeze protein to ice by lowering the barrier for binding and consolidation of the protein:ice interaction surface. Thus, the antifreeze protein can bind to the molecularly rough ice surface by becoming actively involved in the formation of its own binding site. Also, the disruption of water structure around the rest of the protein helps prevent the adsorbed protein becoming covered by further ice growth.

  6. A soft solid surface on Titan as revealed by the Huygens Surface Science Package

    NASA Astrophysics Data System (ADS)

    Zarnecki, John C.; Leese, Mark R.; Hathi, Brijen; Ball, Andrew J.; Hagermann, Axel; Towner, Martin C.; Lorenz, Ralph D.; McDonnell, J. Anthony M.; Green, Simon F.; Patel, Manish R.; Ringrose, Timothy J.; Rosenberg, Philip D.; Atkinson, Karl R.; Paton, Mark D.; Banaszkiewicz, Marek; Clark, Benton C.; Ferri, Francesca; Fulchignoni, Marcello; Ghafoor, Nadeem A. L.; Kargl, Günter; Svedhem, Håkan; Delderfield, John; Grande, Manuel; Parker, David J.; Challenor, Peter G.; Geake, John E.

    2005-12-01

    The surface of Saturn's largest satellite-Titan-is largely obscured by an optically thick atmospheric haze, and so its nature has been the subject of considerable speculation and discussion. The Huygens probe entered Titan's atmosphere on 14 January 2005 and descended to the surface using a parachute system. Here we report measurements made just above and on the surface of Titan by the Huygens Surface Science Package. Acoustic sounding over the last 90m above the surface reveals a relatively smooth, but not completely flat, surface surrounding the landing site. Penetrometry and accelerometry measurements during the probe impact event reveal that the surface was neither hard (like solid ice) nor very compressible (like a blanket of fluffy aerosol); rather, the Huygens probe landed on a relatively soft solid surface whose properties are analogous to wet clay, lightly packed snow and wet or dry sand. The probe settled gradually by a few millimetres after landing.

  7. A soft solid surface on Titan as revealed by the Huygens Surface Science Package.

    PubMed

    Zarnecki, John C; Leese, Mark R; Hathi, Brijen; Ball, Andrew J; Hagermann, Axel; Towner, Martin C; Lorenz, Ralph D; McDonnell, J Anthony M; Green, Simon F; Patel, Manish R; Ringrose, Timothy J; Rosenberg, Philip D; Atkinson, Karl R; Paton, Mark D; Banaszkiewicz, Marek; Clark, Benton C; Ferri, Francesca; Fulchignoni, Marcello; Ghafoor, Nadeem A L; Kargl, Günter; Svedhem, Håkan; Delderfield, John; Grande, Manuel; Parker, David J; Challenor, Peter G; Geake, John E

    2005-12-01

    The surface of Saturn's largest satellite--Titan--is largely obscured by an optically thick atmospheric haze, and so its nature has been the subject of considerable speculation and discussion. The Huygens probe entered Titan's atmosphere on 14 January 2005 and descended to the surface using a parachute system. Here we report measurements made just above and on the surface of Titan by the Huygens Surface Science Package. Acoustic sounding over the last 90 m above the surface reveals a relatively smooth, but not completely flat, surface surrounding the landing site. Penetrometry and accelerometry measurements during the probe impact event reveal that the surface was neither hard (like solid ice) nor very compressible (like a blanket of fluffy aerosol); rather, the Huygens probe landed on a relatively soft solid surface whose properties are analogous to wet clay, lightly packed snow and wet or dry sand. The probe settled gradually by a few millimetres after landing. PMID:16319828

  8. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated.

    PubMed

    Chung, Daehwan; Young, Jenna; Bomble, Yannick J; Vander Wall, Todd A; Groom, Joseph; Himmel, Michael E; Westpheling, Janet

    2015-01-01

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure

  9. Homologous Expression of the Caldicellulosiruptor bescii CelA Reveals that the Extracellular Protein Is Glycosylated

    PubMed Central

    Bomble, Yannick J.; Vander Wall, Todd A.; Groom, Joseph; Himmel, Michael E.; Westpheling, Janet

    2015-01-01

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure

  10. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated

    DOE PAGESBeta

    Chung, Daehwan; Young, Jenna; Bomble, Yannick J.; Vander Wall, Todd A.; Groom, Joseph; Himmel, Michael E.; Westpheling, Janet

    2015-03-23

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm formore » cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. Finally, it will also allow the study of glycosylation of CelA itself and its role

  11. Quantification of Plasmodium falciparum malaria from complex infections in the Peruvian Amazon using quantitative PCR of the merozoite surface protein 1, block 2 (PfMSP1-B2): in vitro dynamics reveal density-dependent interactions.

    PubMed

    Zervos, Thomas M; Hernandez, Jean N; Sutton, Patrick L; Branch, Oralee H

    2012-05-01

    The majority of Plasmodium falciparum field isolates are defined as complex infections because they contain multiple genetically distinct clones. Studying interactions between clones in complex infections in vivo and in vitro could elucidate important phenomena in malaria infection, transmission and treatment. Using quantitative PCR (qPCR) of the P. falciparum merozoite surface protein 1, block 2 (PfMSP1-B2), we provide a sensitive and efficient genotyping method. This is important for epidemiological studies because it makes it possible to study genotype-specific growth dynamics. We compared 3 PfMSP1-B2 genotyping methods by analysing 79 field isolates from the Peruvian Amazon. In vivo observations from other studies using these techniques led to the hypothesis that clones within complex infections interact. By co-culturing clones with different PfMSP1-B2 genotypes, and measuring parasitaemia using qPCR, we found that suppression of clonal expansion was a factor of the collective density of all clones present in a culture. PfMSP1-B2 qPCR enabled us to find in vitro evidence for parasite-parasite interactions and could facilitate future investigations of growth trends in naturally occurring complex infections. PMID:22339946

  12. Visualizing Validation of Protein Surface Classifiers.

    PubMed

    Sarikaya, A; Albers, D; Mitchell, J; Gleicher, M

    2014-06-01

    Many bioinformatics applications construct classifiers that are validated in experiments that compare their results to known ground truth over a corpus. In this paper, we introduce an approach for exploring the results of such classifier validation experiments, focusing on classifiers for regions of molecular surfaces. We provide a tool that allows for examining classification performance patterns over a test corpus. The approach combines a summary view that provides information about an entire corpus of molecules with a detail view that visualizes classifier results directly on protein surfaces. Rather than displaying miniature 3D views of each molecule, the summary provides 2D glyphs of each protein surface arranged in a reorderable, small-multiples grid. Each summary is specifically designed to support visual aggregation to allow the viewer to both get a sense of aggregate properties as well as the details that form them. The detail view provides a 3D visualization of each protein surface coupled with interaction techniques designed to support key tasks, including spatial aggregation and automated camera touring. A prototype implementation of our approach is demonstrated on protein surface classifier experiments. PMID:25342867

  13. Protein adsorption kinetics in different surface potentials

    NASA Astrophysics Data System (ADS)

    Quinn, A.; Mantz, H.; Jacobs, K.; Bellion, M.; Santen, L.

    2008-03-01

    We have studied the adsorption kinetics of the protein amylase at solid/liquid interfaces. Offering substrates with tailored properties, we are able to separate the impact of short- and long-range interactions. By means of a colloidal Monte Carlo approach including conformational changes of the adsorbed proteins induced by density fluctuations, we develop a scenario that is consistent with the experimentally observed three-step kinetics on specific substrates. Our observations show that not only the surface chemistry determines the properties of an adsorbed protein layer but also the van der Waals contributions of a composite substrate may lead to non-negligible effects.

  14. Surface energetics and protein-protein interactions: analysis and mechanistic implications.

    PubMed

    Peri, Claudio; Morra, Giulia; Colombo, Giorgio

    2016-01-01

    Understanding protein-protein interactions (PPI) at the molecular level is a fundamental task in the design of new drugs, the prediction of protein function and the clarification of the mechanisms of (dis)regulation of biochemical pathways. In this study, we use a novel computational approach to investigate the energetics of aminoacid networks located on the surface of proteins, isolated and in complex with their respective partners. Interestingly, the analysis of individual proteins identifies patches of surface residues that, when mapped on the structure of their respective complexes, reveal regions of residue-pair couplings that extend across the binding interfaces, forming continuous motifs. An enhanced effect is visible across the proteins of the dataset forming larger quaternary assemblies. The method indicates the presence of energetic signatures in the isolated proteins that are retained in the bound form, which we hypothesize to determine binding orientation upon complex formation. We propose our method, BLUEPRINT, as a complement to different approaches ranging from the ab-initio characterization of PPIs, to protein-protein docking algorithms, for the physico-chemical and functional investigation of protein-protein interactions. PMID:27050828

  15. Surface energetics and protein-protein interactions: analysis and mechanistic implications

    PubMed Central

    Peri, Claudio; Morra, Giulia; Colombo, Giorgio

    2016-01-01

    Understanding protein-protein interactions (PPI) at the molecular level is a fundamental task in the design of new drugs, the prediction of protein function and the clarification of the mechanisms of (dis)regulation of biochemical pathways. In this study, we use a novel computational approach to investigate the energetics of aminoacid networks located on the surface of proteins, isolated and in complex with their respective partners. Interestingly, the analysis of individual proteins identifies patches of surface residues that, when mapped on the structure of their respective complexes, reveal regions of residue-pair couplings that extend across the binding interfaces, forming continuous motifs. An enhanced effect is visible across the proteins of the dataset forming larger quaternary assemblies. The method indicates the presence of energetic signatures in the isolated proteins that are retained in the bound form, which we hypothesize to determine binding orientation upon complex formation. We propose our method, BLUEPRINT, as a complement to different approaches ranging from the ab-initio characterization of PPIs, to protein-protein docking algorithms, for the physico-chemical and functional investigation of protein-protein interactions. PMID:27050828

  16. Cation specific binding with protein surface charges.

    PubMed

    Hess, Berk; van der Vegt, Nico F A

    2009-08-11

    Biological organization depends on a sensitive balance of noncovalent interactions, in particular also those involving interactions between ions. Ion-pairing is qualitatively described by the law of "matching water affinities." This law predicts that cations and anions (with equal valence) form stable contact ion pairs if their sizes match. We show that this simple physical model fails to describe the interaction of cations with (molecular) anions of weak carboxylic acids, which are present on the surfaces of many intra- and extracellular proteins. We performed molecular simulations with quantitatively accurate models and observed that the order K(+) < Na(+) < Li(+) of increasing binding affinity with carboxylate ions is caused by a stronger preference for forming weak solvent-shared ion pairs. The relative insignificance of contact pair interactions with protein surfaces indicates that thermodynamic stability and interactions between proteins in alkali salt solutions is governed by interactions mediated through hydration water molecules. PMID:19666545

  17. 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

  18. Structure and sequence analyses of Bacteroides proteins BVU_4064 and BF1687 reveal presence of two novel predominantly-beta domains, predicted to be involved in lipid and cell surface interactions

    DOE PAGESBeta

    Natarajan, Padmaja; Punta, Marco; Kumar, Abhinav; Yeh, Andrew P; Godzik, Adam; Aravind, L.

    2015-01-16

    N-terminal domains of BVU_4064 and BF1687 proteins from Bacteroides vulgatus and Bacteroides fragilis respectively are members of the Pfam family PF12985 (DUF3869). Proteins containing a domain from this family can be found in most Bacteroides species and, in large numbers, in all human gut microbiome samples. Both BVU_4064 and BF1687 proteins have a consensus lipobox motif implying they are anchored to the membrane, but their functions are otherwise unknown. The C-terminal half of BVU_4064 is assigned to protein family PF12986 (DUF3870); the equivalent part of BF1687 was unclassified.

  19. Biomimetic surface coatings from modular amphiphilic proteins

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  20. Denaturation of proteins near polar surfaces.

    PubMed

    Starzyk, Anna; Cieplak, Marek

    2011-12-21

    All-atom molecular dynamics simulations for proteins placed near a model mica surface indicate existence of two types of evolution. One type leads to the surface-induced unfolding and the other just to a deformation. The two behaviors are characterized by distinct properties of the radius of gyration and of a novel distortion parameter that distinguishes between elongated, globular, and planar shapes. They also differ in the nature of their single site diffusion and two-site distance fluctuations. The four proteins chosen for the studies, the tryptophan cage, protein G, hydrophobin and lyzozyme, are small to allow for a fair determination of the forces generated by the surface as the effects of finite cutoffs in the Coulombic interactions are thus minimized. When the net charge on the surface is set to zero artificially, infliction of deformation is seen to persists but no unfolding takes place. Unfolding may also be prevented by a cluster of disulfide bonds, as we observe in simulations of hydrophobin. PMID:22191903

  1. Denaturation of proteins near polar surfaces

    NASA Astrophysics Data System (ADS)

    Starzyk, Anna; Cieplak, Marek

    2011-12-01

    All-atom molecular dynamics simulations for proteins placed near a model mica surface indicate existence of two types of evolution. One type leads to the surface-induced unfolding and the other just to a deformation. The two behaviors are characterized by distinct properties of the radius of gyration and of a novel distortion parameter that distinguishes between elongated, globular, and planar shapes. They also differ in the nature of their single site diffusion and two-site distance fluctuations. The four proteins chosen for the studies, the tryptophan cage, protein G, hydrophobin and lyzozyme, are small to allow for a fair determination of the forces generated by the surface as the effects of finite cutoffs in the Coulombic interactions are thus minimized. When the net charge on the surface is set to zero artificially, infliction of deformation is seen to persists but no unfolding takes place. Unfolding may also be prevented by a cluster of disulfide bonds, as we observe in simulations of hydrophobin.

  2. Electronic structure of bacterial surface protein layers

    SciTech Connect

    Maslyuk, Volodymyr V.; Mertig, Ingrid; Bredow, Thomas; Mertig, Michael; Vyalikh, Denis V.; Molodtsov, Serguei L.

    2008-01-15

    We report an approach for the calculation of the electronic density of states of the dried two-dimensional crystalline surface protein layer (S layer) of the bacterium Bacillus sphaericus NCTC 9602. The proposed model is based on the consideration of individual amino acids in the corresponding conformation of the peptide chain which additively contribute to the electronic structure of the entire protein complex. The derived results agree well with the experimental data obtained by means of photoemission (PE), resonant PE, and near-edge x-ray absorption spectroscopy.

  3. Stellar Spectroscopy during Exoplanet Transits: Revealing structures across stellar surfaces

    NASA Astrophysics Data System (ADS)

    Dravins, Dainis; Ludwig, Hans-Günter; Dahlén, Erik

    2015-08-01

    Exoplanet transits permit to study stellar surface portions that successively become hidden behind the planet. Differential spectroscopy between various transit phases reveals spectra of those stellar surface segments that were hidden. The deduced center-to-limb behavior of stellar spectral line shapes, asymmetries and wavelength shifts enables detailed tests of 3-dimensional hydrodynamic models of stellar atmospheres, such that are required for any precise determination of abundances or seismic properties. Such models can now be computed for widely different classes of stars (including metal-poor ones and white dwarfs), but have been feasible to test and verify only for the Sun with its resolved surface structure. Exoplanet transits may also occur across features such as starspots, whose magnetic signatures will be retrieved from spectra of sufficient fidelity.Knowing the precise background stellar spectra, also properties of exoplanet atmospheres are better constrained: e.g., the Rossiter-McLaughlin effect becomes resolved as not only a simple change of stellar wavelength, but as a variation of the full line profiles and their asymmetries.Such studies are challenging since exoplanets cover only a tiny fraction of the stellar disk. Current work, analyzing sequences of high-fidelity ESO UVES spectra, demonstrate that such spatially resolved stellar spectra can already be (marginally) retrieved in a few cases with the brightest host stars. Already in a near future, ongoing exoplanet surveys are likely to find further bright hosts that will enable such studies for various stellar types. http://arxiv.org/abs/1408.1402

  4. Characterization of the Eimeria maxima sporozoite surface protein IMP1.

    PubMed

    Jenkins, M C; Fetterer, R; Miska, K; Tuo, W; Kwok, O; Dubey, J P

    2015-07-30

    The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection. PMID:26012860

  5. Global transcriptional analysis reveals surface remodeling of Anaplasma marginale in the tick vector

    PubMed Central

    2014-01-01

    Background Pathogens dependent upon vectors for transmission to new hosts undergo environment specific changes in gene transcription dependent on whether they are replicating in the vector or the mammalian host. Differential gene transcription, especially of potential vaccine candidates, is of interest in Anaplasma marginale, the tick-borne causative agent of bovine anaplasmosis. Methods RNA-seq technology allowed a comprehensive analysis of the transcriptional status of A. marginale genes in two conditions: bovine host blood and tick derived cell culture, a model for the tick vector. Quantitative PCR was used to assess transcription of a set of genes in A. marginale infected tick midguts and salivary glands at two time points during the transmission cycle. Results Genes belonging to fourteen pathways or component groups were found to be differentially transcribed in A. marginale in the bovine host versus the tick vector. One of the most significantly altered groups was composed of surface proteins. Of the 56 genes included in the surface protein group, eight were up regulated and 26 were down regulated. The down regulated surface protein encoding genes include several that are well studied due to their immunogenicity and function. Quantitative PCR of a set of genes demonstrated that transcription in tick cell culture most closely approximates transcription in salivary glands of recently infected ticks. Conclusions The ISE6 tick cell culture line is an acceptable model for early infection in tick salivary glands, and reveals disproportionate down regulation of surface protein genes in the tick. Transcriptional profiling in other cell lines may help us simulate additional microenvironments. Understanding vector-specific alteration of gene transcription, especially of surface protein encoding genes, may aid in the development of vaccines or transmission blocking therapies. PMID:24751137

  6. Cell surface receptors for CCN proteins.

    PubMed

    Lau, Lester F

    2016-06-01

    The CCN family (CYR61; CTGF; NOV; CCN1-6; WISP1-3) of matricellular proteins in mammals is comprised of six homologous members that play important roles in development, inflammation, tissue repair, and a broad range of pathological processes including fibrosis and cancer. Despite considerable effort to search for a high affinity CCN-specific receptor akin to growth factor receptors, no such receptor has been found. Rather, CCNs bind several groups of multi-ligand receptors as characteristic of other matricellular proteins. The most extensively documented among CCN-binding receptors are integrins, including αvβ3, αvβ5, α5β1, α6β1, αIIbβ3, αMβ2, and αDβ2, which mediate diverse CCN functions in various cell types. CCNs also bind cell surface heparan sulfate proteoglycans (HSPGs), low density liproprotein receptor-related proteins (LRPs), and the cation-independent mannose-6-phosphate (M6P) receptor, which are endocytic receptors that may also serve as co-receptors in cooperation with other cell surface receptors. CCNs have also been reported to bind FGFR-2, Notch, RANK, and TrkA, potentially altering the affinities of these receptors for their ligands. The ability of CCNs to bind a multitude of receptors in various cell types may account for the remarkable versatility of their functions, and underscore the diverse signaling pathways that mediate their activities. PMID:27098435

  7. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    PubMed Central

    Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

    2015-01-01

    Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

  8. Identification and characterization of the surface proteins of Clostridium difficile

    SciTech Connect

    Dailey, D.C.

    1988-01-01

    Several clostridial proteins were detected on the clostridial cell surface by sensitive radioiodination techniques. Two major proteins and six minor proteins comprised the radioiodinated proteins on the clostridial cell surface. Cellular fractionation of surface radiolabeled C. difficile determined that the radioiodinated proteins were found in the cell wall fraction of C. difficile and surprisingly were also present in the clostridial membrane. Furthermore, an interesting phenomenon of disulfide-crosslinking of the cell surface proteins of C. difficile was observed. Disulfide-linked protein complexes were found in both the membrane and cell wall fractions. In addition, the cell surface proteins of C. difficile were found to be released into the culture medium. In attempts to further characterize the clostridial proteins recombinant DNA techniques were employed. In addition, the role of the clostridial cell surface proteins in the interactions of C. difficile with human PMNs was also investigated.

  9. Protein Flexibility in Docking and Surface Mapping

    PubMed Central

    Lexa, Katrina W.; Carlson, Heather A.

    2012-01-01

    Structure-based drug design has become an essential tool for rapid lead discovery and optimization. As available structural information has increased, researchers have become increasingly aware of the importance of protein flexibility for accurate description of the native state. Typical protein–ligand docking efforts still rely on a single rigid receptor, which is an incomplete representation of potential binding conformations of the protein. These rigid docking efforts typically show the best performance rates between 50 and 75%, while fully flexible docking methods can enhance pose prediction up to 80–95%. This review examines the current toolbox for flexible protein–ligand docking and receptor surface mapping. Present limitations and possibilities for future development are discussed. PMID:22569329

  10. Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions

    PubMed Central

    Shashikanth, Nitesh; Kisting, Meridith A.; Leckband, Deborah E.

    2016-01-01

    The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions. PMID:27009566

  11. Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions.

    PubMed

    Shashikanth, Nitesh; Kisting, Meridith A; Leckband, Deborah E

    2016-01-01

    The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions. PMID:27009566

  12. Activation of Nanoscale Allosteric Protein Domain Motion Revealed by Neutron Spin Echo Spectroscopy

    PubMed Central

    Farago, Bela; Li, Jianquan; Cornilescu, Gabriel; Callaway, David J.E.; Bu, Zimei

    2010-01-01

    NHERF1 is a multidomain scaffolding protein that assembles signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by the membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 Ångstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length-scales and on submicrosecond timescales upon forming a complex with ezrin. We show that a much-simplified coarse-grained model suffices to describe interdomain motion of a multidomain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. Our results demonstrate that the dynamic propagation of allosteric signals to distal sites involves changes in long-range coupled domain motions on submicrosecond timescales, and that these coupled motions can be distinguished and characterized by NSE. PMID:21081097

  13. Structural Analysis of a Complex between Small Ubiquitin-like Modifier 1 (SUMO1) and the ZZ Domain of CREB-binding Protein (CBP/p300) Reveals a New Interaction Surface on SUMO.

    PubMed

    Diehl, Carl; Akke, Mikael; Bekker-Jensen, Simon; Mailand, Niels; Streicher, Werner; Wikström, Mats

    2016-06-10

    We have recently discovered that the ZZ zinc finger domain represents a novel small ubiquitin-like modifier (SUMO) binding motif. In this study we identify the binding epitopes in the ZZ domain of CBP (CREB-binding protein) and SUMO1 using NMR spectroscopy. The binding site on SUMO1 represents a unique epitope for SUMO interaction spatially opposite to that observed for canonical SUMO interaction motifs (SIMs). HADDOCK docking simulations using chemical shift perturbations and residual dipolar couplings was employed to obtain a structural model for the ZZ domain-SUMO1 complex. Isothermal titration calorimetry experiments support this model by showing that the mutation of key residues in the binding site abolishes binding and that SUMO1 can simultaneously and non-cooperatively bind both the ZZ domain and a canonical SIM motif. The binding dynamics of SUMO1 was further characterized using (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersions, which define the off rates for the ZZ domain and SIM motif and show that the dynamic binding process has different characteristics for the two cases. Furthermore, in the absence of bound ligands SUMO1 transiently samples a high energy conformation, which might be involved in ligand binding. PMID:27129204

  14. The Electrophoretic Mobility of Proteins near Surfaces

    NASA Astrophysics Data System (ADS)

    Ramasamy, Perumal; Singh, Avtar; Rafailovich, Miriam; Sokolov, Jonathan

    2004-03-01

    We have attempted to apply the methods developed for surface DNA electrophoresis (1) for proteomics. Droplets of FITC stained Abumin, Poly- L-Lysine, or Casein purchased from Sigma were deposited on glass cover slips. The droplets were then place in contact with a TBE buffer solution contained in a cell molded from PDMS. Pt electrodes were inserted into the cell and a voltage was a applied. The motion of the protein was then imaged with a Leica Confocal microscope as a function of buffer concentration, distance from the surface, and applied voltage. The mobilities were then compared with those of uncharged one micron florescent Polystyrene beads. References: 1)Henzel WJ, Watanabe C, Stults JT., !0 Protein Identification: The Origins of Peptide Mass Fingerprinting. !1 J. American Society for Mass Spectrometry. 14 (September 2003): 931-942 2)Mathesius U, Imin N, Natera SH, Rolfe BG., !0 Proteomics as a functional genomics tool. !1 Methods of Molecular Biology 236: 395-414. *Work supported in part by the NSF-MRSEC program

  15. Applications of yeast surface display for protein engineering

    PubMed Central

    Cherf, Gerald M.; Cochran, Jennifer R.

    2015-01-01

    The method of displaying recombinant proteins on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein, a technology known as yeast surface display, or simply, yeast display, has become a valuable protein engineering tool for a broad spectrum of biotechnology and biomedical applications. This review focuses on the use of yeast display for engineering protein affinity, stability, and enzymatic activity. Strategies and examples for each protein engineering goal are discussed. Additional applications of yeast display are also briefly presented, including protein epitope mapping, identification of protein-protein interactions, and uses of displayed proteins in industry and medicine. PMID:26060074

  16. Screening protein refolding using surface plasmon resonance.

    PubMed

    Jones, Daniel B; Hutchinson, Matthew H; Middelberg, Anton P J

    2004-04-01

    Surface plasmon resonance (SPR) measurements were used to screen refolding conditions to identify a physicochemical environment which gives an acceptable refolding yield for samples of glutathione-S-transferase (GST) denatured in 6 M guanidine hydrochloride and 32 mM dithiothreitol. The SPR measurements were performed on carboxymethylcellulose coated chips that could accommodate two separate flow paths. One side of the chip was derivatized with immobilized glutathione and the other with goat anti-GST antibody. This created a dual-derivatized chip capable of showing both the presence of GST and providing a measure of enzyme activity. The dual-derivatized chip could be regenerated using a two-step washing procedure and reused to analyze multiple samples from a screening study of protein refolding conditions. SPR measurements have been shown to be suitable for screening protein refolding conditions due to the high sensitivity, ease of chip regeneration and the ability to incorporate a control in the experimental design. The combination of such advantages with the high-throughput automated SPR systems currently available may be a valuable approach to determine conditions suitable for protein refolding following insoluble expression in a bacterial host. PMID:15048982

  17. Hepatocellular carcinoma and hepatitis B surface protein

    PubMed Central

    Li, Yong-Wei; Yang, Feng-Cai; Lu, Hui-Qiong; Zhang, Jiong-Shan

    2016-01-01

    The tumorigenesis of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) has been widely studied. HBV envelope proteins are important for the structure and life cycle of HBV, and these proteins are useful for judging the natural disease course and guiding treatment. Truncated and mutated preS/S are produced by integrated viral sequences that are defective for replication. The preS/S mutants are considered “precursor lesions” of HCC. Different preS/S mutants induce various mechanisms of tumorigenesis, such as transactivation of transcription factors and an immune inflammatory response, thereby contributing to HCC. The preS2 mutants and type II “Ground Glass” hepatocytes represent novel biomarkers of HBV-associated HCC. The preS mutants may induce the unfolded protein response and endoplasmic reticulum stress-dependent and stress-independent pathways. Treatments to inhibit hepatitis B surface antigen (HBsAg) and damage secondary to HBsAg or the preS/S mutants include antivirals and antioxidants, such as silymarin, resveratrol, and glycyrrhizin acid. Methods for the prevention and treatment of HCC should be comprehensive. PMID:26877602

  18. Revealing the role of oxidation state in interaction between nitro/amino-derived particulate matter and blood proteins

    NASA Astrophysics Data System (ADS)

    Liu, Zhen; Li, Ping; Bian, Weiwei; Yu, Jingkai; Zhan, Jinhua

    2016-05-01

    Surface oxidation states of ultrafine particulate matter can influence the proinflammatory responses and reactive oxygen species levels in tissue. Surface active species of vehicle-emission soot can serve as electron transfer-mediators in mitochondrion. Revealing the role of surface oxidation state in particles-proteins interaction will promote the understanding on metabolism and toxicity. Here, the surface oxidation state was modeled by nitro/amino ligands on nanoparticles, the interaction with blood proteins were evaluated by capillary electrophoresis quantitatively. The nitro shown larger affinity than amino. On the other hand, the affinity to hemoglobin is 103 times larger than that to BSA. Further, molecular docking indicated the difference of binding intensity were mainly determined by hydrophobic forces and hydrogen bonds. These will deepen the quantitative understanding of protein-nanoparticles interaction from the perspective of surface chemical state.

  19. Revealing the role of oxidation state in interaction between nitro/amino-derived particulate matter and blood proteins

    PubMed Central

    Liu, Zhen; Li, Ping; Bian, Weiwei; Yu, Jingkai; Zhan, Jinhua

    2016-01-01

    Surface oxidation states of ultrafine particulate matter can influence the proinflammatory responses and reactive oxygen species levels in tissue. Surface active species of vehicle-emission soot can serve as electron transfer-mediators in mitochondrion. Revealing the role of surface oxidation state in particles-proteins interaction will promote the understanding on metabolism and toxicity. Here, the surface oxidation state was modeled by nitro/amino ligands on nanoparticles, the interaction with blood proteins were evaluated by capillary electrophoresis quantitatively. The nitro shown larger affinity than amino. On the other hand, the affinity to hemoglobin is 103 times larger than that to BSA. Further, molecular docking indicated the difference of binding intensity were mainly determined by hydrophobic forces and hydrogen bonds. These will deepen the quantitative understanding of protein-nanoparticles interaction from the perspective of surface chemical state. PMID:27181651

  20. Revealing the role of oxidation state in interaction between nitro/amino-derived particulate matter and blood proteins.

    PubMed

    Liu, Zhen; Li, Ping; Bian, Weiwei; Yu, Jingkai; Zhan, Jinhua

    2016-01-01

    Surface oxidation states of ultrafine particulate matter can influence the proinflammatory responses and reactive oxygen species levels in tissue. Surface active species of vehicle-emission soot can serve as electron transfer-mediators in mitochondrion. Revealing the role of surface oxidation state in particles-proteins interaction will promote the understanding on metabolism and toxicity. Here, the surface oxidation state was modeled by nitro/amino ligands on nanoparticles, the interaction with blood proteins were evaluated by capillary electrophoresis quantitatively. The nitro shown larger affinity than amino. On the other hand, the affinity to hemoglobin is 10(3) times larger than that to BSA. Further, molecular docking indicated the difference of binding intensity were mainly determined by hydrophobic forces and hydrogen bonds. These will deepen the quantitative understanding of protein-nanoparticles interaction from the perspective of surface chemical state. PMID:27181651

  1. Are protein-protein interfaces special regions on a protein's surface?

    NASA Astrophysics Data System (ADS)

    Tonddast-Navaei, Sam; Skolnick, Jeffrey

    2015-12-01

    Protein-protein interactions (PPIs) are involved in many cellular processes. Experimentally obtained protein quaternary structures provide the location of protein-protein interfaces, the surface region of a given protein that interacts with another. These regions are termed half-interfaces (HIs). Canonical HIs cover roughly one third of a protein's surface and were found to have more hydrophobic residues than the non-interface surface region. In addition, the classical view of protein HIs was that there are a few (if not one) HIs per protein that are structurally and chemically unique. However, on average, a given protein interacts with at least a dozen others. This raises the question of whether they use the same or other HIs. By copying HIs from monomers with the same folds in solved quaternary structures, we introduce the concept of geometric HIs (HIs whose geometry has a significant match to other known interfaces) and show that on average they cover three quarters of a protein's surface. We then demonstrate that in some cases, these geometric HI could result in real physical interactions (which may or may not be biologically relevant). The composition of the new HIs is on average more charged compared to most known ones, suggesting that the current protein interface database is biased towards more hydrophobic, possibly more obligate, complexes. Finally, our results provide evidence for interface fuzziness and PPI promiscuity. Thus, the classical view of unique, well defined HIs needs to be revisited as HIs are another example of coarse-graining that is used by nature.

  2. Protein-RNA networks revealed through covalent RNA marks.

    PubMed

    Lapointe, Christopher P; Wilinski, Daniel; Saunders, Harriet A J; Wickens, Marvin

    2015-12-01

    Protein-RNA networks are ubiquitous and central in biological control. We present an approach termed RNA Tagging that enables the user to identify protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or cross-linking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA via high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The results showed that although RNA-binding proteins productively bind specific RNAs to control their function, they also 'sample' RNAs without exerting a regulatory effect. We used the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. RNA Tagging is well suited to detect and analyze protein-RNA networks in vivo. PMID:26524240

  3. Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces

    PubMed Central

    Engin, H. Billur; Kreisberg, Jason F.; Carter, Hannah

    2016-01-01

    Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10−4) and oncogenes (Odds Ratio 1.17, P-value < 10−3). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10−8). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer

  4. Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces.

    PubMed

    Engin, H Billur; Kreisberg, Jason F; Carter, Hannah

    2016-01-01

    Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10-4) and oncogenes (Odds Ratio 1.17, P-value < 10-3). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10-8). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer genes

  5. Principal Component Analysis reveals correlation of cavities evolution and functional motions in proteins.

    PubMed

    Desdouits, Nathan; Nilges, Michael; Blondel, Arnaud

    2015-02-01

    Protein conformation has been recognized as the key feature determining biological function, as it determines the position of the essential groups specifically interacting with substrates. Hence, the shape of the cavities or grooves at the protein surface appears to drive those functions. However, only a few studies describe the geometrical evolution of protein cavities during molecular dynamics simulations (MD), usually with a crude representation. To unveil the dynamics of cavity geometry evolution, we developed an approach combining cavity detection and Principal Component Analysis (PCA). This approach was applied to four systems subjected to MD (lysozyme, sperm whale myoglobin, Dengue envelope protein and EF-CaM complex). PCA on cavities allows us to perform efficient analysis and classification of the geometry diversity explored by a cavity. Additionally, it reveals correlations between the evolutions of the cavities and structures, and can even suggest how to modify the protein conformation to induce a given cavity geometry. It also helps to perform fast and consensual clustering of conformations according to cavity geometry. Finally, using this approach, we show that both carbon monoxide (CO) location and transfer among the different xenon sites of myoglobin are correlated with few cavity evolution modes of high amplitude. This correlation illustrates the link between ligand diffusion and the dynamic network of internal cavities. PMID:25424655

  6. Protein structural ensembles are revealed by redefining X-ray electron density noise.

    PubMed

    Lang, P Therese; Holton, James M; Fraser, James S; Alber, Tom

    2014-01-01

    To increase the power of X-ray crystallography to determine not only the structures but also the motions of biomolecules, we developed methods to address two classic crystallographic problems: putting electron density maps on the absolute scale of e(-)/Å(3) and calculating the noise at every point in the map. We find that noise varies with position and is often six to eight times lower than thresholds currently used in model building. Analyzing the rescaled electron density maps from 485 representative proteins revealed unmodeled conformations above the estimated noise for 45% of side chains and a previously hidden, low-occupancy inhibitor of HIV capsid protein. Comparing the electron density maps in the free and nucleotide-bound structures of three human protein kinases suggested that substrate binding perturbs distinct intrinsic allosteric networks that link the active site to surfaces that recognize regulatory proteins. These results illustrate general approaches to identify and analyze alternative conformations, low-occupancy small molecules, solvent distributions, communication pathways, and protein motions. PMID:24363322

  7. Revealing Higher Order Protein Structure Using Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Chait, Brian T.; Cadene, Martine; Olinares, Paul Dominic; Rout, Michael P.; Shi, Yi

    2016-04-01

    The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope.

  8. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. PMID:26659058

  9. Revealing Higher Order Protein Structure Using Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Chait, Brian T.; Cadene, Martine; Olinares, Paul Dominic; Rout, Michael P.; Shi, Yi

    2016-06-01

    The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope.

  10. Revealing Higher Order Protein Structure Using Mass Spectrometry.

    PubMed

    Chait, Brian T; Cadene, Martine; Olinares, Paul Dominic; Rout, Michael P; Shi, Yi

    2016-06-01

    The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope. Graphical Abstract ᅟ. PMID:27080007

  11. Network analysis reveals common host protein/s modulating pathogenesis of neurotropic viruses.

    PubMed

    Ghosh, Sourish; Mukherjee, Sriparna; Sengupta, Nabonita; Roy, Arunava; Dey, Dhritiman; Chakraborty, Surajit; Chattopadhyay, Dhrubajyoti; Banerjee, Arpan; Basu, Anirban

    2016-01-01

    Network analysis through graph theory provides a quantitative approach to characterize specific proteins and their constituent assemblies that underlie host-pathogen interactions. In the present study, graph theory was used to analyze the interactome designed out of 50 differentially expressing proteins from proteomic analysis of Chandipura Virus (CHPV, Family: Rhabdoviridae) infected mouse brain tissue to identify the primary candidates for intervention. Using the measure of degree centrality, that quantifies the connectedness of a single protein within a milieu of several other interacting proteins, DJ-1 was selected for further molecular validation. To elucidate the generality of DJ-1's role in propagating infection its role was also monitored in another RNA virus, Japanese Encephalitis Virus (JEV, Family: Flaviviridae) infection. Concurrently, DJ-1 got over-expressed in response to reactive oxygen species (ROS) generation following viral infection which in the early phase of infection migrated to mitochondria to remove dysfunctional mitochondria through the process of mitophagy. DJ-1 was also observed to modulate the viral replication and interferon responses along with low-density lipoprotein (LDL) receptor expression in neurons. Collectively these evidences reveal a comprehensive role for DJ-1 in neurotropic virus infection in the brain. PMID:27581498

  12. Network analysis reveals common host protein/s modulating pathogenesis of neurotropic viruses

    PubMed Central

    Ghosh, Sourish; Mukherjee, Sriparna; Sengupta, Nabonita; Roy, Arunava; Dey, Dhritiman; Chakraborty, Surajit; Chattopadhyay, Dhrubajyoti; Banerjee, Arpan; Basu, Anirban

    2016-01-01

    Network analysis through graph theory provides a quantitative approach to characterize specific proteins and their constituent assemblies that underlie host-pathogen interactions. In the present study, graph theory was used to analyze the interactome designed out of 50 differentially expressing proteins from proteomic analysis of Chandipura Virus (CHPV, Family: Rhabdoviridae) infected mouse brain tissue to identify the primary candidates for intervention. Using the measure of degree centrality, that quantifies the connectedness of a single protein within a milieu of several other interacting proteins, DJ-1 was selected for further molecular validation. To elucidate the generality of DJ-1’s role in propagating infection its role was also monitored in another RNA virus, Japanese Encephalitis Virus (JEV, Family: Flaviviridae) infection. Concurrently, DJ-1 got over-expressed in response to reactive oxygen species (ROS) generation following viral infection which in the early phase of infection migrated to mitochondria to remove dysfunctional mitochondria through the process of mitophagy. DJ-1 was also observed to modulate the viral replication and interferon responses along with low-density lipoprotein (LDL) receptor expression in neurons. Collectively these evidences reveal a comprehensive role for DJ-1 in neurotropic virus infection in the brain. PMID:27581498

  13. Improved solubilization of surface proteins from Listeria monocytogenes for 2-DE.

    PubMed

    Mujahid, Sana; Pechan, Tibor; Wang, Chinling

    2007-11-01

    Solubilization of bacterial surface (cell wall and membrane-associated) proteins for 2-DE is challenging, particularly in the case of Gram-positive bacteria. This is primarily due to strong protein association with the cell wall peptidoglycan and protein hydrophobicity. We solubilized surface proteins for 2-DE from the Gram-positive pathogen Listeria monocytogenes using mutanolysin, which digests cell wall peptidoglycan, and one of three different mixtures of zwitterionic detergent and chaotropes: (i) CHAPS/urea, (ii) amidosulfobetaine-14 (ASB-14)/urea/thiourea (iii) N-decyl-N,N'-dimethyl-3-ammonio-1-propanesulfonate/urea/thiourea. Cell lysis with mutanolysin followed by solubilization with ASB-14/urea/thiourea gave the highest overall protein yield with the best 2-DE resolution. Protein spot identification by MALDI-TOF/TOF-MS analysis revealed 29 characterized surface proteins of L. monocytogenes, 17 of which have not previously been reported on the surface proteome map. This is the first report describing the successful solubilization and 2-DE of L. monocytogenes proteins bound to the cell surface via an LPXTG motif or by a hydrophobic tail. The increase in surface proteome coverage obtained by mutanolysin and ASB-14/urea/thiourea solubilization suggests the utility of this method for future analytical and comparative studies of surface proteins from Listeria, and possibly other Gram-positive bacteria, using 2-DE proteomic analysis. An updated 2-DE reference map of L. monocytogenes surface proteins is presented. PMID:17922522

  14. Ribosomal History Reveals Origins of Modern Protein Synthesis

    PubMed Central

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  15. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

    PubMed

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D

    2016-03-22

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  16. Development, Characterization, and Optimization of Protein Level in Date Bars Using Response Surface Methodology

    PubMed Central

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM). Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD) with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables. PMID:22792044

  17. Development, characterization, and optimization of protein level in date bars using response surface methodology.

    PubMed

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM). Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD) with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables. PMID:22792044

  18. Graphlet-based edge clustering reveals pathogen-interacting proteins

    PubMed Central

    Solava, R. W.; Michaels, R. P.; Milenković, T.

    2012-01-01

    Motivation: Prediction of protein function from protein interaction networks has received attention in the post-genomic era. A popular strategy has been to cluster the network into functionally coherent groups of proteins and assign the entire cluster with a function based on functions of its annotated members. Traditionally, network research has focused on clustering of nodes. However, clustering of edges may be preferred: nodes belong to multiple functional groups, but clustering of nodes typically cannot capture the group overlap, while clustering of edges can. Clustering of adjacent edges that share many neighbors was proposed recently, outperforming different node clustering methods. However, since some biological processes can have characteristic ‘signatures’ throughout the network, not just locally, it may be of interest to consider edges that are not necessarily adjacent. Results: We design a sensitive measure of the ‘topological similarity’ of edges that can deal with edges that are not necessarily adjacent. We cluster edges that are similar according to our measure in different baker's yeast protein interaction networks, outperforming existing node and edge clustering approaches. We apply our approach to the human network to predict new pathogen-interacting proteins. This is important, since these proteins represent drug target candidates. Availability: Software executables are freely available upon request. Contact: tmilenko@nd.edu Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:22962470

  19. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

    In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins. PMID:25230046

  20. Hepatitis B virus large surface protein: function and fame

    PubMed Central

    Churin, Yuri; Roderfeld, Martin

    2015-01-01

    Chronic infection with hepatitis B virus (HBV) is the leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. HBV life cycle begins with viral attachment to hepatocytes, mediated by the large HBV surface protein (LHBs). Identification of the sodium-taurocholate cotransporting polypeptide (NTCP) as a HBV receptor has revealed a suitable target for viral entry inhibition. Analysis of serum hepatitis B surface antigen (HBsAg) level is a non-invasive diagnostic parameter that improves HBV treatment opportunities. Furthermore, HBsAg plays an important role in manipulation of host immune response by HBV. However, observations in patients with chronic hepatitis B under conditions of immune suppression and in transgenic mouse models of HBV infection suggest, that in absence of adaptive immune responses cellular mechanisms induced by HBV may also lead to the development of liver diseases. Thus, the multifaceted pathological aspects of HBsAg predetermine the design of new therapeutical options modulating associated biological implications. PMID:25713800

  1. Hepatitis B virus large surface protein: function and fame.

    PubMed

    Churin, Yuri; Roderfeld, Martin; Roeb, Elke

    2015-02-01

    Chronic infection with hepatitis B virus (HBV) is the leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. HBV life cycle begins with viral attachment to hepatocytes, mediated by the large HBV surface protein (LHBs). Identification of the sodium-taurocholate cotransporting polypeptide (NTCP) as a HBV receptor has revealed a suitable target for viral entry inhibition. Analysis of serum hepatitis B surface antigen (HBsAg) level is a non-invasive diagnostic parameter that improves HBV treatment opportunities. Furthermore, HBsAg plays an important role in manipulation of host immune response by HBV. However, observations in patients with chronic hepatitis B under conditions of immune suppression and in transgenic mouse models of HBV infection suggest, that in absence of adaptive immune responses cellular mechanisms induced by HBV may also lead to the development of liver diseases. Thus, the multifaceted pathological aspects of HBsAg predetermine the design of new therapeutical options modulating associated biological implications. PMID:25713800

  2. Effect of fullerenol surface chemistry on nanoparticle binding-induced protein misfolding

    NASA Astrophysics Data System (ADS)

    Radic, Slaven; Nedumpully-Govindan, Praveen; Chen, Ran; Salonen, Emppu; Brown, Jared M.; Ke, Pu Chun; Ding, Feng

    2014-06-01

    Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and dynamics of ubiquitin. We found that all derivatives bound to the model protein. Specifically, the more hydrophilic nanoparticles with a higher number of hydroxyl groups bound to the surface of the protein via hydrogen bonds, which stabilized the protein without inducing large conformational changes in the protein structure. In contrast, fullerene derivatives with a smaller number of hydroxyl groups buried their hydrophobic surface inside the protein, thereby causing protein denaturation. Overall, our results revealed a distinct role of surface chemistry on nanoparticle-protein binding and binding-induced protein misfolding.Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and

  3. Protein interactome reveals converging molecular pathways among autism disorders.

    PubMed

    Sakai, Yasunari; Shaw, Chad A; Dawson, Brian C; Dugas, Diana V; Al-Mohtaseb, Zaina; Hill, David E; Zoghbi, Huda Y

    2011-06-01

    To uncover shared pathogenic mechanisms among the highly heterogeneous autism spectrum disorders (ASDs), we developed a protein interaction network that identified hundreds of new interactions among proteins encoded by ASD-associated genes. We discovered unexpectedly high connectivity between SHANK and TSC1, previously implicated in syndromic autism, suggesting that common molecular pathways underlie autistic phenotypes in distinct syndromes. ASD patients were more likely to harbor copy number variations that encompass network genes than were control subjects. We also identified, in patients with idiopathic ASD, three de novo lesions (deletions in 16q23.3 and 15q22 and one duplication in Xq28) that involve three network genes (NECAB2, PKM2, and FLNA). The protein interaction network thus provides a framework for identifying causes of idiopathic autism and for understanding molecular pathways that underpin both syndromic and idiopathic ASDs. PMID:21653829

  4. A Superhydrophobic Surface Templated by Protein Self-Assembly and Emerging Application toward Protein Crystallization.

    PubMed

    Gao, Aiting; Wu, Qian; Wang, Dehui; Ha, Yuan; Chen, Zhijun; Yang, Peng

    2016-01-20

    A proteinaceous superhydrophobic material for facile protein crystallization is reported. The lysozyme phase transition is rationally manipulated to form a reliable superhydrophobic coating on virtually arbitrary material surfaces with good thermostability and mechanical robustness. Such a surface exhibits a fascinating capability to drive protein crystallization, and the protein crystal array can be facilitated in a large area at an ultralow protein concentration. PMID:26607764

  5. Solution NMR Structure of Lin0431 Protein from Listeria innocua Reveals High Structural Similarity with Domain II of Bacterial Transcription Antitermination Protein NusG

    PubMed Central

    Tang, Yuefeng; Xiao, Rong; Ciccosanti, Colleen; Janjua, Haleema; Lee, Dong Yup; Everett, John K.; Swapna, G.V.T.; Acton, Thomas B.; Rost, Burkhard; Montelione, Gaetano T.

    2010-01-01

    Lin0431 protein from Listeria innocua (UniProtKB/TrEMBL ID Q92EM7/Q92EM7_LISIN) was selected as a target of the Northeast Structural Genomics Consortium (target ID: LkR112). Here, we present the high-quality NMR solution structure of this protein which is the first representative for a member of DUF1312 domain family. Lin0431 protein exhibits a β-sandwich topology. Four anti-parallel β-strands form one face of the sandwich and the other three anti-parallel β-strands together with a short α-helix form the other face of the sandwich. Structure alignment by Dali reveals an unexpected structural similarity with domain II of NusG from Aquifex aeolicus. Analyses of the electrostatic protein surface potential and searches for protein surface cavities reveal the conserved basic charged surface cavities of both the Lin0431 and domain II of AaeNusG, suggesting they may bind the negatively charged nucleic acids and/or and other binding partners. The high structural similarity and similar surface features, despite the lack of recognizable sequence similarity, between Lin0431 and AaeNusG domain II suggest that the domain II of NusG and DUF1312 domains have a homologous relationship and may share similar biochemical functions. PMID:20602357

  6. The dark energy of proteins comes to light: conformational entropy and its role in protein function revealed by NMR relaxation.

    PubMed

    Wand, A Joshua

    2013-02-01

    Historically it has been virtually impossible to experimentally determine the contribution of residual protein entropy to fundamental protein activities such as the binding of ligands. Recent progress has illuminated the possibility of employing NMR relaxation methods to quantitatively determine the role of changes in conformational entropy in molecular recognition by proteins. The method rests on using fast internal protein dynamics as a proxy. Initial results reveal a large and variable role for conformational entropy in the binding of ligands by proteins. Such a role for conformational entropy in molecular recognition has significant implications for enzymology, signal transduction, allosteric regulation and the development of protein-directed pharmaceuticals. PMID:23246280

  7. RPE cell surface proteins in normal and dystrophic rats

    SciTech Connect

    Clark, V.M.; Hall, M.O.

    1986-02-01

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE.

  8. High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface.

    PubMed

    Li, Zixuan; Moniz, Heather; Wang, Shuo; Ramiah, Annapoorani; Zhang, Fuming; Moremen, Kelley W; Linhardt, Robert J; Sharp, Joshua S

    2015-04-24

    Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes. PMID:25752613

  9. High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface*

    PubMed Central

    Li, Zixuan; Moniz, Heather; Wang, Shuo; Ramiah, Annapoorani; Zhang, Fuming; Moremen, Kelley W.; Linhardt, Robert J.; Sharp, Joshua S.

    2015-01-01

    Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes. PMID:25752613

  10. Changes on Pluto's Surface Revealed with Long Timebase Photometry

    NASA Astrophysics Data System (ADS)

    George, Erin; Buie, M.

    2013-10-01

    We are continuing to monitor the long-term photometric behavior of Pluto in order to constrain volatile surface migration. As Pluto passes near the center of the galaxy, the fields are too crowded for normal aperture photometric techniques. We approached this problem with a combination of point-spread function (PSF) photometry and optimal image subtraction (OIS). Our data are from the 0.8-m robotic telescope at Lowell Observatory, the 1-m robotic telescope at New Mexico State Observatory, and the Faulkes 2-m robotic telescope at Siding Spring, part of Las Cumbres Observatory. Our latest results add photometric data up through 2012 to the data collected since discovery. Our new reduction scheme consists of background catalogs, image subtraction using deep templates, and Pluto photometry extraction. We also use the known photometric properties of Charon determined with HST to remove Charon's contribution from old and new data and compare these results with the HST data where Pluto is measured by itself. Data since 2002 show marked departures from the behavior prior to that time. These results provide clear evidence for time evolution of Pluto's surface albedo. We will present these results along with implications for present-day processes that are altering the surface of Pluto. This work also provides crucial insight into the effort required to provide ground-based support observations for the upcoming New Horizons flyby of Pluto in 2015. Support for this work was provided by NASA Planetary Astronomy Program, grant number NNX09AB43G.

  11. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated

    SciTech Connect

    Chung, Daehwan; Young, Jenna; Bomble, Yannick J.; Vander Wall, Todd A.; Groom, Joseph; Himmel, Michael E.; Westpheling, Janet

    2015-03-23

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. Finally, it will also allow the study of glycosylation of CelA itself and its role in the

  12. Protein surface representation and analysis by dimension reduction

    PubMed Central

    2012-01-01

    Background Protein structures are better conserved than protein sequences, and consequently more functional information is available in structures than in sequences. However, proteins generally interact with other proteins and molecules via their surface regions and a backbone-only analysis of protein structures may miss many of the functional and evolutionary features. Surface information can help better elucidate proteins' functions and their interactions with other proteins. Computational analysis and comparison of protein surfaces is an important challenge to overcome to enable efficient and accurate functional characterization of proteins. Methods In this study we present a new method for representation and comparison of protein surface features. Our method is based on mapping the 3-D protein surfaces onto 2-D maps using various dimension reduction methods. We have proposed area and neighbor based metrics in order to evaluate the accuracy of this surface representation. In order to capture functionally relevant information, we encode geometric and biochemical features of the protein, such as hydrophobicity, electrostatic potential, and curvature, into separate color channels in the 2-D map. The resulting images can then be compared using efficient 2-D image registration methods to identify surface regions and features shared by proteins. Results We demonstrate the utility of our method and characterize its performance using both synthetic and real data. Among the dimension reduction methods investigated, SNE, LandmarkIsomap, Isomap, and Sammon's mapping provide the best performance in preserving the area and neighborhood properties of the original 3-D surface. The enriched 2-D representation is shown to be useful in characterizing the functional site of chymotrypsin and able to detect structural similarities in heat shock proteins. A texture mapping using the 2-D representation is also proposed as an interesting application to structure visualization. PMID

  13. Proteomics and glycoproteomics of pluripotent stem-cell surface proteins.

    PubMed

    Sun, Bingyun

    2015-03-01

    Pluripotent stem cells are a unique cell type with promising potential in regenerative and personalized medicine. Yet the difficulty to understand and coax their seemingly stochastic differentiation and spontaneous self-renewal have largely limited their clinical applications. A call has been made by numerous researchers for a better characterization of surface proteins on these cells, in search of biomarkers that can dictate developmental stages and lineage specifications, and can help formulate mechanistic insight of stem-cell fate choices. In the past two decades, proteomics has gained significant recognition in profiling surface proteins at high throughput. This review will summarize the impact of these studies on stem-cell biology, and discuss the used proteomic techniques. A systematic comparison of all the techniques and their results is also attempted here to help reveal pros, cons, and the complementarity of the existing methods. This awareness should assist in selecting suitable strategies for stem-cell related research, and shed light on technical improvements that can be explored in the future. PMID:25211708

  14. Dynamics of natural killer cell receptor revealed by quantitative analysis of photoswitchable protein.

    PubMed

    Pageon, Sophie V; Aquino, Gerardo; Lagrue, Kathryn; Köhler, Karsten; Endres, Robert G; Davis, Daniel M

    2013-11-01

    Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies that rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report an apparently novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, after a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 ± 0.06 μm(2) s(-1)) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse, and remains surprisingly stable there. Unexpectedly, however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected intersynaptic exchange of protein. PMID:24209843

  15. Surface modification of graphene nanopores for protein translocation

    PubMed Central

    Shan, Y. P.; Tiwari, P. B.; Krishnakumar, P.; Vlassiouk, I.; Li, W.Z.; Wang, X.W.; Darici, Y.; Lindsay, S.M.; Wang, H. D.; Smirnov, S.; He, J.

    2014-01-01

    Studies of DNA translocation through graphene nanopores have revealed their potential for DNA sequencing. Here we report a study of protein translocation through chemically modified graphene nanopores. A transmission electron microscope (TEM) was used to cut nanopores with diameters between 5-20 nm in multilayer graphene prepared by chemical vapor deposition (CVD). After oxygen plasma treatment, the dependence of the measured ionic current on salt concentration and pH was consistent with a small surface charge induced by the formation of carboxyl groups. While translocation of gold nanoparticles (10 nm) was readily detected through such treated pores of a larger diameter, translocation of protein ferritin was not observed either for oxygen plasma treated pores, or for pores modified with mercaptohexadecanoic acid. Ferritin translocation events were reliably observed after the pores were modified with the phospholipid-PEG (DPPE-PEG750) amphiphile. The ion current signature of translocation events was complex, suggesting that a series of interactions between the protein and pore occur during the process. PMID:24231385

  16. Surface modification of graphene nanopores for protein translocation.

    PubMed

    Shan, Y P; Tiwari, P B; Krishnakumar, P; Vlassiouk, I; Li, W Z; Wang, X W; Darici, Y; Lindsay, S M; Wang, H D; Smirnov, S; He, J

    2013-12-13

    Studies of DNA translocation through graphene nanopores have revealed their potential for DNA sequencing. Here we report a study of protein translocation through chemically modified graphene nanopores. A transmission electron microscope (TEM) was used to cut nanopores with diameters between 5 and 20 nm in multilayer graphene prepared by chemical vapor deposition (CVD). After oxygen plasma treatment, the dependence of the measured ionic current on salt concentration and pH was consistent with a small surface charge induced by the formation of carboxyl groups. While translocation of gold nanoparticles (10 nm) was readily detected through such treated pores of a larger diameter, translocation of the protein ferritin was not observed either for oxygen plasma treated pores, or for pores modified with mercaptohexadecanoic acid. Ferritin translocation events were reliably observed after the pores were modified with the phospholipid-PEG (DPPE-PEG750) amphiphile. The ion current signature of translocation events was complex, suggesting that a series of interactions between the protein and pores occurs during the process. PMID:24231385

  17. Nuclear envelope-associated endosomes deliver surface proteins to the nucleus.

    PubMed

    Chaumet, Alexandre; Wright, Graham D; Seet, Sze Hwee; Tham, Keit Min; Gounko, Natalia V; Bard, Frederic

    2015-01-01

    Endocytosis directs molecular cargo along three main routes: recycling to the cell surface, transport to the Golgi apparatus or degradation in endolysosomes. Pseudomonas exotoxin A (PE) is a bacterial protein that typically traffics to the Golgi and then the endoplasmic reticulum before translocating to the cytosol. Here we show that a substantial fraction of internalized PE is also located in nuclear envelope-associated endosomes (NAE), which display limited mobility, exhibit a propensity to undergo fusion and readily discharge their contents into the nuclear envelope. Electron microscopy and protein trapping in the nucleus indicate that NAE mediate PE transfer into the nucleoplasm. RNAi screening further revealed that NAE-mediated transfer depends on the nuclear envelope proteins SUN1 and SUN2, as well as the Sec61 translocon complex. These data reveal a novel endosomal route from the cell surface to the nucleoplasm that facilitates the accumulation of extracellular and cell surface proteins in the nucleus. PMID:26356418

  18. Mechanical Unfolding of an Autotransporter Passenger Protein Reveals the Secretion Starting Point and Processive Transport Intermediates.

    PubMed

    Baclayon, Marian; Ulsen, Peter van; Mouhib, Halima; Shabestari, Maryam Hashemi; Verzijden, Timo; Abeln, Sanne; Roos, Wouter H; Wuite, Gijs J L

    2016-06-28

    The backbone of secreted autotransporter passenger proteins generally attains a stable β-helical structure. The secretion of passengers across the outer membrane was proposed to be driven by sequential folding of this structure at the cell surface. This mechanism would require a relatively stable intermediate as starting point. Here, we investigated the mechanics of secreted truncated versions of the autotransporter hemoglobin protease (Hbp) of Escherichia coli using atomic force microscopy. The data obtained reveal a β-helical structure at the C terminus that is very stable. In addition, several other distinct metastable intermediates are found which are connected during unfolding by multiroute pathways. Computational analysis indicates that these intermediates correlate to the β-helical rungs in the Hbp structure which are clamped by stacked aromatic residues. Our results suggest a secretion mechanism that is initiated by a stable C-terminal structure and driven forward by several folding intermediates that build up the β-helical backbone. PMID:27219538

  19. In Silico Study of Variable Surface Proteins in Plasmodium Species: Perspectives in Drug Design.

    PubMed

    Yadav, Manoj Kumar; Swati, D

    2016-09-01

    The variable surface proteins expressed by P. falciparum and P. vivax are transported to the surface of infected erythrocyte and are exposed to the host immune system. The possibility of using variable surface proteins as a common drug target has been analyzed in both the Plasmodium species. Sequence analysis of variable surface proteins showed a low-level conservation within as well as between the species. Amino acid composition analysis revealed higher frequency of hydrophilic amino acids as compared with that of hydrophobic residues. In order to gain more insight into their diverse functional role, the three-dimensional structure was predicted using comparative modeling approach. These models were evaluated and validated by checking stereochemistry of underlying amino acids. Structural alignment of variable surface proteins by superimposing them shows less conservation. Due to differences at sequence as well as structural level, the variable surface proteins are expected to show difference in their degree of invasiveness. These differences were also cross-examined by evolutionary study, and the results obtained were in accordance with the aforesaid study. The existence of structural differences noticed in the present study showed that the variable surface proteins could not be used as a common drug target in both the malarial species. Therefore, species-specific strategy may be followed for drug targeting against variable surface proteins of P. falciparum and P. vivax. PMID:26253721

  20. Surface Passivation for Single-molecule Protein Studies

    PubMed Central

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

    2014-01-01

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

  1. Structural Determinants for Protein adsorption/non-adsorption to Silica Surface

    PubMed Central

    Mathé, Christelle; Devineau, Stéphanie; Aude, Jean-Christophe; Lagniel, Gilles; Chédin, Stéphane; Legros, Véronique; Mathon, Marie-Hélène; Renault, Jean-Philippe; Pin, Serge; Boulard, Yves; Labarre, Jean

    2013-01-01

    The understanding of the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest in both fundamental research and applications such as nanotechnology. However, despite intense research, the mechanisms and the structural determinants of protein/surface interactions are still unclear. We developed a strategy consisting in identifying, in a mixture of hundreds of soluble proteins, those proteins that are adsorbed on the surface and those that are not. If the two protein subsets are large enough, their statistical comparative analysis must reveal the physicochemical determinants relevant for adsorption versus non-adsorption. This methodology was tested with silica nanoparticles. We found that the adsorbed proteins contain a higher number of charged amino acids, particularly arginine, which is consistent with involvement of this basic amino acid in electrostatic interactions with silica. The analysis also identified a marked bias toward low aromatic amino acid content (phenylalanine, tryptophan, tyrosine and histidine) in adsorbed proteins. Structural analyses and molecular dynamics simulations of proteins from the two groups indicate that non-adsorbed proteins have twice as many π-π interactions and higher structural rigidity. The data are consistent with the notion that adsorption is correlated with the flexibility of the protein and with its ability to spread on the surface. Our findings led us to propose a refined model of protein adsorption. PMID:24282583

  2. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

    SciTech Connect

    Wise K.S.; Kim, M.F.

    1987-12-01

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface /sup 125/I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with (/sup 35/S) methionine, /sup 14/C-amino acids, or (/sup 3/H) palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11.

  3. Effect of fullerenol surface chemistry on nanoparticle binding-induced protein misfolding.

    PubMed

    Radic, Slaven; Nedumpully-Govindan, Praveen; Chen, Ran; Salonen, Emppu; Brown, Jared M; Ke, Pu Chun; Ding, Feng

    2014-07-21

    Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation - a common strategy for solubilizing and functionalizing fullerene - on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and dynamics of ubiquitin. We found that all derivatives bound to the model protein. Specifically, the more hydrophilic nanoparticles with a higher number of hydroxyl groups bound to the surface of the protein via hydrogen bonds, which stabilized the protein without inducing large conformational changes in the protein structure. In contrast, fullerene derivatives with a smaller number of hydroxyl groups buried their hydrophobic surface inside the protein, thereby causing protein denaturation. Overall, our results revealed a distinct role of surface chemistry on nanoparticle-protein binding and binding-induced protein misfolding. PMID:24934397

  4. Real-time Redox Measurements during Endoplasmic Reticulum Stress Reveal Interlinked Protein Folding Functions

    PubMed Central

    Merksamer, Philip I.; Trusina, Ala; Papa, Feroz R.

    2008-01-01

    SUMMARY Disruption of protein folding in the endoplasmic reticulum (ER) causes unfolded proteins to accumulate, triggering the unfolded protein response (UPR). UPR outputs in turn decrease ER unfolded proteins to close a negative feedback loop. However, because it is infeasible to directly measure the concentration of unfolded proteins in vivo, cells are generically described as experiencing “ER stress” whenever the UPR is active. Because ER redox potential is optimized for oxidative protein folding, we reasoned that measureable redox changes should accompany unfolded protein accumulation. To test this concept, we employed fluorescent protein reporters to dynamically measure ER redox status and UPR activity in single cells. Using these tools, we show that diverse stressors, both experimental and physiological, compromise ER protein oxidation when UPR-imposed homeostatic control is lost. Using genetic analysis we uncovered redox heterogeneities in isogenic cell populations, and revealed functional interlinks between ER protein folding, modification, and quality control systems. PMID:19026441

  5. Yeast surface display for protein engineering and characterization.

    PubMed

    Gai, S Annie; Wittrup, K Dane

    2007-08-01

    Yeast surface display is being employed to engineer desirable properties into proteins for a broad variety of applications. Labeling with soluble ligands enables rapid and quantitative analysis of yeast-displayed libraries by flow cytometry, while cell-surface selections allow screening of libraries with insoluble or even as-yet-uncharacterized binding targets. In parallel, the utilization of yeast surface display for protein characterization, including in particular the mapping of functional epitopes mediating protein-protein interactions, represents a significant recent advance. PMID:17870469

  6. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity.

    PubMed

    Bobrovsky, Pavel; Manuvera, Valentin; Polina, Nadezhda; Podgorny, Oleg; Prusakov, Kirill; Govorun, Vadim; Lazarev, Vassili

    2016-07-01

    Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis Thus, we cloned the regions containing the human Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection. PMID:27160295

  7. Conformational kinetics reveals affinities of protein conformational states

    PubMed Central

    Daniels, Kyle G.; Suo, Yang; Oas, Terrence G.

    2015-01-01

    Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein’s affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states. PMID:26162682

  8. Competitive Protein Adsorption on Polysaccharide and Hyaluronate Modified Surfaces

    PubMed Central

    Ombelli, Michela; Costello, Lauren; Postle, Corinne; Anantharaman, Vinod; Meng, Qing Cheng; Composto, Russell J.; Eckmann, David M.

    2011-01-01

    We measured adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) onto six distinct bare and dextran- and hyaluronate-modified silicon surfaces created using two dextran grafting densities and three hyaluronic acid (HA) sodium salts derived from human umbilical cord, rooster comb and streptococcus zooepidemicus. Film thickness and surface morphology depended on HA molecular weight and concentration. BSA coverage was enhanced on surfaces upon competitive adsorption of BSA:Fg mixtures. Dextranization differentially reduced protein adsorption onto surfaces based on oxidation state. Hyaluronization was demonstrated to provide the greatest resistance to protein coverage, equivalent to that of the most resistant dextranized surface. Resistance to protein adsorption was independent of the type of hyaluronic acid utilized. With changing bulk protein concentration from 20 to 40 µg ml−1 for each species, Fg coverage on silicon increased by 4×, whereas both BSA and Fg adsorption on dextran and HA were far less dependent of protein bulk concentration. PMID:21623481

  9. Proteomic Approach to Reveal the Proteins Associated with Encystment of the Ciliate Euplotes encysticus

    PubMed Central

    Chen, Jiwu; Gao, Xiuxia; Wang, Bangzheng; Chen, Fenfen; Wu, Na; Zhang, Yuanyuan

    2014-01-01

    In order to identify and reveal the proteins related to encystment of the ciliate Euplotes encysticus, we analyzed variation in the abundance of the proteins isolated from the resting cyst comparing with proteins in the vegetative cell. 2-D electrophoresis, MALDI-TOF MS techniques and Bioinformatics were used for proteome separation, quantification and identification. The comparative proteomics studies revealed 26 proteins with changes on the expression in the resting cysts, including 12 specific proteins and 14 differential proteins. 12 specific proteins and 10 out of the 14 differential proteins were selected and identified by MALDI-TOF MS. The identified specific proteins with known functions included type II cytoskeletal 1, keratin, Nop16 domain containing protein, protein arginine n-methyltransferase, epsilon-trimethyllysine hydroxylase and calpain-like protein. The identified differential proteins with known functions included Lysozyme C, keratinocyte growth factor, lysozyme homolog AT-2, formate acetyltransferase, alpha S1 casein and cold-shock protein. We discussed the functions of these proteins as well as their contribution in the process of encystment. These identified proteins covered a wide range of molecular functions, including gene regulation, RNA regulation, proteins degradation and oxidation resistance, stress response, material transport and cytoskeleton organization. Therefore, differential expression of these proteins was essential for cell morphological and physiological changes during encystment. This suggested that the peculiar proteins and differential proteins might play important roles in the process of the vegetative cells transforming into the resting cysts. These observations may be novel findings that bring new insights into the detailed mechanisms of dormancy. PMID:24837719

  10. Mass spectrometric analysis reveals remnants of host-pathogen molecular interactions at the starch granule surface in wheat endosperm.

    PubMed

    Wall, Michael L; Wheeler, Heather L; Smith, Jeffrey; Figeys, Daniel; Altosaar, Illimar

    2010-09-01

    The starch granules of wheat seed are solar energy-driven deposits of fixed carbon and, as such, present themselves as targets of pathogen attack. The seed's array of antimicrobial proteins, peptides, and small molecules comprises a molecular defense against penetrating pathogens. In turn, pathogens exhibit an arsenal of enzymes to facilitate the degradation of the host's endosperm. In this context, the starch granule surface is a relatively unexplored domain in which unique molecular barriers may be deployed to defend against and inhibit the late stages of infection. Therefore, it was compelling to explore the starch granule surface in mature wheat seed, which revealed evidence of host-pathogen molecular interactions that may have occurred during grain development. In this study, starch granules from the soft wheat Triticum aestivum cv. AC Andrew and hard wheat T. turgidum durum were isolated and water washed 20 times, and their surface proteins were digested in situ with trypsin. The peptides liberated into the supernatant and the peptides remaining at the starch granule surface were separately examined. In this way, we demonstrated that the identified proteins have a strong affinity for the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Although most of these peptides corresponded to uncharacterized hypothetical proteins of fungal pathogens, several peptide fragments were identical to cytosolic and membrane proteins of specific microbial pathogens. During development and maturation, wheat seed appeared to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. PMID:20701481

  11. Surface Association of Pht Proteins of Streptococcus pneumoniae

    PubMed Central

    Plumptre, Charles D.; Ogunniyi, Abiodun D.

    2013-01-01

    Streptococcus pneumoniae is a major human pathogen responsible for massive global morbidity and mortality. The pneumococcus attaches a variety of proteins to its cell surface, many of which contribute to virulence; one such family are the polyhistidine triad (Pht) proteins PhtA, PhtB, PhtD, and PhtE. In this study, we have examined the mechanism of Pht surface attachment using PhtD as a model. Analysis of deletion and point mutants identified a three-amino-acid region of PhtD (Q27-H28-R29) that is critical for the process. The analogous region in PhtE was also necessary for its attachment to the cell surface. Furthermore, we show that a large proportion of the total amount of each Pht protein is released into bacterial culture supernatants. Other surface proteins were also released, albeit to lesser extents, and this was not due to pneumococcal autolysis. The extent of release of surface proteins was strain dependent and was not affected by the capsule. Lastly, we compared the fitness of wild-type and ΔphtABDE pneumococci in vivo in a mouse coinfection model. Release of Pht proteins by the wild type did not complement the mutant strain, consistent with surface-attached rather than soluble forms of the Pht proteins playing the major role in virulence. The significant degree of release of Pht proteins from intact bacteria may have implications for the use of these proteins in novel vaccines. PMID:23876799

  12. Accuracy of functional surfaces on comparatively modeled protein structures

    PubMed Central

    Zhao, Jieling; Dundas, Joe; Kachalo, Sema; Ouyang, Zheng; Liang, Jie

    2012-01-01

    Identification and characterization of protein functional surfaces are important for predicting protein function, understanding enzyme mechanism, and docking small compounds to proteins. As the rapid speed of accumulation of protein sequence information far exceeds that of structures, constructing accurate models of protein functional surfaces and identify their key elements become increasingly important. A promising approach is to build comparative models from sequences using known structural templates such as those obtained from structural genome projects. Here we assess how well this approach works in modeling binding surfaces. By systematically building three-dimensional comparative models of proteins using Modeller, we determine how well functional surfaces can be accurately reproduced. We use an alpha shape based pocket algorithm to compute all pockets on the modeled structures, and conduct a large-scale computation of similarity measurements (pocket RMSD and fraction of functional atoms captured) for 26,590 modeled enzyme protein structures. Overall, we find that when the sequence fragment of the binding surfaces has more than 45% identity to that of the tempalte protein, the modeled surfaces have on average an RMSD of 0.5 Å, and contain 48% or more of the binding surface atoms, with nearly all of the important atoms in the signatures of binding pockets captured. PMID:21541664

  13. Conformal Nanopatterning of Extracellular Matrix Proteins onto Topographically Complex Surfaces

    PubMed Central

    Sun, Yan; Jallerat, Quentin; Szymanski, John M.

    2015-01-01

    We report a method for conformal nanopatterning of extracellular matrix proteins onto engineered surfaces independent of underlying microtopography. This enables fibronectin, laminin, and other proteins to be applied to biomaterial surfaces in complex geometries inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface, used here to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment. PMID:25506720

  14. Interactions of proteins in gels, solutions and on surfaces

    NASA Astrophysics Data System (ADS)

    Ramasamy, Radha Perumal

    2006-12-01

    The study of protein interaction, identification and separation has applications in various fields relating to Biotechnology. In this research these aspects were investigated. The proteins albumin, casein, poly-L-lysine were studied. FITC and TRITC were used to fluorescently tag the proteins. Confocal microscopy was used to image the interaction of proteins. The migration of fluorescently tagged protein-salt aggregates on solid surfaces during electrophoresis was investigated using Confocal microscopy. The secondary structural modifications of proteins in solutions were investigated using FTIR micro spectroscopic imaging. The size of the colloids formed due to protein-protein interactions as a function of the protein concentrations were studied using DLS and their charges were found using zeta potential measurements. Based on DL.S and zeta potential measurements, a model is proposed for interactions of oppositely charged proteins. The nature of interaction was found using UV - Visual spectroscopy. It was found that oppositely charged proteins formed ionic bonds. It was also found that FITC molecule influenced the surface charge of albumin more than TRITC molecule. The effects of the influence of cell geometries upon Electro Osmotic Flow (EOF) were studied using neutrally charged fluorescent Polystyrene beads. Results showed that tagging proteins with fluorescent molecules influenced their mobility and interactions with other proteins. However no secondary structural modifications of the proteins were observed when oppositely charged proteins interacted. It was also observed that electrostatic interactions made oppositely charged proteins form large aggregates. The EOF was found to be dependent upon the ionic strength of the buffer, conductivity of the solid surfaces, distance from the surface and position of the electrodes in the electrophoretic cell.

  15. DARC: Mapping Surface Topography by Ray-Casting for Effective Virtual Screening at Protein Interaction Sites.

    PubMed

    Gowthaman, Ragul; Miller, Sven A; Rogers, Steven; Khowsathit, Jittasak; Lan, Lan; Bai, Nan; Johnson, David K; Liu, Chunjing; Xu, Liang; Anbanandam, Asokan; Aubé, Jeffrey; Roy, Anuradha; Karanicolas, John

    2016-05-12

    Protein-protein interactions represent an exciting and challenging target class for therapeutic intervention using small molecules. Protein interaction sites are often devoid of the deep surface pockets presented by "traditional" drug targets, and crystal structures reveal that inhibitors typically engage these sites using very shallow binding modes. As a consequence, modern virtual screening tools developed to identify inhibitors of traditional drug targets do not perform as well when they are instead deployed at protein interaction sites. To address the need for novel inhibitors of important protein interactions, here we introduce an alternate docking strategy specifically designed for this regime. Our method, termed DARC (Docking Approach using Ray-Casting), matches the topography of a surface pocket "observed" from within the protein to the topography "observed" when viewing a potential ligand from the same vantage point. We applied DARC to carry out a virtual screen against the protein interaction site of human antiapoptotic protein Mcl-1 and found that four of the top-scoring 21 compounds showed clear inhibition in a biochemical assay. The Ki values for these compounds ranged from 1.2 to 21 μM, and each had ligand efficiency comparable to promising small-molecule inhibitors of other protein-protein interactions. These hit compounds do not resemble the natural (protein) binding partner of Mcl-1, nor do they resemble any known inhibitors of Mcl-1. Our results thus demonstrate the utility of DARC for identifying novel inhibitors of protein-protein interactions. PMID:26126123

  16. Minimalist explicit solvation models for surface loops in proteins.

    PubMed

    White, Ronald P; Meirovitch, Hagai

    2006-01-01

    We have performed molecular dynamics simulations of protein surface loops solvated by explicit water, where a prime focus of the study is the small numbers (e.g., ~100) of explicit water molecules employed. The models include only part of the protein (typically 500 - 1000 atoms), and the water molecules are restricted to a region surrounding the loop. In this study, the number of water molecules (N(w)) is systematically varied, and convergence with large N(w) is monitored to reveal N(w)(min), the minimum number required for the loop to exhibit realistic (fully hydrated) behavior. We have also studied protein surface coverage, as well as diffusion and residence times for water molecules as a function of N(w). A number of other modeling parameters are also tested. These include the number of environmental protein atoms explicitly considered in the model, as well as two ways to constrain the water molecules to the vicinity of the loop (where we find one of these methods to perform better when N(w) is small). The results (for RMSD and its fluctuations for four loops) are further compared to much larger, fully solvated systems (using ~10,000 water molecules under periodic boundary conditions and Ewald electrostatics), and to results for the GBSA implicit solvation model. We find that the loop backbone can stabilize with a surprisingly small number of water molecules (as low as 5 molecules per amino acid residue). The side chains of the loop require somewhat larger N(w), where the atomic fluctuations become too small if N(w) is further reduced. Thus, in general, we find adequate hydration to occur at roughly 12 water molecules per residue. This is an important result, because at this hydration level, computational times are comparable to those required for GBSA. Therefore these "minimalist explicit models" can provide a viable and potentially more accurate alternative. The importance of protein loop modeling is discussed in the context of these, and other, loop models

  17. Simulations of HIV Capsid Protein Dimerization Reveal the Effect of Chemistry and Topography on the Mechanism of Hydrophobic Protein Association

    NASA Astrophysics Data System (ADS)

    Yu, Naiyin; Hagan, Michael F.

    2012-09-01

    Recent work has shown that the hydrophobic protein surfaces in aqueous solution sit near a drying transition. The tendency for these surfaces to expel water from their vicinity leads to self assembly of macromolecular complexes. In this article we show with a realistic model for a biologically pertinent system how this phenomenon appears at the molecular level. We focus on the association of the C-terminal domain (CA-C) of the human immunodeficiency virus (HIV) capsid protein. By combining all-atom simulations with specialized sampling techniques we measure the water density distribution during the approach of two CA-C proteins as a function of separation and amino acid sequence in the interfacial region. The simulations demonstrate that CA-C protein-protein interactions sit at the edge of a dewetting transition and that this mesoscopic manifestation of the underlying liquid-vapor phase transition can be readily manipulated by biology or protein engineering to significantly affect association behavior. While the wild type protein remains wet until contact, we identify a set of in silico mutations, in which three hydrophilic amino acids are replaced with nonpolar residues, that leads to dewetting prior to association. The existence of dewetting depends on the size and relative locations of substituted residues separated by nm length scales, indicating long range cooperativity and a sensitivity to surface topography. These observations identify important details which are missing from descriptions of protein association based on buried hydrophobic surface area.

  18. Selective characterization of proteins on nanoscale concave surfaces.

    PubMed

    Qian, Xi; Rameshbabu, Utthara; Dordick, Jonathan S; Siegel, Richard W

    2016-01-01

    Nanoscale curvature plays a critical role in nanostructure-biomolecule interactions, yet the understanding of such effects in concave nanostructures is still very limited. Because concave nanostructures usually possess convex surface curvatures as well, it is challenging to selectively study the proteins on concave surfaces alone. In this work, we have developed a novel and facile method to address this issue by desorbing proteins on the external surfaces of hollow gold nanocages (AuNG), allowing the selective characterization of retained proteins immobilized on their internal concave surfaces. The selective desorption of proteins was achieved via varying the solution ionic strength, and was demonstrated by both calculation and experimental comparison with non-hollow nanoparticles. This method has created a new platform for the discrete observation of proteins adsorbed inside AuNG hollow cores, and this work suggests an expanded biomedical application space for hollow nanomaterials. PMID:26513422

  19. Pretreatment of amphiphilic comb polymer surfaces dramatically affects protein adsorption.

    PubMed

    Zhang, Zhanping; Ma, Hongwei; Hausner, Douglas B; Chilkoti, Ashutosh; Beebe, Thomas P

    2005-01-01

    New applications in regenerative biotechnology require the ability to understand and control protein-surface interactions on micrometer and submicrometer length scales. Evidence presented here shows that micropatterned amphiphilic comb polymer films exhibit a pretreatment-dependent behavior with respect to protein adsorption for the proteins fibronectin, laminin, and for serum. A micropatterned surface, consisting of protein-reactive regions, separated by comb polymer, was created and tested for protein adsorption using the surface-sensitive imaging tool TOF-SIMS. Immersion of micropatterned surfaces in solutions of fibronectin or laminin resulted in uniform protein coverage on both the comb polymer and protein-reactive regions. However, preimmersion of similarly patterned surfaces in water for 2 h prior to protein incubation was found to dramatically improve the protein-resistant properties of the comb polymer regions. These results are consistent with poly(ethylene glycol) (PEG) side chain reorientation and/or hydration and poly(methyl methacrylate) (PMMA) backbone segregation away from the interface region. PMID:16283770

  20. Network Analysis of Circular Permutations in Multidomain Proteins Reveals Functional Linkages for Uncharacterized Proteins

    PubMed Central

    Adjeroh, Donald; Jiang, Yue; Jiang, Bing-Hua; Lin, Jie

    2014-01-01

    Various studies have implicated different multidomain proteins in cancer. However, there has been little or no detailed study on the role of circular multidomain proteins in the general problem of cancer or on specific cancer types. This work represents an initial attempt at investigating the potential for predicting linkages between known cancer-associated proteins with uncharacterized or hypothetical multidomain proteins, based primarily on circular permutation (CP) relationships. First, we propose an efficient algorithm for rapid identification of both exact and approximate CPs in multidomain proteins. Using the circular relations identified, we construct networks between multidomain proteins, based on which we perform functional annotation of multidomain proteins. We then extend the method to construct subnetworks for selected cancer subtypes, and performed prediction of potential link-ages between uncharacterized multidomain proteins and the selected cancer types. We include practical results showing the performance of the proposed methods. PMID:25741177

  1. Surface Functionalization for Protein and Cell Patterning

    NASA Astrophysics Data System (ADS)

    Colpo, Pascal; Ruiz, Ana; Ceriotti, Laura; Rossi, François

    The interaction of biological systems with synthetic material surfaces is an important issue for many biological applications such as implanted devices, tissue engineering, cell-based sensors and assays, and more generally biologic studies performed ex vivo. To ensure reliable outcomes, the main challenge resides in the ability to design and develop surfaces or artificial micro-environment that mimic 'natural environment' in interacting with biomolecules and cells without altering their function and phenotype. At this effect, microfabrication, surface chemistry and material science play a pivotal role in the design of advanced in-vitro systems for cell culture applications. In this chapter, we discuss and describe different techniques enabling the control of cell-surface interactions, including the description of some techniques for immobilization of ligands for controlling cell-surface interactions and some methodologies for the creation of well confined cell rich areas.

  2. A script to highlight hydrophobicity and charge on protein surfaces

    PubMed Central

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

    2015-01-01

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

  3. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

    SciTech Connect

    Bricker, T.M.; Boyer, M.J.; Keith, J.; Watson-McKown, R.; Wise, K.S.

    1988-02-01

    Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 (/sup 35/S)methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of (/sup 3/H)palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from /sup 125/I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of (/sup 3/H)palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins.

  4. Streptococcal cysteine proteinase releases biologically active fragments of streptococcal surface proteins.

    PubMed

    Berge, A; Björck, L

    1995-04-28

    Streptococcus pyogenes are important pathogenic bacteria which produce an extracellular cysteine proteinase contributing to their virulence and pathogenicity. S. pyogenes also express surface molecules, M proteins, that are major virulence determinants due to their antiphagocytic property. In the present work live S. pyogenes bacteria of the M1 serotype were incubated with purified cysteine proteinase. Several peptides were solubilized, and analysis of their protein-binding properties and amino acid sequences revealed two internal fibrinogen-binding fragments of M1 protein (17 and 21 kDa, respectively), and a 36-kDa IgG-binding NH2-terminal fragment of protein H, an IgGFc-binding surface molecule. M protein also plays a role in streptococcal adherence, and removal of this and other surface proteins could promote bacterial dissemination, whereas the generation of soluble complexes between immunoglobulins and immunoglobulin-binding streptococcal surface proteins could be an etiological factor in the development of glomerulonephritis and rheumatic fever. Thus, in these serious complications to S. pyogenes infections immune complexes are found in affected organs. The cysteine proteinase also solubilized a 116-kDa internal fragment of C5a peptidase, another streptococcal surface protein. Activation of the complement system generates C5a, a peptide stimulating leukocyte chemotaxis. C5a-mediated granulocyte migration was blocked by the 116-kDa fragment. This mechanism, by which phagocytes could be prevented from reaching the site of infection, may also contribute to the pathogenicity and virulence of S. pyogenes. PMID:7730368

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

    PubMed Central

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

    2001-01-01

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

  6. Crystal Structure of West Nile Virus Envelope Glycoprotein Reveals Viral Surface Epitopes

    SciTech Connect

    Kanai,R.; Kar, K.; Anthony, K.; Gould, L.; Ledizet, M.; Fikrig, E.; Marasco, W.; Koski, R.; Modis, Y.

    2006-01-01

    West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.

  7. Hydration dynamics near a model protein surface

    SciTech Connect

    Russo, Daniela; Hura, Greg; Head-Gordon, Teresa

    2003-09-01

    The evolution of water dynamics from dilute to very high concentration solutions of a prototypical hydrophobic amino acid with its polar backbone, N-acetyl-leucine-methylamide (NALMA), is studied by quasi-elastic neutron scattering and molecular dynamics simulation for both the completely deuterated and completely hydrogenated leucine monomer. We observe several unexpected features in the dynamics of these biological solutions under ambient conditions. The NALMA dynamics shows evidence of de Gennes narrowing, an indication of coherent long timescale structural relaxation dynamics. The translational water dynamics are analyzed in a first approximation with a jump diffusion model. At the highest solute concentrations, the hydration water dynamics is significantly suppressed and characterized by a long residential time and a slow diffusion coefficient. The analysis of the more dilute concentration solutions takes into account the results of the 2.0M solution as a model of the first hydration shell. Subtracting the first hydration layer based on the 2.0M spectra, the translational diffusion dynamics is still suppressed, although the rotational relaxation time and residential time are converged to bulk-water values. Molecular dynamics analysis shows spatially heterogeneous dynamics at high concentration that becomes homogeneous at more dilute concentrations. We discuss the hydration dynamics results of this model protein system in the context of glassy systems, protein function, and protein-protein interfaces.

  8. Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli.

    PubMed

    Johnson, Brant R; Hymes, Jeffrey; Sanozky-Dawes, Rosemary; Henriksen, Emily DeCrescenzo; Barrangou, Rodolphe; Klaenhammer, Todd R

    2016-01-01

    The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa. PMID:26475115

  9. Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli

    PubMed Central

    Johnson, Brant R.; Hymes, Jeffrey; Sanozky-Dawes, Rosemary; Henriksen, Emily DeCrescenzo

    2015-01-01

    The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa. PMID:26475115

  10. Enhanced microcontact printing of proteins on nanoporous silica surface

    NASA Astrophysics Data System (ADS)

    Blinka, Ellen; Loeffler, Kathryn; Hu, Ye; Gopal, Ashwini; Hoshino, Kazunori; Lin, Kevin; Liu, Xuewu; Ferrari, Mauro; Zhang, John X. J.

    2010-10-01

    We demonstrate porous silica surface modification, combined with microcontact printing, as an effective method for enhanced protein patterning and adsorption on arbitrary surfaces. Compared to conventional chemical treatments, this approach offers scalability and long-term device stability without requiring complex chemical activation. Two chemical surface treatments using functionalization with the commonly used 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were compared with the nanoporous silica surface on the basis of protein adsorption. The deposited thickness and uniformity of porous silica films were evaluated for fluorescein isothiocyanate (FITC)-labeled rabbit immunoglobulin G (R-IgG) protein printed onto the substrates via patterned polydimethlysiloxane (PDMS) stamps. A more complete transfer of proteins was observed on porous silica substrates compared to chemically functionalized substrates. A comparison of different pore sizes (4-6 nm) and porous silica thicknesses (96-200 nm) indicates that porous silica with 4 nm diameter, 57% porosity and a thickness of 96 nm provided a suitable environment for complete transfer of R-IgG proteins. Both fluorescence microscopy and atomic force microscopy (AFM) were used for protein layer characterizations. A porous silica layer is biocompatible, providing a favorable transfer medium with minimal damage to the proteins. A patterned immunoassay microchip was developed to demonstrate the retained protein function after printing on nanoporous surfaces, which enables printable and robust immunoassay detection for point-of-care applications.

  11. Cleaning of biomaterial surfaces: protein removal by different solvents.

    PubMed

    Kratz, Fabian; Grass, Simone; Umanskaya, Natalia; Scheibe, Christian; Müller-Renno, Christine; Davoudi, Neda; Hannig, Matthias; Ziegler, Christiane

    2015-04-01

    The removal of biofilms or protein films from biomaterials is still a challenging task. In particular, for research investigations on real (applied) surfaces the reuse of samples is of high importance, because reuse allows the comparison of the same sample in different experiments. The aim of the present study was to evaluate the cleaning efficiency of different solvents (SDS, water, acetone, isopropanol, RIPA-buffer and Tween-20) on five different biomaterials (titanium, gold, PMMA (no acetone used), ceramic, and PTFE) with different wettability which were covered by layers of two different adsorbed proteins (BSA and lysozyme). The presence of a protein film after adsorption was confirmed by transmission electron microscopy (TEM). After treatment of the surfaces with the different solvents, the residual proteins on the surface were determined by BCA-assay (bicinchoninic acid assay). Data of the present study indicate that SDS is an effective solvent, but for several protein-substrate combinations it does not show the cleaning efficiency often mentioned in literature. RIPA-buffer and Tween-20 were more effective. They showed very low residual protein amounts after cleaning on all examined material surfaces and for both proteins, however, with small differences for the respective substrate-protein combinations. RIPA-buffer in combination with ultrasonication completely removed the protein layer as confirmed by TEM. PMID:25725311

  12. Lipid Interaction Networks of Peripheral Membrane Proteins Revealed by Data-Driven Micelle Docking

    PubMed Central

    Dancea, Felician; Kami, Keiichiro; Overduin, Michael

    2008-01-01

    Many signaling and trafficking proteins contain modular domains that bind reversibly to cellular membranes. The structural basis of the intermolecular interactions which mediate these membrane-targeting events remains elusive since protein-membrane complexes are not directly accessible to standard structural biology techniques. Here we report a fast protein-micelle docking methodology that yields three-dimensional model structures of proteins inserted into micelles, revealing energetically favorable orientations, convergent insertion angles, and an array of protein-lipid interactions at atomic resolution. The method is applied to two peripheral membrane proteins, the early endosome antigen 1 (EEA1) FYVE (a zinc finger domain found in the proteins Fab1, YOTB/ZK632.12, Vac1, and EEA1) and Vam7p phagocyte oxidase homology domains, which are revealed to form extensive networks of interactions with multiple phospholipid headgroups and acyl chains. The resulting structural models explain extensive published mutagenesis data and reveal novel binding determinants. The docking restraints used here were based on NMR data, but can be derived from any technique that detects insertion of protein residues into a membrane, and can be applied to virtually any peripheral membrane protein or membrane-like structure. PMID:17890395

  13. Effect of phosphorus levels on the protein profiles of secreted protein and root surface protein of rice.

    PubMed

    Shinano, Takuro; Yoshimura, Tomoko; Watanabe, Toshihiro; Unno, Yusuke; Osaki, Mitsuru; Nanjo, Yohei; Komatsu, Setsuko

    2013-11-01

    Plant roots are complicated organs that absorb water and nutrients from the soil. Roots also play an essential role in protecting plants from attack by soil pathogens and develop a beneficial role with some soil microorganisms. Plant-derived rhizosphere proteins (e.g., root secretory proteins and root surface binding proteins) are considered to play important roles in developing mutual relationships in the rhizosphere. In the rhizosphere, where plant roots meet the surrounding environment, it has been suggested that root secretory protein and root surface binding protein are important factors. Furthermore, it is not known how the physiological status of the plant affects the profile of these proteins. In this study, rice plants were grown aseptically, with or without phosphorus nutrition, and proteins were obtained from root bathing solution (designated as root secretory proteins) and obtained using 0.2 M CaCl2 solution (designated as root surface binding proteins). The total number of identified proteins in the root bathing solution was 458, and the number of root surface binding proteins was 256. More than half of the proteins were observed in both fractions. Most of the proteins were categorized as either having signal peptides or no membrane transport helix sites. The functional categorization suggested that most of the proteins seemed to have secretory pathways and were involved in defense/disease-related functions. These characteristics seem to be unique to rhizosphere proteins, and the latter might be part of the plants strategy to defeat pathogens in the soil. The low phosphorus treatment significantly increased the number of pathogenesis-related proteins in the root secretory proteins, whereas the change was small in the case of the root surface binding proteins. The results suggested that the roots are actively and selectively secreting protein into the rhizosphere. PMID:24083427

  14. Identification of Surface Proteins from Lactobacillus casei BL23 Able to Bind Fibronectin and Collagen.

    PubMed

    Muñoz-Provencio, Diego; Pérez-Martínez, Gaspar; Monedero, Vicente

    2011-03-01

    Strains of lactobacilli show the capacity to attach to extracellular matrix proteins. Cell-wall fractions of Lactobacillus casei BL23 enriched in fibronectin, and collagen-binding proteins were isolated. Mass spectrometry analysis of their protein content revealed the presence of stress-related proteins (GroEL, ClpL), translational elongation factors (EF-Tu, EF-G), oligopeptide solute-binding proteins, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The latter two enzymes were expressed in Escherichia coli and purified as glutathione-S-transferase (GST) fusion proteins, and their in vitro binding activity to fibronectin and collagen was confirmed. These results reinforce the idea that lactobacilli display on their surfaces a variety of moonlighting proteins that can be important in their adaptation to survive at intestinal mucosal sites and in the interaction with host cells. PMID:26781495

  15. Identification of Renibacterium salmoninarum surface proteins by radioiodination.

    PubMed

    Fredriksen, A; Bakken, V

    1994-09-01

    Surface exposed proteins of Renibacterium salmoninarum were identified by radiolabelling whole bacterial cells with 125I, followed by SDS-PAGE and autoradiography. The most prominent bands had molecular masses of approximately 57 kDa and 22 kDa; in addition, some less intensively labelled bands were detected. Polyclonal sera raised against the 22 kDa protein did not react with the 57 kDa protein. N-terminal amino acid sequence analysis of the purified 22 kDa protein showed no similarity with the sequence of the 57 kDa protein. PMID:7926685

  16. Grafting zwitterionic polymer onto cryogel surface enhances protein retention in steric exclusion chromatography on cryogel monolith.

    PubMed

    Tao, Shi-Peng; Zheng, Jie; Sun, Yan

    2015-04-10

    Cryogel monoliths with interconnected macropores (10-100μm) and hydrophilic surfaces can be employed as chromatography media for protein retention in steric exclusion chromatography (SXC). SXC is based on the principle that the exclusion of polyethylene glycol (PEG) on both a hydrophilic chromatography surface and a protein favors their association, leading to the protein retention on the chromatography surface. Elution of the retained protein can be achieved by reducing PEG concentration. In this work, the surface of polyacrylamide-based cryogel monolith was modified by grafting zwitterionic poly(carboxybetaine methacrylate) (pCBMA), leading the increase in the surface hydrophilicity. Observation by scanning electron microscopy revealed the presence of the grafted pCBMA chain clusters on the cryogel surface, but pCBMA grafting did not result in the changes of the physical properties of the monolith column, and the columns maintained good recyclability in SXC. The effect of the surface grafting on the SXC behavior of γ-globulin was investigated in a wide flow rate range (0.6-12cm/min). It was found that the dynamic retention capacity increased 1.4-1.8 times by the zwitterionic polymer grafting in the flow rate range of 1.5-12cm/min. The mechanism of enhanced protein retention on the zwitterionic polymer-grafted surface was proposed. The research proved that zwitterionic polymer modification was promising for the development of new materials for SXC applications. PMID:25757821

  17. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

    SciTech Connect

    Klopffleisch, Karsten; Phan, Nguyen; Chen, Jay; Panstruga, Ralph; Uhrig, Joachim; Jones, Alan M

    2011-01-01

    The heterotrimeric G-protein complex is minimally composed of G{alpha}, G{beta}, and G{gamma} subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

  18. Proteomics Analysis Reveals Distinct Corona Composition on Magnetic Nanoparticles with Different Surface Coatings: Implications for Interactions with Primary Human Macrophages

    PubMed Central

    Vogt, Carmen; Pernemalm, Maria; Kohonen, Pekka; Laurent, Sophie; Hultenby, Kjell; Vahter, Marie; Lehtiö, Janne; Toprak, Muhammet S.; Fadeel, Bengt

    2015-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising contrast agents for magnetic resonance imaging. The influence of different surface coatings on the biocompatibility of SPIONs has been addressed, but the potential impact of the so-called corona of adsorbed proteins on the surface of SPIONs on their biological behavior is less well studied. Here, we determined the composition of the plasma protein corona on silica-coated versus dextran-coated SPIONs using mass spectrometry-based proteomics approaches. Notably, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed distinct protein corona compositions for the two different SPIONs. Relaxivity of silica-coated SPIONs was modulated by the presence of a protein corona. Moreover, the viability of primary human monocyte-derived macrophages was influenced by the protein corona on silica-coated, but not dextran-coated SPIONs, and the protein corona promoted cellular uptake of silica-coated SPIONs, but did not affect internalization of dextran-coated SPIONs. PMID:26444829

  19. Analysis of protein transport in the Brassica oleracea vasculature reveals protein-specific destinations.

    PubMed

    Niu, Chenxing; Anstead, James; Verchot, Jeanmarie

    2012-03-01

    We investigated the vascular transport properties of exogenously applied proteins to Brassica oleracea plants and compared their delivery to various aerial parts of the plant with carboxy fluorescein (CF) dye. We identified unique properties for each protein. Alexafluor-tagged bovine serum albumin (Alexa-BSA) and Alexafluor-tagged Histone H1 (Alexa-Histone) moved slower than CF dye throughout the plant. Interestingly, Alexa-Histone was retained in the phloem and phloem parenchyma while Alexa-BSA moved into the apoplast. One possibility is that Alexa-Histone sufficiently resembles plant endogenous proteins and is retained in the vascular stream, while Alexa-BSA is exported from the cell as a foreign protein. Both proteins diffuse from the leaf veins into the leaf lamina. Alexa-BSA accumulated in the leaf epidermis while Alexa-Histone accumulated mainly in the mesophyll layers. Fluorescein-tagged hepatitis C virus core protein (fluorescein-HCV) was also delivered to B. oleracea plants and is larger than Alexa-BSA. This protein moves more rapidly than BSA through the plant and was restricted to the leaf veins. Fluorescein-HCV failed to unload to the leaf lamina. These combined data suggest that there is not a single default pathway for the vascular transfer of exogenous proteins in B. oleracea plants. Specific protein properties appear to determine their destination and transport properties within the phloem. PMID:22476467

  20. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid.

    PubMed Central

    Wise, K S; Kim, M F

    1987-01-01

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125I-labeled proteins with a series of monoclonal antibodies (MAbs). Surface proteins p70, p65, p50, and p44 were shown to be integral membrane components by selective partitioning into the hydrophobic phase during Triton X-114 (TX-114)-phase fractionation, whereas p41 was concomitantly identified as a surface protein exclusively partitioning into the aqueous phase. Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [35S]methionine, 14C-amino acids, or [3H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Covalent lipid attachment was established by high-performance liquid chromatography identification of [3H]methyl palmitate after acid methanolysis of delipidated proteins. An additional, unidentified methanolysis product suggested conversion of palmitate to another form of lipid also attached to these proteins. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a larger p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11. These studies establish that a highly restricted set of distinct, lipid

  1. Mechanism of Bacterial Cell-Surface Attachment Revealed by the Structure of Cellulosomal Type II Cohesin-dockerin Complex

    SciTech Connect

    Adams,J.; Pal, G.; Jia, Z.; Smith, S.

    2006-01-01

    Bacterial cell-surface attachment of macromolecular complexes maintains the microorganism in close proximity to extracellular substrates and allows for optimal uptake of hydrolytic byproducts. The cellulosome is a large multienzyme complex used by many anaerobic bacteria for the efficient degradation of plant cell-wall polysaccharides. The mechanism of cellulosome retention to the bacterial cell surface involves a calcium-mediated protein-protein interaction between the dockerin (Doc) module from the cellulosomal scaffold and a cohesin (Coh) module of cell-surface proteins located within the proteoglycan layer. Here, we report the structure of an ultra-high-affinity (K{sub a} = 1.44 x 10{sup 10} M{sup 1-}) complex between type II Doc, together with its neighboring X module from the cellulosome scaffold of Clostridium thermocellum, and a type II Coh module associated with the bacterial cell surface. Identification of X module-Doc and X module-Coh contacts reveal roles for the X module in Doc stability and enhanced Coh recognition. This extremely tight interaction involves one face of the Coh and both helices of the Doc and comprises significant hydrophobic character and a complementary extensive hydrogen-bond network. This structure represents a unique mechanism for cell-surface attachment in anaerobic bacteria and provides a rationale for discriminating between type I and type II Coh modules.

  2. Electro-induced protein deposition on low-fouling surfaces

    NASA Astrophysics Data System (ADS)

    Cole, M. A.; Voelcker, N. H.; Thissen, H.

    2007-12-01

    Control over protein adsorption is a key issue for numerous biomedical applications ranging from diagnostic microarrays to tissue-engineered medical devices. Here, we describe a method for creating surfaces that prevent non-specific protein adsorption, which upon application of an external trigger can be transformed into surfaces showing high protein adsorption on demand. Silicon wafers were used as substrate materials upon which thin functional coatings were constructed by the deposition of an allylamine plasma polymer followed by high-density grafting of poly(ethylene oxide) aldehyde, resulting in a low-fouling surface. When the underlying highly doped silicon substrate was used as an electrode, the resulting electrostatic attraction between the electrode and charged proteins in solution induced protein deposition at the low-fouling interface. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were used to characterize the surface modifications. Controlled protein adsorption experiments were carried out using horseradish peroxidase. The amount of protein deposited at the surface was then investigated by means of a colorimetric assay. It is expected that the concept described here will find use in a variety of biotechnological and biomedical applications, particularly in the area of biochips.

  3. 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

  4. Selective and programmed cleavage of GPI-anchored proteins from the surface membrane by phospholipase C.

    PubMed

    Müller, Alexandra; Klöppel, Christine; Smith-Valentine, Megan; Van Houten, Judith; Simon, Martin

    2012-01-01

    Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content. PMID:22024023

  5. Nanoscale periodic modulations on sodium chloride surface revealed by tuning fork atomic force microscopy.

    PubMed

    Clark, Kendal W; Qin, Shengyong; Zhang, X-G; Li, An-Ping

    2012-05-11

    The sodium chloride surface is one of the most common platforms for the study of catalysts, thin film growth, and atmospheric aerosols. Here we report a nanoscale periodic modulation pattern on the surface of a cleaved NaCl single crystal, revealed by non-contact atomic force microscopy with a tuning fork sensor. The surface pattern shows two orthogonal domains, extending over the entire cleavage surface. The spatial modulations exhibit a characteristic period of 5.4 nm, along <110> crystallographic directions of the NaCl. The modulations are robust in vacuum, not affected by the tip-induced electric field or gentle annealing (<300 °C); however, they are eliminated after exposure to water and an atomically flat surface can be recovered by subsequent thermal annealing after water exposure. A strong electrostatic charging is revealed on the cleavage surface which may facilitate the formation of the observed metastable surface reconstruction. PMID:22513484

  6. Nanoscale periodic modulations on sodium chloride surface revealed by tuning fork atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Clark, Kendal W.; Qin, Shengyong; Zhang, X.-G.; Li, An-Ping

    2012-05-01

    The sodium chloride surface is one of the most common platforms for the study of catalysts, thin film growth, and atmospheric aerosols. Here we report a nanoscale periodic modulation pattern on the surface of a cleaved NaCl single crystal, revealed by non-contact atomic force microscopy with a tuning fork sensor. The surface pattern shows two orthogonal domains, extending over the entire cleavage surface. The spatial modulations exhibit a characteristic period of 5.4 nm, along <110> crystallographic directions of the NaCl. The modulations are robust in vacuum, not affected by the tip-induced electric field or gentle annealing (<300 °C) however, they are eliminated after exposure to water and an atomically flat surface can be recovered by subsequent thermal annealing after water exposure. A strong electrostatic charging is revealed on the cleavage surface which may facilitate the formation of the observed metastable surface reconstruction.

  7. Energy Landscape of All-Atom Protein-Protein Interactions Revealed by Multiscale Enhanced Sampling

    PubMed Central

    Moritsugu, Kei; Terada, Tohru; Kidera, Akinori

    2014-01-01

    Protein-protein interactions are regulated by a subtle balance of complicated atomic interactions and solvation at the interface. To understand such an elusive phenomenon, it is necessary to thoroughly survey the large configurational space from the stable complex structure to the dissociated states using the all-atom model in explicit solvent and to delineate the energy landscape of protein-protein interactions. In this study, we carried out a multiscale enhanced sampling (MSES) simulation of the formation of a barnase-barstar complex, which is a protein complex characterized by an extraordinary tight and fast binding, to determine the energy landscape of atomistic protein-protein interactions. The MSES adopts a multicopy and multiscale scheme to enable for the enhanced sampling of the all-atom model of large proteins including explicit solvent. During the 100-ns MSES simulation of the barnase-barstar system, we observed the association-dissociation processes of the atomistic protein complex in solution several times, which contained not only the native complex structure but also fully non-native configurations. The sampled distributions suggest that a large variety of non-native states went downhill to the stable complex structure, like a fast folding on a funnel-like potential. This funnel landscape is attributed to dominant configurations in the early stage of the association process characterized by near-native orientations, which will accelerate the native inter-molecular interactions. These configurations are guided mostly by the shape complementarity between barnase and barstar, and lead to the fast formation of the final complex structure along the downhill energy landscape. PMID:25340714

  8. Using Surface Curvature to Control the Dimerization of a Surface-Active Protein

    NASA Astrophysics Data System (ADS)

    Kurylowicz, Martin; Giuliani, Maximiliano; Dutcher, John

    2012-02-01

    Understanding the influence of surface geometry on adsorbed proteins promises new possibilities in biophysics, such as topographical catalysis, molecular recognition of geometric cues, and modulations of oligomerization or ligand binding. We have created nano-textured hydrophobic surfaces that are stable in buffer by spin coating polystyrene (PS) nanoparticles (NPs) to form patchy NP monolayers on a PS substrate, yielding flat and highly curved areas on the same sample. Moreover, we have separated surface chemistry from texture by floating a 10 nm thick film of monodisperse PS onto the NP-functionalized surface. Using Single Molecule Force Spectroscopy we have compared in situ the distribution of detachment lengths for proteins on curved surfaces to that measured on flat surfaces. We have shown that β-Lactoglobulin (β-LG), a surface-active protein which helps to stabilize oil droplets in milk, forms dimers on both flat PS surfaces and surfaces with a radius of curvature of 100 nm, whereas β-LG monomers exist for more highly curved surfaces with radii of curvature of 25 and 40 nm. It is surprising that rather large radii of curvature have such a strong influence on proteins whose radius is only ˜2 nm. Furthermore, the transition from dimer to monomer with changes in surface curvature offers promising applications for proteins whose function can be modified by their oligomerization state.

  9. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain.

    PubMed

    Pearson, M A; Reczek, D; Bretscher, A; Karplus, P A

    2000-04-28

    The ezrin-radixin-moesin (ERM) protein family link actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks their binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain. This extended binding mode suggests a novel mechanism for how different signals could produce varying levels of activation. Sequence conservation suggests a similar regulation of the tumor suppressor merlin. PMID:10847681

  10. Protein Function Annotation By Local Binding Site Surface Similarity

    PubMed Central

    Spitzer, Russell; Cleves, Ann E.; Varela, Rocco; Jain, Ajay N.

    2013-01-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against approximately 60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that pre-dated query protein biochemical annotation for five out of the eight query proteins. A panel of twelve currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins. PMID:24166661

  11. Surface Display of Recombinant Proteins on Bacillus subtilis Spores

    PubMed Central

    Isticato, Rachele; Cangiano, Giuseppina; Tran, Hoa T.; Ciabattini, Annalisa; Medaglini, Donata; Oggioni, Marco R.; De Felice, Maurilio; Pozzi, Gianni; Ricca, Ezio

    2001-01-01

    We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 × 103 TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules. PMID:11591673

  12. Intrinsic surface-drying properties of bioadhesive proteins.

    PubMed

    Akdogan, Yasar; Wei, Wei; Huang, Kuo-Ying; Kageyama, Yoshiyuki; Danner, Eric W; Miller, Dusty R; Martinez Rodriguez, Nadine R; Waite, J Herbert; Han, Songi

    2014-10-13

    Sessile marine mussels must "dry" underwater surfaces before adhering to them. Synthetic adhesives have yet to overcome this fundamental challenge. Previous studies of bioinspired adhesion have largely been performed under applied compressive forces, but such studies are poor predictors of the ability of an adhesive to spontaneously penetrate surface hydration layers. In a force-free approach to measuring molecular-level interaction through surface-water diffusivity, different mussel foot proteins were found to have different abilities to evict hydration layers from surfaces-a necessary step for adsorption and adhesion. It was anticipated that DOPA would mediate dehydration owing to its efficacy in bioinspired wet adhesion. Instead, hydrophobic side chains were found to be a critical component for protein-surface intimacy. This direct measurement of interfacial water dynamics during force-free adsorptive interactions at solid surfaces offers guidance for the engineering of wet adhesives and coatings. PMID:25168789

  13. Conformational selection in a protein-protein interaction revealed by dynamic pathway analysis

    SciTech Connect

    Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco; Otten, Renee; Higgins, Matthew K.; Schertler, Gebhard F. X.; Oprian, Daniel D.; Kern, Dorothee

    2015-12-24

    Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Lastly, protein dynamics in free recoverin limits the overall rate of binding.

  14. Conformational selection in a protein-protein interaction revealed by dynamic pathway analysis

    DOE PAGESBeta

    Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco; Otten, Renee; Higgins, Matthew K.; Schertler, Gebhard F. X.; Oprian, Daniel D.; Kern, Dorothee

    2015-12-24

    Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsinmore » kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Lastly, protein dynamics in free recoverin limits the overall rate of binding.« less

  15. Phenotypic mixing between different hepadnavirus nucleocapsid proteins reveals C protein dimerization to be cis preferential.

    PubMed Central

    Chang, C; Zhou, S; Ganem, D; Standring, D N

    1994-01-01

    Hepadnaviruses encode a single core (C) protein which assembles into a nucleocapsid containing the polymerase (P) protein and pregenomic RNA during viral replication in hepatocytes. We examined the ability of heterologous hepadnavirus C proteins to cross-oligomerize. Using a two-hybrid assay in HepG2 cells, we observed cross-oligomerization among the core proteins from hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus. When expressed in Xenopus oocytes, in which hepadnavirus C proteins form capsids, the C polypeptides from woodchuck hepatitis virus and ground squirrel hepatitis virus, but not duck hepatitis B virus, can efficiently coassemble with an epitope-tagged HBV core polypeptide to form mixed capsids. However, when two different core mRNAs are coexpressed in oocytes the core monomers show a strong preference for forming homodimers rather than heterodimers. This holds true even for coexpression of two HBV C proteins differing only by an epitope tag, suggesting that core monomers are not free to diffuse and associate with other monomers. Thus, mixed capsids result from aggregation of different species of homodimers. Images PMID:7518533

  16. Conformational Selection in a Protein-Protein Interaction revealed by Dynamic Pathway Analysis

    PubMed Central

    Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco; Otten, Renee; Higgins, Matthew K.; Schertler, Gebhard F. X.; Oprian, Daniel D.; Kern, Dorothee

    2015-01-01

    SUMMARY Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsin kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Protein dynamics in free recoverin limits the overall rate of binding. PMID:26725117

  17. Formation of Ordered Arrays of Proteins on Surfaces

    NASA Technical Reports Server (NTRS)

    Lenhoff, A. M.

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. While the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models, with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation and crystal growth.

  18. Protein-Induced Surface Structuring in Myelin Membrane Monolayers

    PubMed Central

    Rosetti, Carla M.; Maggio, Bruno

    2007-01-01

    Monolayers prepared from myelin conserve all the compositional complexity of the natural membrane when spread at the air-water interface. They show a complex pressure-dependent surface pattern that, on compression, changes from the coexistence of two liquid phases to a viscous fractal phase embedded in a liquid phase. We dissected the role of major myelin protein components, myelin basic protein (MBP), and Folch-Lees proteolipid protein (PLP) as crucial factors determining the structural dynamics of the interface. By analyzing mixtures of a single protein with the myelin lipids we found that MBP and PLP have different surface pressure-dependent behaviors. MBP stabilizes the segregation of two liquid phases at low pressures and becomes excluded from the film under compression, remaining adjacent to the interface. PLP, on the contrary, organizes a fractal-like pattern at all surface pressures when included in a monolayer of the protein-free myelin lipids but it remains mixed in the MBP-induced liquid phase. The resultant surface topography and dynamics is regulated by combined near to equilibrium and out-of-equilibrium effects. PLP appears to act as a surface skeleton for the whole components whereas MBP couples the structuring to surface pressure-dependent extrusion and adsorption processes. PMID:17905850

  19. Quantitative Fluorescence Correlation Spectroscopy Reveals a 1000-Fold Increase in Lifetime of Protein Functionality

    PubMed Central

    Zhang, Dianwen; Lans, Hannes; Vermeulen, Wim; Lenferink, Aufried; Otto, Cees

    2008-01-01

    We have investigated dilute protein solutions with fluorescence correlation spectroscopy (FCS) and have observed that a rapid loss of proteins occurs from solution. It is commonly assumed that such a loss is the result of protein adsorption to interfaces. A protocol was developed in which this mode of protein loss can be prevented. However, FCS on fluorescent protein (enhanced green fluorescent protein, mCherry, and mStrawberry) solutions enclosed by adsorption-protected interfaces still reveals a decrease of the fluorescent protein concentration, while the diffusion time is stable over long periods of time. We interpret this decay as a loss of protein functionality, probably caused by denaturation of the fluorescent proteins. We show that the typical lifetime of protein functionality in highly dilute, approximately single molecule per femtoliter solutions can be extended more than 1000-fold (typically from a few hours to >40 days) by adding compounds with surfactant behavior. No direct interactions between the surfactant and the fluorescent proteins were observed from the diffusion time measured by FCS. A critical surfactant concentration of more than 23 μM was required to achieve the desired protein stabilization for Triton X-100. The surfactant does not interfere with DNA-protein binding, because similar observations were made using DNA-cutting restriction enzymes. We associate the occurrence of denaturation of proteins with the activity of water at the water-protein interface, which was recently proposed in terms of the “water attack model”. Our observations suggest that soluble biomolecules can extend an influence over much larger distances than suggested by their actual volume. PMID:18586843

  20. Mechanically Untying a Protein Slipknot: Multiple Pathways Revealed by Force Spectroscopy and Steered Molecular Dynamics Simulations

    PubMed Central

    He, Chengzhi; Genchev, Georgi Z.; Lu, Hui; Li, Hongbin

    2013-01-01

    Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechancial unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie, and the polypeptide chain to completely extend. These distinct unfolding pathways proceed either via a two-state or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus provding a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechancial unfolding pathway of AFV3-109, and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding. PMID:22626004

  1. Structural constraints for the binding of short peptides to claudin-4 revealed by surface plasmon resonance.

    PubMed

    Ling, Jun; Liao, Hailing; Clark, Robin; Wong, Mandy Sze Man; Lo, David D

    2008-11-01

    Claudin family transmembrane proteins play an important role in tight junction structure and function in epithelial cells. Among the 24 isoforms identified in mice and humans, claudin-4 and -3 serve as the receptor for Clostridium perfringens enterotoxin (Cpe). The second extracellular loop (Ecl2) of claudin-4 is responsible for the binding to the C-terminal 30 amino acids of Cpe (Cpe30). To define the structural constraints for the claudin-4/Cpe30 interaction, a surface plasmon resonance (SPR) method was developed. GST fusions with claudin-4 revealed that Ecl2 with the downstream transmembrane domain of claudin-4 reconstituted the basic structural requirement for optimal binding activity to Cpe30, with affinity in the nanomolar range. Two 12-mer peptides selected by phage display against claudin-4-transfected CHO cells and a 12-mer Cpe mutant peptide also showed significant affinity for claudin-4 with this SPR assay, suggesting that a short peptide can establish stable contact with Ecl2 with nanomolar affinity. Alignment of these short peptides unveiled a common Ecl2 binding motif: . Whereas the short peptides bound native claudin-4 on transfected CHO cells in pull-down assays, only the larger Cpe30 peptide affected trans-epithelial electrical resistance (TER) in peptide-treated Caco-2BBe monolayers. Importantly, Cpe30 retained its binding to claudin-4 when fused to the C terminus of influenza hemagglutinin, demonstrating that its binding activity can be maintained in a different biochemical context. These studies may help in the design of assays for membrane receptor interactions with soluble ligands, and in applying new targeting ligands to delivering attached "cargo" proteins. PMID:18782762

  2. Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation

    SciTech Connect

    Raymond, Donald D.; Piper, Mary E.; Gerrard, Sonja R.; Smith, Janet L.

    2010-07-13

    Rift Valley fever virus (RVFV) is a negative-sense RNA virus (genus Phlebovirus, family Bunyaviridae) that infects livestock and humans and is endemic to sub-Saharan Africa. Like all negative-sense viruses, the segmented RNA genome of RVFV is encapsidated by a nucleocapsid protein (N). The 1.93-{angstrom} crystal structure of RVFV N and electron micrographs of ribonucleoprotein (RNP) reveal an encapsidated genome of substantially different organization than in other negative-sense RNA virus families. The RNP polymer, viewed in electron micrographs of both virus RNP and RNP reconstituted from purified N with a defined RNA, has an extended structure without helical symmetry. N-RNA species of {approx}100-kDa apparent molecular weight and heterogeneous composition were obtained by exhaustive ribonuclease treatment of virus RNP, by recombinant expression of N, and by reconstitution from purified N and an RNA oligomer. RNA-free N, obtained by denaturation and refolding, has a novel all-helical fold that is compact and well ordered at both the N and C termini. Unlike N of other negative-sense RNA viruses, RVFV N has no positively charged surface cleft for RNA binding and no protruding termini or loops to stabilize a defined N-RNA oligomer or RNP helix. A potential protein interaction site was identified in a conserved hydrophobic pocket. The nonhelical appearance of phlebovirus RNP, the heterogeneous {approx}100-kDa N-RNA multimer, and the N fold differ substantially from the RNP and N of other negative-sense RNA virus families and provide valuable insights into the structure of the encapsidated phlebovirus genome.

  3. Molecular basis for polyol-induced protein stability revealed by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Liu, Fu-Feng; Ji, Luo; Zhang, Lin; Dong, Xiao-Yan; Sun, Yan

    2010-06-01

    Molecular dynamics simulations of chymotrypsin inhibitor 2 in different polyols (glycerol, xylitol, sorbitol, trehalose, and sucrose) at 363 K were performed to probe the molecular basis of the stabilizing effect, and the data in water, ethanol, and glycol were compared. It is found that protein protection by polyols is positively correlated with both the molecular volume and the fractional polar surface area, and the former contributes more significantly to the protein's stability. Polyol molecules have only a few direct hydrogen bonds with the protein, and the number of hydrogen bonds between a polyol and the protein is similar for different polyols. Thus, it is concluded that the direct interactions contribute little to the stabilizing effect. It is clarified that the preferential exclusion of the polyols is the origin of their protective effects, and it increases with increasing polyol size. Namely, there is preferential hydration on the protein surface (2 Å), and polyol molecules cluster around the protein at a distance of about 4 Å. The preferential exclusion of polyols leads to indirect interactions that prevent the protein from thermal unfolding. The water structure becomes more ordered with increasing the polyol size. So, the entropy of water in the first hydration shell decreases, and a larger extent of decrease is observed with increasing polyol size, leading to larger transfer free energy. The findings suggest that polyols protect the protein from thermal unfolding via indirect interactions. The work has thus elucidated the molecular mechanism of structural stability of the protein in polyol solutions.

  4. Molecular basis for polyol-induced protein stability revealed by molecular dynamics simulations.

    PubMed

    Liu, Fu-Feng; Ji, Luo; Zhang, Lin; Dong, Xiao-Yan; Sun, Yan

    2010-06-14

    Molecular dynamics simulations of chymotrypsin inhibitor 2 in different polyols (glycerol, xylitol, sorbitol, trehalose, and sucrose) at 363 K were performed to probe the molecular basis of the stabilizing effect, and the data in water, ethanol, and glycol were compared. It is found that protein protection by polyols is positively correlated with both the molecular volume and the fractional polar surface area, and the former contributes more significantly to the protein's stability. Polyol molecules have only a few direct hydrogen bonds with the protein, and the number of hydrogen bonds between a polyol and the protein is similar for different polyols. Thus, it is concluded that the direct interactions contribute little to the stabilizing effect. It is clarified that the preferential exclusion of the polyols is the origin of their protective effects, and it increases with increasing polyol size. Namely, there is preferential hydration on the protein surface (2 A), and polyol molecules cluster around the protein at a distance of about 4 A. The preferential exclusion of polyols leads to indirect interactions that prevent the protein from thermal unfolding. The water structure becomes more ordered with increasing the polyol size. So, the entropy of water in the first hydration shell decreases, and a larger extent of decrease is observed with increasing polyol size, leading to larger transfer free energy. The findings suggest that polyols protect the protein from thermal unfolding via indirect interactions. The work has thus elucidated the molecular mechanism of structural stability of the protein in polyol solutions. PMID:20550422

  5. Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

    PubMed Central

    Champer, Jackson; Ito, James I.; Clemons, Karl V.; Stevens, David A.; Kalkum, Markus

    2016-01-01

    We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy). Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4), Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here. PMID:26878023

  6. Identification of sporozoite surface proteins and antigens of Eimeria nieschulzi (Apicomplexa)

    SciTech Connect

    Tilley, M.; Upton, S.J. )

    1990-03-01

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and {sup 125}I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed greater than 50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53-54, 45, 28, 23-24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 125I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82-88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic.

  7. Super-resolution Microscopy of Clickable Amino Acids Reveals the Effects of Fluorescent Protein Tagging on Protein Assemblies.

    PubMed

    Vreja, Ingrid C; Nikić, Ivana; Göttfert, Fabian; Bates, Mark; Kröhnert, Katharina; Outeiro, Tiago F; Hell, Stefan W; Lemke, Edward A; Rizzoli, Silvio O

    2015-11-24

    The advent of super-resolution microscopy (nanoscopy) has set high standards for fluorescence tagging. Fluorescent proteins (FPs) are convenient tags in conventional imaging, but their use in nanoscopy has been questioned due to their relatively large size and propensity to form multimers. Here, we compared the nanoscale organization of proteins with or without FP tags by introducing the unnatural amino acid propargyl-L-lysine (PRK) in 26 proteins known to form multimolecular arrangements and into their FP-tagged variants. We revealed the proteins by coupling synthetic fluorophores to PRK via click chemistry and visualized them using ground-state depletion microscopy followed by individual molecule return, as well as stimulated emission depletion microscopy. The arrangements formed by the FP-tagged and nontagged proteins were similar. Mild, but statistically significant differences were observed for only six proteins (23% of all proteins tested). This suggests that FP-based nanoscopy is generally reliable. Unnatural amino acids should be a reliable alternative for the few proteins that are sensitive to FP tagging. PMID:26498474

  8. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

    PubMed

    Kohansal-Nodehi, Mahdokht; Chua, John Je; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. PMID:27115346

  9. Putative protein partners for the human CPI-17 protein revealed by bacterial two-hybrid screening.

    PubMed

    Kim, Kyung-mi; Adyshev, Djanybek M; Kása, Anita; Zemskov, Evgeny A; Kolosova, Irina A; Csortos, Csilla; Verin, Alexander D

    2013-07-01

    We have previously demonstrated that PKC-potentiated inhibitory protein of protein phosphatase-1 (CPI-17) is expressed in lung endothelium. CPI-17, a specific inhibitor of myosin light chain phosphatase (MLCP), is involved in the endothelial cytoskeletal and barrier regulation. In this paper, we report the identification of fourteen putative CPI-17 interacting proteins in the lung using BacterioMatch Two-Hybrid System. Five of them: plectin 1 isoform 1, alpha II spectrin, OK/SW-CL.16, gelsolin isoform a, and junction plakoglobin are involved in actin cytoskeleton organization and cell adhesion, suggesting possible significance of these binding partners in CPI-17-mediated cytoskeletal reorganization of endothelial cells. Furthermore, we confirmed the specific interaction between plakoglobin and CPI-17, which is affected by the phosphorylation status of CPI-17 in human lung microvascular endothelial cells. PMID:23583905

  10. Evaluation of the Effectiveness of Surfactants and Denaturants to Elute and Denature Adsorbed Protein on Different Surface Chemistries.

    PubMed

    Thyparambil, Aby A; Wei, Yang; Latour, Robert A

    2015-11-01

    The elution and/or denaturation of proteins from material surfaces by chemical excipients such as surfactants and denaturants is important for numerous applications including medical implant reprocessing, bioanalyses, and biodefense. The objective of this study was to develop and apply methods to quantitatively assess how surface chemistry and adsorption conditions influence the effectiveness of three commonly used surfactants (sodium dodecyl sulfate, n-octyl-β-d-glucoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and two denaturants (guanidium hydrochloride and urea) to elute protein (hen egg white lysozyme and bovine pancreatic ribonuclease A) from three different surface chemistries (silica glass, poly(methyl methacrylate), and high-density polyethylene). The structure and bioactivity of residual protein on the surface following elution were characterized using circular dichroism spectropolarimetry and enzyme assays to assess the extent of protein denaturation. Our results indicate that the denaturants were generally more effective than the surfactants in removing the adsorbed proteins from each type of surface. Also, the denaturing capacity of these excipients on the residual proteins on the surfaces was distinctly different from their influence on the proteins in solution and was unique for each of the adsorption conditions. Taken altogether, these results reveal that the effectiveness of surfactants and denaturants to elute and denature adsorbed protein is significantly influenced by surface chemistry and the conditions from which the protein was adsorbed. These results provide a basis for the selection, design, and further development of chemical agents for protein elution and surface decontamination. PMID:26449787

  11. Proteomic Profiling of Cereal Aphid Saliva Reveals Both Ubiquitous and Adaptive Secreted Proteins

    PubMed Central

    Wilkinson, Tom L.

    2013-01-01

    The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113. PMID:23460852

  12. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn

    PubMed Central

    Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

    2015-01-01

    The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize. PMID:26587848

  13. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

    PubMed

    Kalescky, Robert; Zhou, Hongyu; Liu, Jin; Tao, Peng

    2016-04-01

    Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery. PMID:27115535

  14. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery

    PubMed Central

    Liu, Jin

    2016-01-01

    Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier’s principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery. PMID:27115535

  15. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

    PubMed

    Dong, Yongbin; Wang, Qilei; Zhang, Long; Du, Chunguang; Xiong, Wenwei; Chen, Xinjian; Deng, Fei; Ma, Zhiyan; Qiao, Dahe; Hu, Chunhui; Ren, Yangliu; Li, Yuling

    2015-01-01

    The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize. PMID:26587848

  16. Quantitative surface studies of protein adsorption by infrared spectroscopy. II. Quantification of adsorbed and bulk proteins

    SciTech Connect

    Fink, D.J.; Hutson, T.B.; Chittur, K.K.; Gendreau, R.M.

    1987-08-15

    Attenuated total reflectance Fourier transform infrared spectra of surface-adsorbed proteins are correlated with concentration measurements determined by /sup 125/I-labeled proteins. This paper demonstrates that linear correlations between the intensity of the major bands of proteins and the quantity of proteins can be obtained for human albumin and immunoglobulin G up to surface concentrations of approximately 0.25 microgram/cm2. A poorer correlation was observed for human fibrinogen. A linear correlation was also observed between the concentration in the bulk solution and the major bands of albumin up to a concentration of 60 mg/ml.

  17. Surface (S)-layer proteins of Deinococcus radiodurans and their utility as vehicles for surface localization of functional proteins.

    PubMed

    Misra, Chitra Seetharam; Basu, Bhakti; Apte, Shree Kumar

    2015-12-01

    The radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi. The association of SlpA with Hpi on one side and peptidoglycan on the other, localizes this protein in the 'interstitial' layer of the deinoccocal cell wall. Gene chimeras of hpi-phoN and slh-phoN were constructed to test efficacy of S-layer proteins, as vehicles for cell surface localization in D. radiodurans. The Hpi-PhoN protein localized exclusively in the membrane fraction, and displayed cell-based phosphatase activity in vivo. The SLH-PhoN, which localized to both cytosolic and membrane fractions, displayed in vitro activity but no cell-based in vivo activity. Hpi, therefore, emerged as an efficient surface localizing protein and can be exploited for suitable applications of this superbug. PMID:26450150

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

    PubMed

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

    2016-08-01

    The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein-protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097-1107. © 2016 Wiley Periodicals, Inc. PMID:27093649

  19. Intrinsic Surface-Drying Properties of Bio-adhesive Proteins

    PubMed Central

    Akdogan, Yasar; Wei, Wei; Huang, Kuo-Ying; Kageyama, Yoshiyuki; Danner, Eric W.; Miller, Dusty R.; Martinez Rodriguez, Nadine R.; Herbert Waite, J.

    2014-01-01

    Sessile marine mussels must “dry” underwater surfaces before adhering to them. Synthetic adhesives have yet to overcome this fundamental challenge. Previous studies of bio-inspired adhesion have largely been performed under applied compressive forces but these are poor predictors of an adhesive’s ability to spontaneously penetrate surface hydration layers. In a force-free approach to measuring molecular-level interaction via the surface water diffusivity, different mussel foot proteins were found to have differential abilities to evict hydration layers from the surfaces—a necessary step for adsorption and adhesion. It was anticipated that Dopa would mediate dehydration given its efficacy forbio-inspired wet adhesion. Instead, hydrophobic side-chains are found to be a critical component in bringing about protein-surface intimacy. This is the first direct measurement of interfacial water dynamics during force-free adsorptive interactions at solid surfaces, and offers guidance for engineering wet adhesives and coatings. PMID:25168789

  20. Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays

    PubMed Central

    Torres, Alexis J.; Vasudevan, Lavanya; Holowka, David; Baird, Barbara A.

    2008-01-01

    Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-FcεRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-FcεRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-FcεRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-FcεRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (α5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-FcεRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-FcεRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the FcεRI β subunit but enhances stimulated Ca2+ release from intracellular stores. The results suggest that paxillin associated with clustered IgE-FcεRI has a net positive effect on FcεRI signaling. PMID:19004813

  1. Cyclic and Macrocyclic Peptides as Chemical Tools To Recognise Protein Surfaces and Probe Protein-Protein Interactions.

    PubMed

    Cardote, Teresa A F; Ciulli, Alessio

    2016-04-19

    Targeting protein surfaces and protein-protein interactions (PPIs) with small molecules is a frontier goal of chemical biology and provides attractive therapeutic opportunities in drug discovery. The molecular properties of protein surfaces, including their shallow features and lack of deep binding pockets, pose significant challenges, and as a result have proved difficult to target. Peptides are ideal candidates for this mission due to their ability to closely mimic many structural features of protein interfaces. However, their inherently low intracellular stability and permeability and high in vivo clearance have thus far limited their biological applications. One way to improve these properties is to constrain the secondary structure of linear peptides by cyclisation. Herein we review various classes of cyclic and macrocyclic peptides as chemical probes of protein surfaces and modulators of PPIs. The growing interest in this area and recent advances provide evidence of the potential of developing peptide-like molecules that specifically target these interactions. PMID:26563831

  2. Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor

    PubMed Central

    Pandini, Alessandro; Kleinjung, Jens; Rasool, Shafqat; Khan, Shahid

    2015-01-01

    Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) “torque” helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could

  3. Combined flow cytometric analysis of surface and intracellular antigens reveals surface molecule markers of human neuropoiesis.

    PubMed

    Turaç, Gizem; Hindley, Christopher J; Thomas, Ria; Davis, Jason A; Deleidi, Michela; Gasser, Thomas; Karaöz, Erdal; Pruszak, Jan

    2013-01-01

    Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, we present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. We have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f(-)/CD200(high) surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology. PMID:23826393

  4. Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

    PubMed

    Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

    2005-07-01

    Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

  5. Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

    PubMed Central

    Sznajder, Anna; Pfeiffer, Daniel

    2014-01-01

    Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism. PMID:25548058

  6. The Protein Architecture of Human Secretory Vesicles Reveals Differential Regulation of Signaling Molecule Secretion by Protein Kinases

    PubMed Central

    Taupenot, Laurent; Ziegler, Michael; O'Connor, Daniel T.; Ma, Qi; Smoot, Michael; Ideker, Trey; Hook, Vivian

    2012-01-01

    Secretory vesicles are required for release of chemical messengers to mediate intercellular signaling among human biological systems. It is necessary to define the organization of the protein architecture of the ‘human’ dense core secretory vesicles (DCSV) to understand mechanisms for secretion of signaling molecules essential for cellular regulatory processes. This study, therefore, conducted extensive quantitative proteomics and systems biology analyses of human DCSV purified from human pheochromocytoma. Over 600 human DCSV proteins were identified with quantitative evaluation of over 300 proteins, revealing that most proteins participate in producing peptide hormones and neurotransmitters, enzymes, and the secretory machinery. Systems biology analyses provided a model of interacting DCSV proteins, generating hypotheses for differential intracellular protein kinases A and C signaling pathways. Activation of cellular PKA and PKC pathways resulted in differential secretion of neuropeptides, catecholamines, and β-amyloid of Alzheimer's disease for mediating cell-cell communication. This is the first study to define a model of the protein architecture of human DCSV for human disease and health. PMID:22916103

  7. Analysis of the cell surface expression of cytokine receptors using the surface protein biotinylation method.

    PubMed

    Pavel, Mahmud Arif; Lam, Clarissa; Kashyap, Parul; Salehi-Najafabadi, Zahra; Singh, Gurpreet; Yu, Yong

    2014-01-01

    Cytokines are pleiotropic, low-molecular-weight proteins that regulate the immune responses to infection and inflammation. They stimulate the immune responses by binding to cytokine receptors on the cell plasma membrane. Thus, knowledge of the expression level of particular cytokine receptors on cell surface is crucial for understanding the cytokine function and regulation. One of the techniques to explore the membrane embedded cytokine receptors is cell surface biotinylation. Biotinylated surface proteins can be rapidly purified through the strong interaction between biotin and streptavidin. Here, we describe the procedure for surface biotinylation and purification of biotinylated cytokine receptors for further downstream analysis. PMID:24908305

  8. Boolean modeling of transcriptome data reveals novel modes of heterotrimeric G-protein action.

    PubMed

    Pandey, Sona; Wang, Rui-Sheng; Wilson, Liza; Li, Song; Zhao, Zhixin; Gookin, Timothy E; Assmann, Sarah M; Albert, Réka

    2010-06-01

    Heterotrimeric G-proteins mediate crucial and diverse signaling pathways in eukaryotes. Here, we generate and analyze microarray data from guard cells and leaves of G-protein subunit mutants of the model plant Arabidopsis thaliana, with or without treatment with the stress hormone, abscisic acid. Although G-protein control of the transcriptome has received little attention to date in any system, transcriptome analysis allows us to search for potentially uncommon yet significant signaling mechanisms. We describe the theoretical Boolean mechanisms of G-protein x hormone regulation, and then apply a pattern matching approach to associate gene expression profiles with Boolean models. We find that (1) classical mechanisms of G-protein signaling are well represented. Conversely, some theoretical regulatory modes of the G-protein are not supported; (2) a new mechanism of G-protein signaling is revealed, in which Gbeta regulates gene expression identically in the presence or absence of Galpha; (3) guard cells and leaves favor different G-protein modes in transcriptome regulation, supporting system specificity of G-protein signaling. Our method holds significant promise for analyzing analogous 'switch-like' signal transduction events in any organism. PMID:20531402

  9. Boolean modeling of transcriptome data reveals novel modes of heterotrimeric G-protein action

    PubMed Central

    Pandey, Sona; Wang, Rui-Sheng; Wilson, Liza; Li, Song; Zhao, Zhixin; Gookin, Timothy E; Assmann, Sarah M; Albert, Réka

    2010-01-01

    Heterotrimeric G-proteins mediate crucial and diverse signaling pathways in eukaryotes. Here, we generate and analyze microarray data from guard cells and leaves of G-protein subunit mutants of the model plant Arabidopsis thaliana, with or without treatment with the stress hormone, abscisic acid. Although G-protein control of the transcriptome has received little attention to date in any system, transcriptome analysis allows us to search for potentially uncommon yet significant signaling mechanisms. We describe the theoretical Boolean mechanisms of G-protein × hormone regulation, and then apply a pattern matching approach to associate gene expression profiles with Boolean models. We find that (1) classical mechanisms of G-protein signaling are well represented. Conversely, some theoretical regulatory modes of the G-protein are not supported; (2) a new mechanism of G-protein signaling is revealed, in which Gβ regulates gene expression identically in the presence or absence of Gα; (3) guard cells and leaves favor different G-protein modes in transcriptome regulation, supporting system specificity of G-protein signaling. Our method holds significant promise for analyzing analogous ‘switch-like' signal transduction events in any organism. PMID:20531402

  10. The interactome of a PTB domain-containing adapter protein, Odin, revealed by SILAC

    PubMed Central

    Zhong, Jun; Chaerkady, Raghothama; Kandasamy, Kumaran; Gucek, Marjan; Cole, Robert N.; Pandey, Akhilesh

    2011-01-01

    Signal transduction pathways are tightly controlled by positive and negative regulators. We have previously identified Odin (also known as ankyrin repeat and sterile alpha motif domain containing 1A; gene symbol AKNS1A) as a negative regulator of growth factor signaling; however, the mechanisms through which Odin regulates these pathways remain to be elucidated. To determine how Odin negatively regulates growth factor signaling, we undertook a proteomic approach to systematically identify proteins that interact with Odin using the SILAC strategy. In this study, we identified 18 molecules that were specifically associated in a protein complex with Odin. Our study established that the complete family of 14-3-3 proteins occur in a protein complex with Odin, which is also supported by earlier reports that identified a few members of the 14-3-3 family as Odin interactors. Among the novel protein interactors of Odin were CD2-associated protein, SH3 domain kinase binding protein 1 and DAB2 interacting protein. We confirmed 8 of the eighteen interactions identified in the Odin protein complex by co-immunoprecipitation experiments. Finally, a literature-based network analysis revealed that Odin interacting partners are involved in various cellular processes, some of which are key molecules in regulating receptor endocytosis. PMID:21081186

  11. Cardiomyocyte degeneration with calpain deficiency reveals a critical role in protein homeostasis.

    PubMed

    Galvez, Anita S; Diwan, Abhinav; Odley, Amy M; Hahn, Harvey S; Osinska, Hanna; Melendez, Jaime G; Robbins, Jeffrey; Lynch, Roy A; Marreez, Yehia; Dorn, Gerald W

    2007-04-13

    Regulating the balance between synthesis and proteasomal degradation of cellular proteins is essential for tissue growth and maintenance, but the critical pathways regulating protein ubiquitination and degradation are incompletely defined. Although participation of calpain calcium-activated proteases in post-necrotic myocardial autolysis is well characterized, their importance in homeostatic turnover of normal cardiac tissue is controversial. Hence, we evaluated the consequences of physiologic calpain (calcium-activated protease) activity in cultured cardiomyocytes and unstressed mouse hearts. Comparison of in vitro proteolytic activities of cardiac-expressed calpains 1 and 2 revealed calpain 1, but not calpain 2, activity at physiological calcium concentrations. Physiological calpain 1 activation was evident in adenoviral transfected cultured cardiomyocytes as proteolysis of specific substrates, generally increased protein ubiquitination, and accelerated protein turnover, that were each inhibited by coexpression of the inhibitor protein calpastatin. Conditional forced expression of calpain 1, but not calpain 2, in mouse hearts demonstrated substrate-specific proteolytic activity under basal conditions, with hyperubiquitination of cardiac proteins and increased 26S proteasome activity. Loss of myocardial calpain activity by forced expression of calpastatin diminished ubiquitination of 1 or more specific myocardial proteins, without affecting overall ubiquitination or proteasome activity, and resulted in a progressive dilated cardiomyopathy characterized by accumulation of intracellular protein aggregates, formation of autophagosomes, and degeneration of sarcomeres. Thus, calpain 1 is upstream of, and necessary for, ubiquitination and proteasomal degradation of a subset of myocardial proteins whose abnormal accumulation produces autophagosomes and degeneration of cardiomyocytes with functional decompensation. PMID:17332428

  12. Interaction of Serum Proteins with Surface of Hemodialysis Fiber Membranes

    NASA Astrophysics Data System (ADS)

    Afrin, Rehana; Shirako, Yuji; Kishimoto, Kikuo; Ikai, Atsushi

    2012-08-01

    The poly(vinyl pyrrolidone)-covered hydrophilic surface of hollow-fiber membranes (fiber membrane, hereafter) for hemodialysis was mechanically probed using modified tips on an atomic force microscope (AFM) with covalent crosslinkers and several types of serum protein. The retraction part of many of the force extension (F-E) curves obtained with AFM tips coated with serum albumin had a long and smooth extension up to 200-300 nm indicating forced elongation of poly(vinyl pyrrolidone) chains. When fibrinogen-coated tips were used, long extension F-E curves up to 500 nm with multiple peaks were obtained in addition to smooth curves most likely reflecting the unfolding of fibrinogen molecules. The results indicated that individual polymer chains had a significant affinity toward serum proteins. The adhesion frequency of tips coated with serum proteins was lower on the poly(vinyl pyrrolidone) surface than on the uncoated hydrophobic polysulfone surface.

  13. Using the RosettaSurface Algorithm to Predict Protein Structure at Mineral Surfaces

    PubMed Central

    Pacella, Michael S.; Koo, Da Chen Emily; Thottungal, Robin A.; Gray, Jeffrey J.

    2014-01-01

    Determination of protein structure on mineral surfaces is necessary to understand biomineralization processes toward better treatment of biomineralization diseases and design of novel protein-synthesized materials. To date, limited atomic-resolution data have hindered experimental structure determination for proteins on mineral surfaces. Molecular simulation represents a complementary approach. In this chapter, we review RosettaSurface, a computational structure prediction-based algorithm designed to broadly sample conformational space to identify low-energy structures. We summarize the computational approaches, the published applications, and the new releases of the code in the Rosetta 3 framework. In addition, we provide a protocol capture to demonstrate the practical steps to employ RosettaSurface. As an example, we provide input files and output data analysis for a previously unstudied mineralization protein, osteocalcin. Finally, we summarize ongoing challenges in energy function optimization and conformational searching and suggest that the fusion between experiment and calculation is the best route forward. PMID:24188775

  14. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

    PubMed Central

    Kohansal-Nodehi, Mahdokht; Chua, John JE; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. DOI: http://dx.doi.org/10.7554/eLife.14530.001 PMID:27115346

  15. Selectivity by Small-Molecule Inhibitors of Protein Interactions Can Be Driven by Protein Surface Fluctuations

    PubMed Central

    Johnson, David K.; Karanicolas, John

    2015-01-01

    Small-molecules that inhibit interactions between specific pairs of proteins have long represented a promising avenue for therapeutic intervention in a variety of settings. Structural studies have shown that in many cases, the inhibitor-bound protein adopts a conformation that is distinct from its unbound and its protein-bound conformations. This plasticity of the protein surface presents a major challenge in predicting which members of a protein family will be inhibited by a given ligand. Here, we use biased simulations of Bcl-2-family proteins to generate ensembles of low-energy conformations that contain surface pockets suitable for small molecule binding. We find that the resulting conformational ensembles include surface pockets that mimic those observed in inhibitor-bound crystal structures. Next, we find that the ensembles generated using different members of this protein family are overlapping but distinct, and that the activity of a given compound against a particular family member (ligand selectivity) can be predicted from whether the corresponding ensemble samples a complementary surface pocket. Finally, we find that each ensemble includes certain surface pockets that are not shared by any other family member: while no inhibitors have yet been identified to take advantage of these pockets, we expect that chemical scaffolds complementing these “distinct” pockets will prove highly selective for their targets. The opportunity to achieve target selectivity within a protein family by exploiting differences in surface fluctuations represents a new paradigm that may facilitate design of family-selective small-molecule inhibitors of protein-protein interactions. PMID:25706586

  16. Surface imprinting of proteins: from mechanism to application

    NASA Astrophysics Data System (ADS)

    Wang, Yantian; Mueller, Steffen; Sokolov, Jonathan; Levon, Kalle; Rigas, Basil; Rafailovich, Miriam

    2009-03-01

    Protein adsorption properties on different surfaces have been of great interest due to their importance in biomedical applications. In this study, adsorption of proteins on gold, thiol self-assembled monolayer (SAM), and molecularly imprinted thiol SAM was studied. Alkaline phosphatase (AP), an enzyme that can catalyze p-nitrophenyl phosphate and produce a yellow end product which has light absorbance at 405nm, was co-adsorbed with 11-mercapto-1-undecanol to fabricate the imprinted surface. Different washing methods were used to remove AP and create re-adsorption sites. The adsorption amount of AP before and after washing was measured by spectrophotometer after enzyme reaction. Re-adsorption of AP onto the three surfaces was compared and showed that the imprinted surface re-bound the protein molecules at the template site. Potentiometric response of the three substrates to AP was measured at different pH, the charge effect on the potential response was studied. The selective binding of the template proteins made it a useful technique as a protein sensor.

  17. Plasmodium falciparum Merozoite Surface Protein 6 Is a Dimorphic Antigen

    PubMed Central

    Pearce, J. Andrew; Triglia, Tony; Hodder, Anthony N.; Jackson, David C.; Cowman, Alan F.; Anders, Robin F.

    2004-01-01

    Merozoite surface protein 1 (MSP1) is a highly polymorphic Plasmodium falciparum merozoite surface protein implicated in the invasion of human erythrocytes during the asexual cycle. It forms a complex with MSP6 and MSP7 on the merozoite surface, and this complex is released from the parasite around the time of erythrocyte invasion. MSP1 and many other merozoite surface proteins contain dimorphic elements in their protein structures, and here we show that MSP6 is also dimorphic. The sequences of eight MSP6 genes indicate that the alleles of each dimorphic form of MSP6 are highly conserved. The smaller 3D7-type MSP6 alleles are detected in parasites from all malarious regions of the world, whereas K1-type MSP6 alleles have only been detected in parasites from mainland Southeast Asia. Cleavage of MSP6, which produces the p36 fragment in 3D7-type MSP6 and associates with MSP1, also occurs in K1-type MSP6 but at a different site in the protein. Anti-3D7 MSP6 antibodies weakly inhibited erythrocyte invasion by homologous 3D7 merozoites but did not inhibit a parasite line expressing the K1-type MSP6 allele. Antibodies from hyperimmune individuals affinity purified on an MSP3 peptide cross-reacted with MSP6; therefore, MSP6 may also be a target of antibody-dependent cellular inhibition. PMID:15039357

  18. Characterization of the Eimeria maxima sporozoite surface protein IMP1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of this study was to characterize Eimeria maxima immunoprotective protein IMP1 that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transc...

  19. Protein secretion and surface display in Gram-positive bacteria

    PubMed Central

    Schneewind, Olaf; Missiakas, Dominique M.

    2012-01-01

    The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983

  20. Analysis of Protein Phosphatase-1 and Aurora Protein Kinase Suppressors Reveals New Aspects of Regulatory Protein Function in Saccharomyces cerevisiae

    PubMed Central

    Ghosh, Anuprita; Cannon, John F.

    2013-01-01

    Protein phosphatase-1 (PP1) controls many processes in eukaryotic cells. Modulation of mitosis by reversing phosphorylation of proteins phosphorylated by aurora protein kinase is a critical function for PP1. Overexpression of the sole PP1, Glc7, in budding yeast, Saccharomyces cerevisiae, is lethal. This work shows that lethality requires the function of Glc7 regulatory proteins Sds22, Reg2, and phosphorylated Glc8. This finding shows that Glc7 overexpression induced cell death requires a specific subset of the many Glc7-interacting proteins and therefore is likely caused by promiscuous dephosphorylation of a variety of substrates. Additionally, suppression can occur by reducing Glc7 protein levels by high-copy Fpr3 without use of its proline isomerase domain. This divulges a novel function of Fpr3. Most suppressors of GLC7 overexpression also suppress aurora protein kinase, ipl1, temperature-sensitive mutations. However, high-copy mutant SDS22 genes show reciprocal suppression of GLC7 overexpression induced cell death or ipl1 temperature sensitivity. Sds22 binds to many proteins besides Glc7. The N-terminal 25 residues of Sds22 are sufficient to bind, directly or indirectly, to seven proteins studied here including the spindle assembly checkpoint protein, Bub3. These data demonstrate that Sds22 organizes several proteins in addition to Glc7 to perform functions that counteract Ipl1 activity or lead to hyper Glc7 induced cell death. These data also emphasize that Sds22 targets Glc7 to nuclear locations distinct from Ipl1 substrates. PMID:23894419

  1. Self-assembling triblock proteins for biofunctional surface modification

    NASA Astrophysics Data System (ADS)

    Fischer, Stephen E.

    Despite the tremendous promise of cell/tissue engineering, significant challenges remain in engineering functional scaffolds to precisely regulate the complex processes of tissue growth and development. As the point of contact between the cells and the scaffold, the scaffold surface plays a major role in mediating cellular behaviors. In this dissertation, the development and utility of self-assembling, artificial protein hydrogels as biofunctional surface modifiers is described. The design of these recombinant proteins is based on a telechelic triblock motif, in which a disordered polyelectrolyte central domain containing embedded bioactive ligands is flanked by two leucine zipper domains. Under moderate conditions of temperature and pH, the leucine zipper end domains form amphiphilic alpha-helices that reversibly associate into homo-trimeric aggregates, driving hydrogel formation. Moreover, the amphiphilic nature of these helical domains enables surface adsorption to a variety of scaffold materials to form biofunctional protein coatings. The nature and stability of these coatings in various solution conditions, and their interaction with mammalian cells is the primary focus of this dissertation. In particular, triblock protein coatings functionalized with cell recognition sequences are shown to produce well-defined surfaces with precise control over ligand density. The impact of this is demonstrated in multiple cell types through ligand density-dependent cell-substrate interactions. To improve the stability of these physically self-assembled coatings, two covalent crosslinking strategies are described---one in which a zero-length chemical crosslinker (EDC) is utilized and a second in which disulfide bonds are engineered into the recombinant proteins. These targeted crosslinking approaches are shown to increase the stability of surface adsorbed protein layers with minimal effect on the presentation of many bioactive ligands. Finally, to demonstrate the versatility

  2. Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans

    PubMed Central

    Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C. G.; Benavente, Ricardo

    2012-01-01

    The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed no sequence homology. This discrepancy challenged the hypothesis that the SC arose only once in evolution. To pursue this matter we focused on the evolution of SYCP1 and SYCP3, the two major structural SC proteins of mammals. Remarkably, our comparative bioinformatic and expression studies revealed that SYCP1 and SYCP3 are also components of the SC in the basal metazoan Hydra. In contrast to previous assumptions, we therefore conclude that SYCP1 and SYCP3 form monophyletic groups of orthologous proteins across metazoans. PMID:23012415

  3. Revealing the Restructured Surface of Li[Mn2]O4

    DOE PAGESBeta

    Amos, Charles D.; Roldan, Manuel A.; Varela, Maria; Goodenough, John B.; Ferreira, Paulo J.

    2016-03-29

    The spinel Revealing the Restructured Surface of Li[Mn2]O4 is a candidate cathode for a Li-ion battery, but its capacity fades over a charge/discharge cycle of Li1–x[Mn2]O4 (0 < x < 1) that is associated with a loss of Mn to the organic-liquid electrolyte. It is known that the disproportionation reaction 2Mn3+ = Mn2+ + Mn4+ occurs at the surface of a Mn spinel, and it is important to understand the atomic structure and composition of the surface of Revealing the Restructured Surface of Li[Mn2]O4 in order to understand how Mn loss occurs. We report a study of the surface reconstructionmore » of Revealing the Restructured Surface of Li[Mn2]O4 by aberration-corrected scanning transmission electron microscopy. The atomic structure coupled with Mn-valence and the distribution of the atomic ratio of oxygen obtained by electron energy loss spectroscopy reveals a thin, stable surface layer of Mn3O4, a subsurface region of Li1+x[Mn2]O4 with retention of bulk Li[Mn2]O4. We conclude that this observation is compatible with the disproportionation reaction coupled with oxygen deficiency and a displacement of surface Li+ from the Mn3O4 surface phase. These results provide a critical step toward understanding how Mn is lost from Li[Mn2]O4, once inside a battery.« less

  4. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles

    SciTech Connect

    Furlong, D.B.; Nibert, M.L.; Fields, B.N.

    1988-01-01

    Electron microscopy revealed structures consisting of long fibers topped with knobs extending from the surfaces of virions of mammalian reoviruses. The morphology of these structures was reminiscent of the fiber protein of adenovirus. Fibers were also seen extending from the reovirus top component and intermediate subviral particles but not from cores, suggesting that the fibers consist of either the ..mu..1C or sigma1 outer capsid protein. Amino acid sequence analysis predicts that the reovirus cell attachment protein sigma1 contains an extended fiber domain. When sigma1 protein was released from viral particles with mild heat and subsequently obtained in isolation, it was found to have a morphology identical to that of the fiber structures seen extending from the viral particles. The identification of an extended form of sigma1 has important implications for its function in cell attachment. Other evidence suggest that sigma1 protein may occur in virions in both an extended and an unextended state.

  5. Protein-Protein Interaction Network could reveal the relationship between the breast and colon cancer

    PubMed Central

    Zamanian-Azodi, Mona; Rezaei-Tavirani, Mostafa; Rahmati-Rad, Sara; Hasanzadeh, Hadi; Rezaei Tavirani, Majid; Seyyedi, Samaneh Sadat

    2015-01-01

    Aim: This study is aimed to elicit the possible correlation between breast and colon cancer from molecular prospective by analyzing and comparing pathway-based biomarkers. Background: Breast and colon cancer are known to be frequent causes of morbidity and mortality in men and women around the world. There is some evidence that while the incident of breast cancer in young women is high, it is reported lower in the aged women. In fact, aged women are more prone to colorectal cancer than older men. . In addition, many studies showed that several biomarkers are common among these malignancies. Patients and methods: The genes were retrieved and compared from KEGG database and WikiPathway, and subsequently, protein-protein interaction (PPI) network was constructed and analyzed using Cytoscape v:3.2.1 software and related algorithms. Results: More than forty common genes were identified among these malignancies; however, by pathways comparison, twenty genes are related to both breast and colon cancer. Centrality and cluster screening identified hub genes, including SMAD2, SMAD3, (SMAD4, MYC), JUN, BAD, TP53. These seven genes are enriched in regulation of transforming growth factor beta receptor signaling pathway, positive regulation of Rac protein signal transduction, positive regulation of mitochondrial outer membrane permeabilization involved in apoptotic signaling pathway, and positive regulation of mitotic metaphase/anaphase transition respectively. Conclusion: As there are numerous genes frequent between colorectal cancer and breast cancer, there may be a common molecular origin for these malignancies occurrences. It seems that breast cancer in females interferes with the rate of colorectal cancer incidence. PMID:26328044

  6. Protein-surface interactions on stimuli-responsive polymeric biomaterials.

    PubMed

    Cross, Michael C; Toomey, Ryan G; Gallant, Nathan D

    2016-04-01

    Responsive surfaces: a review of the dependence of protein adsorption on the reversible volume phase transition in stimuli-responsive polymers. Specifically addressed are a widely studied subset: thermoresponsive polymers. Findings are also generalizable to other materials which undergo a similarly reversible volume phase transition. As of 2015, over 100,000 articles have been published on stimuli-responsive polymers and many more on protein-biomaterial interactions. Significantly, fewer than 100 of these have focused specifically on protein interactions with stimuli-responsive polymers. These report a clear trend of increased protein adsorption in the collapsed state compared to the swollen state. This control over protein interactions makes stimuli-responsive polymers highly useful in biomedical applications such as wound repair scaffolds, on-demand drug delivery, and antifouling surfaces. Outstanding questions are whether the protein adsorption is reversible with the volume phase transition and whether there is a time-dependence. A clear understanding of protein interactions with stimuli-responsive polymers will advance theoretical models, experimental results, and biomedical applications. PMID:26942693

  7. On the surface interactions of proteins with lignin.

    PubMed

    Salas, Carlos; Rojas, Orlando J; Lucia, Lucian A; Hubbe, Martin A; Genzer, Jan

    2013-01-01

    Lignins are used often in formulations involving proteins but little is known about the surface interactions between these important biomacromolecules. In this work, we investigate the interactions at the solid-liquid interface of lignin with the two main proteins in soy, glycinin (11S) and β-conglycinin (7S). The extent of adsorption of 11S and 7S onto lignin films and the degree of hydration of the interfacial layers is quantified via Quartz crystal microgravimetry (QCM) and surface plasmon resonance (SPR). Solution ionic strength and protein denaturation (2-mercaptoethanol and urea) critically affect the adsorption process as protein molecules undergo conformational changes and their hydrophobic or hydrophilic amino acid residues interact with the surrounding medium. In general, the adsorption of the undenatured proteins onto lignin is more extensive compared to that of the denatured biomolecules and a large amount of water is coupled to the adsorbed molecules. The reduction in water contact angle after protein adsorption (by ~40° and 35° for undenatured 11S and 7S, respectively) is explained by strong nonspecific interactions between soy proteins and lignin. PMID:23234476

  8. Switchable surface coatings for control over protein adsorption

    NASA Astrophysics Data System (ADS)

    Cole, Martin A.; Jasieniak, Marek; Voelcker, Nicolas H.; Thissen, Helmut; Horn, Roger; Griesser, Hans J.

    2007-12-01

    Control over biomolecule interactions at interfaces is becoming an increasingly important goal for a range of scientific fields and is being intensively studied in areas of biotechnological, biomedical and materials science. Improvement in the control over materials and biomolecules is particularly important to applications such as arrays, biosensors, tissue engineering, drug delivery and 'lab on a chip' devices. Further development of these devices is expected to be achieved with thin coatings of stimuli responsive materials that can have their chemical properties 'switched' or tuned to stimulate a certain biological response such as adsorption/desorption of proteins. Switchable coatings show great potential for the realisation of spatial and temporal immobilisation of cells and biomolecules such as DNA and proteins. This study focuses on protein adsorption onto coatings of the thermosensitive polymer poly(N-isopropylacrylamide) (pNIPAM) which can exhibit low and high protein adsorption properties based on its temperature dependent conformation. At temperatures above its lower critical solution temperature (LCST) pNIPAM polymer chains are collapsed and protein adsorbing whilst below the LCST they are hydrated and protein repellent. Coatings of pNIPAM on silicon wafers were prepared by free radical polymerisation in the presence of surface bound polymerisable groups. Surface analysis and protein adsorption was carried out using X-ray photoelectron spectroscopy, time of flight secondary ion mass spectrometry and contact angle measurements. This study is expected to aid the development of stimuli-responsive coatings for biochips and biodevices.

  9. Surface-based morphometry reveals distinct cortical thickness and surface area profiles in Williams syndrome.

    PubMed

    Green, Tamar; Fierro, Kyle C; Raman, Mira M; Saggar, Manish; Sheau, Kristen E; Reiss, Allan L

    2016-04-01

    Morphometric investigations of brain volumes in Williams syndrome (WS) consistently show significant reductions in gray matter volume compared to controls. Cortical thickness (CT) and surface area (SA) are two constituent parts of cortical gray matter volume that are considered genetically distinguishable features of brain morphology. Yet, little is known about the independent contribution of cortical CT and SA to these volumetric differences in WS. Thus, our objectives were: (i) to evaluate whether the microdeletion in chromosome 7 associated with WS has a distinct effect on CT and SA, and (ii) to evaluate age-related variations in CT and SA within WS. We compared CT and SA values in 44 individuals with WS to 49 age- and sex-matched typically developing controls. Between-group differences in CT and SA were evaluated across two age groups: young (age range 6.6-18.9 years), and adults (age range 20.2-51.5 years). Overall, we found contrasting effects of WS on cortical thickness (increases) and surface area (decreases). With respect to brain topography, the between-group pattern of CT differences showed a scattered pattern while the between-group surface area pattern was widely distributed throughout the brain. In the adult subgroup, we observed a cluster of increases in cortical thickness in WS across the brain that was not observed in the young subgroup. Our findings suggest that extensive early reductions in surface area are the driving force for the overall reduction in brain volume in WS. The age-related cortical thickness findings might reflect delayed or even arrested development of specific brain regions in WS. PMID:26852730

  10. Systematic Analysis of Essential Genes Reveals New Regulators of G protein Signaling

    PubMed Central

    Cappell, Steven D.; Baker, Rachael; Skowyra, Dorota; Dohlman, Henrik G.

    2010-01-01

    SUMMARY The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify new signaling components we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly-represented. Included in this group are members of the SCF (Skp-Cullin-F-Box) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCFCdc4 acts together with the Cdc34 ubiquitin conjugating enzyme at the level of the G protein, promotes degradation of the G protein α subunit, Gpa1, in vivo and catalyzes Gpa1 ubiquitination in vitro. These new insights to the G protein signaling network reveal the essential-genome as an untapped resource for identifying new components and regulators of signal transduction pathways. PMID:20542006

  11. Proteomic profiling of maize opaque endosperm mutants reveals selective accumulation of lysine-enriched proteins

    PubMed Central

    Morton, Kyla J.; Jia, Shangang; Zhang, Chi; Holding, David R.

    2016-01-01

    Reduced prolamin (zein) accumulation and defective endoplasmic reticulum (ER) body formation occurs in maize opaque endosperm mutants opaque2 (o2), floury2 (fl2), defective endosperm*B30 (DeB30), and Mucronate (Mc), whereas other opaque mutants such as opaque1 (o1) and floury1 (fl1) are normal in these regards. This suggests that other factors contribute to kernel texture. A liquid chromatography approach coupled with tandem mass spectrometry (LC-MS/MS) proteomics was used to compare non-zein proteins of nearly isogenic opaque endosperm mutants. In total, 2762 proteins were identified that were enriched for biological processes such as protein transport and folding, amino acid biosynthesis, and proteolysis. Principal component analysis and pathway enrichment suggested that the mutants partitioned into three groups: (i) Mc, DeB30, fl2 and o2; (ii) o1; and (iii) fl1. Indicator species analysis revealed mutant-specific proteins, and highlighted ER secretory pathway components that were enriched in selected groups of mutants. The most significantly changed proteins were related to stress or defense and zein partitioning into the soluble fraction for Mc, DeB30, o1, and fl1 specifically. In silico dissection of the most significantly changed proteins revealed novel qualitative changes in lysine abundance contributing to the overall lysine increase and the nutritional rebalancing of the o2 and fl2 endosperm. PMID:26712829

  12. Quantitative proteomics reveals the effect of protein glycosylation in soybean root under flooding stress

    PubMed Central

    Mustafa, Ghazala; Komatsu, Setsuko

    2014-01-01

    Flooding stress has a negative impact on soybean cultivation because it severely impairs growth and development. To understand the flooding responsive mechanism in early stage soybeans, a glycoproteomic technique was used. Two-day-old soybeans were treated with flooding for 2 days and roots were collected. Globally, the accumulation level of glycoproteins, as revealed by cross-reaction with concanavalin A decreased by 2 days of flooding stress. Glycoproteins were enriched from total protein extracts using concanavalin A lectin resin and analyzed using a gel-free proteomic technique. One-hundred eleven and 69 glycoproteins were identified without and with 2 days of flooding stress, respectively. Functional categorization of these identified glycoproteins indicated that the accumulation level of proteins related to protein degradation, cell wall, and glycolysis increased, while stress-related proteins decreased under flooding stress. Also the accumulation level of glycoproteins localized in the secretory pathway decreased under flooding stress. Out of 23 common glycoproteins between control and flooding conditions, peroxidases and glycosyl hydrolases were decreased by 2 days of flooding stress. mRNA expression levels of proteins in the endoplasmic reticulum and N-glycosylation related proteins were downregulated by flooding stress. These results suggest that flooding might negatively affect the process of N-glycosylation of proteins related to stress and protein degradation; however glycoproteins involved in glycolysis are activated. PMID:25477889

  13. Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors

    PubMed Central

    Midde, Krishna K.; Aznar, Nicolas; Laederich, Melanie B.; Ma, Gary S.; Kunkel, Maya T.; Newton, Alexandra C.; Ghosh, Pradipta

    2015-01-01

    Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK–GIV–Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states. PMID:25713130

  14. Proteomics Reveals Plastid- and Periplastid-Targeted Proteins in the Chlorarachniophyte Alga Bigelowiella natans

    PubMed Central

    Hopkins, Julia F.; Spencer, David F.; Laboissiere, Sylvie; Neilson, Jonathan A.D.; Eveleigh, Robert J.M.; Durnford, Dion G.; Gray, Michael W.; Archibald, John M.

    2012-01-01

    Chlorarachniophytes are unicellular marine algae with plastids (chloroplasts) of secondary endosymbiotic origin. Chlorarachniophyte cells retain the remnant nucleus (nucleomorph) and cytoplasm (periplastidial compartment, PPC) of the green algal endosymbiont from which their plastid was derived. To characterize the diversity of nucleus-encoded proteins targeted to the chlorarachniophyte plastid, nucleomorph, and PPC, we isolated plastid–nucleomorph complexes from the model chlorarachniophyte Bigelowiella natans and subjected them to high-pressure liquid chromatography-tandem mass spectrometry. Our proteomic analysis, the first of its kind for a nucleomorph-bearing alga, resulted in the identification of 324 proteins with 95% confidence. Approximately 50% of these proteins have predicted bipartite leader sequences at their amino termini. Nucleus-encoded proteins make up >90% of the proteins identified. With respect to biological function, plastid-localized light-harvesting proteins were well represented, as were proteins involved in chlorophyll biosynthesis. Phylogenetic analyses revealed that many, but by no means all, of the proteins identified in our proteomic screen are of apparent green algal ancestry, consistent with the inferred evolutionary origin of the plastid and nucleomorph in chlorarachniophytes. PMID:23221610

  15. Essential Strategies for Revealing Nanoscale Protein Dynamics by Neutron Spin Echo Spectroscopy.

    PubMed

    Callaway, David J E; Bu, Zimei

    2016-01-01

    Determining the internal motions of a protein on nanosecond-to-microsecond timescales and on nanometer length scales is challenging by experimental biophysical techniques. Neutron spin echo spectroscopy (NSE) offers a unique opportunity to determine such nanoscale protein domain motions. However, the major hurdle in applying NSE to determine nanoscale protein motion is that the time and length scales of internal protein motions tend to be comparable to that of the global motions of a protein. The signals detected by NSE tend to be dominated by rigid-body translational and rotational diffusion. Using theoretical analyses, our laboratory showed that selective deuteration of a protein domain or a subunit can enhance the capability of NSE to reveal the internal motions in a protein complex. Here, we discuss the essential theoretical analysis and experimental methodology in detail. Protein nanomachines are far more complex than any molecular motors that have been artificially constructed, and their skillful utilization likely represents the future of medicine. With selective deuteration, NSE will allow us to see these nanomachines in motion. PMID:26791982

  16. Crystal Structures of Flax Rust Avirulence Proteins AvrL567-A and -D Reveal Details of the Structural Basis for Flax Disease Resistance Specificity[W

    PubMed Central

    Wang, Ching-I A.; Gunčar, Gregor; Forwood, Jade K.; Teh, Trazel; Catanzariti, Ann-Maree; Lawrence, Gregory J.; Loughlin, Fionna E.; Mackay, Joel P.; Schirra, Horst Joachim; Anderson, Peter A.; Ellis, Jeffrey G.; Dodds, Peter N.; Kobe, Boštjan

    2007-01-01

    The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a β-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities. PMID:17873095

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

    NASA Astrophysics Data System (ADS)

    Song, Sheng

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

  18. Comparative proteomic analysis of surface proteins of Trichinella spiralis muscle larvae and intestinal infective larvae.

    PubMed

    Liu, Ruo Dan; Cui, Jing; Liu, Xiao Lin; Jiang, Peng; Sun, Ge Ge; Zhang, Xi; Long, Shao Rong; Wang, Li; Wang, Zhong Quan

    2015-10-01

    The critical step for Trichinella spiralis infection is that muscle larvae (ML) are activated to intestinal infective larvae (IIL) and invade intestinal epithelium to further develop. The IIL is its first invasive stage, surface proteins are directly exposed to host environment and are crucial for larval invasion and development. In this study, shotgun LC-MS/MS was used to analyze surface protein profiles of ML and IIL. Totally, 41 proteins common to both larvae, and 85 ML biased and 113 IIL biased proteins. Some proteins (e.g., putative scavenger receptor cysteine-rich domain protein and putative onchocystatin) were involved in host-parasite interactions. Gene ontology analysis revealed that proteins involved in generation of precursor metabolites and energy; and nucleobase, nucleoside, nucleotide and nucleic acid metabolic process were enriched in IIL at level 4. Some IIL biased proteins might play important role in larval invasion and development. qPCR results confirmed the high expression of some genes in IIL. Our study provides new insights into larval invasion, host-Trichinella interaction and for screening vaccine candidate antigens. PMID:26184560

  19. Spectrofluorimetric assessment of the surface hydrophobicity of proteins.

    PubMed Central

    Cardamone, M; Puri, N K

    1992-01-01

    The equilibrium binding of the apolar fluorescent dye 1-anilinonaphthalene-8-sulphonate (ANS) to bacteriorhodopsin, BSA, chicken egg lysozyme, ovalbumin, porcine somatotrophin (PST) and bovine pancreatic ribonuclease (RNAase) was quantitatively evaluated using Scatchard- and Klotz-plot analyses. On the basis of the average association constant for ANS binding sites (Ka), the proteins could be ranked in order of surface hydrophobicity as: Bacteriorhodopsin greater than BSA greater than ovalbumin greater than PST greater than lysozyme greater than RNAase. The number of protein-ANS binding sites was determined as 54, 10, 3, 1, 2 and 1 respectively. The ANS-based assessment of the surface hydrophobicity of these proteins was generally in agreement with the average hydrophobicity based on amino acid sequence [Bigelow (1967) J. Theor. Biol. 16, 187-211], except for results with PST and ovalbumin. The proteins were also analysed by reversed-phase h.p.l.c. using C1 and C8 columns. There was no significant correlation between ANS and reversed-phase-h.p.l.c. assessment of hydrophobicity, with the results obtained by h.p.l.c. being dependent upon the column used. ANS-based measurement of surface hydrophobicity appears to be the most appropriate means for assessing proteins such as to reflect their overall three-dimensional structure in solution. PMID:1546973

  20. A 75 kd merozoite surface protein of Plasmodium falciparum which is related to the 70 kd heat-shock proteins.

    PubMed Central

    Ardeshir, F; Flint, J E; Richman, S J; Reese, R T

    1987-01-01

    Proteins on the merozoite surface of the human malarial parasite Plasmodium falciparum are targets of the host's immune response. The merozoite surface location of p75, a 75 kd P. falciparum protein, was established by immunoelectron microscopy using antisera raised to the expressed product of a cDNA clone. Immunoprecipitation from protein extracts biosynthetically labeled during different periods of the asexual cycle showed that p75 is made continuously, although ring-stage parasites appear to synthesize larger quantities. p75 is conserved and invariant in size in eight isolates of P. falciparum. The 880 bp cDNA sequence encoding part of p75 reveals one open reading frame containing a repetitive sequence unit of four amino acids. The predicted reading frame is correct since antisera to a synthetic peptide corresponding to the repetitive region recognize p75 in immunoblots. The sequence of p75 is homologous with the sequences of proteins from the ubiquitous, highly conserved family of 70 kd heat-shock proteins, suggesting an important physiological function for p75. The cDNA fragment encoding part of p75 hybridizes with multiple genomic fragments, whose sizes are identical in DNA from nine P. falciparum strains, suggesting that the gene for p75 is well conserved and may be part of a gene family. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. PMID:3556166

  1. Unexpected Link between Lipooligosaccharide Biosynthesis and Surface Protein Release in Mycobacterium marinum*

    PubMed Central

    van der Woude, Aniek D.; Sarkar, Debasmita; Bhatt, Apoorva; Sparrius, Marion; Raadsen, Susanne A.; Boon, Louis; Geurtsen, Jeroen; van der Sar, Astrid M.; Luirink, Joen; Houben, Edith N. G.; Besra, Gurdyal S.; Bitter, Wilbert

    2012-01-01

    The mycobacterial cell envelope is characterized by the presence of a highly impermeable second membrane, which is composed of mycolic acids intercalated with different unusual free lipids, such as lipooligosaccharides (LOS). Transport across this cell envelope requires a dedicated secretion system for extracellular proteins, such as PE_PGRS proteins, which are specific mycobacterial proteins with polymorphic GC-rich sequence (PGRS). In this study, we set out to identify novel components involved in the secretion of PE_PGRS proteins by screening Mycobacterium marinum transposon mutants for secretion defects. Interestingly, most mutants were not affected in secretion but in the release of PE_PGRS proteins from the cell surface. These mutants had insertions in a gene cluster associated with LOS biosynthesis. Lipid analysis of these mutants revealed a role at different stages of LOS biosynthesis for 10 novel genes. Furthermore, we show that regulatory protein WhiB4 is involved in LOS biosynthesis. The absence of the most extended LOS molecule, i.e. LOS-IV, and a concomitant accumulation of LOS-III was already sufficient to reduce the release of PE_PGRS proteins from the mycobacterial cell surface. A similar effect was observed for major surface protein EspE. These results show that the attachment of surface proteins is strongly influenced by the glycolipid composition of the mycobacterial cell envelope. Finally, we tested the virulence of a LOS-IV-deficient mutant in our zebrafish embryo infection model. This mutant showed a marked increase in virulence as compared with the wild-type strain, suggesting that LOS-IV plays a role in the modulation of mycobacterial virulence. PMID:22505711

  2. Protein crystallization on liquid surfaces: Forced versus natural crystallization

    NASA Astrophysics Data System (ADS)

    Hirsa, A.

    2005-11-01

    Two-dimensional crystallization of proteins has recently been reported where streptavidin protein dissolved in the bulk liquid anchors to binding sites on a biotinylated lipid monolayer initially spread on the liquid surface. Thermodynamic aspects investigated include the effects of subphase buffer and pH, dilution of bulk protein and monolayer. Here, we investigate three possible avenues where flow can influence protein crystallization: i) change the initial state of monolayer, ii) advect dissolved protein to the interface, iii) apply direct hydrodynamic force on the crystals at the interface. The flow system consists of a stationary open cylinder driven by constant rotation of the floor, in the axisymmetric flow regime with inertia. Direct imaging of the interface illuminated by forward scattering of a laser was utilized to avoid labeling proteins for conventional fluorescence microscopy. These images provide greater detail than Brewster angle microscopy. Scientific motivation is to use flow to probe protein structure, and the application is to make designer protein thin-films, e.g. for biosensors.

  3. Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein

    PubMed Central

    Kankainen, Matti; Paulin, Lars; Tynkkynen, Soile; von Ossowski, Ingemar; Reunanen, Justus; Partanen, Pasi; Satokari, Reetta; Vesterlund, Satu; Hendrickx, Antoni P. A.; Lebeer, Sarah; De Keersmaecker, Sigrid C. J.; Vanderleyden, Jos; Hämäläinen, Tuula; Laukkanen, Suvi; Salovuori, Noora; Ritari, Jarmo; Alatalo, Edward; Korpela, Riitta; Mattila-Sandholm, Tiina; Lassig, Anna; Hatakka, Katja; Kinnunen, Katri T.; Karjalainen, Heli; Saxelin, Maija; Laakso, Kati; Surakka, Anu; Palva, Airi; Salusjärvi, Tuomas; Auvinen, Petri; de Vos, Willem M.

    2009-01-01

    To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues. PMID:19805152

  4. Surface-protein interactions on different stainless steel grades: effects of protein adsorption, surface changes and metal release.

    PubMed

    Hedberg, Y; Wang, X; Hedberg, J; Lundin, M; Blomberg, E; Wallinder, I Odnevall

    2013-04-01

    Implantation using stainless steels (SS) is an example where an understanding of protein-induced metal release from SS is important when assessing potential toxicological risks. Here, the protein-induced metal release was investigated for austenitic (AISI 304, 310, and 316L), ferritic (AISI 430), and duplex (AISI 2205) grades in a phosphate buffered saline (PBS, pH 7.4) solution containing either bovine serum albumin (BSA) or lysozyme (LSZ). The results show that both BSA and LSZ induce a significant enrichment of chromium in the surface oxide of all stainless steel grades. Both proteins induced an enhanced extent of released iron, chromium, nickel and manganese, very significant in the case of BSA (up to 40-fold increase), whereas both proteins reduced the corrosion resistance of SS, with the reverse situation for iron metal (reduced corrosion rates and reduced metal release in the presence of proteins). A full monolayer coverage is necessary to induce the effects observed. PMID:23378148

  5. Surface plasmon resonance applied to G protein-coupled receptors

    PubMed Central

    Locatelli-Hoops, Silvia; Yeliseev, Alexei A.; Gawrisch, Klaus; Gorshkova, Inna

    2013-01-01

    G protein-coupled receptors (GPCR) are integral membrane proteins that transmit signals from external stimuli to the cell interior via activation of GTP-binding proteins (G proteins) thereby mediating key sensorial, hormonal, metabolic, immunological, and neurotransmission processes. Elucidating their structure and mechanism of interaction with extracellular and intracellular binding partners is of fundamental importance and highly relevant to rational design of new effective drugs. Surface plasmon resonance (SPR) has become a method of choice for studying biomolecular interactions at interfaces because measurements take place in real-time and do not require labeling of any of the interactants. However, due to the particular challenges imposed by the high hydrophobicity of membrane proteins and the great diversity of receptor-stimulating ligands, the application of this technique to characterize interactions of GPCR is still in the developmental phase. Here we give an overview of the principle of SPR and analyze current approaches for the preparation of the sensor chip surface, capture and stabilization of GPCR, and experimental design to characterize their interaction with ligands, G proteins and specific antibodies. PMID:24466506

  6. Oligosaccharide Binding Proteins from Bifidobacterium longum subsp. infantis Reveal a Preference for Host Glycans

    PubMed Central

    Garrido, Daniel; Kim, Jae Han; German, J. Bruce; Raybould, Helen E.; Mills, David A.

    2011-01-01

    Bifidobacterium longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota, and it has been characterized by its foraging capacity for human milk oligosaccharides (HMO). Its genome sequence revealed an overabundance of the Family 1 of solute binding proteins (F1SBPs), part of ABC transporters and associated with the import of oligosaccharides. In this study we have used the Mammalian Glycan Array to determine the specific affinities of these proteins. This was correlated with binding protein expression induced by different prebiotics including HMO. Half of the F1SBPs in B. infantis were determined to bind mammalian oligosaccharides. Their affinities included different blood group structures and mucin oligosaccharides. Related to HMO, other proteins were specific for oligomers of lacto-N-biose (LNB) and polylactosamines with different degrees of fucosylation. Growth on HMO induced the expression of specific binding proteins that import HMO isomers, but also bind blood group and mucin oligosaccharides, suggesting coregulated transport mechanisms. The prebiotic inulin induced other family 1 binding proteins with affinity for intestinal glycans. Most of the host glycan F1SBPs in B. infantis do not have homologs in other bifidobacteria. Finally, some of these proteins were found to be adherent to intestinal epithelial cells in vitro. In conclusion, this study represents further evidence for the particular adaptations of B. infantis to the infant gut environment, and helps to understand the molecular mechanisms involved in this process. PMID:21423604

  7. Structure and function of a mitochondrial late embryogenesis abundant protein are revealed by desiccation.

    PubMed

    Tolleter, Dimitri; Jaquinod, Michel; Mangavel, Cécile; Passirani, Catherine; Saulnier, Patrick; Manon, Stephen; Teyssier, Emeline; Payet, Nicole; Avelange-Macherel, Marie-Hélène; Macherel, David

    2007-05-01

    Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into alpha-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes. PMID:17526751

  8. Proteomic analysis of a segregant population reveals candidate proteins linked to mealiness in peach.

    PubMed

    Almeida, Andréa Miyasaka; Urra, Claudio; Moraga, Carol; Jego, Marcela; Flores, Alejandra; Meisel, Lee; González, Mauricio; Infante, Rodrigo; Defilippi, Bruno G; Campos-Vargas, Reinaldo; Orellana, Ariel

    2016-01-10

    Peaches are stored at low temperatures to delay ripening and increase postharvest life. However some varieties are susceptible to chilling injury,which leads to fruit mealiness, browning and flesh bleeding. In order to identify potentialmarkers associated with chilling injury,we performed proteomic analyses on a segregating population with contrasting susceptibility to chilling-induced mealiness. Chilling-induced mealiness was assessed by measuring juiciness in fruits that have been stored in cold and then allowed to ripen. Fruitmesocarp and leaf proteome from contrasting segregants were analyzed using 2-DE gels. Comparison of protein abundance between segregants revealed 133 spots from fruit mesocarp and 36 from leaf. Thirty four fruit mesocarp proteins were identified from these spots. Most of these proteins were related to ethylene synthesis, ABA response and stress response. Leaf protein analyses identified 22 proteins, most of which related to energy metabolism. Some of the genes that code for these proteins have been previously correlated with chilling injury through transcript analyses and co-segregation with mealiness QTLs. The results from this study, further deciphers the molecular mechanisms associated with chilling response in peach fruit, and identifies candidate proteins linked to mealiness in peach which may be used as putative markers for this trait. PMID:26459401

  9. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations

    PubMed Central

    Hertig, Samuel

    2016-01-01

    Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein’s constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery—the fact that the two sites involved influence one another in a symmetrical manner—can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest. PMID:27285999

  10. Genome-wide search for eliminylating domains reveals novel function for BLES03-like proteins.

    PubMed

    Khater, Shradha; Mohanty, Debasisa

    2014-08-01

    Bacterial phosphothreonine lyases catalyze a novel posttranslational modification involving formation of dehydrobutyrine/dehyroalanine by β elimination of the phosphate group of phosphothreonine or phosphoserine residues in their substrate proteins. Though there is experimental evidence for presence of dehydro amino acids in human proteins, no eukaryotic homologs of these lyases have been identified as of today. A comprehensive genome-wide search for identifying phosphothreonine lyase homologs in eukaryotes was carried out. Our fold-based search revealed structural and catalytic site similarity between bacterial phosphothreonine lyases and BLES03 (basophilic leukemia-expressed protein 03), a human protein with unknown function. Ligand induced conformational changes similar to bacterial phosphothreonine lyases, and movement of crucial arginines in the loop region to the catalytic pocket upon binding of phosphothreonine-containing peptides was seen during docking and molecular dynamics studies. Genome-wide search for BLES03 homologs using sensitive profile-based methods revealed their presence not only in eukaryotic classes such as chordata and fungi but also in bacterial and archaebacterial classes. The synteny of these archaebacterial BLES03-like proteins was remarkably similar to that of type IV lantibiotic synthetases which harbor LanL-like phosphothreonine lyase domains. Hence, context-based analysis reinforced our earlier sequence/structure-based prediction of phosphothreonine lyase catalytic function for BLES03. Our in silico analysis has revealed that BLES03-like proteins with previously unknown function are novel eukaryotic phosphothreonine lyases involved in biosynthesis of dehydro amino acids, whereas their bacterial and archaebacterial counterparts might be involved in biosynthesis of natural products similar to lantibiotics. PMID:25062915

  11. Systematic protein interactome analysis of glycosaminoglycans revealed YcbS as a novel bacterial virulence factor.

    PubMed

    Hsiao, Felix Shih-Hsiang; Sutandy, Fx Reymond; Syu, Guan-Da; Chen, Yi-Wen; Lin, Jun-Mu; Chen, Chien-Sheng

    2016-01-01

    Microbial pathogens have evolved several strategies for interacting with host cell components, such as glycosaminoglycans (GAGs). Some microbial proteins involved in host-GAG binding have been described; however, a systematic study on microbial proteome-mammalian GAG interactions has not been conducted. Here, we used Escherichia coli proteome chips to probe four typical mammalian GAGs, heparin, heparan sulphate (HS), chondroitin sulphate B (CSB), and chondroitin sulphate C (CSC), and identified 185 heparin-, 62 HS-, 98 CSB-, and 101 CSC-interacting proteins. Bioinformatics analyses revealed the unique functions of heparin- and HS-specific interacting proteins in glycine, serine, and threonine metabolism. Among all the GAG-interacting proteins, three were outer membrane proteins (MbhA, YcbS, and YmgH). Invasion assays confirmed that mutant E. coli lacking ycbS could not invade the epithelial cells. Introducing plasmid carrying ycbS complemented the invading defects at ycbS lacking E. coli mutant, that can be further improved by overexpressing ycbS. Preblocking epithelial cells with YcbS reduced the percentage of E. coli invasions. Moreover, we observed that whole components of the ycb operon were crucial for invasion. The displacement assay revealed that YcbS binds to the laminin-binding site of heparin and might affect the host extracellular matrix structure by displacing heparin from laminin. PMID:27323865

  12. Systematic protein interactome analysis of glycosaminoglycans revealed YcbS as a novel bacterial virulence factor

    PubMed Central

    Hsiao, Felix Shih-Hsiang; Sutandy, FX Reymond; Syu, Guan-Da; Chen, Yi-Wen; Lin, Jun-Mu; Chen, Chien-Sheng

    2016-01-01

    Microbial pathogens have evolved several strategies for interacting with host cell components, such as glycosaminoglycans (GAGs). Some microbial proteins involved in host–GAG binding have been described; however, a systematic study on microbial proteome–mammalian GAG interactions has not been conducted. Here, we used Escherichia coli proteome chips to probe four typical mammalian GAGs, heparin, heparan sulphate (HS), chondroitin sulphate B (CSB), and chondroitin sulphate C (CSC), and identified 185 heparin-, 62 HS-, 98 CSB-, and 101 CSC-interacting proteins. Bioinformatics analyses revealed the unique functions of heparin- and HS-specific interacting proteins in glycine, serine, and threonine metabolism. Among all the GAG-interacting proteins, three were outer membrane proteins (MbhA, YcbS, and YmgH). Invasion assays confirmed that mutant E. coli lacking ycbS could not invade the epithelial cells. Introducing plasmid carrying ycbS complemented the invading defects at ycbS lacking E. coli mutant, that can be further improved by overexpressing ycbS. Preblocking epithelial cells with YcbS reduced the percentage of E. coli invasions. Moreover, we observed that whole components of the ycb operon were crucial for invasion. The displacement assay revealed that YcbS binds to the laminin-binding site of heparin and might affect the host extracellular matrix structure by displacing heparin from laminin. PMID:27323865

  13. Coarse grained simulation reveals antifreeze properties of hyperactive antifreeze protein from Antarctic bacterium Colwellia sp.

    NASA Astrophysics Data System (ADS)

    Nguyen, Hung; Van, Thanh Dac; Le, Ly

    2015-10-01

    The novel hyperactive antifreeze protein (AFP) of Antarctic sea ice bacterium Colwellia sp. provides a target for studying the protection of psychrophilic microgoranisms against freezing environment. Interestingly, the Colwellia sp. hyperactive antifreeze protein (ColAFP) was crystallized without the structural dynamic characteristics. Here, the result indicated, through coarse grained simulation of ColAFP under various subfreezing temperature, that ColAFP remains active at temperature of equal and greater than 275 K (∼2 °C). Extensive simulation analyses also revealed the adaptive mechanism of ColAFP in subfreezing environment. Our result provides a structural dynamic understanding of the ColAFP.

  14. Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry.

    PubMed

    Asara, John M; Schweitzer, Mary H; Freimark, Lisa M; Phillips, Matthew; Cantley, Lewis C

    2007-04-13

    Fossilized bones from extinct taxa harbor the potential for obtaining protein or DNA sequences that could reveal evolutionary links to extant species. We used mass spectrometry to obtain protein sequences from bones of a 160,000- to 600,000-year-old extinct mastodon (Mammut americanum) and a 68-million-year-old dinosaur (Tyrannosaurus rex). The presence of T. rex sequences indicates that their peptide bonds were remarkably stable. Mass spectrometry can thus be used to determine unique sequences from ancient organisms from peptide fragmentation patterns, a valuable tool to study the evolution and adaptation of ancient taxa from which genomic sequences are unlikely to be obtained. PMID:17431180

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Korwin-Edson, Michelle Lynn

    Previous works have shown that cells proliferate differently depending on the chemistry of the glass on which they are growing. Since proteins form the bonds between cells and glass, the hypothesis of this study is that proteins can distinguish between surface chemical variations of glass. This theory was examined through the use of various silica forms, a few select proteins, four surface treatment procedures, and a variety of characterization techniques. The silica forms include amorphous slides, cane, fiber, microspheres, fumed silica and quartz crystal terminals. The proteins selected were human serum albumin, mouse Immunoglobulin G, streptavidin, antimouse IgG, and biotin. The surface treatments utilized to bring about chemical variation on the silica surface were HF acid etching, ethanol cleaning, water plasma treatments, and 1000°C heat treatments. The characterization techniques encompassed both traditional material techniques and biological methods. The techniques studied were atomic force microscopy (AFM), chemical force microscopy (CFM), glancing incidence X-ray analysis (GIXA), fluorescence spectrometry, polyacrylamide gel electrophoresis (SDS-PAGE), and bicinchoninic acid (BCA) assay. It was the main goal of this project to determine the feasibility of these techniques in utilizing proteins as glass surface probes. Proteins were adsorbed to all of the various forms and the binding ability was studied by either stripping off the protein and quantifying them, or by deductive reasoning through the use of "depleted" protein solutions. Fluorimetry and BCA assay both utilized the depleted solutions, but the high error associated with this protocol was prohibitive. SDS-PAGE with streptavidin was very difficult due to staining problems, however the IgG proteins were able to be quantified with some success. GIXA showed that the protein layer thickness is monolayer in nature, which agrees well with the AFM fluid tapping data on protein height, but in addition

  18. Surface modification on microfluidic devices with 2-methacryloyloxyethyl phosphorylcholine polymers for reducing unfavorable protein adsorption.

    PubMed

    Sibarani, James; Takai, Madoka; Ishihara, Kazuhiko

    2007-01-15

    Surface modification of polymer materials for preparing microfluidic devices including poly(dimethyl siloxane) (PDMS) was investigated with phospholipids polymers such as poly(2-methacryloyloxylethyl phosphorylcholine(MPC)-co-n-butyl methacrylate) (PMB) and poly(MPC-co-2-ethylhexyl methacrylate-co-2-(N,N-dimethylamino)ethyl methacrylate) (PMED). The hydrophilicity of every surface on the polymer materials modified with these MPC polymers increased and the value of zeta-potential became close to zero. The protein adsorption on the polymer materials with and without the surface modification was evaluated using a protein mixture of human plasma fibrinogen and serum albumin. Amount of proteins adsorbed on these polymeric materials showed significant reduction by the surface modification with the MPC polymers compared to the uncoated surfaces ranging from 56 to 90%. Furthermore, we successfully prepared PDMS-based microchannel which was modified by simple coating with the PMB and PMED. The modified microchannel also revealed a significant reduction of adsorption of serum albumin. We conclude that the MPC polymers are useful for reducing unfavorable protein adsorption on microfluidic devices. PMID:17112710

  19. The leucine rich amelogenin protein (LRAP) adsorbs as monomers or dimers onto surfaces

    SciTech Connect

    Tarasevich, Barbara J.; Lea, Alan S.; Shaw, Wendy J.

    2010-03-15

    Amelogenin and amelogenin splice variants are believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin proteins onto substrates is very important because protein-surface interactions are critical to it’s function. We have studied the adsorption of LRAP, a splice variant of amelogenin which may also contribute to enamel function, onto model self-assembled monolayers on gold containing of COOH, CH3, and NH2 end groups. Dynamic light scattering (DLS) experiments indicated that LRAP in phosphate buffered saline (PBS) and solutions at saturation with calcium phosphate contained aggregates of nanospheres. Null ellipsometry and atomic force microscopy (AFM) were used to study protein adsorption amounts and structures. Relatively high amounts of adsorption occurred onto the CH3 and NH2 surfaces from both calcium phosphate and PBS solutions. Adsorption was also promoted onto COOH surfaces when calcium was present in the solutions suggesting an interaction that involves calcium bridging with the negatively charged C-terminus. The ellipsometry and AFM studies suggested that the protein adsorbed onto all surfaces as LRAP monomers. We propose that the monomers adsorb onto the surfaces by disassembling or “shedding” from the nanospheres that are present in solution. This work reveals the importance of small subnanosphere-sized structures of LRAP at interfaces, structures that may be important in the biomineralization of tooth enamel.

  20. An extracellular interactome of Immunoglobulin and LRR proteins reveals receptor-ligand networks

    PubMed Central

    Özkan, Engin; Carrillo, Robert A.; Eastman, Catharine L.; Weiszmann, Richard; Waghray, Deepa; Johnson, Karl G.; Zinn, Kai; Celniker, Susan E.; Garcia, K. Christopher

    2013-01-01

    Extracellular domains of cell-surface receptors and ligands mediate cell-cell communication, adhesion, and initiation of signaling events, but most existing protein-protein “interactome” datasets lack information for extracellular interactions. We probed interactions between receptor extracellular domains, focusing on the Immunoglobulin Superfamily (IgSF), Fibronectin type-III (FnIII) and Leucine-rich repeat (LRR) families of Drosophila, a set of 202 proteins, many of which are known to be important in neuronal and developmental functions. Out of 20503 candidate protein pairs tested, we observed 106 interactions, 83 of which were previously unknown. We ‘deorphanized’ the 20-member subfamily of defective in proboscis IgSF proteins, showing that they selectively interact with an 11-member subfamily of previously uncharacterized IgSF proteins. Both subfamilies interact with a single common ‘orphan’ LRR protein. We also observed new interactions between Hedgehog and EGFR pathway components. Several of these interactions could be visualized in live-dissected embryos, demonstrating that this approach can identify physiologically relevant receptor-ligand pairs. PMID:23827685

  1. The heterogeneity of human antibody responses to vaccinia virus revealed through use of focused protein arrays.

    PubMed

    Duke-Cohan, Jonathan S; Wollenick, Kristin; Witten, Elizabeth A; Seaman, Michael S; Baden, Lindsey R; Dolin, Raphael; Reinherz, Ellis L

    2009-02-18

    The renewed interest in strategies to combat infectious agents with epidemic potential has led to a re-examination of vaccination protocols against smallpox. To help define which antigens elicit a human antibody response, we have targeted proteins known or predicted to be presented on the surface of the intracellular mature virion (IMV) or the extracellular enveloped virion (EEV). The predicted ectodomains were expressed in a mammalian in vitro coupled transcription/translation reaction using tRNA(lys) precharged with lysine-epsilon-biotin followed by solid phase immobilization on 384-well neutravidin-coated plates. The generated array is highly specific and sensitive in a micro-ELISA format. By comparison of binding of vaccinia-immune sera to the reticulocyte lysate-produced proteins and to secreted post-translationally modified proteins, we demonstrate that for several proteins including the EEV proteins B5 and A33, proper recognition is dependent upon appropriate folding, with little dependence upon glycosylation per se. We further demonstrate that the humoral immune response to vaccinia among different individuals is not uniform in specificity or strength, as different IMV and EEV targets predominate within the group of immunogenic proteins. This heterogeneity likely results from the diversity of HLA Class II alleles and CD4 T helper cell epitopes stimulating B cell antibody production. Our findings have important implications both for design of new recombinant subunit vaccines as well as for methods of assaying the human antibody response utilizing recombinant proteins produced in vitro. PMID:19146908

  2. Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA

    PubMed Central

    Kim, Sanghyun; Zbaida, David; Elbaum, Michael; Leh, Hervé; Nogues, Claude; Buckle, Malcolm

    2015-01-01

    VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein–protein interactions. In order to isolate the protein–DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1–VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1–VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1. PMID:26044711

  3. Bacterial populations on brewery filling hall surfaces as revealed by next-generation sequencing.

    PubMed

    Priha, Outi; Raulio, Mari; Maukonen, Johanna; Vehviläinen, Anna-Kaisa; Storgårds, Erna

    2016-01-01

    Due to the presence of moisture and nutrients, brewery filling line surfaces are susceptible to unwanted microbial attachment. Knowledge of the attaching microbes will aid in designing hygienic control of the process. In this study the bacterial diversity present on brewery filling line surfaces was revealed by next generation sequencing. The two filling lines studied maintained their characteristic bacterial community throughout three sampling times (13-163 days). On the glass bottle line, γ-proteobacteria dominated (35-82% of all OTUs), whereas on the canning line α-, β- and γ-proteobacteria and actinobacteria were most common. The most frequently detected genera were Acinetobacter, Propinobacterium and Pseudomonas. The halophilic genus Halomonas was commonly detected, which might be due to its tolerance to alkaline foam cleaners. This study has revealed a detailed overall picture of the bacterial groups present on filling line surfaces. Further effort should be given to determine the efficacy of washing procedures on different bacterial groups. PMID:27064426

  4. Organic bioelectronics probing conformational changes in surface confined proteins

    PubMed Central

    Macchia, Eleonora; Alberga, Domenico; Manoli, Kyriaki; Mangiatordi, Giuseppe F.; Magliulo, Maria; Palazzo, Gerardo; Giordano, Francesco; Lattanzi, Gianluca; Torsi, Luisa

    2016-01-01

    The study of proteins confined on a surface has attracted a great deal of attention due to its relevance in the development of bio-systems for laboratory and clinical settings. In this respect, organic bio-electronic platforms can be used as tools to achieve a deeper understanding of the processes involving protein interfaces. In this work, biotin-binding proteins have been integrated in two different organic thin-film transistor (TFT) configurations to separately address the changes occurring in the protein-ligand complex morphology and dipole moment. This has been achieved by decoupling the output current change upon binding, taken as the transducing signal, into its component figures of merit. In particular, the threshold voltage is related to the protein dipole moment, while the field-effect mobility is associated with conformational changes occurring in the proteins of the layer when ligand binding occurs. Molecular Dynamics simulations on the whole avidin tetramer in presence and absence of ligands were carried out, to evaluate how the tight interactions with the ligand affect the protein dipole moment and the conformation of the loops surrounding the binding pocket. These simulations allow assembling a rather complete picture of the studied interaction processes and support the interpretation of the experimental results. PMID:27312768

  5. Organic bioelectronics probing conformational changes in surface confined proteins.

    PubMed

    Macchia, Eleonora; Alberga, Domenico; Manoli, Kyriaki; Mangiatordi, Giuseppe F; Magliulo, Maria; Palazzo, Gerardo; Giordano, Francesco; Lattanzi, Gianluca; Torsi, Luisa

    2016-01-01

    The study of proteins confined on a surface has attracted a great deal of attention due to its relevance in the development of bio-systems for laboratory and clinical settings. In this respect, organic bio-electronic platforms can be used as tools to achieve a deeper understanding of the processes involving protein interfaces. In this work, biotin-binding proteins have been integrated in two different organic thin-film transistor (TFT) configurations to separately address the changes occurring in the protein-ligand complex morphology and dipole moment. This has been achieved by decoupling the output current change upon binding, taken as the transducing signal, into its component figures of merit. In particular, the threshold voltage is related to the protein dipole moment, while the field-effect mobility is associated with conformational changes occurring in the proteins of the layer when ligand binding occurs. Molecular Dynamics simulations on the whole avidin tetramer in presence and absence of ligands were carried out, to evaluate how the tight interactions with the ligand affect the protein dipole moment and the conformation of the loops surrounding the binding pocket. These simulations allow assembling a rather complete picture of the studied interaction processes and support the interpretation of the experimental results. PMID:27312768

  6. Organic bioelectronics probing conformational changes in surface confined proteins

    NASA Astrophysics Data System (ADS)

    Macchia, Eleonora; Alberga, Domenico; Manoli, Kyriaki; Mangiatordi, Giuseppe F.; Magliulo, Maria; Palazzo, Gerardo; Giordano, Francesco; Lattanzi, Gianluca; Torsi, Luisa

    2016-06-01

    The study of proteins confined on a surface has attracted a great deal of attention due to its relevance in the development of bio-systems for laboratory and clinical settings. In this respect, organic bio-electronic platforms can be used as tools to achieve a deeper understanding of the processes involving protein interfaces. In this work, biotin-binding proteins have been integrated in two different organic thin-film transistor (TFT) configurations to separately address the changes occurring in the protein-ligand complex morphology and dipole moment. This has been achieved by decoupling the output current change upon binding, taken as the transducing signal, into its component figures of merit. In particular, the threshold voltage is related to the protein dipole moment, while the field-effect mobility is associated with conformational changes occurring in the proteins of the layer when ligand binding occurs. Molecular Dynamics simulations on the whole avidin tetramer in presence and absence of ligands were carried out, to evaluate how the tight interactions with the ligand affect the protein dipole moment and the conformation of the loops surrounding the binding pocket. These simulations allow assembling a rather complete picture of the studied interaction processes and support the interpretation of the experimental results.

  7. Protein Surface Matching by Combining Local and Global Geometric Information

    PubMed Central

    Ellingson, Leif; Zhang, Jinfeng

    2012-01-01

    Comparison of the binding sites of proteins is an effective means for predicting protein functions based on their structure information. Despite the importance of this problem and much research in the past, it is still very challenging to predict the binding ligands from the atomic structures of protein binding sites. Here, we designed a new algorithm, TIPSA (Triangulation-based Iterative-closest-point for Protein Surface Alignment), based on the iterative closest point (ICP) algorithm. TIPSA aims to find the maximum number of atoms that can be superposed between two protein binding sites, where any pair of superposed atoms has a distance smaller than a given threshold. The search starts from similar tetrahedra between two binding sites obtained from 3D Delaunay triangulation and uses the Hungarian algorithm to find additional matched atoms. We found that, due to the plasticity of protein binding sites, matching the rigid body of point clouds of protein binding sites is not adequate for satisfactory binding ligand prediction. We further incorporated global geometric information, the radius of gyration of binding site atoms, and used nearest neighbor classification for binding site prediction. Tested on benchmark data, our method achieved a performance comparable to the best methods in the literature, while simultaneously providing the common atom set and atom correspondences. PMID:22815760

  8. Protein surface matching by combining local and global geometric information.

    PubMed

    Ellingson, Leif; Zhang, Jinfeng

    2012-01-01

    Comparison of the binding sites of proteins is an effective means for predicting protein functions based on their structure information. Despite the importance of this problem and much research in the past, it is still very challenging to predict the binding ligands from the atomic structures of protein binding sites. Here, we designed a new algorithm, TIPSA (Triangulation-based Iterative-closest-point for Protein Surface Alignment), based on the iterative closest point (ICP) algorithm. TIPSA aims to find the maximum number of atoms that can be superposed between two protein binding sites, where any pair of superposed atoms has a distance smaller than a given threshold. The search starts from similar tetrahedra between two binding sites obtained from 3D Delaunay triangulation and uses the Hungarian algorithm to find additional matched atoms. We found that, due to the plasticity of protein binding sites, matching the rigid body of point clouds of protein binding sites is not adequate for satisfactory binding ligand prediction. We further incorporated global geometric information, the radius of gyration of binding site atoms, and used nearest neighbor classification for binding site prediction. Tested on benchmark data, our method achieved a performance comparable to the best methods in the literature, while simultaneously providing the common atom set and atom correspondences. PMID:22815760

  9. Antigenic characterization of dimorphic surface protein in Mycobacterium tuberculosis.

    PubMed

    Matsuba, Takashi; Siddiqi, Umme Ruman; Hattori, Toshio; Nakajima, Chie; Fujii, Jun; Suzuki, Yasuhiko

    2016-05-01

    The Mycobacterium tuberculosis Rv0679c protein is a surface protein that contributes to host cell invasion. We previously showed that a single nucleotide transition of the Rv0679c gene leads to a single amino acid substitution from asparagine to lysine at codon 142 in the Beijing genotype family. In this study, we examined the immunological effect of this substitution. Several recombinant proteins were expressed in Escherichia coli and Mycobacterium smegmatis and characterized with antisera and two monoclonal antibodies named 5D4-C2 and 8G10-H2. A significant reduction of antibody binding was detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis in the Lys142-type protein. This reduction of 8G10-H2 binding was more significant, with the disappearance of a signal in the proteins expressed by recombinant mycobacteria in western blot analysis. In addition, epitope mapping analysis of the recombinant proteins showed a linear epitope by 5D4-C2 and a discontinuous epitope by 8G10-H2. The antibody recognizing the conformational epitope detected only mycobacterial Asn142-type recombinant protein. Our results suggest that a single amino acid substitution of Rv0679c has potency for antigenic change in Beijing genotype strains. PMID:27190237

  10. Dynamic Coupling among Protein Binding, Sliding, and DNA Bending Revealed by Molecular Dynamics.

    PubMed

    Tan, Cheng; Terakawa, Tsuyoshi; Takada, Shoji

    2016-07-13

    Protein binding to DNA changes the DNA's structure, and altered DNA structure can, in turn, modulate the dynamics of protein binding. This mutual dependency is poorly understood. Here we investigated dynamic couplings among protein binding to DNA, protein sliding on DNA, and DNA bending by applying a coarse-grained simulation method to the bacterial architectural protein HU and 14 other DNA-binding proteins. First, we verified our method by showing that the simulated HU exhibits a weak preference for A/T-rich regions of DNA and a much higher affinity for gapped and nicked DNA, consistent with biochemical experiments. The high affinity was attributed to a local DNA bend, but not the specific chemical moiety of the gap/nick. The long-time dynamic analysis revealed that HU sliding is associated with the movement of the local DNA bending site. Deciphering single sliding steps, we found the coupling between HU sliding and DNA bending is akin to neither induced-fit nor population-shift; instead they moved concomitantly. This is reminiscent of a cation transfer on DNA and can be viewed as a protein version of polaron-like sliding. Interestingly, on shorter time scales, HU paused when the DNA was highly bent at the bound position and escaped from pauses once the DNA spontaneously returned to a less bent structure. The HU sliding is largely regulated by DNA bending dynamics. With 14 other proteins, we explored the generality and versatility of the dynamic coupling and found that 6 of the 15 assayed proteins exhibit the polaron-like sliding. PMID:27309278

  11. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    SciTech Connect

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-13

    Re-engineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. The context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

  12. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    DOE PAGESBeta

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-13

    Re-engineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test thismore » strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. The context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.« less

  13. The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

    SciTech Connect

    Zhang, Yangli; Gao, Zengqiang; Guo, Zhen; Zhang, Hongpeng; Zhang, Zhenzhen; Luo, Miao; Hou, Haifeng; Huang, Ailong; Dong, Yuhui; Wang, Deqiang

    2013-09-27

    Highlights: •We determined the first structure of human α1M with heavy electron density of the chromophore. •We proposed a new structural model of the chromophore. •We first revealed that the two conserved surface regions of α1M are proposed as putative protein–protein interface sites. -- Abstract: Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners.

  14. Determination of Protein Surface Hydration by Systematic Charge Mutations

    NASA Astrophysics Data System (ADS)

    Yang, Jin; Jia, Menghui; Qin, Yangzhong; Wang, Dihao; Pan, Haifeng; Wang, Lijuan; Xu, Jianhua; Zhong, Dongping; Dongping Zhong Collaboration; Jianhua Xu Collaboration

    Protein surface hydration is critical to its structural stability, flexibility, dynamics and function. Recent observations of surface solvation on picosecond time scales have evoked debate on the origin of such relatively slow motions, from hydration water or protein charged sidechains, especially with molecular dynamics simulations. Here, we used a unique nuclease with a single tryptophan as a local probe and systematically mutated neighboring three charged residues to differentiate the contributions from hydration water and charged sidechains. By mutations of alternative one and two and all three charged residues, we observed slight increases in the total tryptophan Stokes shifts with less neighboring charged residue(s) and found insensitivity of charged sidechains to the relaxation patterns. The dynamics is correlated with hydration water relaxation with the slowest time in a dense charged environment and the fastest time at a hydrophobic site. On such picosecond time scales, the protein surface motion is restricted. The total Stokes shifts are dominantly from hydration water relaxation and the slow dynamics is from water-driven relaxation, coupled with local protein fluctuations.

  15. NSP-Cas protein structures reveal a promiscuous interaction module in cell signaling

    SciTech Connect

    Mace, P.D.; Robinson, H.; Wallez, Y.; Dobaczewska, M. K.; Lee, J. J.; Pasquale, E. B.; Riedl, S. J.

    2011-12-01

    Members of the novel SH2-containing protein (NSP) and Crk-associated substrate (Cas) protein families form multidomain signaling platforms that mediate cell migration and invasion through a collection of distinct signaling motifs. Members of each family interact via their respective C-terminal domains, but the mechanism of this association has remained enigmatic. Here we present the crystal structures of the C-terminal domain from the NSP protein BCAR3 and the complex of NSP3 with p130Cas. BCAR3 adopts the Cdc25-homology fold of Ras GTPase exchange factors, but it has a 'closed' conformation incapable of enzymatic activity. The structure of the NSP3-p130Cas complex reveals that this closed conformation is instrumental for interaction of NSP proteins with a focal adhesion-targeting domain present in Cas proteins. This enzyme-to-adaptor conversion enables high-affinity, yet promiscuous, interactions between NSP and Cas proteins and represents an unprecedented mechanistic paradigm linking cellular signaling networks.

  16. Developmental Staging of Maize Microspores Reveals a Transition in Developing Microspore Proteins 1

    PubMed Central

    Bedinger, Patricia A.; Edgerton, Michael D.

    1990-01-01

    A method for the preparation of developmentally staged microspores and young pollen from maize (Zea mays) has been devised. The preparations are of sufficient purity and quantity for biochemical analysis, including the analysis of steady-state protein and RNA populations associated with each stage. A major transition in protein populations occurs during the developmental period that encompasses microspore mitosis, the asymmetric nuclear division producing the vegetative and generative nuclei. Several differences between early and late stage proteins can be detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional gel electrophoresis of proteins reveals that over half of the steady-state proteins differ between the younger and older stages, either quantitative or qualitative. One protein that increases in relative abundance about fourfold is actin. In vitro translation of RNA isolated from staged microspores demonstrates changes in microspore gene expression during the same developmental period. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:16667300

  17. Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

    PubMed

    Hamsten, C; Häggmark, A; Grundström, J; Mikus, M; Lindskog, C; Konradsen, J R; Eklund, A; Pershagen, G; Wickman, M; Grunewald, J; Melén, E; Hedlin, G; Nilsson, P; van Hage, M

    2016-09-01

    Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma. PMID:27145233

  18. Coupled protein domain motion in Taq polymerase revealed by neutron spin-echo spectroscopy

    PubMed Central

    Bu, Zimei; Biehl, Ralf; Monkenbusch, Michael; Richter, Dieter; Callaway, David J. E.

    2005-01-01

    Long-range conformational changes in proteins are ubiquitous in biology for the transmission and amplification of signals; such conformational changes can be triggered by small-amplitude, nanosecond protein domain motion. Understanding how conformational changes are initiated requires the characterization of protein domain motion on these timescales and on length scales comparable to protein dimensions. Using neutron spin-echo spectroscopy (NSE), normal mode analysis, and a statistical-mechanical framework, we reveal overdamped, coupled domain motion within DNA polymerase I from Thermus aquaticus (Taq polymerase). This protein utilizes correlated domain dynamics over 70 Å to coordinate nucleotide synthesis and cleavage during DNA synthesis and repair. We show that NSE spectroscopy can determine the domain mobility tensor, which determines the degree of dynamical coupling between domains. The mobility tensor defines the domain velocity response to a force applied to it or to another domain, just as the sails of a sailboat determine its velocity given the applied wind force. The NSE results provide insights into the nature of protein domain motion that are not appreciated by conventional biophysical techniques. PMID:16306270

  19. Proteome analysis of Paenibacillus larvae reveals the existence of a putative S-layer protein.

    PubMed

    Fünfhaus, Anne; Genersch, Elke

    2012-04-01

    Honey bee pathology has attracted much interest recently due to the problems with honey bee declines in many regions of the world. American Foulbrood (AFB) caused by Paenibacillus larvae is the most devastating bacterial brood disease of the Western honey bee (Apis mellifera) causing considerable economic losses to beekeepers worldwide. AFB outbreaks are mainly caused by two differentially virulent genotypes of P. larvae, P. larvae ERIC I and ERIC II. To better understand AFB pathogenesis and to complement already existing data from the genetic level we aimed at obtaining expression data from the protein level. We successfully developed a protocol for two-dimensional proteome analysis of P. larvae with subsequent mass-spectrometry based protein sequencing. Based on the obtained master protein maps of P. larvae genotypes ERIC I and II we identified the dominantly expressed cytosolic proteins of both genotypes, some of them presumably linked to pathogenesis and virulence. Comparing the master maps of both genotypes revealed differentially expressed proteins, i.e. a putative S-layer protein which is expressed by P. larvae ERIC II but absent from the proteome of P. larvae ERIC I. The implications of our findings for pathogenesis of AFB and virulence of P. larvae will be discussed. PMID:23757273

  20. A Human Pluripotent Stem Cell Surface N-Glycoproteome Resource Reveals Markers, Extracellular Epitopes, and Drug Targets

    PubMed Central

    Boheler, Kenneth R.; Bhattacharya, Subarna; Kropp, Erin M.; Chuppa, Sandra; Riordon, Daniel R.; Bausch-Fluck, Damaris; Burridge, Paul W.; Wu, Joseph C.; Wersto, Robert P.; Chan, Godfrey Chi Fung; Rao, Sridhar; Wollscheid, Bernd; Gundry, Rebekah L.

    2014-01-01

    Summary Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures. PMID:25068131

  1. Analysis of rat plasma proteins desorbed from gold and methyl- and hydroxyl-terminated alkane thiols on gold surfaces.

    PubMed

    Källtorp, M; Carlén, A; Thomsen, P; Olsson, J; Tengvall, P

    2000-03-01

    It is believed that adsorbed blood or plasma components, such as water, peptides, carbohydrates and proteins, determine key events in the concomitant inflammatory tissue response close to implants. The aim of the present study was to develop a procedure for the collection and analysis of minor amounts of proteins bound to solid metal implant surfaces. The combination of a sodium dodecyl sulfate washing method coupled with a polyacylamide gel electrophoretic protein separation technique (SDS-PAGE), Western blot and image analysis enabled the desorption, identification and semiquantification of specific proteins. The analyzed proteins were albumin, immunoglobulin G, fibrinogen and fibronectin. Concentration procedures of proteins were not required with this method despite the small area of the test surfaces. The plasma proteins were adsorbed to pure gold and hydroxylated and methylated gold surfaces, which elicit different tissue responses in vivo and plasma protein adsorption patterns in vitro. The image analysis revealed that the pure gold surfaces adsorbed the largest amount of total and specific proteins. This is in accordance with previous ellipsometry/antibody experiments in vitro. Further, the principles described for the protein analysis can be applied on implant surfaces ex vivo. PMID:15348048

  2. Cdon, a cell surface protein, mediates oligodendrocyte differentiation and myelination.

    PubMed

    Wang, Li-Chun; Almazan, Guillermina

    2016-06-01

    During central nervous system development, oligodendrocyte progenitors (OLPs) establish multiple branched processes and axonal contacts to initiate myelination. A complete understanding of the molecular signals implicated in cell surface interaction to initiate myelination/remyelination is currently lacking. The objective of our study was to assess whether Cdon, a cell surface protein that was shown to participate in muscle and neuron cell development, is involved in oligodendrocyte (OLG) differentiation and myelination. Here, we demonstrate that endogenous Cdon protein is expressed in OLPs, increasing in the early differentiation stages and decreasing in mature OLGs. Immunocytochemistry of endogenous Cdon showed localization on both OLG cell membranes and cellular processes exhibiting puncta- or varicosity-like structures. Cdon knockdown with siRNA decreased protein levels by 62% as well as two myelin-specific proteins, MBP and MAG. Conversely, overexpression of full-length rat Cdon increased myelin proteins in OLGs. The complexity of OLGs branching and contact point numbers with axons were also increased in Cdon overexpressing cells growing alone or in coculture with dorsal root ganglion neurons (DRGNs). Furthermore, myelination of DRGNs was decreased when OLPs were transfected with Cdon siRNA. Altogether, our results suggest that Cdon participates in OLG differentiation and myelination, most likely in the initial stages of development. GLIA 2016;64:1021-1033. PMID:26988125

  3. 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.

  4. The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function

    PubMed Central

    Das, Debanu; Finn, Robert D.; Carlton, Dennis; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins. PMID:20944221

  5. Kinetic and Conformational Insights of Protein Adsorption onto Montmorillonite Revealed Using in Situ ATR-FTIR/2D-COS.

    PubMed

    Schmidt, Michael P; Martínez, Carmen Enid

    2016-08-01

    Protein adsorption onto clay minerals is a process with wide-ranging impacts on the environmental cycling of nutrients and contaminants. This process is influenced by kinetic and conformational factors that are often challenging to probe in situ. This study represents an in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopic investigation of the adsorption of a model protein (bovine serum albumin (BSA)) onto a clay mineral (montmorillonite) at four concentrations (1.50, 3.75, 7.50, and 15.0 μM) under environmentally relevant conditions. At all concentrations probed, FTIR spectra show that BSA readily adsorbs onto montmorillonite. Adsorption kinetics follow an Elovich model, suggesting that primary limitations on adsorption rates are surface-related heterogeneous energetic restrictions associated with protein rearrangement and lateral protein-protein interaction. BSA adsorption onto montmorillonite fits the Langmuir model, yielding K = 5.97 × 10(5) M(-1). Deconvolution and curve fitting of the amide I band at the end of the adsorption process (∼120 min) shows a large extent of BSA unfolding upon adsorption at 1.50 μM, with extended chains and turns increasing at the expense of α-helices. At higher concentrations/surface coverages, BSA unfolding is less pronounced and a more compact structure is assumed. Two-dimensional correlation spectroscopic (2D-COS) analysis reveals three different pathways corresponding to adsorbed conformations. At 1.50 μM, adsorption increases extended chains, followed by a loss in α-helices and a subsequent increase in turns. At 3.75 μM, extended chains decrease and then aggregated strands increase and side chains decrease, followed by a decrease in turns. With 7.50 and 15.0 μM BSA, the loss of side-chain vibrations is followed by an increase in aggregated strands and a subsequent decrease in turns and extended chains. Overall, the BSA concentration and resultant surface coverage have a profound

  6. Translocation of signalling proteins to the plasma membrane revealed by a new bioluminescent procedure

    PubMed Central

    2011-01-01

    Background Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors. High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive. Results Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca2+)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca2+ concentration [Ca2+] difference between bulk cytosolic and the sub-plasma membrane rim. Conclusion This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane. Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation. Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs

  7. The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

    SciTech Connect

    Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.; Garboczi, David N.

    2010-11-03

    The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

  8. Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group.

    PubMed

    Nunoura, Takuro; Takaki, Yoshihiro; Kakuta, Jungo; Nishi, Shinro; Sugahara, Junichi; Kazama, Hiromi; Chee, Gab-Joo; Hattori, Masahira; Kanai, Akio; Atomi, Haruyuki; Takai, Ken; Takami, Hideto

    2011-04-01

    The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the 'Thaumarchaeota' and 'Korarchaeota'. Here, we show the genome sequence of Candidatus 'Caldiarchaeum subterraneum' that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea. PMID:21169198

  9. Secretory protein profiling reveals TNFα inactivation by selective and promiscuous Sec61 modulators

    PubMed Central

    Maifeld, Sarah V.; MacKinnon, Andrew L.; Garrison, Jennifer L.; Sharma, Ajay; Kunkel, Eric J.; Hegde, Ramanujan S.; Taunton, Jack

    2013-01-01

    Summary Cotransins are cyclic heptadepsipeptides that bind the Sec61 translocon to inhibit cotranslational translocation of a subset of secreted and type I transmembrane proteins. The few known cotransin-sensitive substrates are all targeted to the translocon by a cleavable signal sequence, previously shown to be a critical determinant of cotransin sensitivity. By profiling two cotransin variants against a panel of secreted and transmembrane proteins, we demonstrate that cotransin side-chain differences profoundly affect substrate selectivity. Among the most sensitive substrates we identified is the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα). Like all type II transmembrane proteins, TNFα is targeted to the translocon by its membrane-spanning domain, indicating that a cleavable signal sequence is not strictly required for cotransin sensitivity. Our results thus reveal an unanticipated breadth of translocon substrates whose expression is inhibited by Sec61 modulators. PMID:21944747

  10. Mass Spectrometry and Next-Generation Sequencing Reveal an Abundant and Rapidly Evolving Abalone Sperm Protein

    PubMed Central

    Palmer, Melody R.; McDowall, Margo H.; Stewart, Lia; Ouaddi, Aleena; MacCoss, Michael J.; Swanson, Willie J.

    2014-01-01

    SUMMARY Abalone, a broadcast spawning marine mollusk, is an important model for molecular interactions and positive selection in fertilization, but the focus has previously been on only two sperm proteins, lysin and sp18.We used genomic and proteomic techniques to bring new insights to this model by characterizing the testis transcriptome and sperm proteome of the Red abalone Haliotis rufescens. One pair of homologous, testis-specific proteins contains a secretion signal and is small, abundant, and associated with the acrosome. Comparative analysis revealed that homologs are extremely divergent between species, and show strong evidence for positive selection. The acrosomal localization and rapid evolution of these proteins indicates that they play an important role in fertilization, and could be involved in the species-specificity of sperm-egg interactions in abalone. Our genomic and proteomic characterization of abalone fertilization resulted in the identification of interesting, novel peptides that have eluded detection in this important model system for 20 years. PMID:23585193

  11. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans

    PubMed Central

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W.; Grubert, Fabian; Candille, Sophie I.; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L.; Tang, Hua; Ricci, Emiliano; Snyder, Michael P.

    2015-01-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy—many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. PMID:26297486

  12. Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic

    PubMed Central

    Zhang, Hai-nan; Yang, Lina; Ling, Jian-ya; Czajkowsky, Daniel M.; Wang, Jing-Fang; Zhang, Xiao-Wei; Zhou, Yi-Ming; Ge, Feng; Yang, Ming-kun; Xiong, Qian; Guo, Shu-Juan; Le, Huang-Ying; Wu, Song-Fang; Yan, Wei; Liu, Bingya; Zhu, Heng; Chen, Zhu; Tao, Sheng-ce

    2015-01-01

    Arsenic is highly effective for treating acute promyelocytic leukemia (APL) and has shown significant promise against many other tumors. However, although its mechanistic effects in APL are established, its broader anticancer mode of action is not understood. In this study, using a human proteome microarray, we identified 360 proteins that specifically bind arsenic. Among the most highly enriched proteins in this set are those in the glycolysis pathway, including the rate-limiting enzyme in glycolysis, hexokinase-1. Detailed biochemical and metabolomics analyses of the highly homologous hexokinase-2 (HK2), which is overexpressed in many cancers, revealed significant inhibition by arsenic. Furthermore, overexpression of HK2 rescued cells from arsenic-induced apoptosis. Our results thus strongly implicate glycolysis, and HK2 in particular, as a key target of arsenic. Moreover, the arsenic-binding proteins identified in this work are expected to serve as a valuable resource for the development of synergistic antitumor therapeutic strategies. PMID:26598702

  13. Targeted Protein Destabilization Reveals an Estrogen-mediated ER Stress Response

    PubMed Central

    Raina, Kanak; Noblin, Devin J.; Serebrenik, Yevgeniy V.; Adams, Alison; Zhao, Connie; Crews, Craig M.

    2014-01-01

    Accumulation of unfolded proteins within the endoplasmic reticulum (ER) of eukaryotic cells leads to an unfolded protein response (UPR) that either restores homeostasis or commits the cells to apoptosis. Tools traditionally used to study the UPR are pro-apoptotic and thus confound analysis of long-term cellular responses to ER stress. Here, we describe an Endoplasmic Reticulum-localized HaloTag (ERHT) protein that can be conditionally destabilized using a small molecule hydrophobic tag (HyT36). Treatment of ERHT-expressing cells with HyT36 induces an acute, resolvable ER stress that results in transient UPR activation without induction of apoptosis. Transcriptome analysis of late-stage responses to this UPR stimulus reveals a link between UPR activity and estrogen signaling. PMID:25242550

  14. Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

    PubMed

    De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

    2016-02-01

    Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. PMID:26031839

  15. Structures of replication initiation proteins from staphylococcal antibiotic resistance plasmids reveal protein asymmetry and flexibility are necessary for replication.

    PubMed

    Carr, Stephen B; Phillips, Simon E V; Thomas, Christopher D

    2016-03-18

    Antibiotic resistance in pathogenic bacteria is a continual threat to human health, often residing in extrachromosomal plasmid DNA. Plasmids of the pT181 family are widespread and confer various antibiotic resistances to Staphylococcus aureus. They replicate via a rolling circle mechanism that requires a multi-functional, plasmid-encoded replication protein to initiate replication, recruit a helicase to the site of initiation and terminate replication after DNA synthesis is complete. We present the first atomic resolution structures of three such replication proteins that reveal distinct, functionally relevant conformations. The proteins possess a unique active site and have been shown to contain a catalytically essential metal ion that is bound in a manner distinct from that of any other rolling circle replication proteins. These structures are the first examples of the Rep_trans Pfam family providing insights into the replication of numerous antibiotic resistance plasmids from Gram-positive bacteria, Gram-negative phage and the mobilisation of DNA by conjugative transposons. PMID:26792891

  16. Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions

    PubMed Central

    Beld, Joris; Blatti, Jillian L.; Behnke, Craig; Mendez, Michael; Burkart, Michael D.

    2014-01-01

    The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes. PMID:25110394

  17. Structures of replication initiation proteins from staphylococcal antibiotic resistance plasmids reveal protein asymmetry and flexibility are necessary for replication

    PubMed Central

    Carr, Stephen B.; Phillips, Simon E.V.; Thomas, Christopher D.

    2016-01-01

    Antibiotic resistance in pathogenic bacteria is a continual threat to human health, often residing in extrachromosomal plasmid DNA. Plasmids of the pT181 family are widespread and confer various antibiotic resistances to Staphylococcus aureus. They replicate via a rolling circle mechanism that requires a multi-functional, plasmid-encoded replication protein to initiate replication, recruit a helicase to the site of initiation and terminate replication after DNA synthesis is complete. We present the first atomic resolution structures of three such replication proteins that reveal distinct, functionally relevant conformations. The proteins possess a unique active site and have been shown to contain a catalytically essential metal ion that is bound in a manner distinct from that of any other rolling circle replication proteins. These structures are the first examples of the Rep_trans Pfam family providing insights into the replication of numerous antibiotic resistance plasmids from Gram-positive bacteria, Gram-negative phage and the mobilisation of DNA by conjugative transposons. PMID:26792891

  18. Properties of X-ray resonant scattering in the Bragg case revealed on the Riemann surface.

    PubMed

    Saka, Takashi

    2016-07-01

    Continuing the work described in the previous paper [Saka (2016). Acta Cryst. A72, 338-348], the dynamical theory for perfect crystals in the Bragg case is reformulated using the Riemann surface. In particular, diffraction under resonant scattering conditions is investigated. The characteristic features of the dispersion surface and the rocking curve are analytically revealed using four parameters, which are the real and imaginary parts of two quantities specifying the degree of departure from the exact Bragg conditions and the reflection strength. Characteristic properties that have been deduced through numerical analysis are derived analytically using these four parameters. Visualization of the geometric relationships between the four parameters on the Riemann surface is useful for understanding many properties such as symmetry and sharpness of the rocking curve under special conditions. Therefore, employing the Riemann surface is instructive for numerical analysis and useful for understanding dynamical diffraction in the Bragg case. PMID:27357849

  19. Retinol-binding protein 4 and its potential roles in hypercholesterolemia revealed by proteomics

    PubMed Central

    Jugnam-ang, Watcharapong; Pannengpetch, Supitcha; Isarankura-Na-Ayudhya, Patcharee; Thippakorn, Chadinee; Isarankura-Na-Ayudhya, Chartchalerm; Lawung, Ratana; Prachayasittiku, Virapong

    2015-01-01

    Effects of hypercholesterolemia on alterations of serum proteins have not been fully elucidated. Herein, using two-dimensional gel electrophoresis (2-DE) in conjunction with LC-MS searching has successfully been carried out to investigate the change of protein expression profiles as consequences of raised blood cholesterol at different levels (normal group: total cholesterol 200 mg/dL; borderline high group: total cholesterol 200-239 mg/dL; and high group: total cholesterol ≥ 240 mg/dL) (n = 45). Results revealed that down-regulation of retinol-binding protein 4 (RBP4) (-2.26 fold), transthyretin (-1.25 fold) and gelsolin (-1.47 fold) was observed in the high group. Meanwhile, the other proteins such as haptoglobin, complement factor B and CD5 antigen-like protein were up-regulated upto +3.24, +1.96 and +2.04 fold, respectively. Confirmation by Western blotting revealed a significant reduction of RBP4 (approximately 50 %) in individual samples derived from the high group. Presumptive conclusion can be drawn that down-regulation of RBP4 might be attributable to the inflammation of adipocytes caused by the release of proinflammatory cytokines (e.g. tumor necrosis factor α and interleukin-1β) from adipose tissues. Moreover, the decrease of transthyretin might also be taken into accounts since it is known that the transthyretin usually forms complex with RBP4 to prevent glomerular filtration and excretion through the kidney. The suppressing effect on RBP4 should be potentiated by the increase of complement factor B and CD5 antigen-like protein, which rendered the adipose tissues to overwhelm the liberation of RBP4 to blood circulation by metabolic and inflammatory processes. Such inflammation could further modulate the induction of cytokine release (e.g. IL-6 and IL-1β), resulting in the synthesis of acute phase protein, in particular, haptoglobin and C-reactive proteins from hepatocytes. However, the mechanism of gelsolin reduction remains unclear. Among these

  20. Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains.

    PubMed

    Rychli, Kathrin; Grunert, Tom; Ciolacu, Luminita; Zaiser, Andreas; Razzazi-Fazeli, Ebrahim; Schmitz-Esser, Stephan; Ehling-Schulz, Monika; Wagner, Martin

    2016-02-01

    surface virulence associated protein SvpA. Furthermore proteins involved in cell wall modification, such as the lipoteichonic acid primase LtaP and the N-acetylmuramoyl-l-alanine amidase (Lmo2591) are more abundant in EGDe than in the persistent strains and could indirectly contribute to virulence. In conclusion this study provides information about a set of proteins that could potentially support survival of L. monocytogenes in abiotic niches in food processing environments. Based on these data, a more detailed analysis of the role of the identified proteins under stresses mimicking conditions in food producing environment is essential for further elucidate the mechanism of the phenomenon of persistence of L. monocytogenes. PMID:26594790

  1. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    PubMed Central

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; Johns, Douglas G.; Charles, M. Arthur; Ren, Gang

    2015-01-01

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL. PMID:25737239

  2. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    DOE PAGESBeta

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; et al

    2015-03-04

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobicmore » environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.« less

  3. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    SciTech Connect

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; Johns, Douglas G.; Charles, M. Arthur; Ren, Gang

    2015-03-04

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.

  4. HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Charles, River; Tong, Huimin; Zhang, Lei; Patel, Mili; Wang, Francis; Rames, Matthew J.; Ren, Amy; Rye, Kerry-Anne; Qiu, Xiayang; Johns, Douglas G.; Charles, M. Arthur; Ren, Gang

    2015-03-01

    Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.

  5. Identification of vitronectin as a major plasma protein adsorbed on polymer surfaces of different copolymer composition.

    PubMed

    Bale, M D; Wohlfahrt, L A; Mosher, D F; Tomasini, B; Sutton, R C

    1989-12-01

    The arrays of proteins adsorbed from plasma onto a series of polystyrene copolymeric latexes were analyzed by enzyme-linked immunosorbent assay (ELISA) of washed beads and immunoblotting of proteins desorbed from the beads and separated by polyacrylamide gel electrophoresis (PAGE). Beads were prepared by continuous emulsion polymerization in the absence of surfactant. Coomassie brilliant blue staining of gel electropherograms of desorbed proteins indicated that the presence of small amounts of comonomers (1 to 10 mole %) significantly influenced the composition of the adsorbed protein layer. Immunoblotting revealed that fibrinogen, fibronectin, and vitronectin were adsorbed by all surfaces investigated. C3 and Clq adsorption varied significantly with copolymer composition. The ELISAs revealed that although the concentrations of vitronectin and fibronectin in plasma are similar, the extent of vitronectin adsorption from 70% to 85% plasma was greater by two orders of magnitude than fibronectin adsorption. Vitronectin adsorbed on carboxylic acid-containing copolymers reacted more strongly with a conformationally sensitive antivitronectin monoclonal antibody (MoAb) than vitronectin adsorbed to polystyrene and was more susceptible to cleavage by plasma proteases(s). The results show that vitronectin is a major protein adsorbed from concentrated plasma and that small changes in the chemical composition of a copolymer profoundly affects the extent and nature of protein adsorption from complex mixtures such as plasma. PMID:2479428

  6. A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR.

    PubMed

    Sgourakis, Nikolaos G; May, Nathan A; Boyd, Lisa F; Ying, Jinfa; Bax, Ad; Margulies, David H

    2015-11-27

    As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent Ig-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full-length and truncated forms of the MHC-I molecule H2-L(d) by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-L(d), "mini-H2-L(d)," consisting of only the α1α2 platform domain. Mini-H2-L(d) refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the β2-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and β2-microglobulin-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the picosecond-nanosecond dynamics of the free mini-H2-L(d) MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway. PMID:26463211

  7. Kinetic evidence for protein clustering at a surface

    SciTech Connect

    Ramsden, J.J. ); Bachmanova, G.I.; Archakov, A.I. )

    1994-12-01

    The molecules designated 1A2 and 2B4 belong to the cytochrome P450 protein superfamily. They can interact specifically with lipid bilayers via the penetration of part of their amino acid chains into the bilayer. The kinetics of their irreversible adsorption from solution to phospholipid bilayers, accurately measured in the low to intermediate coverage range using an integrated optics reflectance technique, differ significantly: at intermediate bulk solution concentrations, 2B4 shows typical random sequential adsorption (RSA) kinetics, whereas 1A2 shows Langmuir kinetics. At higher bulk concentration the behavior of 1[ital A]2 switches to RSA kinetics, and at very low concentrations 2[ital B]4 switches to Langmuir kinetics. The Langmuir kinetics provides strong evidence for clustering of the molecules at the bilayer surface, and the observed concentration dependence of the kinetics is consistent with the clusters arising through lateral diffusion of the proteins on the surface.

  8. SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell.

    PubMed

    Kim, Henry S; Martel, Anne; Girard, Eric; Moulin, Martine; Härtlein, Michael; Madern, Dominique; Blackledge, Martin; Franzetti, Bruno; Gabel, Frank

    2016-05-24

    Water molecules in the immediate vicinity of biomacromolecules, including proteins, constitute a hydration layer characterized by physicochemical properties different from those of bulk water and play a vital role in the activity and stability of these structures, as well as in intermolecular interactions. Previous studies using solution scattering, crystallography, and molecular dynamics simulations have provided valuable information about the properties of these hydration shells, including modifications in density and ionic concentration. Small-angle scattering of x-rays (SAXS) and neutrons (SANS) are particularly useful and complementary techniques to study biomacromolecular hydration shells due to their sensitivity to electronic and nuclear scattering-length density fluctuations, respectively. Although several sophisticated SAXS/SANS programs have been developed recently, the impact of physicochemical surface properties on the hydration layer remains controversial, and systematic experimental data from individual biomacromolecular systems are scarce. Here, we address the impact of physicochemical surface properties on the hydration shell by a systematic SAXS/SANS study using three mutants of a single protein, green fluorescent protein (GFP), with highly variable net charge (+36, -6, and -29). The combined analysis of our data shows that the hydration shell is locally denser in the vicinity of acidic surface residues, whereas basic and hydrophilic/hydrophobic residues only mildly modify its density. Moreover, the data demonstrate that the density modifications result from the combined effect of residue-specific recruitment of ions from the bulk in combination with water structural rearrangements in their vicinity. Finally, we find that the specific surface-charge distributions of the different GFP mutants modulate the conformational space of flexible parts of the protein. PMID:27224484

  9. Cell-surface MHC density profiling reveals instability of autoimmunity-associated HLA

    PubMed Central

    Miyadera, Hiroko; Ohashi, Jun; Lernmark, Åke; Kitamura, Toshio; Tokunaga, Katsushi

    2014-01-01

    Polymorphisms within HLA gene loci are strongly associated with susceptibility to autoimmune disorders; however, it is not clear how genetic variations in these loci confer a disease risk. Here, we devised a cell-surface MHC expression assay to detect allelic differences in the intrinsic stability of HLA-DQ proteins. We found extreme variation in cell-surface MHC density among HLA-DQ alleles, indicating a dynamic allelic hierarchy in the intrinsic stability of HLA-DQ proteins. Using the case-control data for type 1 diabetes (T1D) for the Swedish and Japanese populations, we determined that T1D risk–associated HLA-DQ haplotypes, which also increase risk for autoimmune endocrinopathies and other autoimmune disorders, encode unstable proteins, whereas the T1D–protective haplotypes encode the most stable HLA-DQ proteins. Among the amino acid variants of HLA-DQ, alterations in 47α, the residue that is located on the outside of the peptide-binding groove and acts as a key stability regulator, showed strong association with T1D. Evolutionary analysis suggested that 47α variants have been the target of positive diversifying selection. Our study demonstrates a steep allelic hierarchy in the intrinsic stability of HLA-DQ that is associated with T1D risk and protection, suggesting that HLA instability mediates the development of autoimmune disorders. PMID:25485681

  10. Tissue-Based Proteogenomics Reveals that Human Testis Endows Plentiful Missing Proteins.

    PubMed

    Zhang, Yao; Li, Qidan; Wu, Feilin; Zhou, Ruo; Qi, Yingzi; Su, Na; Chen, Lingsheng; Xu, Shaohang; Jiang, Tao; Zhang, Chengpu; Cheng, Gang; Chen, Xinguo; Kong, Degang; Wang, Yujia; Zhang, Tao; Zi, Jin; Wei, Wei; Gao, Yuan; Zhen, Bei; Xiong, Zhi; Wu, Songfeng; Yang, Pengyuan; Wang, Quanhui; Wen, Bo; He, Fuchu; Xu, Ping; Liu, Siqi

    2015-09-01

    Investigations of missing proteins (MPs) are being endorsed by many bioanalytical strategies. We proposed that proteogenomics of testis tissue was a feasible approach to identify more MPs because testis tissues have higher gene expression levels. Here we combined proteomics and transcriptomics to survey gene expression in human testis tissues from three post-mortem individuals. Proteins were extracted and separated with glycine- and tricine-SDS-PAGE. A total of 9597 protein groups were identified; of these, 166 protein groups were listed as MPs, including 138 groups (83.1%) with transcriptional evidence. A total of 2948 proteins are designated as MPs, and 5.6% of these were identified in this study. The high incidence of MPs in testis tissue indicates that this is a rich resource for MPs. Functional category analysis revealed that the biological processes that testis MPs are mainly involved in are sexual reproduction and spermatogenesis. Some of the MPs are potentially involved in tumorgenesis in other tissues. Therefore, this proteogenomics analysis of individual testis tissues provides convincing evidence of the discovery of MPs. All mass spectrometry data from this study have been deposited in the ProteomeXchange (data set identifier PXD002179). PMID:26282447

  11. 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

  12. Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction

    PubMed Central

    Teramoto, Takamasa; Fujikawa, Yukari; Kawaguchi, Yoshirou; Kurogi, Katsuhisa; Soejima, Masayuki; Adachi, Rumi; Nakanishi, Yuichi; Mishiro-Sato, Emi; Liu, Ming-Cheh; Sakakibara, Yoichi; Suiko, Masahito; Kimura, Makoto; Kakuta, Yoshimitsu

    2013-01-01

    Post-translational protein modification by tyrosine-sulfation plays an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalyzed by the Golgi-enzyme called the tyrosylprotein sulfotransferase (TPST). To date, no crystal structure is available for TPST. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human TPST isoform 2 (TPST2) complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3’-phosphoadenosine-5’-phosphate (PAP) at 1.9Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. TPST2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel β-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the TPST2 structure resembles that observed for the receptor type tyrosine kinases. PMID:23481380

  13. Effects of pressure on the dynamics of a hyperthermophilic protein revealed by quasielastic neutron scattering

    NASA Astrophysics Data System (ADS)

    Shrestha, U. R.; Bhowmik, D.; Copley, J. R. D.; Tyagi, M.; Leao, J. B.; Chu, X.-Q.

    Inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens is a large oligomeric protein derived from hyperthermophilic microorganism that is found near hydrothermal vents deep under the sea, where the pressure is nearly 100 MPa. Here we study the effects of pressure on the conformational flexibility and relaxation dynamics of IPPase over a wide temperature range using quasielastic neutron scattering (QENS) technique. Two spectrometers were used to investigate the β-relaxation dynamics of proteins in time ranges from 2 to 25 ps, and from 100 ps to 2 ns. Our results reveal that, under the pressure of 100 MPa, IPPase displays much faster relaxation dynamics than a mesophilic model protein, hen egg white lysozyme (HEWL), opposite to what we observed previously under the ambient pressure. These contradictory observations imply that high pressure affects the dynamical properties of proteins by distorting their energy landscapes. Accordingly, we derived a general schematic denaturation phase diagram that can be used as a general picture to understand the effects of pressure on protein dynamics and activities Wayne State Univ Startup Fund.

  14. The Plasmodium berghei translocon of exported proteins reveals spatiotemporal dynamics of tubular extensions.

    PubMed

    Matz, Joachim M; Goosmann, Christian; Brinkmann, Volker; Grützke, Josephine; Ingmundson, Alyssa; Matuschewski, Kai; Kooij, Taco W A

    2015-01-01

    The erythrocyte is an extraordinary host cell for intracellular pathogens and requires extensive remodelling to become permissive for infection. Malaria parasites modify their host red blood cells through protein export to acquire nutrients and evade immune responses. Endogenous fluorescent tagging of three signature proteins of the Plasmodium berghei translocon of exported proteins (PTEX), heat shock protein 101, exported protein 2 (EXP2), and PTEX88, revealed motile, tubular extensions of the parasitophorous vacuole that protrude from the parasite far into the red blood cell. EXP2 displays a more prominent presence at the periphery of the parasite, consistent with its proposed role in pore formation. The tubular compartment is most prominent during trophozoite growth. Distinct spatiotemporal expression of individual PTEX components during sporogony and liver-stage development indicates additional functions and tight regulation of the PTEX translocon during parasite life cycle progression. Together, live cell imaging and correlative light and electron microscopy permitted previously unrecognized spatiotemporal and subcellular resolution of PTEX-containing tubules in murine malaria parasites. These findings further refine current models for Plasmodium-induced erythrocyte makeover. PMID:26219962

  15. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

    PubMed

    Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

    2016-06-01

    Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria. PMID:27183143

  16. The Plasmodium berghei translocon of exported proteins reveals spatiotemporal dynamics of tubular extensions

    PubMed Central

    Matz, Joachim M.; Goosmann, Christian; Brinkmann, Volker; Grützke, Josephine; Ingmundson, Alyssa; Matuschewski, Kai; Kooij, Taco W. A.

    2015-01-01

    The erythrocyte is an extraordinary host cell for intracellular pathogens and requires extensive remodelling to become permissive for infection. Malaria parasites modify their host red blood cells through protein export to acquire nutrients and evade immune responses. Endogenous fluorescent tagging of three signature proteins of the Plasmodium berghei translocon of exported proteins (PTEX), heat shock protein 101, exported protein 2 (EXP2), and PTEX88, revealed motile, tubular extensions of the parasitophorous vacuole that protrude from the parasite far into the red blood cell. EXP2 displays a more prominent presence at the periphery of the parasite, consistent with its proposed role in pore formation. The tubular compartment is most prominent during trophozoite growth. Distinct spatiotemporal expression of individual PTEX components during sporogony and liver-stage development indicates additional functions and tight regulation of the PTEX translocon during parasite life cycle progression. Together, live cell imaging and correlative light and electron microscopy permitted previously unrecognized spatiotemporal and subcellular resolution of PTEX-containing tubules in murine malaria parasites. These findings further refine current models for Plasmodium-induced erythrocyte makeover. PMID:26219962

  17. Modelling NifH2 protein of Clostridium pasteurianum reveals clues about its physiological function.

    PubMed

    Kasap, Murat

    2006-11-01

    Clostridium pasteurianum is an anaerobic free-living nitrogen fixer. As a unique feature, the organism contains five extra nifH-like genes in addition to nifH1. Detailed analysis with respect to the structure and function of the nifH-like gene products is missing due to the lack of information about the presence of their translation products in the cell. Recent work indicates that the nifH2 gene is transcribed and translated into a polypeptide of expected size in nitrogen-fixing cells of C. pasteurianum and is regulated both at the transcriptional and translational levels. However, the current data do not reveal the physiological function of the NifH2 protein. In this study, we have used computer tools and the NifH1 of C. pasteurianum as the template to predict a possible tertiary structure and assign a putative function for NifH2 protein. A comparison of the structures of the NifH1 and modelled NifH2 revealed similarities in the polypeptide conformations for both monomers. Analysis of the properties of nucleotide binding, dimer interacting and cluster containing regions did not reveal major differences between NifH1 and modelled NifH2, although minor differences were observed. Rigid docking results revealed the possibility of formation of a NifH1-NifH2 heterodimer as well as formation of a NifH2 homodimer. We, therefore, propose that NifH2 can form a dimer with NifH1, albeit less efficiently and may function as a regulatory Fe-protein. PMID:16495101

  18. Acid Cleavable Surface enhanced Raman Tagging for Protein Detection

    PubMed Central

    Zhang, Dongmao; Vangala, Karthikeshwar; Li, Shaoyong; Yanney, Michael; Xia, Hao; Zou, Sige; Sygula, Andrzej

    2010-01-01

    Dye conjugation is a common strategy improving the surface enhanced Raman detection sensitivity of biomolecules. Reported is a proof-of-concept study of a novel surface enhanced Raman spectroscopic tagging strategy termed as acid-cleavable SERS tag (ACST) method. Using Rhodamine B as the starting material, we prepared the first ACST prototype that consisted of, from the distal end, a SERS tag moiety (STM), an acid-cleavable linker, and a protein reactive moiety. Complete acid cleavage of the ACST tags was achieved at a very mild condition that is 1.5% trifluoroacetic acid (TFA) aqueous solution at room temperature. SERS detection of this ACST tagged protein was demonstrated using bovine serum albumin (BSA) as the model protein. While the SERS spectrum of intact ACST-BSA was entirely dominated by the fluorescent signal of STM, quality SERS spectra can be readily obtained with the acid cleaved ACST-BSA conjugates. Separation of the acid cleaved STM from protein further enhances the SERS sensitivity. Current SERS detection sensitivity, achieved with the acid cleaved ACST-BSA conjugate is ~5 nM in terms of the BSA concentration and ~1.5 nM in ACST content. The linear dynamic range of the cleaved ACST-BSA conjugate spans four orders of magnitudes from ~10 nM to ~100 μM in protein concentrations. Further improvement in the SERS sensitivity can be achieved with resonance Raman acquisition. This cleavable tagging strategy may also be used for elimination of protein interference in fluorescence based biomolecule detection. PMID:21109888

  19. Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus

    PubMed Central

    Huang, Mingwei; Hebert, Alexander S.; Coon, Joshua J.; Hull, Christina M.

    2015-01-01

    Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote. PMID:26313153

  20. VASP: A Volumetric Analysis of Surface Properties Yields Insights into Protein-Ligand Binding Specificity

    PubMed Central

    Chen, Brian Y.; Honig, Barry

    2010-01-01

    Many algorithms that compare protein structures can reveal similarities that suggest related biological functions, even at great evolutionary distances. Proteins with related function often exhibit differences in binding specificity, but few algorithms identify structural variations that effect specificity. To address this problem, we describe the Volumetric Analysis of Surface Properties (VASP), a novel volumetric analysis tool for the comparison of binding sites in aligned protein structures. VASP uses solid volumes to represent protein shape and the shape of surface cavities, clefts and tunnels that are defined with other methods. Our approach, inspired by techniques from constructive solid geometry, enables the isolation of volumetrically conserved and variable regions within three dimensionally superposed volumes. We applied VASP to compute a comparative volumetric analysis of the ligand binding sites formed by members of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains and the serine proteases. Within both families, VASP isolated individual amino acids that create structural differences between ligand binding cavities that are known to influence differences in binding specificity. Also, VASP isolated cavity subregions that differ between ligand binding cavities which are essential for differences in binding specificity. As such, VASP should prove a valuable tool in the study of protein-ligand binding specificity. PMID:20814581

  1. Cryovolcanic Features on Titan's Surface as Revealed by the Cassini RADAR

    NASA Technical Reports Server (NTRS)

    Lopes, R. M.; Elachi, C.; Stofan, E.; Paganelli, F.; Wood, C.; Kirk, R.; Lorenz, R.; Fortes, A. D.; Lunine, J.

    2005-01-01

    The Cassini Titan Radar Mapper obtained Synthetic Aperture radar images of about 1.1% of Titan's surface during the spacecraft s first targeted fly-by on October 26, 2004 (referred to as the Ta fly-by). These images revealed that Titan is very complex geologically. Features identified include a possible volcanic dome or shield, craters that appear to be of volcanic origin, and extensive flows. We will discuss these features and others that will likely be revealed during Cassini s T3 Titan fly-by of February 15, 2005, during which a swath covering comparable amount of the surface will be obtained. Additional information is included in the original extended abstract.

  2. Revealing the binding mode between respiratory syncytial virus fusion protein and benzimidazole-based inhibitors.

    PubMed

    Ji, Dingjue; Ye, Wei; Chen, HaiFeng

    2015-07-01

    Human respiratory syncytial virus (HRSV) is a major respiratory pathogen in newborn infants and young children and can also be a threat to some elderly and high-risk adults with chronic pulmonary disease and the severely immunocompromised. The RSV fusion (RSVF) protein has been an attractive target for vaccine and drug development. Experimental results indicate a series of benzimidazole-based inhibitors which target RSVF protein to inhibit the viral entry of RSV. To reveal the binding mode between these inhibitors and RSVF protein, molecular docking and molecular dynamics simulations were used to investigate the interactions between the inhibitors and the core domain of RSVF protein. MD results suggest that the active molecules have stronger π-π stacking, cation-π, and other interactions than less active inhibitors. The binding free energy between the active inhibitor and RSVF protein is also found to be significantly lower than that of the less active one using MM/GBSA. Then, Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) methods were used to construct three dimensional quantitative structure-activity (3D-QSAR) models. The cross-validated q(2) values are found to be 0.821 and 0.795 for CoMFA and CoMSIA, respectively. And the non-cross-validated r(2) values are 0.973 and 0.961. Ninety-two test set compounds validated these models. The results suggest that these models are robust with good prediction abilities. Furthermore, these models reveal possible methods to improve the bioactivity of inhibitors. PMID:25872614

  3. Alluvial Fans on Titan Reveal Atmosphere and Surface Interactions and Material Transport

    NASA Astrophysics Data System (ADS)

    Radebaugh, J.; Ventra, D.; Lorenz, R. D.; Farr, T. G.; Kirk, R. L.; Hayes, A.; Malaska, M. J.; Birch, S.; Liu, Z. Y. C.; Lunine, J. I.; Barnes, J. W.; Le Gall, A. A.; Lopes, R. M. C.; Stofan, E. R.; Wall, S. D.; Paillou, P.

    2015-12-01

    Alluvial fans, important depositional systems that record how sediment is stored and moved on planetary surfaces, are found on the surface of Titan, a body of significantly different materials and process rates than Earth. As seen by Cassini's Synthetic Aperture Radar (SAR) images at 350 m resolution, fans on Titan are found globally and are variable in size, shape and relationship to adjacent landforms. Their morphologies and SAR characteristics, which reveal roughness, textural patterns and other material properties, show similarities with fans in Death Valley seen by SAR and indicate there are regions of high relative relief locally, in the Ganesa, Xanadu and equatorial mountain belt regions. The Leilah Fluctus fans near Ganesa are ~30 km x 15 km, similar to the largest Death Valley fans, and revealing mountainous topography adjacent to plains. Others have gentle slopes over hundreds of kilometers, as in the high southern latitude lakes regions or the Mezzoramia southern midlatitudes, where a fan system is 200 km x 150 km, similar to the Qarn Alam fan emerging into the Rub al Khali in Oman. Additionally, there is evidence for a range of particle sizes, from relatively coarse (~2 cm or more) to fine, revealing long-term duration and variability in erosion by methane rainfall and transport. Some features have morphologies consistent with proximality to high-relief source areas and highly ephemeral runoff, while others appear to draw larger catchment areas and are perhaps characterized by more prolonged episodes of flow. The presence of many fans indicates the longevity of rainfall and erosion in Titan's surface processes and reveals that sediment transport and the precipitation that drives it are strongly episodic. Alluvial fans join rivers, lakes, eroded mountains, sand dunes and dissolution features in the list of surface morphologies derived from atmospheric and fluvial processes similar to those on Earth, strengthening comparisons between the two planetary

  4. A protective surface protein from type V group B streptococci shares N-terminal sequence homology with the alpha C protein.

    PubMed

    Lachenauer, C S; Madoff, L C

    1996-10-01

    Infection by group B streptococci (GBS) is an important cause of bacterial disease in neonates, pregnant women, and nonpregnant adults. Historically, serotypes Ia, Ib, II, and III have been most prevalent among disease cases; recently, type V strains have emerged as important strains in the United States and elsewhere. In addition to type-specific capsular polysaccharides, many GBS strains possess surface proteins which demonstrate a laddering pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and resistance to trypsin digestion. These include the alpha C protein, the R proteins, and protein Rib. Some of these proteins elicit protective antibodies in animals. We demonstrate a trypsin-resistant laddering protein purified from a type V GBS strain by mutanolysin extraction and column chromatography. This protein contains a major 90-kDa band and a series of smaller bands spaced approximately 10 kDa apart on SDS-PAGE. Cross-reactivity of the type V protein with the alpha C protein and with R1 was demonstrated on Western blot (immunoblot). N-terminal sequence analysis of the protein revealed residue identity with 17 of 18 residues at corresponding positions on the alpha protein. Western blot of SDS extracts of 41 clinical type V isolates with rabbit antiserum to the protein demonstrated a homologous protein in 25 isolates (61%); two additional strains exhibited a heterologous pattern which was also demonstrated with 4G8, a monoclonal antibody directed to the alpha C protein repeat region. Rabbit antiserum raised to the type V protein conferred protection in neonatal mice against a type V strain bearing a homologous protein. These data support the hypothesis that there exists a family of trypsin-resistant, laddering GBS surface proteins which may play a role in immunity to GBS infection. PMID:8926097

  5. A protective surface protein from type V group B streptococci shares N-terminal sequence homology with the alpha C protein.

    PubMed Central

    Lachenauer, C S; Madoff, L C

    1996-01-01

    Infection by group B streptococci (GBS) is an important cause of bacterial disease in neonates, pregnant women, and nonpregnant adults. Historically, serotypes Ia, Ib, II, and III have been most prevalent among disease cases; recently, type V strains have emerged as important strains in the United States and elsewhere. In addition to type-specific capsular polysaccharides, many GBS strains possess surface proteins which demonstrate a laddering pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and resistance to trypsin digestion. These include the alpha C protein, the R proteins, and protein Rib. Some of these proteins elicit protective antibodies in animals. We demonstrate a trypsin-resistant laddering protein purified from a type V GBS strain by mutanolysin extraction and column chromatography. This protein contains a major 90-kDa band and a series of smaller bands spaced approximately 10 kDa apart on SDS-PAGE. Cross-reactivity of the type V protein with the alpha C protein and with R1 was demonstrated on Western blot (immunoblot). N-terminal sequence analysis of the protein revealed residue identity with 17 of 18 residues at corresponding positions on the alpha protein. Western blot of SDS extracts of 41 clinical type V isolates with rabbit antiserum to the protein demonstrated a homologous protein in 25 isolates (61%); two additional strains exhibited a heterologous pattern which was also demonstrated with 4G8, a monoclonal antibody directed to the alpha C protein repeat region. Rabbit antiserum raised to the type V protein conferred protection in neonatal mice against a type V strain bearing a homologous protein. These data support the hypothesis that there exists a family of trypsin-resistant, laddering GBS surface proteins which may play a role in immunity to GBS infection. PMID:8926097

  6. Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane

    PubMed Central

    Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

    2016-01-01

    Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers. DOI: http://dx.doi.org/10.7554/eLife.12125.001 PMID:26824389

  7. Molecular Dynamics Simulation Reveals Correlated Inter-Lobe Motion in Protein Lysine Methyltransferase SMYD2

    PubMed Central

    Spellmon, Nicholas; Sun, Xiaonan; Sirinupong, Nualpun; Edwards, Brian; Li, Chunying; Yang, Zhe

    2015-01-01

    SMYD proteins are an exciting field of study as they are linked to many types of cancer-related pathways. Cardiac and skeletal muscle development and function also depend on SMYD proteins opening a possible avenue for cardiac-related treatment. Previous crystal structure studies have revealed that this special class of protein lysine methyltransferases have a bilobal structure, and an open–closed motion may regulate substrate specificity. Here we use the molecular dynamics simulation to investigate the still-poorly-understood SMYD2 dynamics. Cross-correlation analysis reveals that SMYD2 exhibits a negative correlated inter-lobe motion. Principle component analysis suggests that this correlated dynamic is contributed to by a twisting motion of the C-lobe with respect to the N-lobe and a clamshell-like motion between the lobes. Dynamical network analysis defines possible allosteric paths for the correlated dynamics. There are nine communities in the dynamical network with six in the N-lobe and three in the C-lobe, and the communication between the lobes is mediated by a lobe-bridging β hairpin. This study provides insight into the dynamical nature of SMYD2 and could facilitate better understanding of SMYD2 substrate specificity. PMID:26717235

  8. The Volumetric Diversity of Misfolded Prion Protein Oligomers Revealed by Pressure Dissociation*

    PubMed Central

    Torrent, Joan; Lange, Reinhard; Rezaei, Human

    2015-01-01

    Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct β-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation of one specific PrP oligomer, we reveal a large negative activation volume and an increase in both apparent activation enthalpy and entropy. This suggests a transition state ensemble that is less structured and significantly more hydrated than the oligomeric state. Finally, we found that site-specific fluorescent labeling allows monitoring of the transient population of a kinetic intermediate in the dissociation reaction. Our results indicate that defects in atomic packing may deserve consideration as a new factor that influences differences between PrP assemblies and that could be relevant also for explaining the origin of prion strains. PMID:26126829

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

    PubMed

    Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

    2016-10-01

    Various molecular interaction forces are generated during protein adsorption process on material surfaces. Thus, it is necessary to control them to suppress protein adsorption and the subsequent cell and tissue responses. A series of binary copolymer brush layers were prepared via surface-initiated atom transfer radical polymerization, by mixing the cationic monomer unit and anionic monomer unit randomly in various ratios. Surface characterization revealed that the constructed copolymer brush layers exhibited an uniform super-hydrophilic nature and different surface potentials. The strength of the electrostatic interaction forces operating on these mixed-charge copolymer brush surfaces was evaluated quantitatively using force-versus-distance (f-d) curve measurements by atomic force microscopy (AFM) and probes modified by negatively charged carboxyl groups or positively charged amino groups. The electrostatic interaction forces were determined based on the charge ratios of the copolymer brush layers. Notably, the surface containing equivalent cationic/anionic monomer units hardly interacted with both the charged groups. Furthermore, the protein adsorption force and the protein adsorption mass on these surfaces were examined by AFM f-d curve measurement and surface plasmon resonance measurement, respectively. To clarify the influence of the electrostatic interaction on the protein adsorption behavior on the surface, three kinds of proteins having negative, positive, and relatively neutral net charges under physiological conditions were used in this study. We quantitatively demonstrated that the amount of adsorbed proteins on the surfaces would have a strong correlation with the strength of surface-protein interaction forces, and that the strength of surface-protein interaction forces would be determined from the combination between the properties of the electrostatic interaction forces on the surfaces and the charge properties of the proteins. Especially, the

  10. An update on cell surface proteins containing extensin-motifs.

    PubMed

    Borassi, Cecilia; Sede, Ana R; Mecchia, Martin A; Salgado Salter, Juan D; Marzol, Eliana; Muschietti, Jorge P; Estevez, Jose M

    2016-01-01

    In recent years it has become clear that there are several molecular links that interconnect the plant cell surface continuum, which is highly important in many biological processes such as plant growth, development, and interaction with the environment. The plant cell surface continuum can be defined as the space that contains and interlinks the cell wall, plasma membrane and cytoskeleton compartments. In this review, we provide an updated view of cell surface proteins that include modular domains with an extensin (EXT)-motif followed by a cytoplasmic kinase-like domain, known as PERKs (for proline-rich extensin-like receptor kinases); with an EXT-motif and an actin binding domain, known as formins; and with extracellular hybrid-EXTs. We focus our attention on the EXT-motifs with the short sequence Ser-Pro(3-5), which is found in several different protein contexts within the same extracellular space, highlighting a putative conserved structural and functional role. A closer understanding of the dynamic regulation of plant cell surface continuum and its relationship with the downstream signalling cascade is a crucial forthcoming challenge. PMID:26475923

  11. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway.

    PubMed

    Shi, Tujin; Niepel, Mario; McDermott, Jason E; Gao, Yuqian; Nicora, Carrie D; Chrisler, William B; Markillie, Lye M; Petyuk, Vladislav A; Smith, Richard D; Rodland, Karin D; Sorger, Peter K; Qian, Wei-Jun; Wiley, H Steven

    2016-01-01

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling. PMID:27405981

  12. The Structure of the BfrB-Bfd Complex Reveals Protein-Protein Interactions Enabling Iron Release from Bacterioferritin

    SciTech Connect

    Yao, Huili; Wang, Yan; Lovell, Scott; Kumar, Ritesh; Ruvinsky, Anatoly M; Battaile, Kevin P; Vakser, Ilya A; Rivera, Mario

    2012-09-11

    Ferritin-like molecules are unique to cellular iron homeostasis because they can store iron at concentrations much higher than those dictated by the solubility of Fe3+. Very little is known about the protein interactions that deliver iron for storage or promote the mobilization of stored iron from ferritin-like molecules. Here, we report the X-ray crystal structure of Pseudomonas aeruginosa bacterioferritin (Pa-BfrB) in complex with bacterioferritin-associated ferredoxin (Pa-Bfd) at 2.0 Å resolution. As the first example of a ferritin-like molecule in complex with a cognate partner, the structure provides unprecedented insight into the complementary interface that enables the [2Fe-2S] cluster of Pa-Bfd to promote heme-mediated electron transfer through the BfrB protein dielectric (~18 Å), a process that is necessary to reduce the core ferric mineral and facilitate mobilization of Fe2+. The Pa-BfrB-Bfd complex also revealed the first structure of a Bfd, thus providing a first view to what appears to be a versatile metal binding domain ubiquitous to the large Fer2_BFD family of proteins and enzymes with diverse functions. Residues at the Pa-BfrB-Bfd interface are highly conserved in Bfr and Bfd sequences from a number of pathogenic bacteria, suggesting that the specific recognition between Pa-BfrB and Pa-Bfd is of widespread significance to the understanding of bacterial iron homeostasis.

  13. The structure of the BfrB-Bfd complex reveals protein-protein interactions enabling iron release from bacterioferritin

    PubMed Central

    Yao, Huili; Wang, Yan; Lovell, Scott; Kumar, Ritesh; Ruvinsky, Anatoly M.; Battaile, Kevin P.; Vakser, Ilya A.; Rivera, Mario

    2012-01-01

    Ferritin-like molecules are unique to cellular iron homeostasis because they can store iron at concentrations much higher than those dictated by the solubility of Fe3+. Very little is known about the protein interactions that deliver iron for storage, or promote the mobilization of stored iron from ferritin-like molecules. Here, we report the X-ray crystal structure of Pseudomonas aeruginosa bacterioferritin (Pa-BfrB) in complex with bacterioferritin-associated ferredoxin (Pa-Bfd) at 2.0 Å resolution. As the first example of a ferritin-like molecule in complex with a cognate partner, the structure provides unprecedented insight into the complementary interface that enables the [2Fe-2S] cluster of Pa-Bfd to promote heme-mediated electron transfer through the BfrB protein dielectric (~18 Å), a process that is necessary to reduce the core ferric mineral and facilitate mobilization of Fe2+. The Pa-BfrB-Bfd complex also revealed the first structure of a Bfd, thus providing a first view to what appears to be a versatile metal binding domain ubiquitous to the large Fer2_BFD family of proteins and enzymes with diverse functions. Residues at the Pa-BfrB-Bfd interface are highly conserved in Bfr and Bfd sequences from a number of pathogenic bacteria, suggesting that the specific recognition between Pa-BfrB and Pa-Bfd is of widespread significance to the understanding of bacterial iron homeostasis. PMID:22812654

  14. Controlling Protein Oligomerization with Surface Curvature on the Nanoscale

    NASA Astrophysics Data System (ADS)

    Kurylowicz, Marty; Dutcher, John

    2011-03-01

    We investigate the effect of surface curvature on the conformation of beta-lactoglobulin (β LG) using Single Molecule Force Spectroscopy. β LG is a model interfacial protein which stabilizes oil droplets in milk and is known to undergo structural rearrangement when adsorbed onto a surface. We reliably control nanoscale surface curvature by creating close-packed monolayers of monodisperse polystyrene (PS) nanoparticles with diameters of 20, 40, 60, 80 and 140 nm, which are stable in aqueous buffer. By adsorbing β LG onto these hydrophobic surfaces and collecting force-extension curves in the fluid phase we can compare the conformation of β LG on 5 different surface curvatures with that on a flat PS film. We demonstrate a transition from oligomeric to monomeric β LG as the surface curvature is increased. Histograms of contour length from fits to peaks in the force-extension curves show a single maximum near 30 nm for β LG adsorbed onto nanoparticles with diameters less than 80 nm. For the larger nanoparticles, the histogram approaches that observed for β LG adsorbed onto a flat PS film, with maxima indicative of β LG dimers and trimers.

  15. Domains of surfactant protein A that affect protein oligomerization, lipid structure and surface tension.

    PubMed

    Palaniyar, N; Ikegami, M; Korfhagen, T; Whitsett, J; McCormack, F X

    2001-05-01

    Surfactant protein A (SP-A) is an abundant protein found in pulmonary surfactant which has been reported to have multiple functions. In this review, we focus on the structural importance of each domain of SP-A in the functions of protein oligomerization, the structural organization of lipids and the surface-active properties of surfactant, with an emphasis on ultrastructural analyses. The N-terminal domain of SP-A is required for disulfide-dependent protein oligomerization, and for binding and aggregation of phospholipids, but there is no evidence that this domain directly interacts with lipid membranes. The collagen-like domain is important for the stability and oligomerization of SP-A. It also contributes shape and dimension to the molecule, and appears to determine membrane spacing in lipid aggregates such as common myelin and tubular myelin. The neck domain of SP-A is primarily involved in protein trimerization, which is critical for many protein functions, but it does not appear to be directly involved in lipid interactions. The globular C-terminal domain of SP-A clearly plays a central role in lipid binding, and in more complex functions such as the formation and/or stabilization of curved membranes. In recent work, we have determined that the maintenance of low surface tension of surfactant in the presence of serum protein inhibitors requires cooperative interactions between the C-terminal and N-terminal domains of the molecule. This effect of SP-A requires a high degree of oligomeric assembly of the protein, and may be mediated by the activity of the protein to alter the form or physical state of surfactant lipid aggregates. PMID:11369537

  16. The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites

    PubMed Central

    Zhao, Yonghong; Liu, Deqian; Kaluarachchi, Warna D.; Bellamy, Henry D.; White, Mark A.; Fox, Robert O.

    2003-01-01

    The mRNA of Escherichia coli yedU gene is induced 31-fold upon heat shock. The 31-kD YedU protein, also calls Hsp31, is highly conserved in several human pathogens and has chaperone activity. We solved the crystal structure of YedU at 2.2 Å resolution. YedU monomer has an α/β/α sandwich domain and a small α/β domain. YedU is a dimer in solution, and its crystal structure indicates that a significant amount of surface area is buried upon dimerization. There is an extended hydrophobic patch that crosses the dimer interface on the surface of the protein. This hydrophobic patch is likely the substrate-binding site responsible for the chaperone activity. The structure also reveals a potential protease-like catalytic triad composed of Cys184, His185, and Asp213, although no enzymatic activity could be identified. YedU coordinates a metal ion using His85, His122, and Glu90. This 2-His-1-carboxylate motif is present in carboxypeptidase A (a zinc enzyme), and a number of dioxygenases and hydroxylases that utilize iron as a cofactor, suggesting another potential function for YedU. PMID:14500888

  17. Bacterial community analysis of beef cattle feedlots reveals that pen surface is distinct from feces.

    PubMed

    Durso, Lisa M; Harhay, Gregory P; Smith, Timothy P L; Bono, James L; DeSantis, Todd Z; Clawson, Michael L

    2011-05-01

    The surface of beef cattle feedlot pens is commonly conceptualized as being packed uncomposted manure. Despite the important role that the feedlot pen may play in the transmission of veterinary and zoonotic pathogens, the bacterial ecology of feedlot surface material is not well understood. Our present study characterized the bacterial communities of the beef cattle feedlot pen surface material using 3647 full-length 16S rDNA sequences, and we compared the community composition of feedlot pens to the fecal source material. The feedlot surface composite was represented by members of the phylum Actinobacteria (42%), followed by Firmicutes (24%), Bacteroidetes (24%), and Proteobacteria (9%). The feedlot pen surface material bacterial communities were clearly distinct from those of the feces from animals in the same pen. Comparisons with previously published results of feces from the animals in the same pen reveal that, of 139 genera identified, only 25 were present in both habitats. These results indicate that, microbiologically, the feedlot pen surface material is separate and distinct from the fecal source material, suggesting that bacteria that originate in cattle feces face different selection pressures and survival challenges during their tenure in the feedlot pen, as compared to their residence in the gastrointestinal tract. PMID:21214381

  18. Revealing the Reconstructed Surface of Li[Mn2]O4.

    PubMed

    Amos, Charles D; Roldan, Manuel A; Varela, Maria; Goodenough, John B; Ferreira, Paulo J

    2016-05-11

    The spinel Li[Mn2]O4 is a candidate cathode for a Li-ion battery, but its capacity fades over a charge/discharge cycle of Li1-x[Mn2]O4 (0 < x < 1) that is associated with a loss of Mn to the organic-liquid electrolyte. It is known that the disproportionation reaction 2Mn(3+) = Mn(2+) + Mn(4+) occurs at the surface of a Mn spinel, and it is important to understand the atomic structure and composition of the surface of Li[Mn2]O4 in order to understand how Mn loss occurs. We report a study of the surface reconstruction of Li[Mn2]O4 by aberration-corrected scanning transmission electron microscopy. The atomic structure coupled with Mn-valence and the distribution of the atomic ratio of oxygen obtained by electron energy loss spectroscopy reveals a thin, stable surface layer of Mn3O4, a subsurface region of Li1+x[Mn2]O4 with retention of bulk Li[Mn2]O4. This observation is compatible with the disproportionation reaction coupled with oxygen deficiency and a displacement of surface Li(+) from the Mn3O4 surface phase. These results provide a critical step toward understanding how Mn is lost from Li[Mn2]O4, once inside a battery. PMID:27022834

  19. Analysis of the Pseudoalteromonas tunicata Genome Reveals Properties of a Surface-Associated Life Style in the Marine Environment

    PubMed Central

    Thomas, Torsten; Evans, Flavia F.; Schleheck, David; Mai-Prochnow, Anne; Burke, Catherine; Penesyan, Anahit; Dalisay, Doralyn S.; Stelzer-Braid, Sacha; Saunders, Neil; Johnson, Justin; Ferriera, Steve; Kjelleberg, Staffan; Egan, Suhelen

    2008-01-01

    Background Colonisation of sessile eukaryotic host surfaces (e.g. invertebrates and seaweeds) by bacteria is common in the marine environment and is expected to create significant inter-species competition and other interactions. The bacterium Pseudoalteromonas tunicata is a successful competitor on marine surfaces owing primarily to its ability to produce a number of inhibitory molecules. As such P. tunicata has become a model organism for the studies into processes of surface colonisation and eukaryotic host-bacteria interactions. Methodology/Principal Findings To gain a broader understanding into the adaptation to a surface-associated life-style, we have sequenced and analysed the genome of P. tunicata and compared it to the genomes of closely related strains. We found that the P. tunicata genome contains several genes and gene clusters that are involved in the production of inhibitory compounds against surface competitors and secondary colonisers. Features of P. tunicata's oxidative stress response, iron scavenging and nutrient acquisition show that the organism is well adapted to high-density communities on surfaces. Variation of the P. tunicata genome is suggested by several landmarks of genetic rearrangements and mobile genetic elements (e.g. transposons, CRISPRs, phage). Surface attachment is likely to be mediated by curli, novel pili, a number of extracellular polymers and potentially other unexpected cell surface proteins. The P. tunicata genome also shows a utilisation pattern of extracellular polymers that would avoid a degradation of its recognised hosts, while potentially causing detrimental effects on other host types. In addition, the prevalence of recognised virulence genes suggests that P. tunicata has the potential for pathogenic interactions. Conclusions/Significance The genome analysis has revealed several physiological features that would provide P. tunciata with competitive advantage against other members of the surface-associated community

  20. Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

    PubMed

    Borges, Chad R; Rehder, Douglas S

    2016-09-15

    Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. PMID:27503803

  1. The Lipid-Droplet Proteome Reveals that Droplets Are a Protein-Storage Depot

    SciTech Connect

    Cermelli, Silvia; Guo, Yi; Gross, Steven P.; Welte, Michael

    2006-09-19

    Lipid droplets are ubiquitous organelles that are among the basic building blocks of eukaryotic cells. Despite central roles for cholesterol homeostasis and lipid metabolism, their function and protein composition are poorly understood. Results: We purified lipid droplets from Drosophila embryos and analyzed the associated proteins by capillary LC-MS-MS. Important functional groups include enzymes involved in lipid metabolism, signaling molecules, and proteins related to membrane trafficking. Unexpectedly, histones H2A, H2Av, and H2B were present. Using biochemistry, genetics, real-time imaging, and cell biology, we confirm that roughly 50% of certain embryonic histones are physically attached to lipid droplets, a localization conserved in other fly species. Histone association with droplets starts during oogenesis and is prominent in early embryos, but it is undetectable in later stages or in cultured cells. Histones on droplets are not irreversibly trapped; quantitation of droplet histone levels and transplantation experiments suggest that histones are transferred from droplets to nuclei as development proceeds. When this maternal store of histones is unavailable because lipid droplets are mislocalized, zygotic histone production starts prematurely. Conclusions: Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.

  2. Nullspace Sampling with Holonomic Constraints Reveals Molecular Mechanisms of Protein Gαs

    PubMed Central

    Pachov, Dimitar V.; van den Bedem, Henry

    2015-01-01

    Proteins perform their function or interact with partners by exchanging between conformational substates on a wide range of spatiotemporal scales. Structurally characterizing these exchanges is challenging, both experimentally and computationally. Large, diffusional motions are often on timescales that are difficult to access with molecular dynamics simulations, especially for large proteins and their complexes. The low frequency modes of normal mode analysis (NMA) report on molecular fluctuations associated with biological activity. However, NMA is limited to a second order expansion about a minimum of the potential energy function, which limits opportunities to observe diffusional motions. By contrast, kino-geometric conformational sampling (KGS) permits large perturbations while maintaining the exact geometry of explicit conformational constraints, such as hydrogen bonds. Here, we extend KGS and show that a conformational ensemble of the α subunit Gαs of heterotrimeric stimulatory protein Gs exhibits structural features implicated in its activation pathway. Activation of protein Gs by G protein-coupled receptors (GPCRs) is associated with GDP release and large conformational changes of its α-helical domain. Our method reveals a coupled α-helical domain opening motion while, simultaneously, Gαs helix α5 samples an activated conformation. These motions are moderated in the activated state. The motion centers on a dynamic hub near the nucleotide-binding site of Gαs, and radiates to helix α4. We find that comparative NMA-based ensembles underestimate the amplitudes of the motion. Additionally, the ensembles fall short in predicting the accepted direction of the full activation pathway. Taken together, our findings suggest that nullspace sampling with explicit, holonomic constraints yields ensembles that illuminate molecular mechanisms involved in GDP release and protein Gs activation, and further establish conformational coupling between key structural

  3. Revealing divergent evolution, identifying circular permutations and detecting active-sites by protein structure comparison

    PubMed Central

    Chen, Luonan; Wu, Ling-Yun; Wang, Yong; Zhang, Shihua; Zhang, Xiang-Sun

    2006-01-01

    Background Protein structure comparison is one of the most important problems in computational biology and plays a key role in protein structure prediction, fold family classification, motif finding, phylogenetic tree reconstruction and protein docking. Results We propose a novel method to compare the protein structures in an accurate and efficient manner. Such a method can be used to not only reveal divergent evolution, but also identify circular permutations and further detect active-sites. Specifically, we define the structure alignment as a multi-objective optimization problem, i.e., maximizing the number of aligned atoms and minimizing their root mean square distance. By controlling a single distance-related parameter, theoretically we can obtain a variety of optimal alignments corresponding to different optimal matching patterns, i.e., from a large matching portion to a small matching portion. The number of variables in our algorithm increases with the number of atoms of protein pairs in almost a linear manner. In addition to solid theoretical background, numerical experiments demonstrated significant improvement of our approach over the existing methods in terms of quality and efficiency. In particular, we show that divergent evolution, circular permutations and active-sites (or structural motifs) can be identified by our method. The software SAMO is available upon request from the authors, or from and . Conclusion A novel formulation is proposed to accurately align protein structures in the framework of multi-objective optimization, based on a sequence order-independent strategy. A fast and accurate algorithm based on the bipartite matching algorithm is developed by exploiting the special features. Convergence of computation is shown in experiments and is also theoretically proven. PMID:16948858

  4. Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse

    PubMed Central

    Zampieri, Alexandre; Champagne, Julien; Auzemery, Baptiste; Fuentes, Ivanna; Maurel, Benjamin; Bienvenu, Frédéric

    2015-01-01

    We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed “Tandem-HTRF”, can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material. PMID:26503526

  5. The crystal structure of choline kinase reveals a eukaryotic protein kinase fold

    SciTech Connect

    Peisach, D.; Gee, P.; Kent, K.; Xu, Z.

    2010-03-08

    Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 {angstrom} crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.

  6. Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications*

    PubMed Central

    Halim, Adnan; Carlsson, Michael C.; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y.; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L.; Wandall, Hans H.

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM1 characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines. PMID:25389185

  7. Structure of the Yeast DEAD Box Protein Mss116p Reveals Two Wedges that Crimp RNA

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2010-01-12

    The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF{sub 3}, or ADP-AlF{sub 4}{sup -}. The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge {alpha} helix bending RNA 3' of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5' of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which a protein uses two wedges to act as a molecular crimper.

  8. Proteomic Investigation of Aphid Honeydew Reveals an Unexpected Diversity of Proteins

    PubMed Central

    Haubruge, Eric; Hance, Thierry; Thonart, Philippe; De Pauw, Edwin; Francis, Frédéric

    2013-01-01

    Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige) approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora). Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu), and flagellin that might act as mediators in the plant-aphid interaction. In addition to providing the first aphid honeydew proteome analysis, we propose to reconsider the importance of this substance, mainly acknowledged to be a waste product, from the aphid ecology perspective. PMID:24086359

  9. Biointerface: protein enhanced stem cells binding to implant surface.

    PubMed

    Chrzanowski, W; Kondyurin, A; Lee, Jae Ho; Lord, Megan S; Bilek, M M M; Kim, Hae-Won

    2012-09-01

    The number of metallic implantable devices placed every year is estimated at 3.7 million. This number has been steadily increasing over last decades at a rate of around 8 %. In spite of the many successes of the devices the implantation of biomaterial into tissues almost universally leads to the development of an avascular sac, which consists of fibrous tissue around the device and walls off the implant from the body. This reaction can be detrimental to the function of implant, reduces its lifetime, and necessitates repeated surgery. Clearly, to reduce the number of revision surgeries and improve long-term implant function it is necessary to enhance device integration by modulating cell adhesion and function. In this paper we have demonstrated that it is possible to enhance stem cell attachment using engineered biointerfaces. To create this functional interface, samples were coated with polymer (as a precursor) and then ion implanted to create a reactive interface that aids the binding of biomolecules--fibronectin. Both AFM and XPS analyses confirmed the presence of protein layers on the samples. The amount of protein was significant greater for the ion implanted surfaces and was not disrupted upon washing with detergent, hence the formation of strong bonds with the interface was confirmed. While, for non ion implanted surfaces, a decrease of protein was observed after washing with detergent. Finally, the number of stem cells attached to the surface was enhanced for ion implanted surfaces. The studies presented confirm that the developed bionterface with immobilised fibronectin is an effective means to modulate stem cell attachment. PMID:22714559

  10. Lysozyme adsorption at a silica surface using simulation and experiment: effects of pH on protein layer structure.

    PubMed

    Kubiak-Ossowska, Karina; Cwieka, Monika; Kaczynska, Agnieszka; Jachimska, Barbara; Mulheran, Paul A

    2015-10-01

    Hen Egg White Lysozyme (HEWL) is a widely used exemplar to study protein adsorption on surfaces and interfaces. Here we use fully atomistic Molecular Dynamics (MD) simulations, Multi-Parametric Surface Plasmon Resonance (MP-SPR), contact angle and zeta potential measurements to study HEWL adsorption at a silica surface. The simulations provide a detailed description of the adsorption mechanism and indicate that at pH7 the main adsorption driving force is electrostatics, supplemented by weaker hydrophobic forces. Moreover, they reveal the preferred orientation of the adsorbed protein and show that its structure is only slightly altered at the interface with the surface. This provides the basis for interpreting the experimental results, which indicate the surface adsorbs a close-packed monolayer at about pH10 where the surface has a large negative zeta potential and the HEWL is positively charged. At higher pH, the adsorption amount of the protein layer is greatly reduced due to the loss of charge on the protein. At lower pH, the smaller zeta potential of the surface leads to lower HEWL adsorption. These interpretations are complemented by the contact angle measurements that show how the hydrophobicity of the surface is greatest when the surface coverage is highest. The simulations provide details of the hydrophobic residues exposed to solution by the adsorbed HEWL, completing the picture of the protein layer structure. PMID:26315945

  11. Zika virus NS1 structure reveals diversity of electrostatic surfaces among flaviviruses.

    PubMed

    Song, Hao; Qi, Jianxun; Haywood, Joel; Shi, Yi; Gao, George F

    2016-05-01

    The association of Zika virus (ZIKV) infections with microcephaly has resulted in an ongoing public-health emergency. Here we report the crystal structure of a C-terminal fragment of ZIKV nonstructural protein 1 (NS1), a major host-interaction molecule that functions in flaviviral replication, pathogenesis and immune evasion. Comparison with West Nile and dengue virus NS1 structures reveals conserved features but diverse electrostatic characteristics at host-interaction interfaces, thus possibly implying different modes of flavivirus pathogenesis. PMID:27088990

  12. Effects of surface compositional and structural heterogeneity on nanoparticle-protein interactions: different protein configurations.

    PubMed

    Huang, Rixiang; Carney, Randy P; Ikuma, Kaoru; Stellacci, Francesco; Lau, Boris L T

    2014-06-24

    As nanoparticles (NPs) enter into biological systems, they are immediately exposed to a variety and concentration of proteins. The physicochemical interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of NP surface heterogeneity, the interactions between bovine serum albumin (BSA) and gold NPs (AuNPs) with similar chemical composition but different surface structures were investigated. Different interaction modes and BSA conformations were studied by dynamic light scattering, circular dichroism spectroscopy, fluorescence quenching and isothermal titration calorimetry (ITC). Depending on the surface structure of AuNPs, BSA seems to adopt either a "side-on" or an "end-on" conformation on AuNPs. ITC demonstrated that the adsorption of BSA onto AuNPs with randomly distributed polar and nonpolar groups was primarily driven by electrostatic interaction, and all BSA were adsorbed in the same process. The adsorption of BSA onto AuNPs covered with alternating domains of polar and nonpolar groups was a combination of different interactions. Overall, the results of this study point to the potential for utilizing nanoscale manipulation of NP surfaces to control the resulting NP-protein interactions. PMID:24882660

  13. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.

    PubMed

    Gao, Xing-Huang; Krokowski, Dawid; Guan, Bo-Jhih; Bederman, Ilya; Majumder, Mithu; Parisien, Marc; Diatchenko, Luda; Kabil, Omer; Willard, Belinda; Banerjee, Ruma; Wang, Benlian; Bebek, Gurkan; Evans, Charles R; Fox, Paul L; Gerson, Stanton L; Hoppel, Charles L; Liu, Ming; Arvan, Peter; Hatzoglou, Maria

    2015-01-01

    The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. PMID:26595448

  14. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle.

    PubMed

    Uezumi, Akiyoshi; Nakatani, Masashi; Ikemoto-Uezumi, Madoka; Yamamoto, Naoki; Morita, Mitsuhiro; Yamaguchi, Asami; Yamada, Harumoto; Kasai, Takehiro; Masuda, Satoru; Narita, Asako; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi; Fukada, So-Ichiro; Nishino, Ichizo; Tsuchida, Kunihiro

    2016-08-01

    Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases. PMID:27509136

  15. Mapping Hydrophobicity on the Protein Molecular Surface at Atom-Level Resolution

    PubMed Central

    Nicolau Jr., Dan V.; Paszek, Ewa; Fulga, Florin; Nicolau, Dan V.

    2014-01-01

    A precise representation of the spatial distribution of hydrophobicity, hydrophilicity and charges on the molecular surface of proteins is critical for the understanding of the interaction with small molecules and larger systems. The representation of hydrophobicity is rarely done at atom-level, as this property is generally assigned to residues. A new methodology for the derivation of atomic hydrophobicity from any amino acid-based hydrophobicity scale was used to derive 8 sets of atomic hydrophobicities, one of which was used to generate the molecular surfaces for 35 proteins with convex structures, 5 of which, i.e., lysozyme, ribonuclease, hemoglobin, albumin and IgG, have been analyzed in more detail. Sets of the molecular surfaces of the model proteins have been constructed using spherical probes with increasingly large radii, from 1.4 to 20 Å, followed by the quantification of (i) the surface hydrophobicity; (ii) their respective molecular surface areas, i.e., total, hydrophilic and hydrophobic area; and (iii) their relative densities, i.e., divided by the total molecular area; or specific densities, i.e., divided by property-specific area. Compared with the amino acid-based formalism, the atom-level description reveals molecular surfaces which (i) present an approximately two times more hydrophilic areas; with (ii) less extended, but between 2 to 5 times more intense hydrophilic patches; and (iii) 3 to 20 times more extended hydrophobic areas. The hydrophobic areas are also approximately 2 times more hydrophobicity-intense. This, more pronounced “leopard skin”-like, design of the protein molecular surface has been confirmed by comparing the results for a restricted set of homologous proteins, i.e., hemoglobins diverging by only one residue (Trp37). These results suggest that the representation of hydrophobicity on the protein molecular surfaces at atom-level resolution, coupled with the probing of the molecular surface at different geometric resolutions

  16. Using extremely halophilic bacteria to understand the role of surface charge and surface hydration in protein evolution, folding, and function

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Deole, Ratnakar; Osu Collaboration

    2013-03-01

    Halophilic Archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant, and have evolved highly acidic proteomes that only function at high salinity. We examine osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila. We find that H. halophila has an acidic proteome and accumulates molar concentrations of KCl when grown in high salt media. Upon growth of H. halophila in low salt media, its cytoplasmic K + content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. We conclude that proteome acidity is not driven by stabilizing interactions between K + ions and acidic side chains, but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. We propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K + binding sites on an increasingly acidic protein surface.

  17. Single molecule surface enhanced resonance Raman scattering (SERRS) of the enhanced green fluorescent protein (EGFP)

    NASA Astrophysics Data System (ADS)

    Hofkens, Johan; De Schryver, Frans C.; Cotlet, Mircea; Habuchi, Satoshi

    2004-06-01

    One of the most intriguing findings in single molecule spectroscopy (SMS) is the observation of Raman spectra of individual molecules, despite the small cross section of the transitions involved. The observation of the spectra can be explained by the surface enhanced Raman scattering (SERRS) effect. At the single-molecule level, the SERRS-spectra recorded as a function of time reveal inhomogeneous behaviour such as on/off blinking, spectral diffusion, intensity fluctuations of vibrational line, and even splitting of some lines within the spectrum of one molecule. Single-molecule SERRS (SM-SERRS) spectroscopy opens up exciting opportunities in the field of biophysics and biomedical spectroscopy. The first example of single protein SERRS was performed on hemoglobin. However, the possibility of extracting the heme group by silver sols can not be excluded. Here we report on SM-SERRS spectra of enhanced green fluorescent protein (EGFP) in which the chromophore is kept in the protein. The time series of SM-SERRS spectra suggest the conversion of the EGFP chromophore between the deprotonated and the protonated form. Autocorrelation analysis of SM-SERRS trajectory reveals the presence of fast dynamics taking place in the protein. Our findings show the potential of the technique to study structural dynamics of protein molecules.

  18. Protein microarrays based on polymer brushes prepared via surface-initiated atom transfer radical polymerization.

    PubMed

    Barbey, Raphael; Kauffmann, Ekkehard; Ehrat, Markus; Klok, Harm-Anton

    2010-12-13

    Polymer brushes represent an interesting platform for the development of high-capacity protein binding surfaces. Whereas the protein binding properties of polymer brushes have been investigated before, this manuscript evaluates the feasibility of poly(glycidyl methacrylate) (PGMA) and PGMA-co-poly(2-(diethylamino)ethyl methacrylate) (PGMA-co-PDEAEMA) (co)polymer brushes grown via surface-initiated atom transfer radical polymerization (SI-ATRP) as protein reactive substrates in a commercially available microarray system using tantalum-pentoxide-coated optical waveguide-based chips. The performance of the polymer-brush-based protein microarray chips is assessed using commercially available dodecylphosphate (DDP)-modified chips as the benchmark. In contrast to the 2D planar, DDP-coated chips, the polymer-brush-covered chips represent a 3D sampling volume. This was reflected in the results of protein immobilization studies, which indicated that the polymer-brush-based coatings had a higher protein binding capacity as compared to the reference substrates. The protein binding capacity of the polymer-brush-based coatings was found to increase with increasing brush thickness and could also be enhanced by copolymerization of 2-(diethylamino)ethyl methacrylate (DEAEMA), which catalyzes epoxide ring-opening of the glycidyl methacrylate (GMA) units. The performance of the polymer-brush-based microarray chips was evaluated in two proof-of-concept microarray experiments, which involved the detection of biotin-streptavidin binding as well as a model TNFα reverse assay. These experiments revealed that the use of polymer-brush-modified microarray chips resulted not only in the highest absolute fluorescence readouts, reflecting the 3D nature and enhanced sampling volume provided by the brush coating, but also in significantly enhanced signal-to-noise ratios. These characteristics make the proposed polymer brushes an attractive alternative to commercially available, 2D microarray

  19. Attachment of Pathogenic Prion Protein to Model Oxide Surfaces

    PubMed Central

    Jacobson, Kurt H.; Kuech, Thomas R.; Pedersen, Joel A.

    2014-01-01

    Prions are the infectious agents in the class of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies, which affect humans, deer, sheep, and cattle. Prion diseases of deer and sheep can be transmitted via environmental routes, and soil is has been implicated in the transmission of these diseases. Interaction with soil particles is expected to govern the transport, bioavailability and persistence of prions in soil environments. A mechanistic understanding of prion interaction with soil components is critical for understanding the behavior of these proteins in the environment. Here, we report results of a study to investigate the interactions of prions with model oxide surfaces (Al2O3, SiO2) using quartz crystal microbalance with dissipation monitoring and optical waveguide light mode spectroscopy. The efficiency of prion attachment to Al2O3 and SiO2 depended strongly on pH and ionic strength in a manner consistent with electrostatic forces dominating interaction with these oxides. The N-terminal portion of the protein appeared to facilitate attachment to Al2O3 under globally electrostatically repulsive conditions. We evaluated the utility of recombinant prion protein as a surrogate for prions in attachment experiments and found that its behavior differed markedly from that of the infectious agent. Our findings suggest that prions preferentially associate with positively charged mineral surfaces in soils (e.g., iron and aluminum oxides). PMID:23611152

  20. GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export.

    PubMed

    Zhang, Maoxiang; Davis, Jason E; Li, Chunman; Gao, Jie; Huang, Wei; Lambert, Nevin A; Terry, Alvin V; Wu, Guangyu

    2016-01-01

    Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor. PMID:26811329

  1. Combination of hydrogel nanoparticles and proteomics to reveal secreted proteins associated with decidualization of human uterine stromal cells

    PubMed Central

    2011-01-01

    Background Identification of secreted proteins of low abundance is often limited by abundant and high molecular weight (MW) proteins. We have optimised a procedure to overcome this limitation. Results Low MW proteins in the conditioned media of cultured cells were first captured using dual-size exclusion/affinity hydrogel nanoparticles and their identities were then revealed by proteomics. Conclusions This technique enables the analysis of secreted proteins of cultured cells low MW and low abundance. PMID:21884602

  2. Group A streptococcus expresses a trio of surface proteins containing protective epitopes.

    PubMed

    Niedermeyer, Shannon E; Penfound, Thomas A; Hohn, Claudia; Li, Yi; Homayouni, Ramin; Zhao, Jingnan; Dale, James B

    2014-10-01

    Group A streptococci (GAS) (Streptococcus pyogenes) are common causes of infections in humans for which there is no licensed vaccine. Decades of work has focused on the role of the surface M protein in eliciting type-specific protective immunity. Recent studies have identified additional surface proteins of GAS that contain opsonic epitopes. In the present study, we describe a serotype M65 GAS originally isolated during an epidemiologic study in Bamako, Mali, which simultaneously expressed M, M-related protein (Mrp), and streptococcal protective antigen (Spa) on the bacterial surface. The emm, mrp, and spa genes were sequenced from PCR amplicons derived from the M65 chromosome. Rabbit antisera raised against synthetic peptides copying the N-terminal regions of M, Mrp, and Spa were highly specific for each peptide, reacted with the surface of M65 GAS, and promoted bactericidal activity against the organism. A mixture of antisera against all three peptides was most effective in the bactericidal assays. Immunofluorescence microscopy revealed that the M, Mrp, and Spa antisera bound to the bacterial surface in the presence of human plasma proteins and resulted in the deposition of complement. Five additional spa genes were identified in the Mrp-positive GAS serotypes, and their sequences were determined. Our results indicate that there are multiple antigens on the surface of GAS that evoke antibodies that promote bacterial killing. A more complete understanding of the relative contributions of M, Mrp, and Spa in eliciting protective immunity may aid in the development of GAS vaccines with enhanced coverage and efficacy. PMID:25080552

  3. Chemistry-specific surface adsorption of the barnacle settlement-inducing protein complex

    PubMed Central

    Petrone, Luigi; Aldred, Nick; Emami, Kaveh; Enander, Karin; Ederth, Thomas; Clare, Anthony S.

    2015-01-01

    Gregarious settlement in barnacle larvae (cyprids) is induced by a contact pheromone, the settlement-inducing protein complex (SIPC). The SIPC has been identified both in the cuticle of adult barnacles and in the temporary adhesive secretion (footprint) of cyprids. Besides acting as a settlement inducer, the presence of the SIPC in footprints points to its additional involvement in the adhesion process. SIPC adsorption behaviour was therefore investigated on a series of self-assembled monolayers (SAMs) by surface plasmon resonance at the pH of seawater (8.3). Fibrinogen and α2-macroglobulin (A2M) (blood complement protease inhibitors with which the SIPC shares 29% sequence homology) were used in the adsorption experiments as positive and negative standards, respectively. The mass uptake of the SIPC was comparable to that of fibrinogen, with adsorption observed even on the protein-resistant oligo(ethylene glycol) surface. Notably, on the positively charged SAM the SIPC showed a kinetic overshoot, indicating a metastable configuration causing the amount of adsorbed protein to temporarily exceed its equilibrium value. A2M adsorption was low or negligible on all SAMs tested, except for the positively charged surface, indicating that A2M adsorption is mainly driven by electrostatics. Evaluation of SIPC non-specific adsorption kinetics revealed that it adsorbed irreversibly and non-cooperatively on all surfaces tested. PMID:25657832

  4. Chemistry-specific surface adsorption of the barnacle settlement-inducing protein complex.

    PubMed

    Petrone, Luigi; Aldred, Nick; Emami, Kaveh; Enander, Karin; Ederth, Thomas; Clare, Anthony S

    2015-02-01

    Gregarious settlement in barnacle larvae (cyprids) is induced by a contact pheromone, the settlement-inducing protein complex (SIPC). The SIPC has been identified both in the cuticle of adult barnacles and in the temporary adhesive secretion (footprint) of cyprids. Besides acting as a settlement inducer, the presence of the SIPC in footprints points to its additional involvement in the adhesion process. SIPC adsorption behaviour was therefore investigated on a series of self-assembled monolayers (SAMs) by surface plasmon resonance at the pH of seawater (8.3). Fibrinogen and α2-macroglobulin (A2M) (blood complement protease inhibitors with which the SIPC shares 29% sequence homology) were used in the adsorption experiments as positive and negative standards, respectively. The mass uptake of the SIPC was comparable to that of fibrinogen, with adsorption observed even on the protein-resistant oligo(ethylene glycol) surface. Notably, on the positively charged SAM the SIPC showed a kinetic overshoot, indicating a metastable configuration causing the amount of adsorbed protein to temporarily exceed its equilibrium value. A2M adsorption was low or negligible on all SAMs tested, except for the positively charged surface, indicating that A2M adsorption is mainly driven by electrostatics. Evaluation of SIPC non-specific adsorption kinetics revealed that it adsorbed irreversibly and non-cooperatively on all surfaces tested. PMID:25657832

  5. Antibody-like Biorecognition Sites for Proteins from Surface Imprinting on Nanoparticles.

    PubMed

    Bhakta, Snehasis; Seraji, Mohammad Saiful Islam; Suib, Steven L; Rusling, James F

    2015-12-30

    Natural antibodies are used widely for important applications such as biomedical analysis, cancer therapy, and directed drug delivery, but they are expensive and may have limited stability. This study describes synthesis of antibody-like binding sites by molecular imprinting on silica nanoparticles (SiNP) using a combination of four organosilane monomers with amino acid-like side chains providing hydrophobic, hydrophilic, and H-bonding interactions with target proteins. This approach provided artificial antibody (AA) nanoparticles with good selectivity and specificity to binding domains on target proteins in a relatively low-cost synthesis. The AAs were made by polymer grafting onto SiNPs for human serum albumin (HSA) and glucose oxidase (GOx). Binding affinity, selectivity, and specificity was compared to several other proteins using adsorption isotherms and surface plasmon resonance (SPR). The Langmuir-Freundlich adsorption model was used to obtain apparent binding constants (KLF) from binding isotherms of HSA (6.7 × 10(4)) and GOx (4.7 × 10(4)) to their respective AAs. These values were 4-300 fold larger compared to a series of nontemplate proteins. SPR binding studies of AAs with proteins attached to a gold surface confirmed good specificity and revealed faster binding for the target proteins compared to nontarget proteins. Target proteins retained their secondary structures upon binding. Binding capacity of AAHSA for HSA was 5.9 mg HSA/g compared to 1.4 mg/g for previously report imprinted silica beads imprinted with poly(aminophenyl)boronic acid. Also, 90% recovery for HSA spiked into 2% calf serum was found for AAHSA. PMID:26636440

  6. Structured and Unstructured Binding of an Intrinsically Disordered Protein as Revealed by Atomistic Simulations.

    PubMed

    Ithuralde, Raúl Esteban; Roitberg, Adrián Enrique; Turjanski, Adrián Gustavo

    2016-07-20

    Intrinsically disordered proteins (IDPs) are a set of proteins that lack a definite secondary structure in solution. IDPs can acquire tertiary structure when bound to their partners; therefore, the recognition process must also involve protein folding. The nature of the transition state (TS), structured or unstructured, determines the binding mechanism. The characterization of the TS has become a major challenge for experimental techniques and molecular simulations approaches since diffusion, recognition, and binding is coupled to folding. In this work we present atomistic molecular dynamics (MD) simulations that sample the free energy surface of the coupled folding and binding of the transcription factor c-myb to the cotranscription factor CREB binding protein (CBP). This process has been recently studied and became a model to study IDPs. Despite the plethora of available information, we still do not know how c-myb binds to CBP. We performed a set of atomistic biased MD simulations running a total of 15.6 μs. Our results show that c-myb folds very fast upon binding to CBP with no unique pathway for binding. The process can proceed through both structured or unstructured TS's with similar probabilities. This finding reconciles previous seemingly different experimental results. We also performed Go-type coarse-grained MD of several structured and unstructured models that indicate that coupled folding and binding follows a native contact mechanism. To the best of our knowledge, this is the first atomistic MD simulation that samples the free energy surface of the coupled folding and binding processes of IDPs. PMID:27348048

  7. The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

    PubMed Central

    Lai, C F; Gong, S C; Esteban, M

    1991-01-01

    The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor. Images PMID:1985213

  8. Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

    SciTech Connect

    Hast, Michael A.; Nichols, Connie B.; Armstrong, Stephanie M.; Kelly, Shannon M.; Hellinga, Homme W.; Alspaugh, J. Andrew; Beese, Lorena S.

    2012-09-17

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

  9. Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens*

    PubMed Central

    Hast, Michael A.; Nichols, Connie B.; Armstrong, Stephanie M.; Kelly, Shannon M.; Hellinga, Homme W.; Alspaugh, J. Andrew; Beese, Lorena S.

    2011-01-01

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections. PMID:21816822

  10. Dengue Virus Capsid Protein Binding to Hepatic Lipid Droplets (LD) Is Potassium Ion Dependent and Is Mediated by LD Surface Proteins

    PubMed Central

    Carvalho, Filomena A.; Carneiro, Fabiana A.; Martins, Ivo C.; Assunção-Miranda, Iranaia; Faustino, André F.; Pereira, Renata M.; Bozza, Patricia T.; Castanho, Miguel A. R. B.; Mohana-Borges, Ronaldo; Da Poian, Andrea T.

    2012-01-01

    Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of −19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at +13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C protein-functionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na+/K+-ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs. PMID:22130547

  11. Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis.

    PubMed

    Na, Chan Hyun; Jones, Drew R; Yang, Yanling; Wang, Xusheng; Xu, Yanji; Peng, Junmin

    2012-09-01

    Protein ubiquitination is an essential post-translational modification regulating neurodevelopment, synaptic plasticity, learning, and memory, and its dysregulation contributes to the pathogenesis of neurological diseases. Here we report a systematic analysis of ubiquitinated proteome (ubiquitome) in rat brain using a newly developed monoclonal antibody that recognizes the diglycine tag on lysine residues in trypsinized peptides (K-GG peptides). Initial antibody specificity analysis showed that the antibody can distinguish K-GG peptides from linear GG peptides or pseudo K-GG peptides derived from iodoacetamide. To evaluate the false discovery rate of K-GG peptide matches during database search, we introduced a null experiment using bacterial lysate that contains no such peptides. The brain ubiquitome was then analyzed by this antibody enrichment with or without strong cation exchange (SCX) prefractionation. During SCX chromatography, although the vast majority of K-GG peptides were detected in the fractions containing at least three positive charged peptides, specific K-GG peptides with two positive charges (e.g., protein N-terminal acetylated and C-terminal non-K/R peptides) were also identified in early fractions. The reliability of C-terminal K-GG peptides was also extensively investigated. Finally, we collected a data set of 1786 K-GG sites on 2064 peptides in 921 proteins and estimated their abundance by spectral counting. The study reveals a wide range of ubiquitination events on key components in presynaptic region (e.g., Bassoon, NSF, SNAP25, synapsin, synaptotagmin, and syntaxin) and postsynaptic density (e.g., PSD-95, GKAP, CaMKII, as well as receptors for NMDA, AMPA, GABA, serotonin, and acetylcholine). We also determined ubiquitination sites on amyloid precursor protein and alpha synuclein that are thought to be causative agents in Alzhermer's and Parkinson's disorders, respectively. As K-GG peptides can also be produced from Nedd8 or ISG15 modified

  12. Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

    PubMed Central

    Tsytlonok, Maksym; Ibrahim, Shehu M.; Rowling, Pamela J.E.; Xu, Wenshu; Ruedas-Rama, Maria J.; Orte, Angel; Klenerman, David; Itzhaki, Laura S.

    2015-01-01

    Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated. PMID:25565106

  13. Model systems of protein-misfolding diseases reveal chaperone modifiers of proteotoxicity

    PubMed Central

    2016-01-01

    ABSTRACT Chaperones and co-chaperones enable protein folding and degradation, safeguarding the proteome against proteotoxic stress. Chaperones display dynamic responses to exogenous and endogenous stressors and thus constitute a key component of the proteostasis network (PN), an intricately regulated network of quality control and repair pathways that cooperate to maintain cellular proteostasis. It has been hypothesized that aging leads to chronic stress on the proteome and that this could underlie many age-associated diseases such as neurodegeneration. Understanding the dynamics of chaperone function during aging and disease-related proteotoxic stress could reveal specific chaperone systems that fail to respond to protein misfolding. Through the use of suppressor and enhancer screens, key chaperones crucial for proteostasis maintenance have been identified in model organisms that express misfolded disease-related proteins. This review provides a literature-based analysis of these genetic studies and highlights prominent chaperone modifiers of proteotoxicity, which include the HSP70-HSP40 machine and small HSPs. Taken together, these studies in model systems can inform strategies for therapeutic regulation of chaperone functionality, to manage aging-related proteotoxic stress and to delay the onset of neurodegenerative diseases. PMID:27491084

  14. Local axonal protection by WldS as revealed by conditional regulation of protein stability.

    PubMed

    Wang, Jack T; Medress, Zachary A; Vargas, Mauricio E; Barres, Ben A

    2015-08-18

    The expression of the mutant Wallerian degeneration slow (WldS) protein significantly delays axonal degeneration from various nerve injuries and in multiple species; however, the mechanism for its axonal protective property remains unclear. Although WldS is localized predominantly in the nucleus, it also is present in a smaller axonal pool, leading to conflicting models to account for the WldS fraction necessary for axonal protection. To identify where WldS activity is required to delay axonal degeneration, we adopted a method to alter the temporal expression of WldS protein in neurons by chemically regulating its protein stability. We demonstrate that continuous WldS activity in the axonal compartment is both necessary and sufficient to delay axonal degeneration. Furthermore, by specifically increasing axonal WldS expression postaxotomy, we reveal a critical period of 4-5 h postinjury during which the course of Wallerian axonal degeneration can be halted. Finally, we show that NAD(+), the metabolite of WldS/nicotinamide mononucleotide adenylyltransferase enzymatic activity, is sufficient and specific to confer WldS-like axon protection and is a likely molecular mediator of WldS axon protection. The results delineate a therapeutic window in which the course of Wallerian degeneration can be delayed even after injures have occurred and help narrow the molecular targets of WldS activity to events within the axonal compartment. PMID:26209654

  15. QUANTUM DOT SINGLE MOLECULE TRACKING REVEALS A WIDE RANGE OF DIFFUSIVE MOTIONS OF MEMBRANE TRANSPORT PROTEINS.

    PubMed

    Crane, Jonathan M; Haggie, Peter M; Verkman, A S

    2009-03-01

    Single particle tracking (SPT) provides information about the microscopic motions of individual particles in live cells. We applied SPT to study the diffusion of membrane transport proteins in cell plasma membranes in which individual proteins are labeled with quantum dots at engineered extracellular epitopes. Software was created to deduce particle diffusive modes from quantum dot trajectories. SPT of aquaporin (AQP) water channels and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels revealed several types of diffusion. AQP1 was freely mobile in cell membranes, showing rapid, Brownian-type diffusion. The full-length (M1) isoform of AQP4 also diffused rapidly, though the diffusion of a shorter (M23) isoform of AQP4 was highly restricted due to its supermolecular assembly in raft-like orthogonal arrays. CFTR mobility was also highly restricted, in a spring-like potential, due to its tethering to the actin cytoskeleton through PDZ-domain C-terminus interactions. The biological significance of regulated diffusion of membrane transport proteins is a subject of active investigation. PMID:24386532

  16. Local axonal protection by WldS as revealed by conditional regulation of protein stability

    PubMed Central

    Wang, Jack T.; Medress, Zachary A.; Vargas, Mauricio E.; Barres, Ben A.

    2015-01-01

    The expression of the mutant Wallerian degeneration slow (WldS) protein significantly delays axonal degeneration from various nerve injuries and in multiple species; however, the mechanism for its axonal protective property remains unclear. Although WldS is localized predominantly in the nucleus, it also is present in a smaller axonal pool, leading to conflicting models to account for the WldS fraction necessary for axonal protection. To identify where WldS activity is required to delay axonal degeneration, we adopted a method to alter the temporal expression of WldS protein in neurons by chemically regulating its protein stability. We demonstrate that continuous WldS activity in the axonal compartment is both necessary and sufficient to delay axonal degeneration. Furthermore, by specifically increasing axonal WldS expression postaxotomy, we reveal a critical period of 4–5 h postinjury during which the course of Wallerian axonal degeneration can be halted. Finally, we show that NAD+, the metabolite of WldS/nicotinamide mononucleotide adenylyltransferase enzymatic activity, is sufficient and specific to confer WldS-like axon protection and is a likely molecular mediator of WldS axon protection. The results delineate a therapeutic window in which the course of Wallerian degeneration can be delayed even after injures have occurred and help narrow the molecular targets of WldS activity to events within the axonal compartment. PMID:26209654

  17. Quantum dot single molecule tracking reveals a wide range of diffusive motions of membrane transport proteins

    NASA Astrophysics Data System (ADS)

    Crane, Jonathan M.; Haggie, Peter M.; Verkman, A. S.

    2009-02-01

    Single particle tracking (SPT) provides information about the microscopic motions of individual particles in live cells. We applied SPT to study the diffusion of membrane transport proteins in cell plasma membranes in which individual proteins are labeled with quantum dots at engineered extracellular epitopes. Software was created to deduce particle diffusive modes from quantum dot trajectories. SPT of aquaporin (AQP) water channels and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels revealed several types of diffusion. AQP1 was freely mobile in cell membranes, showing rapid, Brownian-type diffusion. The full-length (M1) isoform of AQP4 also diffused rapidly, though the diffusion of a shorter (M23) isoform of AQP4 was highly restricted due to its supermolecular assembly in raft-like orthogonal arrays. CFTR mobility was also highly restricted, in a spring-like potential, due to its tethering to the actin cytoskeleton through PDZ-domain C-terminus interactions. The biological significance of regulated diffusion of membrane transport proteins is a subject of active investigation.

  18. Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes

    PubMed Central

    Wong, Hui Hui; Kumar, Pankaj; Tay, Felicia Pei Ling; Moreau, Dimitri

    2015-01-01

    ABSTRACT Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. IMPORTANCE Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics. PMID:26311884

  19. Deletion of CFTR Translation Start Site Reveals Functional Isoforms of the Protein in CF Patients

    PubMed Central

    Ramalho, Anabela S.; Lewandowska, Marzena A.; Farinha, Carlos M.; Mendes, Filipa; Gonçalves, Juan; Barreto, Celeste; Harris, Ann; Amaral, Margarida D.

    2009-01-01

    Background/Aims: Mutations in the CFTR gene cause Cystic Fibrosis (CF) the most common life-threatening autosomal recessive disease affecting Caucasians. We identified a CFTR mutation (c.120del23) abolishing the normal translation initiation codon, which occurs in two Portuguese CF patients. This study aims at functionally characterizing the effect of this novel mutation. Methods: RNA and protein techniques were applied to both native tissues from CF patients and recombinant cells expressing CFTR constructs to determine whether c.120del23 allows CFTR protein production through usage of alternative internal codons, and to characterize the putative truncated CFTR form(s). Results: Our data show that two shorter forms of CFTR protein are produced when the initiation translation codon is deleted indicating usage of internal initiation codons. The N-truncated CFTR generated by this mutation has decreased stability, very low processing efficiency, and drastically reduced function. Analysis of mutants of four methionine codons downstream to M1 (M82, M150, M152, M156) revealed that each of the codons M150/M152/M156 (exon 4) can mediate CFTR alternative translation. Conclusions: The CFTR N-terminus has an important role in avoiding CFTR turnover and in rendering effective its plasma membrane traffic. These data correlate well with the severe clinical phenotype of CF patients bearing the c.120del23 mutation. PMID:19910674

  20. Cell-surface prion protein interacts with glycosaminoglycans.

    PubMed Central

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

    2002-01-01

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

  1. Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3.

    PubMed

    Bitencourt, Amanda R; Vicentin, Elaine C; Jimenez, Maria C; Ricci, Ricardo; Leite, Juliana A; Costa, Fabio T; Ferreira, Luis C; Russell, Bruce; Nosten, François; Rénia, Laurent; Galinski, Mary R; Barnwell, John W; Rodrigues, Mauricio M; Soares, Irene S

    2013-01-01

    A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3β of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2%) and at least 1 recombinant protein representing PvMSP-3β (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3β, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3α and PvMSP-3β elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential. PMID:23457498

  2. Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3

    PubMed Central

    Bitencourt, Amanda R.; Vicentin, Elaine C.; Jimenez, Maria C.; Ricci, Ricardo; Leite, Juliana A.; Costa, Fabio T.; Ferreira, Luis C.; Russell, Bruce; Nosten, François; Rénia, Laurent; Galinski, Mary R.; Barnwell, John W.; Rodrigues, Mauricio M.; Soares, Irene S.

    2013-01-01

    A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3β of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2%) and at least 1 recombinant protein representing PvMSP-3β (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3β, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3α and PvMSP-3β elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential. PMID:23457498

  3. A crayfish insulin-like-binding protein: another piece in the androgenic gland insulin-like hormone puzzle is revealed.

    PubMed

    Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir

    2013-08-01

    Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a "pulldown" methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23-38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079

  4. Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor

    PubMed Central

    Shao, Xiaohu; Jiang, Mengtian; Yu, Ziniu; Cai, Hao; Li, Lin

    2009-01-01

    Background Previous studies have revealed that the lysin motif (LysM) domains of bacterial cell wall-degrading enzymes are able to bind to peptidoglycan moieties of the cell wall. This suggests an approach for a cell surface display system in Gram-positive bacteria using a LysM-containing protein as the anchoring motif. In this study, we developed a new surface display system in B. thuringiensis using a LysM-containing peptidoglycan hydrolase, endo-β-N-acetylglucosaminidase (Mbg), as the anchor protein. Results Homology searching in the B. thuringiensis YBT-1520 genome revealed a putative peptidoglycan hydrolase gene. The encoded protein, Mbg, exhibited substantial cell-wall binding capacity. The deduced amino acid sequence of Mbg was structurally distinguished as an N-terminal domain with two tandemly aligned LysMs and a C-terminal catalytic domain. A GFP-fusion protein was expressed and used to verify the surface localization by Western blot, flow cytometry, protease accessibility, SDS sensitivity, immunofluorescence, and electron microscopy assays. Low-level constitutive expression of Mbg was elevated by introducing a sporulation-independent promoter of cry3Aa. Truncated Mbg domains with separate N-terminus (Mbgn), C-terminus (Mbgc), LysM1, or LysM2 were further compared for their cell-wall displaying efficiencies. The Mbgn moiety contributed to cell-wall anchoring, while LysM1 was the active domain. Two tandemly repeated Mbgns exhibited the highest display activity, while the activity of three repeated Mbgns was decreased. A heterologous bacterial multicopper oxidase (WlacD) was successfully displayed onto the surface of B. thuringiensis target cells using the optimum (Mbgn)2 anchor, without radically altering its catalytic activity. Conclusion Mbg can be a functional anchor protein to target different heterologous proteins onto the surface of B. thuringiensis cells. Since the LysM domain appears to be universal in Gram-positive bacteria, the strategy

  5. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    PubMed Central

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  6. Interaction with the unfolded protein response reveals a role for stargazin in biosynthetic AMPA receptor transport.

    PubMed

    Vandenberghe, Wim; Nicoll, Roger A; Bredt, David S

    2005-02-01

    The transmembrane protein stargazin enhances levels of functional AMPA receptors at the neuronal plasma membrane and at synapses. To clarify the mechanism for this effect, we studied trafficking of the AMPA receptor subunit glutamate receptor 1 (GluR1) in transfected COS7 cells. GluR1 expressed poorly on the surface of these cells and was primarily retained in the endoplasmic reticulum (ER). Stargazin expression strongly increased the surface fraction of GluR1. This effect was not reduced by a dominant-negative dynamin mutant, suggesting that stargazin does not inhibit AMPA receptor endocytosis. Interestingly, upregulation of ER chaperones as part of the unfolded protein response (UPR) both mimicked and occluded the effect of stargazin, suggesting a role for stargazin in ER processing of AMPA receptors. Consistent with this idea, we detected UPR induction in cerebellar granule cells lacking stargazin. Finally, residual AMPA receptor currents in stargazin-deficient neurons were suppressed by inhibition of the UPR. These findings uncover a role for stargazin in AMPA receptor trafficking through the early compartments of the biosynthetic pathway. Furthermore, they provide evidence for modulation of AMPA receptor trafficking by the UPR. PMID:15689545

  7. Modulation of Protein Fouling and Interfacial Properties at Carbon Surfaces via Immobilization of Glycans Using Aryldiazonium Chemistry.

    PubMed

    Zen, Federico; Angione, M Daniela; Behan, James A; Cullen, Ronan J; Duff, Thomas; Vasconcelos, Joana M; Scanlan, Eoin M; Colavita, Paula E

    2016-01-01

    Carbon materials and nanomaterials are of great interest for biological applications such as implantable devices and nanoparticle vectors, however, to realize their potential it is critical to control formation and composition of the protein corona in biological media. In this work, protein adsorption studies were carried out at carbon surfaces functionalized with aryldiazonium layers bearing mono- and di-saccharide glycosides. Surface IR reflectance absorption spectroscopy and quartz crystal microbalance were used to study adsorption of albumin, lysozyme and fibrinogen. Protein adsorption was found to decrease by 30-90% with respect to bare carbon surfaces; notably, enhanced rejection was observed in the case of the tested di-saccharide vs. simple mono-saccharides for near-physiological protein concentration values. ζ-potential measurements revealed that aryldiazonium chemistry results in the immobilization of phenylglycosides without a change in surface charge density, which is known to be important for protein adsorption. Multisolvent contact angle measurements were used to calculate surface free energy and acid-base polar components of bare and modified surfaces based on the van Oss-Chaudhury-Good model: results indicate that protein resistance in these phenylglycoside layers correlates positively with wetting behavior and Lewis basicity. PMID:27108562

  8. Modulation of Protein Fouling and Interfacial Properties at Carbon Surfaces via Immobilization of Glycans Using Aryldiazonium Chemistry

    NASA Astrophysics Data System (ADS)

    Zen, Federico; Angione, M. Daniela; Behan, James A.; Cullen, Ronan J.; Duff, Thomas; Vasconcelos, Joana M.; Scanlan, Eoin M.; Colavita, Paula E.

    2016-04-01

    Carbon materials and nanomaterials are of great interest for biological applications such as implantable devices and nanoparticle vectors, however, to realize their potential it is critical to control formation and composition of the protein corona in biological media. In this work, protein adsorption studies were carried out at carbon surfaces functionalized with aryldiazonium layers bearing mono- and di-saccharide glycosides. Surface IR reflectance absorption spectroscopy and quartz crystal microbalance were used to study adsorption of albumin, lysozyme and fibrinogen. Protein adsorption was found to decrease by 30–90% with respect to bare carbon surfaces; notably, enhanced rejection was observed in the case of the tested di-saccharide vs. simple mono-saccharides for near-physiological protein concentration values. ζ-potential measurements revealed that aryldiazonium chemistry results in the immobilization of phenylglycosides without a change in surface charge density, which is known to be important for protein adsorption. Multisolvent contact angle measurements were used to calculate surface free energy and acid-base polar components of bare and modified surfaces based on the van Oss-Chaudhury-Good model: results indicate that protein resistance in these phenylglycoside layers correlates positively with wetting behavior and Lewis basicity.

  9. Modulation of Protein Fouling and Interfacial Properties at Carbon Surfaces via Immobilization of Glycans Using Aryldiazonium Chemistry

    PubMed Central

    Zen, Federico; Angione, M. Daniela; Behan, James A.; Cullen, Ronan J.; Duff, Thomas; Vasconcelos, Joana M.; Scanlan, Eoin M.; Colavita, Paula E.

    2016-01-01

    Carbon materials and nanomaterials are of great interest for biological applications such as implantable devices and nanoparticle vectors, however, to realize their potential it is critical to control formation and composition of the protein corona in biological media. In this work, protein adsorption studies were carried out at carbon surfaces functionalized with aryldiazonium layers bearing mono- and di-saccharide glycosides. Surface IR reflectance absorption spectroscopy and quartz crystal microbalance were used to study adsorption of albumin, lysozyme and fibrinogen. Protein adsorption was found to decrease by 30–90% with respect to bare carbon surfaces; notably, enhanced rejection was observed in the case of the tested di-saccharide vs. simple mono-saccharides for near-physiological protein concentration values. ζ-potential measurements revealed that aryldiazonium chemistry results in the immobilization of phenylglycosides without a change in surface charge density, which is known to be important for protein adsorption. Multisolvent contact angle measurements were used to calculate surface free energy and acid-base polar components of bare and modified surfaces based on the van Oss-Chaudhury-Good model: results indicate that protein resistance in these phenylglycoside layers correlates positively with wetting behavior and Lewis basicity. PMID:27108562

  10. Protein Based Localized Surface Plasmon Resonance Gas Sensing

    NASA Astrophysics Data System (ADS)

    Meisam, Omidi; Gh., Amoabediny; Yazdian, F.; Habibi-Rezaei, M.

    2015-01-01

    We apply the localized surface plasmon resonance (LSPR) of gold nanoparticles (GNPs) covalently coupled with cytochrome c (cyt c) to create a nanobiosensor for detecting hydrogen sulfide (H2S) in the range of 15-100 ppb. Monolayer formation of GNPs on glass surface functionalized with 3-aminopropyltrimethoxysilane (APTMS) is performed for fabricating a chip-based format of the optical transducer. By chemical introduction of short-chain thiol derivatives on cyt c protein shell via its lysine residues, a very fast self-assembled monolayer (SAM) of cyt c is formed on the GNPs. Significant shifts in the LSPR peak (ΔλLSPR) are observed by reacting H2S with cyt c. Results show a linear relationship between ΔλLSPR and H2S concentration. Furthermore, shifts in the LSPR peak are reversible and the peak positions return to their pre-exposure values once the H2S is removed. The experimental results strongly indicate that the protein based LSPR chip can be successfully used as a simple, fast, sensitive and quantitative sensor for H2S detection.

  11. Adding Functions to Biomaterial Surfaces through Protein Incorporation.

    PubMed

    Wronska, Małgorzata A; O'Connor, Iain B; Tilbury, Maura A; Srivastava, Akshay; Wall, J Gerard

    2016-07-01

    The concept of biomaterials has evolved from one of inert mechanical supports with a long-term, biologically inactive role in the body into complex matrices that exhibit selective cell binding, promote proliferation and matrix production, and may ultimately become replaced by newly generated tissues in vivo. Functionalization of material surfaces with biomolecules is critical to their ability to evade immunorecognition, interact productively with surrounding tissues and extracellular matrix, and avoid bacterial colonization. Antibody molecules and their derived fragments are commonly immobilized on materials to mediate coating with specific cell types in fields such as stent endothelialization and drug delivery. The incorporation of growth factors into biomaterials has found application in promoting and accelerating bone formation in osteogenerative and related applications. Peptides and extracellular matrix proteins can impart biomolecule- and cell-specificities to materials while antimicrobial peptides have found roles in preventing biofilm formation on devices and implants. In this progress report, we detail developments in the use of diverse proteins and peptides to modify the surfaces of hard biomaterials in vivo and in vitro. Chemical approaches to immobilizing active biomolecules are presented, as well as platform technologies for isolation or generation of natural or synthetic molecules suitable for biomaterial functionalization. PMID:27164952

  12. The Treponema denticola Major Sheath Protein Is Predominantly Periplasmic and Has Only Limited Surface Exposure

    PubMed Central

    Caimano, Melissa J.; Bourell, Kenneth W.; Bannister, Teresa D.; Cox, David L.; Radolf, Justin D.

    1999-01-01

    The recent discovery that the Treponema pallidum genome encodes 12 orthologs of the Treponema denticola major sheath protein (Msp) prompted us to reexamine the cellular location and topology of the T. denticola polypeptide. Experiments initially were conducted to ascertain whether Msp forms an array on or within the T. denticola outer membrane. Transmission electron microscopy (EM) of negatively stained and ultrathin-sectioned organisms failed to identify a typical surface layer, whereas freeze-fracture EM revealed that the T. denticola outer membrane contains heterogeneous transmembrane proteins but no array. In contrast, a lattice-like structure was observed in vesicles released from mildly sonicated treponemes; combined EM and biochemical analyses demonstrated that this structure was the peptidoglycan sacculus. Immunoelectron microscopy (IEM) subsequently was performed to localize Msp in T. denticola. Examination of negatively stained whole mounts identified substantial amounts of Msp in sonicated organisms. IEM of ultrathin-sectioned, intact treponemes also demonstrated that the preponderance of antigen was unassociated with the outer membrane. Lastly, immunofluorescence analysis of treponemes embedded in agarose gel microdroplets revealed that only minor portions of Msp are surface exposed. Taken as a whole, our findings challenge the widely held belief that Msp forms an array within the T. denticola outer membrane and demonstrate, instead, that it is predominantly periplasmic with only limited surface exposure. These findings also have implications for our evolving understanding of the contribution(s) of Msp/Tpr orthologs to treponemal physiology and disease pathogenesis. PMID:10417176

  13. Protein kinase R reveals an evolutionary model for defeating viral mimicry

    PubMed Central

    Elde, Nels C.; Child, Stephanie J.; Geballe, Adam P.; Malik, Harmit S.

    2008-01-01

    Distinguishing self from non-self is a fundamental biological challenge. Many pathogens exploit the challenge of self discrimination by employing mimicry to subvert key cellular processes including the cell cycle, apoptosis, and cytoskeletal dynamics1-5. Other mimics interfere with immunity6, 7. Poxviruses encode K3L, a mimic of eIF2α, which is the substrate of Protein Kinase R (PKR), an important component of innate immunity in vertebrates8, 9. The PKR-K3L interaction exemplifies the conundrum imposed by viral mimicry. To be effective, PKR must recognize a conserved substrate (eIF2α) while avoiding rapidly evolving substrate mimics like K3L. Using the PKR-K3L system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry. We find that PKR has evolved under dramatic episodes of positive selection in primates. The ability of PKR to evade viral mimics is partly due to positive selection at sites most intimately involved in eIF2α recognition. We also find that adaptive changes on multiple surfaces of PKR produce combinations of substitutions that increase the odds of defeating mimicry. Thus, while it can appear that pathogens gain insurmountable advantages by mimicking cellular components, host factors like PKR can compete in molecular ‘arms races’ with mimics because of remarkable evolutionary flexibility at protein interaction interfaces challenged by mimicry. PMID:19043403

  14. Serological characterization of surface-exposed proteins of Coxiella burnetii.

    PubMed

    Jiao, Jun; Xiong, Xiaolu; Qi, Yong; Gong, Wenping; Duan, Changsong; Yang, Xiaomei; Wen, Bohai

    2014-12-01

    The obligate intracellular Gram-negative bacterium Coxiella burnetii causes Q fever, a worldwide zoonosis. Here we labelled Cox. burnetii with biotin and used biotin-streptavidin affinity chromatography to isolate surface-exposed proteins (SEPs). Using two-dimensional electrophoresis combined with mass spectrometry, we identified 37 proteins through bioinformatics analysis. Thirty SEPs expressed in Escherichia coli (recombinant SEPs, rSEPs) were used to generate microarrays, which were probed with sera from mice experimentally infected with Cox. burnetii or sera from Q fever patients. Thirteen rSEPs were recognized as seroreactive, and the majority reacted with at least 50 % of the sera from mice infected with Cox. burnetii but not with sera from mice infected with Rickettsia rickettsii, R. heilongjiangensis, or R. typhi. Further, 13 proteins that reacted with sera from patients with Q fever did not react with sera from patients with brucellosis or mycoplasma pneumonia. Our results suggest that these seroreactive SEPs have potential as serodiagnostic antigens or as subunit vaccine antigens against Q fever. PMID:25298245

  15. G-Protein Coupled Receptors: Surface Display and Biosensor Technology

    NASA Astrophysics Data System (ADS)

    McMurchie, Edward; Leifert, Wayne

    Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

  16. Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis

    PubMed Central

    2010-01-01

    Background Surface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE), a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F) or produced with an old-young smearing process (M). Results TTGE revealed nine bacterial species common to both F and M consortia, but consortium F exhibited a higher diversity than consortium M, with thirteen and ten species, respectively. Population dynamics were studied after application of the consortia on fresh-produced Raclette cheeses. TTGE analyses revealed a similar sequential development of the nine species common to both consortia. Beside common cheese surface bacteria (Staphylococcus equorum, Corynebacterium spp., Brevibacterium linens, Microbacterium gubbeenense, Agrococcus casei), the two consortia contained marine lactic acid bacteria (Alkalibacterium kapii, Marinilactibacillus psychrotolerans) that developed early in ripening (day 14 to 20), shortly after the growth of staphylococci (day 7). A decrease of Listeria counts was observed on cheese surface inoculated at day 7 with 0.1-1 × 102 CFU cm-2, when cheeses were smeared with consortium F or M. Listeria counts went below the detection limit of the method between day 14 and 28 and no subsequent regrowth was detected over 60 to 80 ripening days. In contrast, Listeria grew to high counts (105 CFU cm-2) on cheeses smeared with a defined surface culture. Conclusions This work reports

  17. Structure of the human DNA-repair protein RAD52 containing surface mutations.

    PubMed

    Saotome, Mika; Saito, Kengo; Onodera, Keiichi; Kurumizaka, Hitoshi; Kagawa, Wataru

    2016-08-01

    The Rad52 protein is a eukaryotic single-strand DNA-annealing protein that is involved in the homologous recombinational repair of DNA double-strand breaks. The isolated N-terminal half of the human RAD52 protein (RAD52(1-212)) forms an undecameric ring structure with a surface that is mostly positively charged. In the present study, it was found that RAD52(1-212) containing alanine mutations of the charged surface residues (Lys102, Lys133 and Glu202) is highly amenable to crystallization. The structure of the mutant RAD52(1-212) was solved at 2.4 Å resolution. The structure revealed an association between the symmetry-related RAD52(1-212) rings, in which a partially unfolded, C-terminal region of RAD52 extended into the DNA-binding groove of the neighbouring ring in the crystal. The alanine mutations probably reduced the surface entropy of the RAD52(1-212) ring and stabilized the ring-ring association observed in the crystal. PMID:27487923

  18. Polymer adhesion at surfaces: biological adhesive proteins and their synthetic mimics

    NASA Astrophysics Data System (ADS)

    Messersmith, Phillip

    2008-03-01

    Mussels are famous for their ability to permanently adhere to a wide variety of wet surfaces, such as rocks, metal and polymer ship hulls, and wood structures. They accomplish this through specialized proteins collectively referred to as mussel adhesive proteins (MAPs). The biophysical aspects of MAP adhesion is being revealed through the use of single molecule force measurements. The results provide insight into the adhesive roles of key amino acids found in these proteins, including the magnitude of adhesive forces, cooperative effects, and their self-healing properties. This molecular-level information is being incorporated into designs of biomimetic polymer coatings for a variety of applications. Our biomimetic approach to polymer design will be illustrated by a few examples where adhesive constituents found in MAPs are exploited to make wet-adhesive polymer coatings. In addition, small molecule analogs of MAPs can be used to apply thin functional films onto virtually any material surface using a facile approach. These coatings have a variety of potential uses in microelectronics, water treatment, prevention of environmental biofouling, and for control of biointerfacial phenomena at the surfaces of medical/diagnostic devices.

  19. Evolutionary biochemistry: revealing the historical and physical causes of protein properties

    PubMed Central

    Harms, Michael J.; Thornton, Joseph W.

    2014-01-01

    The repertoire of proteins and nucleic acids in the living world is determined by evolution; their properties are determined by the laws of physics and chemistry. Explanations of these two kinds of causality — the purviews of evolutionary biology and biochemistry, respectively — are typically pursued in isolation, but many fundamental questions fall squarely at the interface of fields. Here we articulate the paradigm of evolutionary biochemistry, which aims to dissect the physical mechanisms and evolutionary processes by which biological molecules diversified and to reveal how their physical architecture facilitates and constrains their evolution. We show how an integration of evolution with biochemistry moves us towards a more complete understanding of why biological molecules have the properties that they do. PMID:23864121

  20. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response

    PubMed Central

    Gao, Xing-Huang; Krokowski, Dawid; Guan, Bo-Jhih; Bederman, Ilya; Majumder, Mithu; Parisien, Marc; Diatchenko, Luda; Kabil, Omer; Willard, Belinda; Banerjee, Ruma; Wang, Benlian; Bebek, Gurkan; Evans, Charles R.; Fox, Paul L.; Gerson, Stanton L.; Hoppel, Charles L.; Liu, Ming; Arvan, Peter; Hatzoglou, Maria

    2015-01-01

    The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. DOI: http://dx.doi.org/10.7554/eLife.10067.001 PMID:26595448

  1. Surface-layer protein from Caulobacter crescentus: expression, purification and X-ray crystallographic analysis.

    PubMed

    Jones, Michael D; Chan, Anson C K; Nomellini, John F; Murphy, Michael E P; Smit, John

    2016-09-01

    Protein surface layers are self-assembling, paracrystalline lattices on the surface of many prokaryotes. Surface-layer proteins have not benefited from widespread structural analysis owing to their resistance to crystallization. Here, the successful expression of a truncated version of RsaA, the surface-layer protein from Caulobacter crescentus, from a Caulobacter protein-expression system is reported. The purification, crystallization and initial X-ray diffraction analysis of the truncated RsaA, the largest surface-layer protein studied to date and the first from a Gram-negative bacterium, are also reported. PMID:27599857

  2. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering

    DOE PAGESBeta

    Close, Devin W.; Paul, Craig Don; Langan, Patricia S.; Wilce, Matthew C. J.; Traore, Daouda A. K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; et al

    2015-05-08

    In this paper, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction ofmore » high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.« less

  3. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering

    SciTech Connect

    Close, Devin W.; Paul, Craig Don; Langan, Patricia S.; Wilce, Matthew C. J.; Traore, Daouda A. K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; Prescott, Mark; Bradbury, Andrew R. M.

    2015-05-08

    In this paper, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.

  4. Automated design of the surface positions of protein helices.

    PubMed Central

    Dahiyat, B. I.; Gordon, D. B.; Mayo, S. L.

    1997-01-01

    Using a protein design algorithm that quantitatively considers side-chain interactions, the design of surface residues of alpha helices was examined. Three scoring functions were tested: a hydrogen-bond potential, a hydrogen-bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen-bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4-p1 were designed by using the Dead-End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 degree C, with melting temperatures from 69-72 degrees C, over 12 degrees C higher than GCN4-p1, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 degrees C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions. PMID:9194194

  5. Proteins in the electric field near the surface of mica.

    PubMed

    Starzyk, Anna; Cieplak, Marek

    2013-07-28

    We elucidate the nature of the electric field produced by a model mica surface and show that above some 0.4 nm it is nearly uniform and of order 12 V/nm. The presence of ions in the solvent above the surface, up to the concentration of about 300 mM, does not modify the nature of the field much. We study the conformational changes of a small protein, the tryptophan cage, as induced by (a) uniform electric field and (b) the electric field near mica. We use all-atom molecular dynamics simulations and provide evidence for the existence of unfolded and deformed conformations in each of these cases. The two behaviors are characterized by distinct properties of the radius of gyration and of the distortion parameter that distinguishes between elongated and globular shapes. The overall geometry of the conformations shifts with the strengths of the uniform field in a manner that depends on the nature of the simulation box--whether it is bounded by neutral walls or not--and on the ionic concentration. Near the mica surface, on the other hand, the fraction of unfolded conformations is close to 1/6 at the ionic strength of 350 mM compared to 1/2 at 20 mM. When the electric charge on the mica is fully neutralized by bringing more ions of the opposite charge then unfolded conformations stay unfolded but an evolution from the native state does not lead to any unfolding. PMID:23902027

  6. Proteins in the electric field near the surface of mica

    NASA Astrophysics Data System (ADS)

    Starzyk, Anna; Cieplak, Marek

    2013-07-01

    We elucidate the nature of the electric field produced by a model mica surface and show that above some 0.4 nm it is nearly uniform and of order 12 V/nm. The presence of ions in the solvent above the surface, up to the concentration of about 300 mM, does not modify the nature of the field much. We study the conformational changes of a small protein, the tryptophan cage, as induced by (a) uniform electric field and (b) the electric field near mica. We use all-atom molecular dynamics simulations and provide evidence for the existence of unfolded and deformed conformations in each of these cases. The two behaviors are characterized by distinct properties of the radius of gyration and of the distortion parameter that distinguishes between elongated and globular shapes. The overall geometry of the conformations shifts with the strengths of the uniform field in a manner that depends on the nature of the simulation box — whether it is bounded by neutral walls or not — and on the ionic concentration. Near the mica surface, on the other hand, the fraction of unfolded conformations is close to 1/6 at the ionic strength of 350 mM compared to 1/2 at 20 mM. When the electric charge on the mica is fully neutralized by bringing more ions of the opposite charge then unfolded conformations stay unfolded but an evolution from the native state does not lead to any unfolding.

  7. Single Molecule FRET of Protein-Nucleic Acid and Protein-Protein complexes: Surface Passivation and Immobilization

    PubMed Central

    Lamichhane, Rajan; Solem, Amanda; Black, Will; Rueda, David

    2010-01-01

    Single-molecule fluorescence spectroscopy reveals the real time dynamics that occur during biomolecular interactions that would otherwise be hidden by the ensemble average. It also removes the requirement to synchronize reactions, thus providing a very intuitive approach to study kinetics of biological systems. Surface immobilization is commonly used to increase observation times to the minute time scale, but it can be detrimental if the sample interacts non-specifically with the surface. Here, we review detailed protocols to prevent such interactions by passivating the surface or by trapping the molecules inside surface immobilized lipid vesicles. Finally, we discuss recent examples where these methods were applied to study the dynamics of important cellular processes at the single molecule level. PMID:20554047

  8. Characterization of the Autophagy Marker Protein Atg8 Reveals Atypical Features of Autophagy in Plasmodium falciparum

    PubMed Central

    Allanki, Aparna Devi; Sijwali, Puran Singh

    2014-01-01

    Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8) employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64) and aspartic (pepstatin) protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine), indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in apicoplast

  9. Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila.

    PubMed Central

    Bannon, G A; Perkins-Dameron, R; Allen-Nash, A

    1986-01-01

    The presence of specific proteins (known as immobilization antigens) on the surface of the ciliated protozoan Tetrahymena thermophila is under environmental regulation. There are five different classes (serotypes) of surface proteins which appear on the cell surface when T. thermophila is cultured under different conditions of temperature or incubation medium; three of these are temperature dependent. The appearance of these proteins on the cell surface is mutually exclusive. We used polyclonal antibodies raised against 30 degrees C (designated SerH3)- and 40 degrees C (designated SerT)-specific surface antigens to study their structure and expression. We showed that these surface proteins contain at least one disulfide bridge. On sodium dodecyl sulfate-denaturing polyacrylamide gels, the nonreduced 30 degrees C- and 40 degrees C-specific surface proteins migrated with molecular sizes of 69 and 36 kilodaltons, respectively. The reduced forms of the proteins migrated with molecular sizes of 58 and 30 kilodaltons, respectively. The synthesis of the surface proteins responded rapidly and with a time course similar to that of the incubation temperature. The synthesis of each surface protein was greatly reduced within 1 h and undetectable by 2 h after a shift to the temperature at which the protein is not expressed. Surface protein synthesis resumed by the end of 1 h after a shift to the temperature at which the protein is expressed. The temperature-dependent induction of these surface proteins appears to be dependent on the synthesis of new mRNA, as indicated by a sensitivity to actinomycin D. Surface protein syntheses were mutually exclusive except at a transition temperature. At 35 degrees C both surface proteins were synthesized by a cell population. These data support the potential of this system as a model for the study of the effects of environmental factors on the genetic regulation of cell surface proteins. Images PMID:3537733

  10. Proteomic Analysis of Proteins Surrounding Occludin and Claudin-4 Reveals Their Proximity to Signaling and Trafficking Networks

    PubMed Central

    Fredriksson, Karin; Van Itallie, Christina M.; Aponte, Angel; Gucek, Marjan; Tietgens, Amber J.; Anderson, James M.

    2015-01-01

    Tight junctions are complex membrane structures that regulate paracellular movement of material across epithelia and play a role in cell polarity, signaling and cytoskeletal organization. In order to expand knowledge of the tight junction proteome, we used biotin ligase (BioID) fused to occludin and claudin-4 to biotinylate their proximal proteins in cultured MDCK II epithelial cells. We then purified the biotinylated proteins on streptavidin resin and identified them by mass spectrometry. Proteins were ranked by relative abundance of recovery by mass spectrometry, placed in functional categories, and compared not only among the N- and C- termini of occludin and the N-terminus of claudin-4, but also with our published inventory of proteins proximal to the adherens junction protein E-cadherin and the tight junction protein ZO-1. When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins. Apart from already known tight junction- proteins, occludin and claudin-4 proximal proteins were enriched in signaling and trafficking proteins, especially endocytic trafficking proteins. However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex. Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure. PMID:25789658

  11. Crystal structure analysis reveals Pseudomonas PilY1 as an essential calcium-dependent regulator of bacterial surface motility

    SciTech Connect

    Orans, Jillian; Johnson, Michael D.L.; Coggan, Kimberly A.; Sperlazza, Justin R.; Heiniger, Ryan W.; Wolfgang, Matthew C.; Redinbo, Matthew R.

    2010-09-21

    Several bacterial pathogens require the 'twitching' motility produced by filamentous type IV pili (T4P) to establish and maintain human infections. Two cytoplasmic ATPases function as an oscillatory motor that powers twitching motility via cycles of pilus extension and retraction. The regulation of this motor, however, has remained a mystery. We present the 2.1 {angstrom} resolution crystal structure of the Pseudomonas aeruginosa pilus-biogenesis factor PilY1, and identify a single site on this protein required for bacterial translocation. The structure reveals a modified {beta}-propeller fold and a distinct EF-hand-like calcium-binding site conserved in pathogens with retractile T4P. We show that preventing calcium binding by PilY1 using either an exogenous calcium chelator or mutation of a single residue disrupts Pseudomonas twitching motility by eliminating surface pili. In contrast, placing a lysine in this site to mimic the charge of a bound calcium interferes with motility in the opposite manner - by producing an abundance of nonfunctional surface pili. Our data indicate that calcium binding and release by the unique loop identified in the PilY1 crystal structure controls the opposing forces of pilus extension and retraction. Thus, PilY1 is an essential, calcium-dependent regulator of bacterial twitching motility.

  12. Crystal structure analysis reveals Pseudomonas PilY1 as an essential calcium-dependent regulator of bacterial surface motility.

    PubMed

    Orans, Jillian; Johnson, Michael D L; Coggan, Kimberly A; Sperlazza, Justin R; Heiniger, Ryan W; Wolfgang, Matthew C; Redinbo, Matthew R

    2010-01-19

    Several bacterial pathogens require the "twitching" motility produced by filamentous type IV pili (T4P) to establish and maintain human infections. Two cytoplasmic ATPases function as an oscillatory motor that powers twitching motility via cycles of pilus extension and retraction. The regulation of this motor, however, has remained a mystery. We present the 2.1 A resolution crystal structure of the Pseudomonas aeruginosa pilus-biogenesis factor PilY1, and identify a single site on this protein required for bacterial translocation. The structure reveals a modified beta-propeller fold and a distinct EF-hand-like calcium-binding site conserved in pathogens with retractile T4P. We show that preventing calcium binding by PilY1 using either an exogenous calcium chelator or mutation of a single residue disrupts Pseudomonas twitching motility by eliminating surface pili. In contrast, placing a lysine in this site to mimic the charge of a bound calcium interferes with motility in the opposite manner--by producing an abundance of nonfunctional surface pili. Our data indicate that calcium binding and release by the unique loop identified in the PilY1 crystal structure controls the opposing forces of pilus extension and retraction. Thus, PilY1 is an essential, calcium-dependent regulator of bacterial twitching motility. PMID:20080557

  13. Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma

    NASA Astrophysics Data System (ADS)

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-11-01

    Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance.

  14. Dynamic Allostery of the Catabolite Activator Protein Revealed by Interatomic Forces

    PubMed Central

    Louet, Maxime; Seifert, Christian; Hensen, Ulf; Gräter, Frauke

    2015-01-01

    The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals. PMID:26244893

  15. The structure of sperm Izumo1 reveals unexpected similarities with Plasmodium invasion proteins.

    PubMed

    Nishimura, Kaoru; Han, Ling; Bianchi, Enrica; Wright, Gavin J; de Sanctis, Daniele; Jovine, Luca

    2016-07-25

    Fertilization, the culminating event in sexual reproduction, occurs when haploid sperm and egg recognize each other and fuse to form a diploid zygote. In mammals this process critically depends on the interaction between Izumo1, a protein exposed on the equatorial segment of acrosome-reacted sperm, and the egg plasma-membrane-anchored receptor Juno [1,2]. The molecular mechanism triggering gamete fusion is unresolved because both Izumo1 and Juno lack sequence similarity to known membrane fusogens. Here we report the crystal structure of Izumo1, which reveals a membrane distal region composed of a four-helix bundle connected to a carboxy-terminal immunoglobulin (Ig)-like domain through a β-hairpin stabilized by disulfide bonds. Remarkably, different regions of Izumo1 display significant structural similarities to two proteins expressed by the invasive sporozoite stage of Plasmodium parasites: SPECT1, which is essential for host cell traversal and hepatocyte invasion [3]; and TRAP, which is necessary for gliding motility and invasion [4]. These observations suggest a link between the molecular mechanisms underlying host cell invasion by the malaria parasite and gamete membrane fusion at fertilization. PMID:27374339

  16. Structural Analysis of Shu Proteins Reveals a DNA Binding Role Essential for Resisting Damage*

    PubMed Central

    Tao, Yuyong; Li, Xu; Liu, Yiwei; Ruan, Jianbin; Qi, Shali; Niu, Liwen; Teng, Maikun

    2012-01-01

    The yeast Shu complex, consisting of the proteins Shu1, Shu2, Psy3, and Csm2, maintains genomic stability by coupling post-replication repair to homologous recombination. However, a lack of biochemical and structural information on the Shu proteins precludes revealing their precise roles within the pathway. Here, we report on the 1.9-Å crystal structure of the Psy3-Csm2 complex. The crystal structure shows that Psy3 forms a heterodimer with Csm2 mainly through a hydrophobic core. Unexpectedly, Psy3 and Csm2 share a similar architecture that closely resembles the ATPase core domain of Rad51. The L2 loop present in Psy3 and Csm2 is similar to that of Rad51 and confers the DNA binding activity of the Shu complex. As with Rad51, the Shu complex appears to form a nucleoprotein filament by binding nonspecifically to DNA. Structure-based mutagenesis studies have demonstrated that the DNA binding activity of the Shu complex is essential for repair of the methyl methanesulfonate-induced DNA damage. Our findings provide good foundations for the understanding of the Srs2 regulation by the Shu complex. PMID:22465956

  17. Mammalian Axoneme Central Pair Complex Proteins: Broader Roles Revealed by Gene Knockout Phenotypes

    PubMed Central

    Teves, Maria E.; Nagarkatti-Gude, David R.; Zhang, Zhibing; Strauss, Jerome F.

    2016-01-01

    The axoneme genes, their encoded proteins, their functions and the structures they form are largely conserved across species. Much of our knowledge of the function and structure of axoneme proteins in cilia and flagella is derived from studies on model organisms like the green algae, Chlamydomonas reinhardtii. The core structure of cilia and flagella is the axoneme, which in most motile cilia and flagella contains a 9 + 2 configuration of microtubules. The two central microtubules are the scaffold of the central pair complex (CPC). Mutations that disrupt CPC genes in Chlamydomonas and other model organisms result in defects in assembly, stability and function of the axoneme, leading to flagellar motility defects. However, targeted mutations generated in mice in the orthologous CPC genes have revealed significant differences in phenotypes of mutants compared to Chlamydomonas. Here we review observations that support the concept of cell-type specific roles for the CPC genes in mice, and an expanded repertoire of functions for the products of these genes in cilia, including non-motile cilia, and other microtubule-associated cellular functions. PMID:26785425

  18. Proteomics profiling reveals novel proteins and functions of the plant stigma exudate

    PubMed Central

    Rejón, Juan David; Delalande, François; Castro, Antonio Jesús

    2013-01-01

    Proteomic analysis of the stigmatic exudate of Lilium longiflorum and Olea europaea led to the identification of 51 and 57 proteins, respectively, most of which are described for the first time in this secreted fluid. These results indicate that the stigmatic exudate is an extracellular environment metabolically active, participating in at least 80 different biological processes and 97 molecular functions. The stigma exudate showed a markedly catabolic profile and appeared to possess the enzyme machinery necessary to degrade large polysaccharides and lipids secreted by papillae to smaller units, allowing their incorporation into the pollen tube during pollination. It may also regulate pollen-tube growth in the pistil through the selective degradation of tube-wall components. Furthermore, some secreted proteins were involved in pollen-tube adhesion and orientation, as well as in programmed cell death of the papillae cells in response to either compatible pollination or incompatible pollen rejection. Finally, the results also revealed a putative cross-talk between genetic programmes regulating stress/defence and pollination responses in the stigma. PMID:24151302

  19. Dynamic Allostery of the Catabolite Activator Protein Revealed by Interatomic Forces.

    PubMed

    Louet, Maxime; Seifert, Christian; Hensen, Ulf; Gräter, Frauke

    2015-08-01

    The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals. PMID:26244893

  20. Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma

    PubMed Central

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-01-01

    Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance. PMID:26582089

  1. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    NASA Astrophysics Data System (ADS)

    He, Yi-Ming; Ma, Bin-Guang

    2016-05-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

  2. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    PubMed Central

    He, Yi-Ming; Ma, Bin-Guang

    2016-01-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions. PMID:27220911

  3. New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

    PubMed Central

    Sehgal, Kapil; Sylvester, Marc; Skubal, Magdalena; Josten, Michele; Steinhäuser, Christian; De Koninck, Paul; Theis, Martin

    2016-01-01

    Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future. PMID:26915047

  4. Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling

    PubMed Central

    Scearce-Levie, Kimberly; Lieberman, Michael D; Elliott, Heather H; Conklin, Bruce R

    2005-01-01

    Background The physiological regulation of G protein-coupled receptors, through desensitization and internalization, modulates the length of the receptor signal and may influence the development of tolerance and dependence in response to chronic drug treatment. To explore the importance of receptor regulation, we engineered a series of Gi-coupled receptors that differ in signal length, degree of agonist-induced internalization, and ability to induce adenylyl cyclase superactivation. All of these receptors, based on the kappa opioid receptor, were modified to be receptors activated solely by synthetic ligands (RASSLs). This modification allows us to compare receptors that have the same ligands and effectors, but differ only in desensitization and internalization. Results Removal of phosphorylation sites in the C-terminus of the RASSL resulted in a mutant that was resistant to internalization and less prone to desensitization. Replacement of the C-terminus of the RASSL with the corresponding portion of the mu opioid receptor eliminated the induction of AC superactivation, without disrupting agonist-induced desensitization or internalization. Surprisingly, removal of phosphorylation sites from this chimera resulted in a receptor that is constitutively internalized, even in the absence of agonist. However, the receptor still signals and desensitizes in response to agonist, indicating normal G-protein coupling and partial membrane expression. Conclusions These studies reveal that internalization, desensitization and adenylyl cyclase superactivation, all processes that decrease chronic Gi-receptor signals, are independently regulated. Furthermore, specific mutations can radically alter superactivation or internalization without affecting the efficacy of acute Gi signaling. These mutant RASSLs will be useful for further elucidating the temporal dynamics of the signaling of G protein-coupled receptors in vitro and in vivo. PMID:15707483

  5. New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.

    PubMed

    Kaczmarczyk, Lech; Labrie-Dion, Étienne; Sehgal, Kapil; Sylvester, Marc; Skubal, Magdalena; Josten, Michele; Steinhäuser, Christian; De Koninck, Paul; Theis, Martin

    2016-01-01

    Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3'UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future. PMID:26915047

  6. Structural analysis and insertion study reveal the ideal sites for surface displaying foreign peptides on a betanodavirus-like particle.

    PubMed

    Xie, Junfeng; Li, Kunpeng; Gao, Yuanzhu; Huang, Runqing; Lai, Yuxiong; Shi, Yan; Yang, Shaowei; Zhu, Guohua; Zhang, Qinfen; He, Jianguo

    2016-01-01

    Betanodavirus infection causes fatal disease of viral nervous necrosis in many cultured marine and freshwater fish worldwide and the virus-like particles (VLP) are effective vaccines against betanodavirus. But vaccine and viral vector designs of betanodavirus VLP based on their structures remain lacking. Here, the three-dimensional structure of orange-spotted grouper nervous necrosis virus (OGNNV) VLP (RBS) at 3.9 Å reveals the organization of capsid proteins (CP). Based on the structural results, seven putative important sites were selected to genetically insert a 6× histidine (His)-tag for VLP formation screen, resulting in four His-tagged VLP (HV) at positions N-terminus, Ala220, Pro292 and C-terminus. The His-tags of N-terminal HV (NHV) were concealed inside virions while those of 220HV and C-terminal HV (CHV) were displayed at the outer surface. NHV, 220HV and CHV maintained the same cell entry ability as RBS in the Asian sea bass (SB) cell line, indicating that their similar surface structures can be recognized by the cellular entry receptor(s). For application of vaccine design, chromatography-purified CHV could provoke NNV-specific antibody responses as strong as those of RBS in a sea bass immunization assay. Furthermore, in carrying capacity assays, N-terminus and Ala220 can only carry short peptides and C-terminus can even accommodate large protein such as GFP to generate fluorescent VLP (CGV). For application of a viral vector, CGV could be real-time visualized to enter SB cells in invasion study. All the results confirmed that the C-terminus of CP is a suitable site to accommodate foreign peptides for vaccine design and viral vector development. PMID:26754256

  7. Survey of activated kinase proteins reveals potential targets for cholangiocarcinoma treatment.

    PubMed

    Dokduang, Hasaya; Juntana, Sirinun; Techasen, Anchalee; Namwat, Nisana; Yongvanit, Puangrat; Khuntikeo, Narong; Riggins, Gregory J; Loilome, Watcharin

    2013-12-01

    Improving therapy for patients with cholangiocarcinoma (CCA) presents a significant challenge. This is made more difficult by a lack of a clear understanding of potential molecular targets, such as deregulated kinases. In this work, we profiled the activated kinases in CCA in order to apply them as the targets for CCA therapy. Human phospho-receptor tyrosine kinases (RTKs) and phospho-kinase array analyses revealed that multiple kinases are activated in both CCA cell lines and human CCA tissues that included cell growth, apoptosis, cell to cell interaction, movement, and angiogenesis RTKs. Predominately, the kinases activated downstream were those in the PI3K/Akt, Ras/MAPK, JAK/STAT, and Wnt/β-catenin signaling pathways. Western blot analysis confirms that Erk1/2 and Akt activation were increased in CCA tissues when compared with their normal adjacent tissue. The inhibition of kinase activation using multi-targeted kinase inhibitors, sorafenib and sunitinib led to significant cell growth inhibition and apoptosis induction via suppression of Erk1/2 and Akt activation, whereas drugs with specificity to a single kinase showed less potency. In conclusion, our study reveals the involvement of multiple kinase proteins in CCA growth that might serve as therapeutic targets for combined kinase inhibition. PMID:23812726

  8. The involvement of proline-rich protein Mus musculus predicted gene 4736 in ocular surface functions

    PubMed Central

    Qi, Xia; Ren, Sheng-Wei; Zhang, Feng; Wang, Yi-Qiang

    2016-01-01

    AIM To research the two homologous predicted proline-rich protein genes, Mus musculus predicted gene 4736 (MP4) and proline-rich protein BstNI subfamily 1 (Prb1) which were significantly upregulated in cultured corneal organs when encountering fungal pathogen preparations. This study was to confirm the expression and potential functions of these two genes in ocular surface. METHODS A Pseudomonas aeruginosa keratitis model was established in Balb/c mice. One day post infection, mRNA level of MP4 was measured using real-time polymerase chain reaction (PCR), and MP4 protein detected by immunohistochemistry (IHC) or Western blot using a customized polyclonal anti-MP4 antibody preparation. Lacrimal glands from normal mice were also subjected to IHC staining for MP4. An online bioinformatics program, BioGPS, was utilized to screen public data to determine other potential locations of MP4. RESULTS One day afte