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Sample records for adhesin complex protein

  1. Discovery of a novel periplasmic protein that forms a complex with a trimeric autotransporter adhesin and peptidoglycan.

    PubMed

    Ishikawa, Masahito; Yoshimoto, Shogo; Hayashi, Ayumi; Kanie, Junichi; Hori, Katsutoshi

    2016-08-01

    Trimeric autotransporter adhesins (TAAs), fibrous proteins on the cell surface of Gram-negative bacteria, have attracted attention as virulence factors. However, little is known about the mechanism of their biogenesis. AtaA, a TAA of Acinetobacter sp. Tol 5, confers nonspecific, high adhesiveness to bacterial cells. We identified a new gene, tpgA, which forms a single operon with ataA and encodes a protein comprising two conserved protein domains identified by Pfam: an N-terminal SmpA/OmlA domain and a C-terminal OmpA_C-like domain with a peptidoglycan (PGN)-binding motif. Cell fractionation and a pull-down assay showed that TpgA forms a complex with AtaA, anchoring it to the outer membrane (OM). Isolation of total PGN-associated proteins showed TpgA binding to PGN. Disruption of tpgA significantly decreased the adhesiveness of Tol 5 because of a decrease in surface-displayed AtaA, suggesting TpgA involvement in AtaA secretion. This is reminiscent of SadB, which functions as a specific chaperone for SadA, a TAA in Salmonella species; however, SadB anchors to the inner membrane, whereas TpgA anchors to the OM through AtaA. The genetic organization encoding the TAA-TpgA-like protein cassette can be found in diverse Gram-negative bacteria, suggesting a common contribution of TpgA homologues to TAA biogenesis. PMID:27074146

  2. Protein secretion systems and adhesins: the molecular armory of Gram-negative pathogens.

    PubMed

    Gerlach, Roman G; Hensel, Michael

    2007-10-01

    Protein secretion is a basic cellular function found in organisms of all kingdoms of life. Gram-negative bacteria have evolved a remarkable number of pathways for the transport of proteins across the cell envelope. The secretion systems fulfill general cellular functions but are also essential for pathogenic bacteria during the interaction with eukaryotic host cells. Secretion systems range from relatively simple structures such as type I secretion systems composed of three subunits that only secrete one substrate protein to complex machines such as type III and IV secretion systems composed of more than 20 subunits that can translocate large sets of effector proteins into eukaryotic target cells. In this review, the main structural and functional features of secretion systems are described. One subgroup of substrate proteins of secretion systems are protein adhesins. Despite the conserved function in binding to host cell ligands or to abiotic surfaces, the assembly of the various bacterial adhesins is highly divergent. Here we give an overview on the recent understanding of the assembly of fimbrial and non-fimbrial adhesins and the role of type I, III and V secretion systems and specialized branches of the general secretion pathway in their biogenesis. PMID:17482513

  3. EHEC Adhesins

    PubMed Central

    McWilliams, Brian D.; Torres, Alfredo G.

    2014-01-01

    Adhesins are a group of proteins in enterohemorrhagic Escherichia coli (EHEC) that are involved in the attachment or colonization of this pathogen to abiotic (plastic or steel) and biological surfaces, such as those found in bovine and human intestines. This review provides the most up-to-date information on these essential adhesion factors, summarizing important historical discoveries and analyzing the current and future state of this research. In doing so, the proteins intimin and Tir are discussed in depth, especially regarding their role in the development of attaching and effacing lesions and in EHEC virulence. Further, a series of fimbrial proteins (Lpf1, Lpf2, curli, ECP, F9, ELF, Sfp, HCP, and type 1 fimbriae) are also described, emphasizing their various contributions to adherence and colonization of different surfaces and their potential use as genetic markers in detection and classification of different EHEC serotypes. This review also discusses the role of several autotransporter proteins (EhaA-D, EspP, Saa and Sab, and Cah), as well as other proteins associated with adherence, such as flagella, EibG, Iha, and OmpA. While these proteins have all been studied to varying degrees, all of the adhesins summarized in this chapter have been linked to different stages of the EHEC life cycle, making them good targets for the development of more effective diagnostics and therapeutics. PMID:25635238

  4. The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation.

    PubMed

    Diaz, Suraya A; Martin, Stephen R; Howell, Steven A; Grainger, Munira; Moon, Robert W; Green, Judith L; Holder, Anthony A

    2016-01-01

    Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity. PMID

  5. Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase

    PubMed Central

    Beaussart, Audrey; Brandhorst, Tristan

    2015-01-01

    ABSTRACT Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. PMID:26396244

  6. Minimum Chemical Requirements for Adhesin Activity of the Acid-Stable Part of Candida albicans Cell Wall Phosphomannoprotein Complex

    PubMed Central

    Kanbe, Toshio; Cutler, Jim E.

    1998-01-01

    This study was conducted to define adhesive characteristics of the acid-stable moiety of the Candida albicans phosphomannoprotein complex (PMPC) on adherence of this fungus to marginal zone macrophages of the mouse spleen. Complete digestion of the acid-stable moiety (Fr.IIS) of the C. albicans PMPC with an α-mannosidase or hydrolysis with 0.6 N sulfuric acid destroyed adhesin activity, as determined by the inability of the soluble digests to inhibit yeast cell adherence to the splenic marginal zone. Fr.IIS adhesin activity was decreased following digestion with an α-1,2-specific mannosidase. Oligomannosyls consisting of one to six mannose units, which were isolated from the acid-stable part of the PMPC, did not inhibit yeast cell binding and thus do not function alone as adhesin sites in the PMPC. To gain more insight into the minimum requirements for adhesin activity, PMPCs were isolated from a Saccharomyces cerevisiae wild-type strain and from mutant strains mnn1, mnn2, and mnn4; the PMPCs were designated scwt/Fr.II, scmn1/Fr.II, scmn2/Fr.II, and scmn4/Fr.II, respectively. S. cerevisiae scmn2/Fr.II lacks oligomannosyl side chain branches from the outer core mannan, and scmn2/Fr.II was the only PMPC without adhesin activity. S. cerevisiae scwt/Fr.II, scmn1/Fr.II, and scmn4/Fr.II showed adhesin activities less than that of C. albicans Fr.II. These three S. cerevisiae PMPCs are generally similar to Fr.IIS, except that the S. cerevisiae structure has fewer and shorter side chains. Immunofluorescence microscopy show that the acid-stable part of the PMPC is displayed homogeneously on the C. albicans yeast cell surface, which would be expected for a surface adhesin. Our results indicate that both the mannan core and the oligomannosyl side chains are responsible for the adhesin activity of the acid-stable part of the PMPC. PMID:9826359

  7. Evidence for adhesin activity in the acid-stable moiety of the phosphomannoprotein cell wall complex of Candida albicans.

    PubMed Central

    Kanbe, T; Cutler, J E

    1994-01-01

    Previously, we showed that Candida albicans hydrophilic yeast cells adhere specifically to mouse splenic marginal-zone macrophages. The adhesins are part of the yeast cell wall phosphomannoprotein complex, and one adhesin site, which reacts with the monoclonal antibody 10G, was identified as a beta-1,2-linked tetramannose in the acid-labile portion of the complex. We report here that the acid-stable part of the complex, which has not been reported previously to have adhesin activity, is in large part responsible for yeast cell binding to the splenic marginal zone. The phosphomannoprotein complex, termed Fr.II, was isolated from C. albicans serotype B yeast cells by beta-mercaptoethanol extraction and concanavalin A-agarose affinity chromatography. Fr.II is devoid of the serotype A-specific antigen factor 6, which functions in yeast cell attachment to epithelial cells. The acid-stable part of Fr.II (i.e., Fr.IIS) was obtained by mild acid hydrolysis and size exclusion fractionation. Fr.IIS was further fractionated into four fractions, Fr.IIS1, Fr.IIS2, Fr.IIS3, and Fr.IIS4, by concanavalin A-agarose column chromatography and elution with a linear gradient of alpha-methyl-D-mannopyranoside. Adhesin activity of these fractions was determined by their ability to block yeast cell binding to the splenic marginal zone. Fr.IIS1 and Fr.IIS2 yielded more material and stronger adhesin activity than either Fr.IIS3 or Fr.IIS4. Only Fr.IIS1 did not react with antibodies (anti-factor 5 and monoclonal antibody 10G) specific for the acid-labile beta-1,2-linked oligosaccharides. Fr.IIS1-coated latex beads attached specifically to the marginal zone in a pattern identical to that of yeast cell binding. Furthermore, Fr.IIS1-latex bead attachment was inhibited by soluble Fr.II or Fr.IIS. Initial chemical analyses indicate that the adhesin site on Fr.IIS1 is a carbohydrate because adhesin activity was destroyed by periodate oxidation but not by proteinase K digestion. Images PMID:8168927

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity

    PubMed Central

    Guo, Shuaiqi; Garnham, Christopher P.; Whitney, John C.; Graham, Laurie A.; Davies, Peter L.

    2012-01-01

    A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice. PMID:23144980

  10. Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

    PubMed

    Guo, Shuaiqi; Garnham, Christopher P; Whitney, John C; Graham, Laurie A; Davies, Peter L

    2012-01-01

    A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca(2+) levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+)-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice. PMID:23144980

  11. Dependence of Bacterial Protein Adhesins on Toll-Like Receptors for Proinflammatory Cytokine Induction

    PubMed Central

    Hajishengallis, George; Martin, Michael; Sojar, Hakimuddin T.; Sharma, Ashu; Schifferle, Robert E.; DeNardin, Ernesto; Russell, Michael W.; Genco, Robert J.

    2002-01-01

    Toll-like receptors (TLRs) are important signal transducers that mediate inflammatory reactions induced by microbes through pattern recognition of virulence molecules such as lipopolysaccharide (LPS) and lipoproteins. We investigated whether proinflammatory cytokine responses induced by certain bacterial protein adhesins may also depend on TLRs. In differentiated THP-1 mononuclear cells stimulated by LPS-free recombinant fimbrillin (rFimA) from Porphyromonas gingivalis, cytokine release was abrogated by monoclonal antibodies (MAbs) to CD14 and TLR4 but not to TLR2. Similar experiments using anti-β2 integrin MAbs suggested that β2 integrins (CD11/CD18) also play a role in cytokine induction by rFimA or native fimbriae. Minor fimbriae (distinct from the fimA-encoded major fimbriae) of P. gingivalis induced proinflammatory cytokine release in a CD14- and TLR2-dependent mode. Cytokine induction by BspA, a leucine-rich repeat protein from Bacteroides forsythus, depended heavily on CD14 and TLR2. We also found that the ability of the streptococcal protein AgI/II to stimulate cytokine release depended partially on CD14 and TLR4, and the AgI/II segment that possibly interacts with these receptors was identified as its N-terminal saliva-binding region. When THP-1 cells were exposed to rFimA for 24 h, surface expression of CD14 and CD18 was decreased and the cells became hyporesponsive to cytokine induction by a second challenge with rFimA. However, tolerance induction was abolished when the THP-1 cells were pretreated with rFimA in the presence of either anti-CD14 MAb or anti-TLR4 MAb. Induction of cross-tolerance between rFimA and LPS correlated with downregulation of the pattern recognition receptors involved. Our data suggest that the CD14-TLR2/4 system is involved in cytokine production and tolerance induction upon interaction with certain proinflammatory bacterial protein adhesins. PMID:11874886

  12. Adhesins of Bartonella spp.

    PubMed

    O'Rourke, Fiona; Schmidgen, Thomas; Kaiser, Patrick O; Linke, Dirk; Kempf, Volkhard A J

    2011-01-01

    Adhesion to host cells represents the first step in the infection process and one of the decisive features in the pathogenicity of Bartonella spp. B. henselae and B. quintana are considered to be the most important human pathogenic species, responsible for cat scratch disease, bacillary angiomatosis, trench fever and other diseases. The ability to cause vasculoproliferative disorders and intraerythrocytic bacteraemia are unique features of the genus Bartonella. Consequently, the interaction with endothelial cells and erythrocytes is a focus in Bartonella research. The genus harbours a variety of trimeric autotransporter adhesins (TAAs) such as the Bartonella adhesin A (BadA) of B. henselae and the variably expressed outer-membrane proteins (Vomps) of B. quintana, which display remarkable variations in length and modular construction. These adhesins mediate many of the biologically-important properties of Bartonella spp. such as adherence to endothelial cells and extracellular matrix proteins and induction of angiogenic gene programming. There is also significant evidence that the laterally acquired Trw-conjugation systems of Bartonella spp. mediate host-specific adherence to erythrocytes. Other potential adhesins are the filamentous haemagglutinins and several outer membrane proteins. The exact molecular functions of these adhesins and their interplay with other pathogenicity factors (e.g., the VirB/D4 type 4 secretion system) need to be analysed in detail to understand how these pathogens adapt to their mammalian hosts. PMID:21557057

  13. Solving the phase problem for carbohydrate-binding proteins using selenium derivatives of their ligands: a case study involving the bacterial F17-G adhesin.

    PubMed

    Buts, Lieven; Loris, Remy; De Genst, Erwin; Oscarson, Stefan; Lahmann, Martina; Messens, Joris; Brosens, Elke; Wyns, Lode; De Greve, Henri; Bouckaert, Julie

    2003-06-01

    The Escherichia coli adhesin F17-G is a carbohydrate-binding protein that allows the bacterium to attach to the intestinal epithelium of young ruminants. The structure of the 17 kDa lectin domain of F17-G was determined using the anomalous dispersion signal of a selenium-containing analogue of the monosaccharide ligand N-acetyl-d-glucosamine in which the anomeric oxygen was replaced by an Se atom. A three-wavelength MAD data set yielded good experimental phases to 2.6 A resolution. The structure was refined to 1.75 A resolution and was used to solve the structures of the ligand-free protein and the F17-G-N-acetyl-d-glucosamine complex. This selenium-carbohydrate phasing method could be of general use for determining the structures of carbohydrate-binding proteins. PMID:12777763

  14. The apicomplexan glideosome and adhesins -- structures and function

    PubMed Central

    Boucher, Lauren E.; Bosch, Jürgen

    2015-01-01

    The apicomplexan family of pathogens, which includes Plasmodium spp. and Toxoplasma gondii, are primarily obligate intracellular parasites and invade multiple cell types. These parasites express extracellular membrane protein receptors, adhesins, to form specific pathogen-host cell interaction complexes. Various adhesins are used to invade a variety of cell types. The receptors are linked to an actomyosin motor, which is part of a complex comprised of many proteins known as the invasion machinery or glideosome. To date, reviews on invasion have focused primarily on the molecular pathways and signals of invasion, with little or no structural information presented. Over 75 structures of parasite receptors and glideosome proteins have been deposited with the Protein Data Bank. These structures include adhesins, motor proteins, bridging proteins, inner membrane complex and cytoskeletal proteins, as well as co-crystal structures with peptides and antibodies. These structures provide information regarding key interactions necessary for target receptor engagement, machinery complex formation, how force is transmitted, and the basis of inhibitory antibodies. Additionally, these structures can provide starting points for the development of antibodies and inhibitory molecules targeting protein-protein interactions, with the aim to inhibit invasion. This review provides an overview of the parasite adhesin protein families, the glideosome components, glideosome architecture, and discuss recent work regarding alternative models. PMID:25764948

  15. The knockdown of each component of the cysteine proteinase-adhesin complex of Entamoeba histolytica (EhCPADH) affects the expression of the other complex element as well as the in vitro and in vivo virulence.

    PubMed

    Ocádiz-Ruiz, Ramón; Fonseca, Wendy; Linford, Alicia S; Yoshino, Timothy P; Orozco, Esther; Rodríguez, Mario A

    2016-01-01

    Entamoeba histolytica is the protozoan parasite causative of human amoebiasis, disease responsible for 40 000-100 000 deaths annually. The cysteine proteinase-adhesin complex of this parasite (EhCPADH) is a heterodimeric protein formed by a cysteine protease (EhCP112) and an adhesin (EhADH) that plays an important role in the cytopathic mechanism of this parasite. The coding genes for EhCP112 and EhADH are adjacent in the E. histolytica genome, suggesting that their expression may be co-regulated, but this hypothesis has not yet been confirmed. Here, we performed the knockdown of EhCP112 and EhADH using gene-specific short-hairpin RNAs (shRNA), and the effect of these knockdowns on the expression of both complex components as well as on the in vitro and in vivo virulence was analysed. Results showed that the knockdown of one of the EhCPADH components produced a simultaneous downregulation of the other protein. Accordingly, a concomitant reduction in the overall expression of the complex was observed. The downregulation of each component also produced a significant decrease in the in vitro and in vivo virulence of trophozoites. These results demonstrated that the expression of EhCP112 and EhADH is co-regulated and confirmed that the EhCPADH complex plays an important role in E. histolytica virulence. PMID:26521708

  16. Identification of a Novel Streptococcal Adhesin P (SadP) Protein Recognizing Galactosyl-α1–4-galactose-containing Glycoconjugates

    PubMed Central

    Kouki, Annika; Haataja, Sauli; Loimaranta, Vuokko; Pulliainen, Arto T.; Nilsson, Ulf J.; Finne, Jukka

    2011-01-01

    Bacterial adhesion is often a prerequisite for infection, and host cell surface carbohydrates play a major role as adhesion receptors. Streptococci are a leading cause of infectious diseases. However, only few carbohydrate-specific streptococcal adhesins are known. Streptococcus suis is an important pig pathogen and a zoonotic agent causing meningitis in pigs and humans. In this study, we have identified an adhesin that mediates the binding of S. suis to galactosyl-α1–4-galactose (Galα1–4Gal)-containing host receptors. A functionally unknown S. suis cell wall protein (SSU0253), designated here as SadP (streptococcal adhesin P), was identified using a Galα1–4Gal-containing affinity matrix and LC-ESI mass spectrometry. Although the function of the protein was not previously known, it was recently identified as an immunogenic cell wall protein in a proteomic study. Insertional inactivation of the sadP gene abolished S. suis Galα1–4Gal-dependent binding. The adhesin gene sadP was cloned and expressed in Escherichia coli. Characterization of its binding specificity showed that SadP recognizes Galα1–4Gal-oligosaccharides and binds its natural glycolipid receptor, GbO3 (CD77). The N terminus of SadP was shown to contain a Galα1-Gal-binding site and not to have apparent sequence similarity to other bacterial adhesins, including the E. coli P fimbrial adhesins, or to E. coli verotoxin or Pseudomonas aeruginosa lectin I also recognizing the same Galα1–4Gal disaccharide. The SadP and E. coli P adhesins represent a unique example of convergent evolution toward binding to the same host receptor structure. PMID:21908601

  17. Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica.

    PubMed Central

    Zhang, T; Stanley, S L

    1994-01-01

    The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with virulent E. histolytica trophozoites. PMID:8188384

  18. Enterohemorrhagic Escherichia coli Adhesins.

    PubMed

    McWilliams, Brian D; Torres, Alfredo G

    2014-06-01

    Adhesins are a group of proteins in enterohemorrhagic Escherichia coli (EHEC) that are involved in the attachment or colonization of this pathogen to abiotic (plastic or steel) and biological surfaces, such as those found in bovine and human intestines. This review provides the most up-to-date information on these essential adhesion factors, summarizing important historical discoveries and analyzing the current and future state of this research. In doing so, the proteins intimin and Tir are discussed in depth, especially regarding their role in the development of attaching and effacing lesions and in EHEC virulence. Further, a series of fimbrial proteins (Lpf1, Lpf2, curli, ECP, F9, ELF, Sfp, HCP, and type 1 fimbria) are also described, emphasizing their various contributions to adherence and colonization of different surfaces and their potential use as genetic markers in detection and classification of different EHEC serotypes. This review also discusses the role of several autotransporter proteins (EhaA-D, EspP, Saa and Sab, and Cah), as well as other proteins associated with adherence, such as flagella, EibG, Iha, and OmpA. While these proteins have all been studied to varying degrees, all of the adhesins summarized in this article have been linked to different stages of the EHEC life cycle, making them good targets for the development of more effective diagnostics and therapeutics. PMID:26103974

  19. A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development

    PubMed Central

    Liu, Ming-Che; Yang, Cheng-Shou; Wang, Co-Shine

    2014-01-01

    The anther-specific gene LLA1271 isolated from lily (Lilium longiflorum Thunb.) anthers is novel and exists in two forms. The protein encoded by LLA1271 may represent an adhesin-like protein first found in higher plants. The protein contains a typical N-terminal signal peptide followed by a highly conserved repeat domain. The LLA1271 gene is temporally expressed at the phase of microspore development. RNA blot and RNA in situ hybridization analyses demonstrated that the gene was expressed both in the tapetum and in the microspore. The gene is endo- and exogenously induced by gibberellin. Studies with the gibberellin biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that LLA1271 is negatively regulated by ethylene, and a cross-talk of regulation between gibberellin and ethylene occurs in young anthers. The treatment with NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development in a state close to that of a tapetum without treatment. The LLA1271 protein is heat stable and heterogeneous. An immunoblot of separated protein fractions of the anther revealed that the LLA1271 protein was detected in protein fraction of the microspore released from the cell wall by treatment with either 0.5% or 2% Triton X-100. Ectopic expression of LLA1271 resulted in impaired stamen and low pollen germination. Scanning electron microscopy of TAP::LLA1271 pollen showed distorted exine formation and patterning. The LLA1271 protein once synthesized in both the tapetum and microspore is secreted and deposited on the surface of microspores, moderately affecting exine formation and patterning. PMID:24591055

  20. The presence of both bone sialoprotein-binding protein gene and collagen adhesin gene as a typical virulence trait of the major epidemic cluster in isolates from orthopedic implant infections.

    PubMed

    Campoccia, Davide; Speziale, Pietro; Ravaioli, Stefano; Cangini, Ilaria; Rindi, Simonetta; Pirini, Valter; Montanaro, Lucio; Arciola, Carla Renata

    2009-12-01

    Staphylococcus aureus is a major, highly clonal, pathogen causing implant infections. This study aimed at investigating the diverse distribution of bacterial adhesins in most prevalent S. aureus strain types causing orthopaedic implant infections. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes. Within the collection of isolates, automated ribotyping detected 98 distinct ribogroups. For many ribogroups, characteristic tandem genes arrangements could be identified. In the predominant S. aureus cluster, enlisting 27 isolates, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. This study suggests that specific adhesins may synergistically act in the onset of implant infections and that anti-adhesin strategies should be targeted to adhesins conjointly present. PMID:19758694

  1. The Staphylococcus aureus lineage-specific markers collagen adhesin and toxic shock syndrome toxin 1 distinguish multilocus sequence typing clonal complexes within spa clonal complexes.

    PubMed

    Deurenberg, Ruud H; Rijnders, Michelle I A; Sebastian, Silvie; Welling, Maaike A; Beisser, Patrick S; Stobberingh, Ellen E

    2009-10-01

    Spa typing/based upon repeat pattern (BURP) sometimes cannot differentiate multilocus sequence typing (MLST) clonal complexes (CCs) within spa-CCs. It has been observed previously that virulence factors, such as collagen adhesin (CNA) and toxic shock syndrome toxin 1 (TSST-1), are associated with certain Staphylococcus aureus lineages. Analysis of methicillin-sensitive and methicillin-resistant S. aureus by spa typing/BURP and detection of CNA and TSST-1 observed an association between CNA and MLST CC1, 12, 22, 30, 45, 51, and 239 and between TSST-1 and MLST CC30. In spa-CC 012, associated with MLST CC7, CC15, and CC30, MLST CC30 could be distinguished from MLST CC7 and CC15 with CNA and TSST-1 as lineage-specific markers. Lineage-specific markers can overcome clustering of nonrelated MLST CCs into 1 spa-CC. PMID:19748421

  2. The Bfp60 surface adhesin is an extracellular matrix and plasminogen protein interacting in Bacteroides fragilis

    PubMed Central

    de Oliveira Ferreira, Eliane; Teixeira, Felipe; Cordeiro, Fabiana; Lobo, Leandro Araujo; Rocha, Edson R.; Smith, Jeffrey C.; Domingues, Regina M C P

    2014-01-01

    Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis can not be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis. PMID:23850366

  3. Lucilia sericata Chymotrypsin Disrupts Protein Adhesin-Mediated Staphylococcal Biofilm Formation

    PubMed Central

    Nigam, Yamni; Sawyer, James; Mack, Dietrich; Pritchard, David I.

    2013-01-01

    Staphylococcus aureus and Staphylococcus epidermidis biofilms cause chronic infections due to their ability to form biofilms. The excretions/secretions of Lucilia sericata larvae (maggots) have effective activity for debridement and disruption of bacterial biofilms. In this paper, we demonstrate how chymotrypsin derived from maggot excretions/secretions disrupts protein-dependent bacterial biofilm formation mechanisms. PMID:23220967

  4. Functional Blocking of Staphylococcus aureus Adhesins following Growth in Ex Vivo Media

    PubMed Central

    Massey, Ruth C.; Dissanayeke, Shobana R.; Cameron, Brian; Ferguson, David; Foster, Timothy J.; Peacock, Sharon J.

    2002-01-01

    Defining the role of Staphylococcus aureus adhesins in disease pathogenesis may depend on the use of bacteria grown in culture media that more closely reflect the human milieu than conventional broth. This study examined the functional effect on S. aureus adhesins following growth in an ex vivo medium containing a complex mixture of human proteins (used peritoneal dialysate) relative to growth in Todd-Hewitt broth. The adherence of S. aureus, cultured in dialysate, to fibronectin and fibrinogen was markedly reduced despite the expresion of full-length ClfA, ClfB, and fibronectin-binding proteins. Growth in dialysate resulted in the acquisition of a surface coat, as visualized by transmission electron microscopy, which was shown to contain fibronectin, fibrinogen, and immunoglobulins. Adherence of S. aureus to fibrinogen following growth in dialysate was significantly reduced by expression of protein A but was restored following growth in immunoglobulin-depleted dialysate. We conclude that bacterial adherence to solid-phase protein is critically dependent on the culture medium, that S. aureus adhesins may become saturated with target protein prior to contact with solid surfaces, and that there is an interaction between fibrinogen-binding proteins and immunoglobulin bound to protein A following contact with host proteins. These findings have important implications for future studies of S. aureus adhesins. PMID:12228257

  5. Characterization of an autotransporter adhesin protein shared by Burkholderia mallei and Burkholderia pseudomallei

    PubMed Central

    2014-01-01

    Background Autotransporters form a large family of outer membrane proteins specifying diverse biological traits of Gram-negative bacteria. In this study, we report the identification and characterization of a novel autotransporter gene product of Burkholderia mallei (locus tag BMA1027 in strain ATCC 23344). Results Database searches identified the gene in at least seven B. mallei isolates and the encoded proteins were found to be 84% identical. Inactivation of the gene encoding the autotransporter in the genome of strain ATCC 23344 substantially reduces adherence to monolayers of HEp-2 laryngeal cells and A549 type II pneumocytes, as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, expression of the autotransporter on the surface of recombinant E. coli bacteria increases adherence to these cell types by 5–7 fold. The gene specifying the autotransporter was identified in the genome of 29 B. pseudomallei isolates and disruption of the gene in strain DD503 reduced adherence to NHBE cultures by 61%. Unlike B. mallei, the mutation did not impair binding of B. pseudomallei to A549 or HEp-2 cells. Analysis of sera from mice infected via the aerosol route with B. mallei and B. pseudomallei revealed that animals inoculated with as few as 10 organisms produce antibodies against the autotransporter, therefore indicating expression in vivo. Conclusions Our data demonstrate that we have identified an autotransporter protein common to the pathogenic species B. mallei and B. pseudomallei which mediates adherence to respiratory epithelial cells and is expressed in vivo during the course of aerosol infection. PMID:24731253

  6. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  7. Carbohydrate Receptors of Bacterial Adhesins: Implications and Reflections

    NASA Astrophysics Data System (ADS)

    Ohlsen, K.; Oelschlaeger, T. A.; Hacker, J.; Khan, A. S.

    Bacteria entering a host depend on adhesins to achieve colonization. Adhesins are bacterial surface structures mediating binding to host surficial areas. Most adhesins are composed of one or several proteins. Usually a single bacterial strain is able to express various adhesins. The adhesion type expressed may influence host-, tissue or even cell tropism of Gram-negative and of Gram-positive bacteria. The binding of fimbrial as well as of afimbrial adhesins of Gram-negative bacteria to host carbohydrate structures (=receptors) has been elucidated in great detail. In contrast, in Gram-positives, most well studied adhesins bind to proteinaceous partners. Nevertheless, for both bacterial groups the binding of bacterial adhesins to eukaryotic carbohydrate receptors is essential for establishing colonization or infection. The characterization of this interaction down to the submolecular level provides the basis for strategies to interfere with this early step of infection which should lead to the prevention of subsequent disease. However, this goal will not be achieved easily because bacterial adherence is not a monocausal event but rather mediated by a variety of adhesins.

  8. Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections.

    PubMed

    Rohde, Holger; Burandt, Eike C; Siemssen, Nicolaus; Frommelt, Lars; Burdelski, Christoph; Wurster, Sabine; Scherpe, Stefanie; Davies, Angharad P; Harris, Llinos G; Horstkotte, Matthias A; Knobloch, Johannes K-M; Ragunath, Chandran; Kaplan, Jeffrey B; Mack, Dietrich

    2007-03-01

    Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. As the use of biofilm-associated factors as vaccines is critically restricted by their prevalence in natural staphylococcal populations we studied the distribution of genes involved in biofilm formation, the biofilm phenotype and production of polysaccharide intercellular adhesin (PIA) in clonally independent Staphylococcus aureus and Staphylococcus epidermidis strains isolated from prosthetic joint infections after total hip or total knee arthroplasty. Biofilm formation was detected in all S. aureus and 69.2% of S. epidermidis strains. Importantly, 27% of biofilm-positive S. epidermidis produced PIA-independent biofilms, in part mediated by the accumulation associated protein (Aap). Protein-dependent biofilms were exclusively found in S. epidermidis strains from total hip arthroplasty (THA). In S. aureus PIA and proteins act cooperatively in biofilm formation regardless of the infection site. PIA and protein factors like Aap are of differential importance for the pathogenesis of S. epidermidis in prosthetic joint infections (PJI) after THA and total knee arthroplasty (TKA), implicating that icaADBC cannot serve as a general virulence marker in this species. In S. aureus biofilm formation proteins are of overall importance and future work should focus on the identification of functionally active molecules. PMID:17187854

  9. Characterization of an Acidic-pH-Inducible Stress Protein (hsp70), a Putative Sulfatide Binding Adhesin, from Helicobacter pylori

    PubMed Central

    Huesca, Mario; Goodwin, Avery; Bhagwansingh, Arianna; Hoffman, Paul; Lingwood, Clifford A.

    1998-01-01

    The in vitro glycolipid binding specificity of the gastric pathogen Helicobacter pylori is altered to include sulfated glycolipids (sulfatides) following brief exposure of the organism to acid pH typical of the stomach. This change is prevented by anti-hsp70 antibodies, suggesting that hsp70 may be a stress-induced surface adhesin, mediating sulfatide recognition. To facilitate investigation of the role of hsp70 in attachment, we have cloned and sequenced the H. pylori hsp70 gene (dnaK). The hsp70 gene was identified by probing a cosmid DNA library made from H. pylori 439 with a PCR amplicon generated with oligonucleotides synthesized to highly conserved regions of dnaK. The 1.9-kb H. pylori hsp70 gene encodes a product of 616 amino acids. Primer extension analysis revealed a single transcription start site, while Northern blot analysis established that hsp70 was preferentially induced by low pH rather than by heat shock. The ability of H. pylori to alter its glycolipid binding specificity following exposure to low pH by upregulating hsp70 and by expressing hsp70 on the bacterial surface may provide a survival advantage during periods of high acid stress. PMID:9712748

  10. Enterococcus faecalis adhesin, ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I.

    PubMed

    Nallapareddy, S R; Qin, X; Weinstock, G M; Höök, M; Murray, B E

    2000-09-01

    Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46 degrees C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46 degrees C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single approximately 105-kDa band of mutanolysin extracts from OG1RF grown at 46 degrees C, while no band was detected in extracts from OG1RF grown at 37 degrees C, nor from the OG1RF ace mutant grown at 37 or 46 degrees C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 microg/ml, and also inhibited the 46 degrees C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I. PMID:10948147

  11. The Extracellular Protein Factor Epf from Streptococcus pyogenes Is a Cell Surface Adhesin That Binds to Cells through an N-terminal Domain Containing a Carbohydrate-binding Module*

    PubMed Central

    Linke, Christian; Siemens, Nikolai; Oehmcke, Sonja; Radjainia, Mazdak; Law, Ruby H. P.; Whisstock, James C.; Baker, Edward N.; Kreikemeyer, Bernd

    2012-01-01

    Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain. PMID:22977243

  12. Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.

    PubMed

    Montanari, Paolo; Bozza, Giuseppe; Capecchi, Barbara; Caproni, Elena; Barrile, Riccardo; Norais, Nathalie; Capitani, Mirco; Sallese, Michele; Cecchini, Paola; Ciucchi, Laura; Gao, Zhenai; Rappuoli, Rino; Pizza, Mariagrazia; Aricò, Beatrice; Merola, Marcello

    2012-03-01

    NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA. PMID:22066472

  13. Identification of glycoprotein receptors within the human salivary proteome for the lectin-like BabA and SabA adhesins of Helicobacter pylori by fluorescence-based 2-D bacterial overlay.

    PubMed

    Walz, Anke; Odenbreit, Stefan; Stühler, Kai; Wattenberg, Andreas; Meyer, Helmut E; Mahdavi, Jafar; Borén, Thomas; Ruhl, Stefan

    2009-03-01

    Because gastric infection by Helicobacter pylori takes place via the oral route, possible interactions of this bacterium with human salivary proteins could occur. By using modified 1- and 2-D bacterial overlay, binding of H. pylori adhesins BabA and SabA to the whole range of salivary proteins was explored. Bound salivary receptor molecules were identified by MALDI-MS and by comparison to previously established proteome maps of whole and glandular salivas. By use of adhesin-deficient mutants, binding of H. pylori to MUC7 and gp-340 could be linked to the SabA and BabA adhesins, respectively, whereas binding to MUC5B was associated with both adhesins. Binding of H. pylori to the proline-rich glycoprotein was newly detected and assigned to BabA adhesin whereas the SabA adhesin was found to mediate binding to newly detected receptor molecules, including carbonic anhydrase VI, secretory component, heavy chain of secretory IgA1, parotid secretory protein and zinc-alpha(2)-glycoprotein. Some of these salivary glycoproteins are known to act as scavenger molecules or are involved in innate immunity whereas others might come to modify the pathogenetic properties of this organism. In general, this 2-D bacterial overlay technique represents a useful supplement in adhesion studies of bacteria with complex protein mixtures. PMID:19253298

  14. Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine

    PubMed Central

    Mizrachi Nebenzahl, Yaffa; Blau, Karin; Kushnir, Tatyana; Shagan, Marilou; Portnoi, Maxim; Cohen, Aviad; Azriel, Shalhevet; Malka, Itai; Adawi, Asad; Kafka, Daniel; Dotan, Shahar; Guterman, Gali; Troib, Shany; Fishilevich, Tali; Gershoni, Jonathan M; Braiman, Alex; Mitchell, Andrea M; Mitchell, Timothy J; Porat, Nurith; Goliand, Inna; Chalifa Caspi, Vered; Swiatlo, Edwin; Tal, Michael; Ellis, Ronald; Elia, Natalie; Dagan, Ron

    2016-01-01

    In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development. PMID:26990554

  15. Lectin-Glycan Interaction Network-Based Identification of Host Receptors of Microbial Pathogenic Adhesins

    PubMed Central

    Ielasi, Francesco S.; Alioscha-Perez, Mitchel; Donohue, Dagmara; Claes, Sandra; Sahli, Hichem; Schols, Dominique

    2016-01-01

    ABSTRACT The first step in the infection of humans by microbial pathogens is their adherence to host tissue cells, which is frequently based on the binding of carbohydrate-binding proteins (lectin-like adhesins) to human cell receptors that expose glycans. In only a few cases have the human receptors of pathogenic adhesins been described. A novel strategy—based on the construction of a lectin-glycan interaction (LGI) network—to identify the potential human binding receptors for pathogenic adhesins with lectin activity was developed. The new approach is based on linking glycan array screening results of these adhesins to a human glycoprotein database via the construction of an LGI network. This strategy was used to detect human receptors for virulent Escherichia coli (FimH adhesin), and the fungal pathogens Candida albicans (Als1p and Als3p adhesins) and C. glabrata (Epa1, Epa6, and Epa7 adhesins), which cause candidiasis. This LGI network strategy allows the profiling of potential adhesin binding receptors in the host with prioritization, based on experimental binding data, of the most relevant interactions. New potential targets for the selected adhesins were predicted and experimentally confirmed. This methodology was also used to predict lectin interactions with envelope glycoproteins of human-pathogenic viruses. It was shown that this strategy was successful in revealing that the FimH adhesin has anti-HIV activity. PMID:27406561

  16. Structural context for protein N-glycosylation in bacteria: The structure of PEB3, an adhesin from Campylobacter jejuni.

    PubMed

    Rangarajan, Erumbi S; Bhatia, Smita; Watson, David C; Munger, Christine; Cygler, Miroslaw; Matte, Allan; Young, N Martin

    2007-05-01

    Campylobacter jejuni is unusual among bacteria in possessing a eukaryotic-like system for N-linked protein glycosylation at Asn residues in sequons of the type Asp/Glu-Xaa-Asn-Xaa-Ser/Thr. However, little is known about the structural context of the glycosylated sequons, limiting the design of novel recombinant glycoproteins. To obtain more information on sequon structure, we have determined the crystal structure of the PEB3 (Cj0289c) dimer. PEB3 has the class II periplasmic-binding protein fold, with each monomer having two domains with a ligand-binding site containing citrate located between them, and overall resembles molybdate- and sulfate-binding proteins. The sequon around Asn90 is located within a surface-exposed loop joining two structural elements. The three key residues are well exposed on the surface; hence, they may be accessible to the PglB oligosaccharyltransferase in the folded state. PMID:17456748

  17. Structural Context for Protein N-glycosylation in Bacteria: The Structure of PEB3, an Adhesin from Campylobacter Jejuni

    SciTech Connect

    Rangarajan,E.; Bhatia, S.; Watson, D.; Munger, C.; Cygler, M.; Matte, A.; Young, N.

    2007-01-01

    Campylobacter jejuni is unusual among bacteria in possessing a eukaryotic-like system for N-linked protein glycosylation at Asn residues in sequons of the type Asp/Glu-Xaa-Asn-Xaa-Ser/Thr. However, little is known about the structural context of the glycosylated sequons, limiting the design of novel recombinant glycoproteins. To obtain more information on sequon structure, we have determined the crystal structure of the PEB3 (Cj0289c) dimer. PEB3 has the class II periplasmic-binding protein fold, with each monomer having two domains with a ligand-binding site containing citrate located between them, and overall resembles molybdate- and sulfate-binding proteins. The sequon around Asn90 is located within a surface-exposed loop joining two structural elements. The three key residues are well exposed on the surface; hence, they may be accessible to the PglB oligosaccharyltransferase in the folded state.

  18. Characterization of a fucoside-binding adhesin of Candida albicans.

    PubMed Central

    Tosh, F D; Douglas, L J

    1992-01-01

    Candida albicans GDH 2346 produces extracellular polymeric material (EP) which contains a mannoprotein adhesin with a lectin-like affinity for fucose-containing glycosides. EP isolated from culture supernatants of this strain was used as starting material for purification of the adhesin. The purification protocol involved a stepwise treatment of EP with N-glycanase, papain, and dilute alkali to cleave the protein and carbohydrate portions of the mannoprotein molecule. Fucoside-binding protein fragments were then recovered by affinity adsorption with the trisaccharide determinant of the H (type 2) blood group antigen which terminates in a residue of L-fucose. The purified adhesin was devoid of carbohydrate and inhibited yeast adhesion to buccal epithelial cells 221 times more efficiently, on a protein weight basis, than did EP. Adhesion inhibition reached a maximum of 78 to 80% at an adhesin concentration of 10 micrograms ml-1. Our results indicate that this protein is the major adhesin of yeast-phase cells of C. albicans GDH 2346 but that one or more secondary adhesion mechanisms may operate. PMID:1398983

  19. Comparative functional genomic analysis of Pasteurellaceae adhesins using phage display.

    PubMed

    Mullen, Lisa M; Nair, Sean P; Ward, John M; Rycroft, Andrew N; Williams, Rachel J; Henderson, Brian

    2007-05-16

    The Pasteurellaceae contain a number of important animal pathogens. Although related, the various members of this family cause a diversity of pathology in a wide variety of organ systems. Adhesion is an important virulence factor in bacterial infections. Surprisingly little is known about the adhesins of the Pasteurellaceae. To attempt to identify the genes coding for adhesins to some key components of the hosts extracellular matrix molecules, phage display libraries of fragmented genomic DNA from Haemophilus influenzae, Actinobacillus pleuropneumoniae, Pasteurella multocida and Aggregatibacter actinomycetemcomitans, were prepared in the phage display vector pG8SAET. The libraries were screened against human or porcine fibronectin, serum albumin or a commercial extracellular matrix containing type IV collagen, laminin and heparin sulphate. Four genes encoding putative adhesins were identified. These genes code for: (i) a 34 kDa human serum albumin binding protein from Haemophilus influenzae; (ii) a 12.8 kDa fibronectin-binding protein from Pasteurella multocida; (iii) a 13.7 kDa fibronectin-binding protein from A. actinomycetemcomitans; (iv) a 9.5 kDa serum albumin-binding protein from A. pleuropneumoniae. None of these genes have previously been proposed to code for adhesins. The applications of phage display with whole bacterial genomes to identify genes encoding novel adhesins in this family of bacteria are discussed. PMID:17258409

  20. Dechlorination of Chloral Hydrate Is Influenced by the Biofilm Adhesin Protein LapA in Pseudomonas putida LF54

    PubMed Central

    Zhang, Wanjun; Huhe; Pan, Yuanbai; Toyofuku, Masanori; Nomura, Nobuhiko; Nakajima, Toshiaki

    2013-01-01

    LapA is the largest surface adhesion protein of Pseudomonas putida that initiates biofilm formation. Here, by using transposon insertion mutagenesis and a conditional lapA mutant, we demonstrate for the first time that LapA influences chloral hydrate (CH) dechlorination in P. putida LF54. PMID:23603683

  1. The multifunctional LigB adhesin binds homeostatic proteins with potential roles in cutaneous infection by pathogenic Leptospira interrogans.

    PubMed

    Choy, Henry A; Kelley, Melissa M; Croda, Julio; Matsunaga, James; Babbitt, Jane T; Ko, Albert I; Picardeau, Mathieu; Haake, David A

    2011-01-01

    Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9-11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react

  2. Highly Conserved Surface Proteins of Oral Spirochetes as Adhesins and Potent Inducers of Proinflammatory and Osteoclastogenic Factors▿

    PubMed Central

    Jun, Hye-Kyoung; Kang, Young-Mi; Lee, Hae-Ri; Lee, Sung-Hoon; Choi, Bong-Kyu

    2008-01-01

    Oral spirochetes include enormously heterogeneous Treponema species, and some have been implicated in the etiology of periodontitis. In this study, we characterized highly conserved surface proteins in four representative oral spirochetes (Treponema denticola, T. lecithinolyticum, T. maltophilum, and T. socranskii subsp. socranskii) that are homologs of T. pallidum Tp92, with opsonophagocytic potential and protective capacity against syphilis. Tp92 homologs of oral spirochetes had predicted signal peptides (20 to 31 amino acids) and molecular masses of 88 to 92 kDa for mature proteins. They showed amino acid sequence identities of 37.9 to 49.3% and similarities of 54.5 to 66.9% to Tp92. The sequence identities and similarities of Tp92 homologs of oral treponemes to one another were 41.6 to 71.6% and 59.9 to 85.6%, respectively. The tp92 gene homologs were successfully expressed in Escherichia coli, and the recombinant proteins were capable of binding to KB cells, an epithelial cell line, and inhibited the binding of the whole bacteria to the cells. Antiserum (the immunoglobulin G fraction) raised against a recombinant form of the T. denticola Tp92 homolog cross-reacted with homologs from three other species of treponemes. The Tp92 homologs stimulated various factors involved in inflammation and osteoclastogenesis, like interleukin-1β (IL-1β), tumor necrosis factor alpha, IL-6, prostaglandin E2, and matrix metalloproteinase 9, in host cells like monocytes and fibroblasts. Our results demonstrate that Tp92 homologs of oral spirochetes are highly conserved and may play an important role in cell attachment, inflammation, and tissue destruction. The coexistence of various Treponema species in a single periodontal pocket and, therefore, the accumulation of multiple Tp92 homologs may amplify the pathological effect in periodontitis. PMID:18390996

  3. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  4. Fibronectin Binding to the Treponema pallidum Adhesin Protein Fragment rTp0483 on Functionalized Self-Assembled Monolayers

    PubMed Central

    Dickerson, Matthew T.; Abney, Morgan B.; Cameron, Caroline E.; Knecht, Marc; Bachas, Leonidas G.; Anderson, Kimberly W.

    2012-01-01

    Past work has shown that Treponema pallidum, the causative agent of syphilis, binds host fibronectin (FN). FN and other host proteins are believed to bind to rare outer membrane proteins (OMPs) of T. pallidum, and it is postulated that this interaction may facilitate cell attachment and mask antigenic targets on the surface. This research seeks to prepare a surface capable of mimicking the FN binding ability of T. pallidum in order to investigate the impact of FN binding with adsorbed Tp0483 on the host response to the surface. By understanding this interaction it may be possible to develop more effective treatments for infection and possibly mimic the stealth properties of the bacteria. Functionalized self-assembled monolayers (SAMs) on0 gold were used to investigate rTp0483 and FN adsorption. Using a quartz crystal microbalance (QCM) rTp0483 adsorption and subsequent FN adsorption onto rTp0483 was determined to be higher on negatively charged carboxylate-terminated self-assembled monolayers (−COO− SAMs) compared to the other surfaces analyzed. Kinetic analysis of rTp0483 adsorption using surface plasmon resonance (SPR) supported this finding. Kinetic analysis of FN adsorption using SPR revealed a multi-step event, where the concentration of immobilized rTp0483 plays a role in FN binding. An examination of relative QCM dissipation energy compared to the shift in frequency showed a correlation between the physical properties of adsorbed rTp0483 and SAM surface chemistry. In addition, AFM images of rTp0483 on selected SAMs illustrated a preference of rTp0483 to bind as aggregates. Adsorption on −COO− SAMs was more uniform across the surface, which may help further explain why FN bound more strongly. rTp0483 antibody studies suggested the involvement of amino acids 274–289 and 316–333 in binding between rTp0483 to FN, while a peptide blocking study only showed inhibition of binding with amino acids 316–333. Finally, surface adsorbed rTp0483 with FN

  5. Algorithmic complexity of a protein

    NASA Astrophysics Data System (ADS)

    Dewey, T. Gregory

    1996-07-01

    The information contained in a protein's amino acid sequence dictates its three-dimensional structure. To quantitate the transfer of information that occurs in the protein folding process, the Kolmogorov information entropy or algorithmic complexity of the protein structure is investigated. The algorithmic complexity of an object provides a means of quantitating its information content. Recent results have indicated that the algorithmic complexity of microstates of certain statistical mechanical systems can be estimated from the thermodynamic entropy. In the present work, it is shown that the algorithmic complexity of a protein is given by its configurational entropy. Using this result, a quantitative estimate of the information content of a protein's structure is made and is compared to the information content of the sequence. Additionally, the mutual information between sequence and structure is determined. It is seen that virtually all the information contained in the protein structure is shared with the sequence.

  6. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  7. Evaluation of Cell Binding Activities of Leptospira ECM Adhesins

    PubMed Central

    Robbins, Gregory T.; Hahn, Beth L.; Evangelista, Karen V.; Padmore, Lavinia; Aranda, Patrick S.; Coburn, Jenifer

    2015-01-01

    Pathogenic spirochetes of the genus Leptospira are the causative agents of leptospirosis, a zoonotic infection that occurs globally. The bacteria colonize the renal proximal tubules of many animals and are shed in the urine. Contact with the urine, or with water contaminated with the urine of infected animals can cause infection of new host animals, including humans. Mechanisms of colonization of the proximal tubule and other tissues are not known, but specific interactions between bacterial adhesins and host substrates are likely to be critical in this process. Several extracellular matrix (ECM) adhesins have been previously identified, but more recently, it has been shown that Leptospira bind more efficiently to cells than ECM. In this work, recombinant forms of five putative Leptospira ECM adhesins, namely LipL32, Loa22, OmpL1, p31/LipL45, and LenA were evaluated for binding to cells as well as an expanded variety of ECM components. Reproducible and significant adhesin activity was demonstrated only for OmpL1, which bound to both mammalian cell lines tested and to glycosaminoglycans (GAGs). While determination of biologically significant bacterial adhesion activity will require generation of site-directed mutant strains, our results suggest that OmpL1 is a strong candidate for future evaluation regarding the roles of the adhesin activity of the protein during L. interrogans infection. PMID:25875373

  8. Adhesins in Human Fungal Pathogens: Glue with Plenty of Stick

    PubMed Central

    de Groot, Piet W. J.; Bader, Oliver; de Boer, Albert D.; Weig, Michael

    2013-01-01

    Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases. PMID:23397570

  9. Binding Efficiency of Protein-Protein Complexes

    PubMed Central

    Day, Eric S.; Cote, Shaun M.; Whitty, Adrian

    2012-01-01

    We examine the relationship between binding affinity and interface size for reversible protein-protein interactions (PPI), using cytokines from the tumor necrosis factor (TNF) superfamily and their receptors as a test case. Using surface plasmon resonance, we measured single-site binding affinities for the large receptor TNFR1 binding to its ligands TNFα (KD = 1.4 ± 0.4 nM) and lymphotoxin-α (KD = 50 ± 10 nM), and also for the small receptor Fn14 binding to TWEAK (KD = 70 ± 10 nM). We additionally assembled data for all other TNF/TNFR family complexes for which reliable single site binding affinities have been reported. We used these values to calculate the binding efficiency – defined as binding energy per Å2 of surface area buried at the contact interface – for the nine of these complexes for which co-crystal structures are available, and compared the results to those for a set of 144 protein-protein complexes with published affinity values. The results show that the most efficient PPI complexes generate ~20 cal.mol−1/Å2 of binding energy. A minimum contact area of ~500 Å2 is required for a stable complex, required to generate sufficient interaction energy to pay the entropic cost of co-localizing two proteins from 1 M solution. The most compact and efficient TNF/TNFR complex was BAFF/BR3, which achieved ~80% of the maximum achievable binding efficiency. Other small receptors also gave high binding efficiencies, while the larger receptors generated only 44-49% of this limit despite interacting primarily through just a single small domain. The results provide new insight into how much binding energy can be generated by a PPI interface of a given size, and establish a quantitative method to predict how large a natural or engineered contact interface must be to achieve a given level of binding affinity. PMID:23088250

  10. Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine.

    PubMed

    Okamba, Faust René; Arella, Maximilien; Music, Nedzad; Jia, Jian Jun; Gottschalk, Marcelo; Gagnon, Carl A

    2010-07-01

    Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 + or - 9.6%) compared to the unvaccinated and challenged animals (45.8 + or - 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 + or - 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 + or - 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines. PMID:20472025

  11. Identification of Cell-Binding Adhesins of Leptospira interrogans

    PubMed Central

    Evangelista, Karen V.; Hahn, Beth; Wunder, Elsio A.; Ko, Albert I.; Haake, David A.; Coburn, Jenifer

    2014-01-01

    Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic Leptospira species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic Leptospira interrogans binds to host cells more efficiently than to ECM components. Using in vitro phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate L. interrogans sv. Copenhageni st. Fiocruz L1–130 adhesins. Recombinant LIC11574, but not its L. biflexa homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for L. interrogans. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in L. interrogans infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and

  12. The Biology of Neisseria Adhesins

    PubMed Central

    Hung, Miao-Chiu; Christodoulides, Myron

    2013-01-01

    Members of the genus Neisseria include pathogens causing important human diseases such as meningitis, septicaemia, gonorrhoea and pelvic inflammatory disease syndrome. Neisseriae are found on the exposed epithelia of the upper respiratory tract and the urogenital tract. Colonisation of these exposed epithelia is dependent on a repertoire of diverse bacterial molecules, extending not only from the surface of the bacteria but also found within the outer membrane. During invasive disease, pathogenic Neisseriae also interact with immune effector cells, vascular endothelia and the meninges. Neisseria adhesion involves the interplay of these multiple surface factors and in this review we discuss the structure and function of these important molecules and the nature of the host cell receptors and mechanisms involved in their recognition. We also describe the current status for recently identified Neisseria adhesins. Understanding the biology of Neisseria adhesins has an impact not only on the development of new vaccines but also in revealing fundamental knowledge about human biology. PMID:24833056

  13. Functional Mapping of an Oligomeric Autotransporter Adhesin of Aggregatibacter actinomycetemcomitans▿

    PubMed Central

    Yu, Chunxiao; Ruiz, Teresa; Lenox, Christopher; Mintz, Keith P.

    2008-01-01

    Extracellular matrix protein adhesin A (EmaA) is a 202-kDa nonfimbrial adhesin, which mediates the adhesion of the oral pathogen Aggregatibacter actinomycetemcomitans to collagen. EmaA oligomers form surface antenna-like protrusions consisting of a long helical rod with an ellipsoidal ending. The functional analysis of in-frame emaA deletion mutants has located the collagen binding activity to the amino terminus of the protein corresponding to amino acids 70 to 386. The level of collagen binding of this deletion mutant was comparable to the emaA mutant strain. Transmission electron microscopy studies indicate that the first 330 amino acids of the mature protein form the ellipsoidal ending of the EmaA protrusions, where the activity resides. Amino acid substitution analysis within this sequence has identified a critical amino acid, which is essential for the formation of the ellipsoidal ending and for collagen binding activity. PMID:18310342

  14. Molecular cloning and characterization of Dr-II, a nonfimbrial adhesin-I-like adhesin isolated from gestational pyelonephritis-associated Escherichia coli that binds to decay-accelerating factor.

    PubMed Central

    Pham, T Q; Goluszko, P; Popov, V; Nowicki, S; Nowicki, B J

    1997-01-01

    Bacterial adhesins play an important role in the colonization of the human urogenital tract. Escherichia coli Dr family adhesins have been found to be frequently expressed in strains associated with pyelonephritis in pregnant females. The tissue receptor for known Dr adhesins has been localized to the short consensus repeat-3 (SCR-3) domain of decay accelerating factor (DAF), a complement regulatory protein. In this report, we identified and cloned draE2, a gene encoding a novel 17-kDa DAF-binding adhesin, Dr-II, from a strain of E. coli associated with acute gestational pyelonephritis. Despite the significant sequence diversity between Dr-II and Dr family adhesins, the receptor of Dr-II was found to be the SCR-3 domain of DAF. Sequence analysis of the 186-amino-acid Dr-II open reading frame revealed significant diversity from other members of the Dr adhesin family, including Dr, AFA-I, AFA-III, and F1845, but only an 8-amino-acid difference in sequence from that of the 17-kDa nonfimbrial adhesin NFA-I of unknown receptor specificity. N-terminal peptide sequencing of the purified adhesin confirmed the identity of the open reading frame and indicated cleavage of a 28-amino-acid signal peptide. Antibodies raised against purified Dr-II adhesin exhibited little or no cross-reactivity to Dr adhesin. Characterization of the biological properties demonstrated that like the Dr adhesins, Dr-II was associated with the ability of E. coli to bind to tubular basement membranes and Bowman's capsule and to be internalized into HeLa cells. PMID:9317041

  15. Suppression subtractive hybridization identifies an autotransporter adhesin gene of E. coli IMT5155 specifically associated with avian pathogenic Escherichia coli (APEC)

    PubMed Central

    2010-01-01

    Background Extraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathoype within the whole ExPEC group. Results Twenty-eight different genomic loci were identified, which are present in IMT5155 but not in CFT073. One of these loci contained a gene encoding a putative autotransporter adhesin. The open reading frame of the gene spans a 3,498 bp region leading to a putative 124-kDa adhesive protein. A specific antibody was raised against this protein and expression of the adhesin was shown under laboratory conditions. Adherence and adherence inhibition assays demonstrated a role for the corresponding protein in adhesion to DF-1 chicken fibroblasts. Sequence analyses revealed that the flanking regions of the chromosomally located gene contained sequences of mobile genetic elements, indicating a probable spread among different strains by horizontal gene transfer. In accordance with this hypothesis, the adhesin was found to be present not only in different phylogenetic groups of extraintestinal pathogenic but also of commensal E. coli strains, yielding a significant association with strains of avian origin. Conclusions We identified a chromosomally located autotransporter gene in a highly virulent APEC

  16. GECluster: a novel protein complex prediction method

    PubMed Central

    Su, Lingtao; Liu, Guixia; Wang, Han; Tian, Yuan; Zhou, Zhihui; Han, Liang; Yan, Lun

    2014-01-01

    Identification of protein complexes is of great importance in the understanding of cellular organization and functions. Traditional computational protein complex prediction methods mainly rely on the topology of protein–protein interaction (PPI) networks but seldom take biological information of proteins (such as Gene Ontology (GO)) into consideration. Meanwhile, the environment relevant analysis of protein complex evolution has been poorly studied, partly due to the lack of high-precision protein complex datasets. In this paper, a combined PPI network is introduced to predict protein complexes which integrate both GO and expression value of relevant protein-coding genes. A novel protein complex prediction method GECluster (Gene Expression Cluster) was proposed based on a seed node expansion strategy, in which a combined PPI network was utilized. GECluster was applied to a training combined PPI network and it predicted more credible complexes than peer methods. The results indicate that using a combined PPI network can efficiently improve protein complex prediction accuracy. In order to study protein complex evolution within cells due to changes in the living environment surrounding cells, GECluster was applied to seven combined PPI networks constructed using the data of a test set including yeast response to stress throughout a wine fermentation process. Our results showed that with the rise of alcohol concentration, protein complexes within yeast cells gradually evolve from one state to another. Besides this, the number of core and attachment proteins within a protein complex both changed significantly. PMID:26019559

  17. The giant adhesin SiiE of Salmonella enterica.

    PubMed

    Barlag, Britta; Hensel, Michael

    2015-01-01

    Salmonella enterica is a Gram-negative, food-borne pathogen, which colonizes the intestinal tract and invades enterocytes. Invasion of polarized cells depends on the SPI1-encoded type III secretion system (T3SS) and the SPI4-encoded type I secretion system (T1SS). The substrate of this T1SS is the non-fimbrial giant adhesin SiiE. With a size of 595 kDa, SiiE is the largest protein of the Salmonella proteome and consists of 53 repetitive bacterial immunoglobulin (BIg) domains, each containing several conserved residues. As known for other T1SS substrates, such as E. coli HlyA, Ca2+ ions bound by conserved D residues within the BIg domains stabilize the protein and facilitate secretion. The adhesin SiiE mediates the first contact to the host cell and thereby positions the SPI1-T3SS to initiate the translocation of a cocktail of effector proteins. This leads to actin remodeling, membrane ruffle formation and bacterial internalization. SiiE binds to host cell apical membranes in a lectin-like manner. GlcNAc and α2-3 linked sialic acid-containing structures are ligands of SiiE. Since SiiE shows repetitive domain architecture, we propose a zipper-like binding mediated by each individual BIg domain. In this review, we discuss the characteristics of the SPI4-T1SS and the giant adhesin SiiE. PMID:25587788

  18. Lipoprotein CD0873 is a novel adhesin of Clostridium difficile.

    PubMed

    Kovacs-Simon, Andrea; Leuzzi, Rosanna; Kasendra, Magdalena; Minton, Nigel; Titball, Richard W; Michell, Stephen L

    2014-07-15

    Clostridium difficile is a cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease. Colonization of the gut is a critical step in the course of infection. The C. difficile lipoprotein CD0873 was identified as a putative adhesin through a bioinformatics approach. Surface exposure of CD0873 was confirmed and a CD0873 mutant was generated. The CD0873 mutant showed a significant reduction in adherence to Caco-2 cells and wild-type bacteria preincubated with anti-CD0873 antibodies showed significantly decreased adherence to Caco-2 cells. In addition, we demonstrated that purified recombinant CD0873 protein alone associates with Caco-2 cells. This is the first definitive identification of a C. difficile adhesin, which now allows work to devise improved measures for preventing and treating disease. PMID:24482399

  19. The Human Disease-Associated Aβ Amyloid Core Sequence Forms Functional Amyloids in a Fungal Adhesin

    PubMed Central

    Rameau, Rachele D.; Jackson, Desmond N.; Beaussart, Audrey; Dufrêne, Yves F.

    2016-01-01

    ABSTRACT There is increasing evidence that many amyloids in living cells have physiological functions. On the surfaces of fungal cells, amyloid core sequences in adhesins can aggregate into 100- to 1,000-nm-wide patches to form high-avidity adhesion nanodomains on the cell surface. The nanodomains form through interactions that have amyloid-like properties: binding of amyloid dyes, perturbation by antiamyloid agents, and interaction with homologous sequences. To test whether these functional interactions are mediated by typical amyloid interactions, we substituted an amyloid core sequence, LVFFA, from human Aβ protein for the native sequence IVIVA in the 1,419-residue Candida albicans adhesin Als5p. The chimeric protein formed cell surface nanodomains and mediated cellular aggregation. The native sequence and chimeric adhesins responded similarly to the amyloid dye thioflavin T and to amyloid perturbants. However, unlike the native protein, the nanodomains formed by the chimeric protein were not force activated and formed less-robust aggregates under flow. These results showed the similarity of amyloid interactions in the amyloid core sequences of native Als5p and Aβ, but they also highlighted emergent properties of the native sequence. Also, a peptide composed of the Aβ amyloid sequence flanked by amino acids from the adhesin formed two-dimensional sheets with sizes similar to the cell surface patches of the adhesins. These results inform an initial model for the structure of fungal cell surface amyloid nanodomains. PMID:26758179

  20. Structure Prediction of Protein Complexes

    NASA Astrophysics Data System (ADS)

    Pierce, Brian; Weng, Zhiping

    Protein-protein interactions are critical for biological function. They directly and indirectly influence the biological systems of which they are a part. Antibodies bind with antigens to detect and stop viruses and other infectious agents. Cell signaling is performed in many cases through the interactions between proteins. Many diseases involve protein-protein interactions on some level, including cancer and prion diseases.

  1. Immunoisolation of Protein Complexes from Xenopus

    PubMed Central

    Conlon, Frank L.; Miteva, Yana; Kaltenbrun, Erin; Waldron, Lauren; Greco, Todd M.; Cristea, Ileana M.

    2013-01-01

    The immunoaffinity isolation of protein complexes is an essential technique for the purification and concentration of protein complexes from cells and tissues. In this chapter we present the methodologies for the purification of proteins and protein complexes from Xenopus laev is and Xenopus tropical is. Specific to this protocol are the techniques for the cryolysis of Xenopus cells and tissues, a procedure that limits contamination from yolk proteins while preserving endogenous protein complexes, the methodologies for immunoaffinity purification of proteins using magnetic beads, and the protocols for western blot analysis. In addition, the procedures in this chapter can be extended to use with proteomic analysis of protein complexes as presented in the following chapter. PMID:22956099

  2. Pseudomonas aeruginosa outer membrane adhesins for human respiratory mucus glycoproteins.

    PubMed Central

    Carnoy, C; Scharfman, A; Van Brussel, E; Lamblin, G; Ramphal, R; Roussel, P

    1994-01-01

    The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cases of cystic fibrosis. For this purpose, microtiter plate adhesion assays have been developed and have suggested that nonpilus adhesins of P. aeruginosa are the most important ones for binding to human respiratory mucins. In order to characterize these mucin-binding adhesins, outer membrane proteins (OMP) from two adhesive strains, 1244-NP and PAK-NP, and their poorly adhesive rpoN mutants, 1244-N3 and PAK-N1, were prepared by a mild extraction with Zwittergent 3-14. Mucin-binding adhesins were detected after polyacrylamide gel electrophoresis and blotting of the OMP on nitrocellulose replicas, using human bronchial mucins labeled with 125I. The binding properties of these OMP with lactotransferrin, another glycoprotein abundant in respiratory mucus, were also studied. Radiolabeled mucins detected four bands at 48, 46, 28, and 25 kDa with strain PAK-NP. With the nonmucoid strain 1244-NP, five bands were observed at 48, 46, 42, 28, and 25 kDa. The bands at 48 and 25 kDa were also visualized by radiolabeled lactotransferrin. These bands were partially or completely displaced by nonradiolabeled respiratory mucin glycopeptides but not by tetramethylurea, suggesting that they recognized carbohydrate sites. In contrast, the poorly adhesive strains showed weakly binding bands. These results demonstrate that outer membranes from two different nonpiliated P. aeruginosa strains express multiple adhesins with an affinity for human respiratory mucins and/or lactotransferrin. Images PMID:8168955

  3. The novel chlamydial adhesin CPn0473 mediates the lipid raft-dependent uptake of Chlamydia pneumoniae.

    PubMed

    Fechtner, Tim; Galle, Jan N; Hegemann, Johannes H

    2016-08-01

    Chlamydiae are Gram-negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down-stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473-coupled fluorescent latex beads adhere to human epithelial HEp-2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp-2 cells with rCPn0473 does not attenuate adhesion but promotes dose-dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473-dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae. PMID:26780295

  4. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

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

    2014-01-01

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

  5. Investigation of a protein complex network

    NASA Astrophysics Data System (ADS)

    Mashaghi, A. R.; Ramezanpour, A.; Karimipour, V.

    2004-09-01

    The budding yeast Saccharomyces cerevisiae is the first eukaryote whose genome has been completely sequenced. It is also the first eukaryotic cell whose proteome (the set of all proteins) and interactome (the network of all mutual interactions between proteins) has been analyzed. In this paper we study the structure of the yeast protein complex network in which weighted edges between complexes represent the number of shared proteins. It is found that the network of protein complexes is a small world network with scale free behavior for many of its distributions. However we find that there are no strong correlations between the weights and degrees of neighboring complexes. To reveal non-random features of the network we also compare it with a null model in which the complexes randomly select their proteins. Finally we propose a simple evolutionary model based on duplication and divergence of proteins.

  6. Trapping mammalian protein complexes in viral particles

    PubMed Central

    Eyckerman, Sven; Titeca, Kevin; Van Quickelberghe, Emmy; Cloots, Eva; Verhee, Annick; Samyn, Noortje; De Ceuninck, Leentje; Timmerman, Evy; De Sutter, Delphine; Lievens, Sam; Van Calenbergh, Serge; Gevaert, Kris; Tavernier, Jan

    2016-01-01

    Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. PMID:27122307

  7. Improved method for protein complex detection using bottleneck proteins

    PubMed Central

    2013-01-01

    Background Detecting protein complexes is one of essential and fundamental tasks in understanding various biological functions or processes. Therefore accurate identification of protein complexes is indispensable. Methods For more accurate detection of protein complexes, we propose an algorithm which detects dense protein sub-networks of which proteins share closely located bottleneck proteins. The proposed algorithm is capable of finding protein complexes which allow overlapping with each other. Results We applied our algorithm to several PPI (Protein-Protein Interaction) networks of Saccharomyces cerevisiae and Homo sapiens, and validated our results using public databases of protein complexes. The prediction accuracy was even more improved over our previous work which used also bottleneck information of the PPI network, but showed limitation when predicting small-sized protein complex detection. Conclusions Our algorithm resulted in overlapping protein complexes with significantly improved F1 score over existing algorithms. This result comes from high recall due to effective network search, as well as high precision due to proper use of bottleneck information during the network search. PMID:23566214

  8. Identification of Novel Laminin- and Fibronectin-binding Proteins by Far-Western Blot: Capturing the Adhesins of Streptococcus suis Type 2

    PubMed Central

    Li, Quan; Liu, Hanze; Du, Dechao; Yu, Yanfei; Ma, Caifeng; Jiao, Fangfang; Yao, Huochun; Lu, Chengping; Zhang, Wei

    2015-01-01

    Bacterial cell wall (CW) and extracellular (EC) proteins are often involved in interactions with extracellular matrix (ECM) proteins such as laminin (LN) and fibronectin (FN), which play important roles in adhesion and invasion. In this study, an efficient method combining proteomic analysis and Far-Western blotting assays was developed to screen directly for bacterial surface proteins with LN- and FN-binding capacity. With this approach, fifteen potential LN-binding proteins and five potential FN-binding proteins were identified from Streptococcus suis serotype 2 (SS2) CW and EC proteins. Nine newly identified proteins, including oligopeptide-binding protein OppA precursor (OppA), elongation factor Tu (EF-Tu), enolase, lactate dehydrogenase (LDH), fructose-bisphosphate aldolase (FBA), 3-ketoacyl-ACP reductase (KAR), Gly ceraldehyde-3-phosphate dehydrogenase (GAPDH), Inosine 5′-monophosphate dehydrogenase (IMPDH), and amino acid ABC transporter permease (ABC) were cloned, expressed, purified and further confirmed by Far-Western blotting and ELISA. Five proteins (OppA, EF-Tu, enolase, LDH, and FBA) exhibited specifically binding activity to both human LN and human FN. Furthermore, seven important recombinant proteins were selected and identified to have the ability to bind Hep-2 cells by the indirect immunofluorescent assay. In addition, four recombinant proteins, and their corresponding polyclonal antibodies, were observed to decrease SS2 adhesion to Hep-2 cells, which indicates that these proteins contribute to the adherence of SS2 to host cell surface. Collectively, these results show that the approach described here represents a useful tool for investigating the host-pathogen interactions. PMID:26636044

  9. Metabolic Adaptation and Protein Complexes in Prokaryotes

    PubMed Central

    Krüger, Beate; Liang, Chunguang; Prell, Florian; Fieselmann, Astrid; Moya, Andres; Schuster, Stefan; Völker, Uwe; Dandekar, Thomas

    2012-01-01

    Protein complexes are classified and have been charted in several large-scale screening studies in prokaryotes. These complexes are organized in a factory-like fashion to optimize protein production and metabolism. Central components are conserved between different prokaryotes; major complexes involve carbohydrate, amino acid, fatty acid and nucleotide metabolism. Metabolic adaptation changes protein complexes according to environmental conditions. Protein modification depends on specific modifying enzymes. Proteins such as trigger enzymes display condition-dependent adaptation to different functions by participating in several complexes. Several bacterial pathogens adapt rapidly to intracellular survival with concomitant changes in protein complexes in central metabolism and optimize utilization of their favorite available nutrient source. Regulation optimizes protein costs. Master regulators lead to up- and downregulation in specific subnetworks and all involved complexes. Long protein half-life and low level expression detaches protein levels from gene expression levels. However, under optimal growth conditions, metabolite fluxes through central carbohydrate pathways correlate well with gene expression. In a system-wide view, major metabolic changes lead to rapid adaptation of complexes and feedback or feedforward regulation. Finally, prokaryotic enzyme complexes are involved in crowding and substrate channeling. This depends on detailed structural interactions and is verified for specific effects by experiments and simulations. PMID:24957769

  10. The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica

    PubMed Central

    Kakoschke, Tamara Katharina; Kakoschke, Sara Carina; Zeuzem, Catharina; Bouabe, Hicham; Adler, Kristin; Heesemann, Jürgen; Rossier, Ombeline

    2016-01-01

    In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies. PMID:27387855

  11. The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica.

    PubMed

    Kakoschke, Tamara Katharina; Kakoschke, Sara Carina; Zeuzem, Catharina; Bouabe, Hicham; Adler, Kristin; Heesemann, Jürgen; Rossier, Ombeline

    2016-01-01

    In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies. PMID:27387855

  12. Predicting Physical Interactions between Protein Complexes*

    PubMed Central

    Clancy, Trevor; Rødland, Einar Andreas; Nygard, Ståle; Hovig, Eivind

    2013-01-01

    Protein complexes enact most biochemical functions in the cell. Dynamic interactions between protein complexes are frequent in many cellular processes. As they are often of a transient nature, they may be difficult to detect using current genome-wide screens. Here, we describe a method to computationally predict physical interactions between protein complexes, applied to both humans and yeast. We integrated manually curated protein complexes and physical protein interaction networks, and we designed a statistical method to identify pairs of protein complexes where the number of protein interactions between a complex pair is due to an actual physical interaction between the complexes. An evaluation against manually curated physical complex-complex interactions in yeast revealed that 50% of these interactions could be predicted in this manner. A community network analysis of the highest scoring pairs revealed a biologically sensible organization of physical complex-complex interactions in the cell. Such analyses of proteomes may serve as a guide to the discovery of novel functional cellular relationships. PMID:23438732

  13. Identification of Coli Surface Antigen 23, a novel adhesin of enterotoxigenic Escherichia coli.

    PubMed

    Del Canto, Felipe; Botkin, Douglas J; Valenzuela, Patricio; Popov, Vsevolod; Ruiz-Perez, Fernando; Nataro, James P; Levine, Myron M; Stine, O Colin; Pop, Mihai; Torres, Alfredo G; Vidal, Roberto

    2012-08-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains. PMID:22645287

  14. Identification of Coli Surface Antigen 23, a Novel Adhesin of Enterotoxigenic Escherichia coli

    PubMed Central

    Del Canto, Felipe; Botkin, Douglas J.; Valenzuela, Patricio; Popov, Vsevolod; Ruiz-Perez, Fernando; Nataro, James P.; Levine, Myron M.; Stine, O. Colin; Pop, Mihai

    2012-01-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains. PMID:22645287

  15. A Protein Complex Map of Trypanosoma brucei

    PubMed Central

    Mehta, Vaibhav; Najafabadi, Hamed S.; Moshiri, Houtan; Jardim, Armando; Salavati, Reza

    2016-01-01

    The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org. PMID:26991453

  16. Proteins as paradigms of complex systems.

    SciTech Connect

    Fenimore, P. W.; Frauenfelder, Hans,; Young, R. D.

    2003-03-26

    The science of complexity has moved to center stage within the past few decades. Complex systems range from glasses to the immune system and the brain. Glasses are too simple to possess all aspects of complexity; brains are too complex to expose common concepts and laws of complexity. Proteins, however, are systems where many concepts and laws of complexity can be explored experimentally, theoretically, and computationally. Such studies have elucidated crucial aspects. The energy landscape has emerged as one central concept; it describes the free energy of a system as a function of temperature and the coordinates of all relevant atoms. A second concept is that of fluctuations. Without fluctuations, proteins would be dead and life impossible. A third concept is slaving. Proteins are not isolated systems; they are embedded in cells and membranes. Slaving arises when the fluctuations in the surroundings of a protein dominate many of the motions of the protein proper.

  17. Complex Reconstitution from Individual Protein Modules.

    PubMed

    Basquin, Jérôme; Taschner, Michael; Lorentzen, Esben

    2016-01-01

    Cellular function relies on protein complexes that work as nano-machines. The structure and function of protein complexes is an outcome of the specific combination of protein subunits, or modules, within the complex. A major focus of molecular biology is thus to understand how protein subunits assemble to form complexes with distinct biological function. To this end, in vitro reconstitution of complexes from individual subunits to study their assembly, structure and activity is of central importance. With purified individual subunits and sub-modules at hand one can systematically dissect the hierarchical assembly of larger complexes using direct protein-protein interaction assays. Furthermore, activity assays can be carried out with individual subunits or smaller sub-complexes and compared to those of the fully assembled complex to precisely map functional sites and provide a molecular basis for in vivo observations. In this chapter we review methods for protein complex assembly from individual subunits and provide examples of advantages and potential pitfalls to this approach. PMID:27165333

  18. Protein Complex Purification by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601

  19. Systematic Characterization of Human Protein Complexes Identifies Chromosome Segregation Proteins

    PubMed Central

    Hutchins, James R.A.; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M.; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A.; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A.; Peters, Jan-Michael

    2010-01-01

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference (RNAi) screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization and tandem affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex (APC/C) and the γ-tubulin ring complex (γ-TuRC), large complexes which are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high throughput follow-up analyses of phenotypic screens in mammalian cells. PMID:20360068

  20. Systematic analysis of human protein complexes identifies chromosome segregation proteins.

    PubMed

    Hutchins, James R A; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A; Peters, Jan-Michael

    2010-04-30

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells. PMID:20360068

  1. Neisseria Adhesin A Variation and Revised Nomenclature Scheme

    PubMed Central

    Bambini, Stefania; De Chiara, Matteo; Muzzi, Alessandro; Mora, Marirosa; Lucidarme, Jay; Brehony, Carina; Borrow, Ray; Masignani, Vega; Comanducci, Maurizio; Maiden, Martin C. J.; Pizza, Mariagrazia; Jolley, Keith A.

    2014-01-01

    Neisseria adhesin A (NadA), involved in the adhesion and invasion of Neisseria meningitidis into host tissues, is one of the major components of Bexsero, a novel multicomponent vaccine licensed for protection against meningococcal serogroup B in Europe, Australia, and Canada. NadA has been identified in approximately 30% of clinical isolates and in a much lower proportion of carrier isolates. Three protein variants were originally identified in invasive meningococci and named NadA-1, NadA-2, and NadA-3, whereas most carrier isolates either lacked the gene or harbored a different variant, NadA-4. Further analysis of isolates belonging to the sequence type 213 (ST-213) clonal complex identified NadA-5, which was structurally similar to NadA-4, but more distantly related to NadA-1, -2, and -3. At the time of this writing, more than 89 distinct nadA allele sequences and 43 distinct peptides have been described. Here, we present a revised nomenclature system, taking into account the complete data set, which is compatible with previous classification schemes and is expandable. The main features of this new scheme include (i) the grouping of the previously named NadA-2 and NadA-3 variants into a single NadA-2/3 variant, (ii) the grouping of the previously assigned NadA-4 and NadA-5 variants into a single NadA-4/5 variant, (iii) the introduction of an additional variant (NadA-6), and (iv) the classification of the variants into two main groups, named groups I and II. To facilitate querying of the sequences and submission of new allele sequences, the nucleotide and amino acid sequences are available at http://pubmlst.org/neisseria/NadA/. PMID:24807056

  2. Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes.

    PubMed

    Angerer, Heike

    2015-01-01

    In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria. PMID:25686363

  3. Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Arnal, L.; Longo, G.; Stupar, P.; Castez, M. F.; Cattelan, N.; Salvarezza, R. C.; Yantorno, O. M.; Kasas, S.; Vela, M. E.

    2015-10-01

    Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract

  4. Surfactant Protein A (SP-A)-mediated Clearance of Staphylococcus aureus Involves Binding of SP-A to the Staphylococcal Adhesin Eap and the Macrophage Receptors SP-A Receptor 210 and Scavenger Receptor Class A*

    PubMed Central

    Sever-Chroneos, Zvjezdana; Krupa, Agnieszka; Davis, Jeremy; Hasan, Misbah; Yang, Ching-Hui; Szeliga, Jacek; Herrmann, Mathias; Hussain, Muzafar; Geisbrecht, Brian V.; Kobzik, Lester; Chroneos, Zissis C.

    2011-01-01

    Staphylococcus aureus causes life-threatening pneumonia in hospitals and deadly superinfection during viral influenza. The current study investigated the role of surfactant protein A (SP-A) in opsonization and clearance of S. aureus. Previous studies showed that SP-A mediates phagocytosis via the SP-A receptor 210 (SP-R210). Here, we show that SP-R210 mediates binding and control of SP-A-opsonized S. aureus by macrophages. We determined that SP-A binds S. aureus through the extracellular adhesin Eap. Consequently, SP-A enhanced macrophage uptake of Eap-expressing (Eap+) but not Eap-deficient (Eap−) S. aureus. In a reciprocal fashion, SP-A failed to enhance uptake of Eap+ S. aureus in peritoneal Raw264.7 macrophages with a dominant negative mutation (SP-R210(DN)) blocking surface expression of SP-R210. Accordingly, WT mice cleared infection with Eap+ but succumbed to sublethal infection with Eap- S. aureus. However, SP-R210(DN) cells compensated by increasing non-opsonic phagocytosis of Eap+ S. aureus via the scavenger receptor scavenger receptor class A (SR-A), while non-opsonic uptake of Eap− S. aureus was impaired. Macrophages express two isoforms: SP-R210L and SP-R210S. The results show that WT alveolar macrophages are distinguished by expression of SP-R210L, whereas SR-A−/− alveolar macrophages are deficient in SP-R210L expressing only SP-R210S. Accordingly, SR-A−/− mice were highly susceptible to both Eap+ and Eap− S. aureus. The lungs of susceptible mice generated abnormal inflammatory responses that were associated with impaired killing and persistence of S. aureus infection in the lung. In conclusion, alveolar macrophage SP-R210L mediates recognition and killing of SP-A-opsonized S. aureus in vivo, coordinating inflammatory responses and resolution of S. aureus pneumonia through interaction with SR-A. PMID:21123169

  5. Binding Forces of Streptococcus mutans P1 Adhesin

    PubMed Central

    Sullan, Ruby May A.; Li, James K.; Crowley, Paula J.; Brady, L. Jeannine; Dufrêne, Yves F.

    2015-01-01

    Streptococcus mutans is a Gram-positive oral bacterium that is a primary etiological agent associated with human dental caries. In the oral cavity, S. mutans adheres to immobilized salivary agglutinin (SAG) contained within the salivary pellicle on the tooth surface. Binding to SAG is mediated by cell surface P1, a multifunctional adhesin that is also capable of interacting with extracellular matrix proteins. This may be of particular importance outside of the oral cavity as S. mutans has been associated with infective endocarditis and detected in atherosclerotic plaque. Despite the biomedical importance of P1, its binding mechanisms are not completely understood. In this work, we use atomic force microscopy-based single-molecule and single-cell force spectroscopy to quantify the nanoscale forces driving P1-mediated adhesion. Single-molecule experiments show that full-length P1, as well as fragments containing only the P1 globular head or C-terminal region, binds to SAG with relatively weak forces (~50 pN). In contrast, single-cell analyses reveal that adhesion of a single S. mutans cell to SAG is mediated by strong (~500 pN) and long-range (up to 6000 nm) forces. This is likely due to the binding of multiple P1 adhesins to self-associated gp340 glycoproteins. Such a cooperative, long-range character of the S. mutans–SAG interaction would therefore dramatically increase the strength and duration of cell adhesion. We also demonstrate, at single-molecule and single-cell levels, the interaction of P1 with fibronectin and collagen, as well as with hydrophobic, but not hydrophilic, substrates. The binding mechanism (strong forces, cooperativity, broad specificity) of P1 provides a molecular basis for its multifunctional adhesion properties. Our methodology represents a valuable approach to probe the binding forces of bacterial adhesins and offers a tractable methodology to assess anti-adhesion therapy. PMID:25671413

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

    PubMed

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

    2016-01-01

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

  7. Protein-protein interaction networks (PPI) and complex diseases

    PubMed Central

    Safari-Alighiarloo, Nahid; Taghizadeh, Mohammad; Rezaei-Tavirani, Mostafa; Goliaei, Bahram

    2014-01-01

    The physical interaction of proteins which lead to compiling them into large densely connected networks is a noticeable subject to investigation. Protein interaction networks are useful because of making basic scientific abstraction and improving biological and biomedical applications. Based on principle roles of proteins in biological function, their interactions determine molecular and cellular mechanisms, which control healthy and diseased states in organisms. Therefore, such networks facilitate the understanding of pathogenic (and physiologic) mechanisms that trigger the onset and progression of diseases. Consequently, this knowledge can be translated into effective diagnostic and therapeutic strategies. Furthermore, the results of several studies have proved that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer and autoimmune disorders. Based on such relationship, a novel paradigm is suggested in order to confirm that the protein interaction networks can be the target of therapy for treatment of complex multi-genic diseases rather than individual molecules with disrespect the network. PMID:25436094

  8. Solid-phase preparation of protein complexes.

    PubMed

    Pengo, Paolo; Veggiani, Gianluca; Rattanamanee, Kwanchai; Gallotta, Andrea; Beneduce, Luca; Fassina, Giorgio

    2010-01-01

    Protein-protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin-biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies. PMID:21038355

  9. Solvation dynamics in a protein surfactant complex

    NASA Astrophysics Data System (ADS)

    Dutta, Partha; Sen, Pratik; Halder, Arnab; Mukherjee, Saptarshi; Sen, Sobhan; Bhattacharyya, Kankan

    2003-08-01

    Solvation dynamics in the denatured state of a protein, lysozyme (denatured by sodium dodecyl sulfate, SDS) is markedly slower than that in the native state. For coumarin 153 bound to lysozyme, the average solvation time, < τs> is 330 ps. In the lysozyme-SDS complex, the solvation dynamics is markedly slower with < τs>=7250 ps. On addition of dithiothreitol (DTT) to the lysozyme-SDS complex, when the di-sulfide bonds are destroyed, < τs> is found to be 1140 ps. The slow dynamics in the denatured protein is attributed to the polymer chain dynamics and the exchange of bound and free water molecules.

  10. Peroxisome protein import: a complex journey

    PubMed Central

    Baker, Alison; Hogg, Thomas Lanyon; Warriner, Stuart L.

    2016-01-01

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor–cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. PMID:27284042

  11. 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

  12. On protein abundance distributions in complex mixtures

    PubMed Central

    2013-01-01

    Mass spectrometry, an analytical technique that measures the mass-to-charge ratio of ionized atoms or molecules, dates back more than 100 years, and has both qualitative and quantitative uses for determining chemical and structural information. Quantitative proteomic mass spectrometry on biological samples focuses on identifying the proteins present in the samples, and establishing the relative abundances of those proteins. Such protein inventories create the opportunity to discover novel biomarkers and disease targets. We have previously introduced a normalized, label-free method for quantification of protein abundances under a shotgun proteomics platform (Griffin et al., 2010). The introduction of this method for quantifying and comparing protein levels leads naturally to the issue of modeling protein abundances in individual samples. We here report that protein abundance levels from two recent proteomics experiments conducted by the authors can be adequately represented by Sichel distributions. Mathematically, Sichel distributions are mixtures of Poisson distributions with a rather complex mixing distribution, and have been previously and successfully applied to linguistics and species abundance data. The Sichel model can provide a direct measure of the heterogeneity of protein abundances, and can reveal protein abundance differences that simpler models fail to show. PMID:23360617

  13. Recognition of bacterial lipopolysaccharide using bacteriophage-adhesin-coated long-period gratings.

    PubMed

    Brzozowska, Ewa; Śmietana, Mateusz; Koba, Marcin; Górska, Sabina; Pawlik, Krzysztof; Gamian, Andrzej; Bock, Wojtek J

    2015-05-15

    In this paper we present a new type of highly sensitive label-free sensor based on long-period gratings (LPG) coated with T4 bacteriophage (phage) adhesin. The adhesin (gp37) binds Escherichia coli B (E. coli B) by recognizing its bacterial lipopolysaccharide (LPS). The LPG biofunctionalization methodology is based on coating LPG surface with nickel ions capable of gp37 histidine tag reversible binding. For the first time recombinant adhesive phage protein has been used as a receptor molecule in biosensing scheme. The specificity of LPS binding by adhesin has been tested with LPG-based device and confirmed using Western blot, Enzyme-Linked Immunosorbent Assay (ELISA) and BIACORE methods. The LPG-based sensor can measure bacterial contamination in real time and with a high accuracy. We show that T4 phage adhesin binds E. coli B LPS in its native or denatured form. The binding is highly specific and irreversible. The applied procedure allows for obtaining reusable biosensors. PMID:25067838

  14. A Biochemical Guide to Yeast Adhesins: Glycoproteins for Social and Antisocial Occasions

    PubMed Central

    Dranginis, Anne M.; Rauceo, Jason M.; Coronado, Juan E.; Lipke, Peter N.

    2007-01-01

    Fungi are nonmotile eukaryotes that rely on their adhesins for selective interaction with the environment and with other fungal cells. Glycosylphosphatidylinositol (GPI)-cross-linked adhesins have essential roles in mating, colony morphology, host-pathogen interactions, and biofilm formation. We review the structure and binding properties of cell wall-bound adhesins of ascomycetous yeasts and relate them to their effects on cellular interactions, with particular emphasis on the agglutinins and flocculins of Saccharomyces and the Als proteins of Candida. These glycoproteins share common structural motifs tailored to surface activity and biological function. After being secreted to the outer face of the plasma membrane, they are covalently anchored in the wall through modified GPI anchors, with their binding domains elevated beyond the wall surface on highly glycosylated extended stalks. N-terminal globular domains bind peptide or sugar ligands, with between millimolar and nanomolar affinities. These affinities and the high density of adhesins and ligands at the cell surface determine microscopic and macroscopic characteristics of cell-cell associations. Central domains often include Thr-rich tandemly repeated sequences that are highly glycosylated. These domains potentiate cell-to-cell binding, but the molecular mechanism of such an association is not yet clear. These repeats also mediate recombination between repeats and between genes. The high levels of recombination and epigenetic regulation are sources of variation which enable the population to continually exploit new niches and resources. PMID:17554046

  15. Assembly reflects evolution of protein complexes.

    PubMed

    Levy, Emmanuel D; Boeri Erba, Elisabetta; Robinson, Carol V; Teichmann, Sarah A

    2008-06-26

    A homomer is formed by self-interacting copies of a protein unit. This is functionally important, as in allostery, and structurally crucial because mis-assembly of homomers is implicated in disease. Homomers are widespread, with 50-70% of proteins with a known quaternary state assembling into such structures. Despite their prevalence, their role in the evolution of cellular machinery and the potential for their use in the design of new molecular machines, little is known about the mechanisms that drive formation of homomers at the level of evolution and assembly in the cell. Here we present an analysis of over 5,000 unique atomic structures and show that the quaternary structure of homomers is conserved in over 70% of protein pairs sharing as little as 30% sequence identity. Where quaternary structure is not conserved among the members of a protein family, a detailed investigation revealed well-defined evolutionary pathways by which proteins transit between different quaternary structure types. Furthermore, we show by perturbing subunit interfaces within complexes and by mass spectrometry analysis, that the (dis)assembly pathway mimics the evolutionary pathway. These data represent a molecular analogy to Haeckel's evolutionary paradigm of embryonic development, where an intermediate in the assembly of a complex represents a form that appeared in its own evolutionary history. Our model of self-assembly allows reliable prediction of evolution and assembly of a complex solely from its crystal structure. PMID:18563089

  16. Structural and Functional Analysis of the N-terminal Domain of the Streptococcus gordonii Adhesin Sgo0707

    PubMed Central

    Nylander, Åsa; Svensäter, Gunnel; Senadheera, Dilani B.; Cvitkovitch, Dennis G.; Davies, Julia R.; Persson, Karina

    2013-01-01

    The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes. PMID:23691093

  17. Dynamic interactions of proteins in complex networks

    SciTech Connect

    Appella, E.; Anderson, C.

    2009-10-01

    Recent advances in techniques such as NMR and EPR spectroscopy have enabled the elucidation of how proteins undergo structural changes to act in concert in complex networks. The three minireviews in this series highlight current findings and the capabilities of new methodologies for unraveling the dynamic changes controlling diverse cellular functions. They represent a sampling of the cutting-edge research presented at the 17th Meeting of Methods in Protein Structure Analysis, MPSA2008, in Sapporo, Japan, 26-29 August, 2008 (http://www.iapsap.bnl.gov). The first minireview, by Christensen and Klevit, reports on a structure-based yeast two-hybrid method for identifying E2 ubiquitin-conjugating enzymes that interact with the E3 BRCA1/BARD1 heterodimer ligase to generate either mono- or polyubiquitinated products. This method demonstrated for the first time that the BRCA1/BARD1 E3 can interact with 10 different E2 enzymes. Interestingly, the interaction with multiple E2 enzymes displayed unique ubiquitin-transfer properties, a feature expected to be common among other RING and U-box E3s. Further characterization of new E3 ligases and the E2 enzymes that interact with them will greatly enhance our understanding of ubiquitin transfer and facilitate studies of roles of ubiquitin and ubiquitin-like proteins in protein processing and trafficking. Stein et al., in the second minireview, describe recent progress in defining the binding specificity of different peptide-binding domains. The authors clearly point out that transient peptide interactions mediated by both post-translational modifications and disordered regions ensure a high level of specificity. They postulate that a regulatory code may dictate the number of combinations of domains and post-translational modifications needed to achieve the required level of interaction specificity. Moreover, recognition alone is not enough to obtain a stable complex, especially in a complex cellular environment. Increasing

  18. Radiolysis of DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Běgusová, Marie; Gillard, Nathalie; Sy, Denise; Castaing, Bertrand; Charlier, Michel; Spotheim-Maurizot, Melanie

    2005-02-01

    We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK.

  19. PCDq: human protein complex database with quality index which summarizes different levels of evidences of protein complexes predicted from H-Invitational protein-protein interactions integrative dataset

    PubMed Central

    2012-01-01

    Background Proteins interact with other proteins or biomolecules in complexes to perform cellular functions. Existing protein-protein interaction (PPI) databases and protein complex databases for human proteins are not organized to provide protein complex information or facilitate the discovery of novel subunits. Data integration of PPIs focused specifically on protein complexes, subunits, and their functions. Predicted candidate complexes or subunits are also important for experimental biologists. Description Based on integrated PPI data and literature, we have developed a human protein complex database with a complex quality index (PCDq), which includes both known and predicted complexes and subunits. We integrated six PPI data (BIND, DIP, MINT, HPRD, IntAct, and GNP_Y2H), and predicted human protein complexes by finding densely connected regions in the PPI networks. They were curated with the literature so that missing proteins were complemented and some complexes were merged, resulting in 1,264 complexes comprising 9,268 proteins with 32,198 PPIs. The evidence level of each subunit was assigned as a categorical variable. This indicated whether it was a known subunit, and a specific function was inferable from sequence or network analysis. To summarize the categories of all the subunits in a complex, we devised a complex quality index (CQI) and assigned it to each complex. We examined the proportion of consistency of Gene Ontology (GO) terms among protein subunits of a complex. Next, we compared the expression profiles of the corresponding genes and found that many proteins in larger complexes tend to be expressed cooperatively at the transcript level. The proportion of duplicated genes in a complex was evaluated. Finally, we identified 78 hypothetical proteins that were annotated as subunits of 82 complexes, which included known complexes. Of these hypothetical proteins, after our prediction had been made, four were reported to be actual subunits of the

  20. The soluble recombinant Neisseria meningitidis adhesin NadA(Δ351-405) stimulates human monocytes by binding to extracellular Hsp90.

    PubMed

    Cecchini, Paola; Tavano, Regina; Polverino de Laureto, Patrizia; Franzoso, Susanna; Mazzon, Cristina; Montanari, Paolo; Papini, Emanuele

    2011-01-01

    The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadA(Δ351-405,) devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadA(Δ351-405) cellular effects in monocytes. We show that NadA(Δ351-405) (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadA(Δ351-405) cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadA(Δ351-405) /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadA(Δ351-405) and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadA(Δ351-405) determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadA(Δ351-405) alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by

  1. Functional Characterization of a Mucus-Specific LPXTG Surface Adhesin from Probiotic Lactobacillus rhamnosus GG ▿

    PubMed Central

    von Ossowski, Ingemar; Satokari, Reetta; Reunanen, Justus; Lebeer, Sarah; De Keersmaecker, Sigrid C. J.; Vanderleyden, Jos; de Vos, Willem M.; Palva, Airi

    2011-01-01

    In spite of the wealth of clinical evidence supporting the health benefits of Lactobacillus rhamnosus GG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover, L. rhamnosus GG contains mucus-binding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) in L. rhamnosus GG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction between L. rhamnosus GG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean that L. rhamnosus GG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic. PMID:21602388

  2. Functional characterization of a mucus-specific LPXTG surface adhesin from probiotic Lactobacillus rhamnosus GG.

    PubMed

    von Ossowski, Ingemar; Satokari, Reetta; Reunanen, Justus; Lebeer, Sarah; De Keersmaecker, Sigrid C J; Vanderleyden, Jos; de Vos, Willem M; Palva, Airi

    2011-07-01

    In spite of the wealth of clinical evidence supporting the health benefits of Lactobacillus rhamnosus GG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover, L. rhamnosus GG contains mucus-binding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) in L. rhamnosus GG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction between L. rhamnosus GG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean that L. rhamnosus GG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic. PMID:21602388

  3. Description of a novel adhesin of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Viale, Mariana Noelia; Echeverria-Valencia, Gabriela; Romasanta, Pablo; Mon, María Laura; Fernandez, Marisa; Malchiodi, Emilio; Romano, María Isabel; Gioffré, Andrea Karina; Santangelo, María de la Paz

    2014-01-01

    The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the μM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding. PMID:25136616

  4. Description of a Novel Adhesin of Mycobacterium avium Subsp. paratuberculosis

    PubMed Central

    Viale, Mariana Noelia; Echeverria-Valencia, Gabriela; Romasanta, Pablo; Mon, María Laura; Fernandez, Marisa; Malchiodi, Emilio; Romano, María Isabel; Gioffré, Andrea Karina; Santangelo, María de la Paz

    2014-01-01

    The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the μM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding. PMID:25136616

  5. A Least Square Method Based Model for Identifying Protein Complexes in Protein-Protein Interaction Network

    PubMed Central

    Dai, Qiguo; Guo, Maozu; Guo, Yingjie; Liu, Xiaoyan; Liu, Yang; Teng, Zhixia

    2014-01-01

    Protein complex formed by a group of physical interacting proteins plays a crucial role in cell activities. Great effort has been made to computationally identify protein complexes from protein-protein interaction (PPI) network. However, the accuracy of the prediction is still far from being satisfactory, because the topological structures of protein complexes in the PPI network are too complicated. This paper proposes a novel optimization framework to detect complexes from PPI network, named PLSMC. The method is on the basis of the fact that if two proteins are in a common complex, they are likely to be interacting. PLSMC employs this relation to determine complexes by a penalized least squares method. PLSMC is applied to several public yeast PPI networks, and compared with several state-of-the-art methods. The results indicate that PLSMC outperforms other methods. In particular, complexes predicted by PLSMC can match known complexes with a higher accuracy than other methods. Furthermore, the predicted complexes have high functional homogeneity. PMID:25405206

  6. A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

    PubMed Central

    Pyburn, Tasia M.; Bensing, Barbara A.; Xiong, Yan Q.; Melancon, Bruce J.; Tomasiak, Thomas M.; Ward, Nicholas J.; Yankovskaya, Victoria; Oliver, Kevin M.; Cecchini, Gary; Sulikowski, Gary A.; Tyska, Matthew J.; Sullam, Paul M.; Iverson, T. M.

    2011-01-01

    GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspBBR), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspBBR structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspBBR-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues. PMID:21765814

  7. A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

    SciTech Connect

    Pyburn, Tasia M.; Bensing, Barbara A.; Xiong, Yan Q.; Melancon, Bruce J.; Tomasiak, Thomas M.; Ward, Nicholas J.; Yankovskaya, Victoria; Oliver, Kevin M.; Cecchini, Gary; Sulikowski, Gary A.; Tyska, Matthew J.; Sullam, Paul M.; Iverson, T.M.

    2014-10-02

    GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIb{alpha}. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB{sub BR}), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB{sub BR} structure revealed that it is comprised of three independently folded subdomains or modules: (1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; (2) a second Ig-fold resembling the binding region of mammalian Siglecs; (3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIb{alpha}. Further examination of purified GspB{sub BR}-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

  8. Quality Control of a Cytoplasmic Protein Complex

    PubMed Central

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H.

    2015-01-01

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2. PMID:25564609

  9. Oxygen promotes biofilm formation of Shewanella putrefaciens CN32 through a diguanylate cyclase and an adhesin

    PubMed Central

    Wu, Chao; Cheng, Yuan-Yuan; Yin, Hao; Song, Xiang-Ning; Li, Wen-Wei; Zhou, Xian-Xuan; Zhao, Li-Ping; Tian, Li-Jiao; Han, Jun-Cheng; Yu, Han-Qing

    2013-01-01

    Although oxygen has been reported to regulate biofilm formation by several Shewanella species, the exact regulatory mechanism mostly remains unclear. Here, we identify a direct oxygen-sensing diguanylate cyclase (DosD) and reveal its regulatory role in biofilm formation by Shewanella putrefaciens CN32 under aerobic conditions. In vitro and in vivo analyses revealed that the activity of DosD culminates to synthesis of cyclic diguanylate (c-di-GMP) in the presence of oxygen. DosD regulates the transcription of bpfA operon which encodes seven proteins including a large repetitive adhesin BpfA and its cognate type I secretion system (TISS). Regulation of DosD in aerobic biofilms is heavily dependent on an adhesin BpfA and the TISS. This study offers an insight into the molecular mechanism of oxygen-stimulated biofilm formation by S. putrefaciens CN32. PMID:23736081

  10. The fimbrial adhesin F17-G of enterotoxigenic Escherichia coli has an immunoglobulin-like lectin domain that binds N-acetylglucosamine.

    PubMed

    Buts, Lieven; Bouckaert, Julie; De Genst, Erwin; Loris, Remy; Oscarson, Stefan; Lahmann, Martina; Messens, Joris; Brosens, Elke; Wyns, Lode; De Greve, Henri

    2003-08-01

    The F17-G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N-acetyl-beta-D-glucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 A resolution crystal structure of the lectin domain of F17-G, both free and in complex with N-acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid-shaped protein in a conserved site around which all natural variations of F17-G are clustered. A model is proposed for the interaction between F17-fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17-G binding to N-acetylglucosamine (0.85 mM-1). Unexpectedly, the F17-G structure reveals that the lectin domains of the F17-G, PapGII and FimH fimbrial adhesins all share the immunoglobulin-like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C-terminal beta-strand G of the immunoglobulin-like fold and provides new insights into pilus assembly, function and adhesion. PMID:12864853

  11. Proteolytic processing of the cilium adhesin MHJ_0194 (P123J ) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules.

    PubMed

    Raymond, Benjamin B A; Jenkins, Cheryl; Seymour, Lisa M; Tacchi, Jessica L; Widjaja, Michael; Jarocki, Veronica M; Deutscher, Ania T; Turnbull, Lynne; Whitchurch, Cynthia B; Padula, Matthew P; Djordjevic, Steven P

    2015-03-01

    Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells. PMID:25293691

  12. Infection of human mucosal tissue by Pseudomonas aeruginosa requires sequential and mutually dependent virulence factors and a novel pilus-associated adhesin

    PubMed Central

    Heiniger, Ryan W.; Winther-Larsen, Hanne C.; Pickles, Raymond J.; Koomey, Michael; Wolfgang, Matthew C.

    2010-01-01

    Summary Tissue damage predisposes humans to life-threatening disseminating infection by the opportunistic pathogen Pseudomonas aeruginosa. Bacterial adherence to host tissue is a critical first step in this infection process. It is well established that P. aeruginosa attachment to host cells involves type IV pili (TFP), which are retractile surface fibers. The molecular details of attachment and the identity of the bacterial adhesin and host receptor remain controversial. Using a mucosal epithelium model system derived from primary human tissue, we show that the pilus-associated protein PilY1 is required for bacterial adherence. We establish that P. aeruginosa preferentially binds to exposed basolateral host cell surfaces, providing a mechanistic explanation for opportunistic infection of damaged tissue. Further, we demonstrate that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus fiber retraction and the host cell intoxication system, termed type III secretion. Our findings offer new and important insights into the complex interactions between a pathogen and its human host and provide compelling evidence that PilY1 serves as the principal P. aeruginosa adhesin for human tissue and that it specifically recognizes a host receptor localized or enriched on basolateral epithelial cell surfaces. PMID:20331639

  13. Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA.

    PubMed

    Subedi, Suresh; Moonens, Kristof; Romão, Ema; Lo, Alvin; Vandenbussche, Guy; Bugaytsova, Jeanna; Muyldermans, Serge; Borén, Thomas; Remaut, Han

    2014-12-01

    Helicobacter pylori is a human pathogen that colonizes about 50% of the world's population, causing chronic gastritis, duodenal ulcers and even gastric cancer. A steady emergence of multiple antibiotic resistant strains poses an important public health threat and there is an urgent requirement for alternative therapeutics. The blood group antigen-binding adhesin BabA mediates the intimate attachment to the host mucosa and forms a major candidate for novel vaccine and drug development. Here, the recombinant expression and crystallization of a soluble BabA truncation (BabA(25-460)) corresponding to the predicted extracellular adhesin domain of the protein are reported. X-ray diffraction data for nanobody-stabilized BabA(25-460) were collected to 2.25 Å resolution from a crystal that belonged to space group P21, with unit-cell parameters a = 50.96, b = 131.41, c = 123.40 Å, α = 90.0, β = 94.8, γ = 90.0°, and which was predicted to contain two BabA(25-460)-nanobody complexes per asymmetric unit. PMID:25484214

  14. GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking

    PubMed Central

    Heo, Lim; Lee, Hasup; Seok, Chaok

    2016-01-01

    Protein-protein docking methods have been widely used to gain an atomic-level understanding of protein interactions. However, docking methods that employ low-resolution energy functions are popular because of computational efficiency. Low-resolution docking tends to generate protein complex structures that are not fully optimized. GalaxyRefineComplex takes such low-resolution docking structures and refines them to improve model accuracy in terms of both interface contact and inter-protein orientation. This refinement method allows flexibility at the protein interface and in the overall docking structure to capture conformational changes that occur upon binding. Symmetric refinement is also provided for symmetric homo-complexes. This method was validated by refining models produced by available docking programs, including ZDOCK and M-ZDOCK, and was successfully applied to CAPRI targets in a blind fashion. An example of using the refinement method with an existing docking method for ligand binding mode prediction of a drug target is also presented. A web server that implements the method is freely available at http://galaxy.seoklab.org/refinecomplex. PMID:27535582

  15. GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking.

    PubMed

    Heo, Lim; Lee, Hasup; Seok, Chaok

    2016-01-01

    Protein-protein docking methods have been widely used to gain an atomic-level understanding of protein interactions. However, docking methods that employ low-resolution energy functions are popular because of computational efficiency. Low-resolution docking tends to generate protein complex structures that are not fully optimized. GalaxyRefineComplex takes such low-resolution docking structures and refines them to improve model accuracy in terms of both interface contact and inter-protein orientation. This refinement method allows flexibility at the protein interface and in the overall docking structure to capture conformational changes that occur upon binding. Symmetric refinement is also provided for symmetric homo-complexes. This method was validated by refining models produced by available docking programs, including ZDOCK and M-ZDOCK, and was successfully applied to CAPRI targets in a blind fashion. An example of using the refinement method with an existing docking method for ligand binding mode prediction of a drug target is also presented. A web server that implements the method is freely available at http://galaxy.seoklab.org/refinecomplex. PMID:27535582

  16. Probing nanoparticle effect in protein-surfactant complexes

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Aswal, V. K.; Kohlbrecher, J.

    2015-06-01

    SANS experiments have been carried to probe the role of anionic silica nanoparticles in the anionic BSA protein-cationic DTAB surfactant complexes. In protein-surfactant complex, surfactant molecules aggregate to form micelle-like clusters along the unfolded polypeptide chains of the protein. The nanoparticle aggregation mediated by oppositely charged protein-surfactant complex coexists with the free protein-surfactant complexes in the nanoparticle-protein-surfactant system. There is rearrangement of micelles in adsorbed protein-surfactant complex on nanoparticles in leading to their (nanoparticle) aggregation. On the other hand, the unfolding of protein in free protein-surfactant complex is found to be significantly enhanced in presence of nanoparticles.

  17. Adhesin receptors of human oral bacteria and modeling of putative adhesin-binding domains.

    PubMed

    Cassels, F J; Hughes, C V; Nauss, J L

    1995-09-01

    Adherence by bacteria to a surface is critical to their survival in the human oral cavity. Many types of molecules are present in the saliva and serous exudates that form the acquired pellicle, a coating on the tooth surface, and serve as receptor molecules for adherent bacteria. The primary colonizing bacteria utilize adhesins to adhere to specific pellicle receptor molecules, then may adhere to other primary colonizers via adhesins, or may present receptor molecules to be utilized by secondary colonizing species. The most common primary colonizing bacteria are streptococci, and six streptococcal cell wall polysaccharide receptor molecules have been structurally characterized. A comparison of the putative adhesin disaccharide-binding regions of the six polysaccharides suggests three groups. A representative of each group was modeled in molecular dynamics simulations. In each case it was found that a loop formed between the galactofuranose beta (Galf beta) and an oxygen of the nearest phosphate group on the reducing side of the Galf beta, that this loop was stabilized by hydrogen bonds, and that within each loop resides the putative disaccharide-binding domain. PMID:8519475

  18. Characterization of Protein Complexes and Subcomplexes in Protein-Protein Interaction Databases

    PubMed Central

    Zaki, Nazar; Mohamed, Elfadil A.; Mora, Antonio

    2015-01-01

    The identification and characterization of protein complexes implicated in protein-protein interaction data are crucial to the understanding of the molecular events under normal and abnormal physiological conditions. This paper provides a novel characterization of subcomplexes in protein interaction databases, stressing definition and representation issues, quantification, biological validation, network metrics, motifs, modularity, and gene ontology (GO) terms. The paper introduces the concept of “nested group” as a way to represent subcomplexes and estimates that around 15% of those nested group with the higher Jaccard index may be a result of data artifacts in protein interaction databases, while a number of them can be found in biologically important modular structures or dynamic structures. We also found that network centralities, enrichment in essential proteins, GO terms related to regulation, imperfect 5-clique motifs, and higher GO homogeneity can be used to identify proteins in nested complexes. PMID:25722891

  19. Identification and analysis of multi-protein complexes in placenta.

    PubMed

    Wang, Fuqiang; Wang, Ling; Xu, Zhiyang; Liang, Gaolin

    2013-01-01

    Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders. PMID:23638173

  20. HKC: an algorithm to predict protein complexes in protein-protein interaction networks.

    PubMed

    Wang, Xiaomin; Wang, Zhengzhi; Ye, Jun

    2011-01-01

    With the availability of more and more genome-scale protein-protein interaction (PPI) networks, research interests gradually shift to Systematic Analysis on these large data sets. A key topic is to predict protein complexes in PPI networks by identifying clusters that are densely connected within themselves but sparsely connected with the rest of the network. In this paper, we present a new topology-based algorithm, HKC, to detect protein complexes in genome-scale PPI networks. HKC mainly uses the concepts of highest k-core and cohesion to predict protein complexes by identifying overlapping clusters. The experiments on two data sets and two benchmarks show that our algorithm has relatively high F-measure and exhibits better performance compared with some other methods. PMID:22174556

  1. Engineering of complex protein sialylation in plants.

    PubMed

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-08-23

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  2. Engineering of complex protein sialylation in plants

    PubMed Central

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-01-01

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  3. Detecting overlapping protein complexes by rough-fuzzy clustering in protein-protein interaction networks.

    PubMed

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks. PMID:24642838

  4. Detecting Overlapping Protein Complexes by Rough-Fuzzy Clustering in Protein-Protein Interaction Networks

    PubMed Central

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks. PMID:24642838

  5. FimH adhesin from host unrestricted Salmonella Enteritidis binds to different glycoprotein ligands expressed by enterocytes from sheep, pig and cattle than FimH adhesins from host restricted Salmonella Abortus-ovis, Salmonella Choleraesuis and Salmonella Dublin.

    PubMed

    Grzymajło, Krzysztof; Ugorski, Maciej; Kolenda, Rafał; Kędzierska, Anna; Kuźmińska-Bajor, Marta; Wieliczko, Alina

    2013-10-25

    Adhesion to gut tissues and colonization of the alimentary tract, two important stages in the pathogenesis of Salmonella, are mediated by FimH adhesin of type 1 fimbriae. It was suggested that minor differences in the structure of FimH are most likely associated with differences in adhesion specificities, and may determine the tropism of various Salmonella serovars to different species and tissues. We investigated this hypothesis by comparing the binding properties of FimH proteins from three Salmonella enterica serovars with limited (Choleraesuis, Dublin) or restricted (Abortusovis) host ranges to FimH from broad host range S. Enteritidis and mannose inactive FimH from S. Gallinarum. Although all active variants of FimH protein were able to bind mannose-rich glycoproteins (RNase B, HRP and Man-BSA) with comparable affinity measured by surface plasmon resonance, there were significant differences in the binding profiles of the FimH proteins from host restricted serovars and host unrestricted serovar Enteritidis, to glycoproteins from enterocyte cell lines established in vitro and derived from sheep, pig and cattle. When low-binding FimH adhesin from S. Enteritidis was subjected to Western blot analysis, it bound to surface membrane protein of about 130 kDa, and high-binding FimH adhesins from S. Abortusovis, S. Choleraesuis and S. Dublin bound to surface membrane protein of about 55 kDa present in each cell line. Differential binding of FimH proteins from host-restricted and broad-host-range Salmonella to intestinal receptors was confirmed using mutant FimH adhesins obtained by site-directed mutagenesis. It was found that the low-binding variant of FimH from S. Choleraesuis with mutation Leu57Pro lost the ability to bind protein band of 55 kDa, but instead interacted with glycoprotein of about 130 kDa. On the other hand, the high-binding variant of FimH adhesin from S. Enteritids with mutation Asn101Ser did not bind to its receptor of 130 kDa, but instead it

  6. Interaction graph mining for protein complexes using local clique merging.

    PubMed

    Li, Xiao-Li; Tan, Soon-Heng; Foo, Chuan-Sheng; Ng, See-Kiong

    2005-01-01

    While recent technological advances have made available large datasets of experimentally-detected pairwise protein-protein interactions, there is still a lack of experimentally-determined protein complex data. To make up for this lack of protein complex data, we explore the mining of existing protein interaction graphs for protein complexes. This paper proposes a novel graph mining algorithm to detect the dense neighborhoods (highly connected regions) in an interaction graph which may correspond to protein complexes. Our algorithm first locates local cliques for each graph vertex (protein) and then merge the detected local cliques according to their affinity to form maximal dense regions. We present experimental results with yeast protein interaction data to demonstrate the effectiveness of our proposed method. Compared with other existing techniques, our predicted complexes can match or overlap significantly better with the known protein complexes in the MIPS benchmark database. Novel protein complexes were also predicted to help biologists in their search for new protein complexes. PMID:16901108

  7. Study of the structural and dynamic effects in the FimH adhesin upon α-d-heptyl mannose binding.

    PubMed

    Vanwetswinkel, Sophie; Volkov, Alexander N; Sterckx, Yann G J; Garcia-Pino, Abel; Buts, Lieven; Vranken, Wim F; Bouckaert, Julie; Roy, René; Wyns, Lode; van Nuland, Nico A J

    2014-02-27

    Uropathogenic Escherichia coli cause urinary tract infections by adhering to mannosylated receptors on the human urothelium via the carbohydrate-binding domain of the FimH adhesin (FimHL). Numerous α-d-mannopyranosides, including α-d-heptyl mannose (HM), inhibit this process by interacting with FimHL. To establish the molecular basis of the high-affinity HM binding, we solved the solution structure of the apo form and the crystal structure of the FimHL-HM complex. NMR relaxation analysis revealed that protein dynamics were not affected by the sugar binding, yet HM addition promoted protein dimerization, which was further confirmed by small-angle X-ray scattering. Finally, to address the role of Y48, part of the "tyrosine gate" believed to govern the affinity and specificity of mannoside binding, we characterized the FimHL Y48A mutant, whose conformational, dynamical, and HM binding properties were found to be very similar to those of the wild-type protein. PMID:24476493

  8. An Acinetobacter trimeric autotransporter adhesin reaped from cells exhibits its nonspecific stickiness via a highly stable 3D structure.

    PubMed

    Yoshimoto, Shogo; Nakatani, Hajime; Iwasaki, Keita; Hori, Katsutoshi

    2016-01-01

    Trimeric autotransporter adhesins (TAAs), cell surface proteins of Gram-negative bacteria, mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific, high adhesiveness to abiotic material surfaces as well as to biotic surfaces. AtaA is a homotrimer of polypeptides comprising 3,630 amino acids and forms long nanofibers; therefore, it is too large and structurally complex to be produced as a recombinant protein. In this study, we isolated AtaA's passenger domain (AtaA PSD), which is translocated to the cell surface through the C-terminal transmembrane domain and exhibits biological functions, using a new method. We introduced a protease recognition site and reaped AtaA nanofibers 225 nm in length from the cell surface through proteolytic cleavage with a specific protease. Biochemical and biophysical analyses of the purified native AtaA PSD revealed that it has a stable structure under alkaline and acidic conditions. Temperatures above 80 °C, which disrupted AtaA's higher-order structure but maintained the full-length AtaA polypeptide, inactivated AtaA's nonspecific adhesiveness, suggesting that the stickiness of AtaA requires its 3D structure. This finding refutes the widespread but vague speculation that large unfolded polypeptides readily stick to various surfaces. PMID:27305955

  9. An Acinetobacter trimeric autotransporter adhesin reaped from cells exhibits its nonspecific stickiness via a highly stable 3D structure

    PubMed Central

    Yoshimoto, Shogo; Nakatani, Hajime; Iwasaki, Keita; Hori, Katsutoshi

    2016-01-01

    Trimeric autotransporter adhesins (TAAs), cell surface proteins of Gram-negative bacteria, mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific, high adhesiveness to abiotic material surfaces as well as to biotic surfaces. AtaA is a homotrimer of polypeptides comprising 3,630 amino acids and forms long nanofibers; therefore, it is too large and structurally complex to be produced as a recombinant protein. In this study, we isolated AtaA’s passenger domain (AtaA PSD), which is translocated to the cell surface through the C-terminal transmembrane domain and exhibits biological functions, using a new method. We introduced a protease recognition site and reaped AtaA nanofibers 225 nm in length from the cell surface through proteolytic cleavage with a specific protease. Biochemical and biophysical analyses of the purified native AtaA PSD revealed that it has a stable structure under alkaline and acidic conditions. Temperatures above 80 °C, which disrupted AtaA’s higher-order structure but maintained the full-length AtaA polypeptide, inactivated AtaA’s nonspecific adhesiveness, suggesting that the stickiness of AtaA requires its 3D structure. This finding refutes the widespread but vague speculation that large unfolded polypeptides readily stick to various surfaces. PMID:27305955

  10. Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22.

    PubMed Central

    Kinder, S A; Holt, S C

    1993-01-01

    Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22. Images PMID:8380804

  11. Identification of Mycobacterium tuberculosis adherence-mediating components: a review of key methods to confirm adhesin function.

    PubMed

    Ramsugit, Saiyur; Pillay, Manormoney

    2016-06-01

    Anti-adhesion therapy represents a potentially promising avenue for the treatment and prevention of tuberculosis in a post-antibiotic era. Adhesins are surface-exposed microbial structures or molecules that enable pathogenic organisms to adhere to host surfaces, a fundamental step towards host infection. Although several Mycobacterium tuberculosis adhesins have been identified, it is predicted that numerous additional adherence-mediating components contribute to the virulence and success of this pathogen. Significant further research to discern and characterize novel M. tuberculosis adhesins is, therefore, required to gain a holistic account of M. tuberculosis adhesion to the host. This would enable the identification of potential drug and vaccine targets for attenuating M. tuberculosis adherence and infectivity. Several methods have been successfully applied to the study and identification of M. tuberculosis adhesins. In this manuscript, we review these methods, which include adherence assays that utilize wild-type and gene knockout mutant strains, epitope masking and competitive inhibition analyses, extracellular matrix protein binding assays, microsphere adhesion assays, M. tuberculosis auto-aggregation assays, and in silico analyses. PMID:27482337

  12. Identification of Mycobacterium tuberculosis adherence-mediating components: a review of key methods to confirm adhesin function

    PubMed Central

    Ramsugit, Saiyur; Pillay, Manormoney

    2016-01-01

    Anti-adhesion therapy represents a potentially promising avenue for the treatment and prevention of tuberculosis in a post-antibiotic era. Adhesins are surface-exposed microbial structures or molecules that enable pathogenic organisms to adhere to host surfaces, a fundamental step towards host infection. Although several Mycobacterium tuberculosis adhesins have been identified, it is predicted that numerous additional adherence-mediating components contribute to the virulence and success of this pathogen. Significant further research to discern and characterize novel M. tuberculosis adhesins is, therefore, required to gain a holistic account of M. tuberculosis adhesion to the host. This would enable the identification of potential drug and vaccine targets for attenuating M. tuberculosis adherence and infectivity. Several methods have been successfully applied to the study and identification of M. tuberculosis adhesins. In this manuscript, we review these methods, which include adherence assays that utilize wild-type and gene knockout mutant strains, epitope masking and competitive inhibition analyses, extracellular matrix protein binding assays, microsphere adhesion assays, M. tuberculosis auto-aggregation assays, and in silico analyses. PMID:27482337

  13. Magnetic Resonance Access to Transiently Formed Protein Complexes**

    PubMed Central

    Sára, Tomáš; Schwarz, Thomas C; Kurzbach, Dennis; Wunderlich, Christoph H; Kreutz, Christoph; Konrat, Robert

    2014-01-01

    Protein–protein interactions are of utmost importance to an understanding of biological phenomena since non-covalent and therefore reversible couplings between basic proteins leads to the formation of complex regulatory and adaptive molecular systems. Such systems are capable of maintaining their integrity and respond to external stimuli, processes intimately related to living organisms. These interactions, however, span a wide range of dissociation constants, from sub-nanomolar affinities in tight complexes to high-micromolar or even millimolar affinities in weak, transiently formed protein complexes. Herein, we demonstrate how novel NMR and EPR techniques can be used for the characterization of weak protein–protein (ligand) complexes. Applications to intrinsically disordered proteins and transiently formed protein complexes illustrate the potential of these novel techniques to study hitherto unobserved (and unobservable) higher-order structures of proteins. PMID:25050230

  14. T4 Phage Tail Adhesin Gp12 Counteracts LPS-Induced Inflammation In Vivo.

    PubMed

    Miernikiewicz, Paulina; Kłopot, Anna; Soluch, Ryszard; Szkuta, Piotr; Kęska, Weronika; Hodyra-Stefaniak, Katarzyna; Konopka, Agnieszka; Nowak, Marcin; Lecion, Dorota; Kaźmierczak, Zuzanna; Majewska, Joanna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2016-01-01

    Bacteriophages that infect Gram-negative bacteria often bind to the bacterial surface by interaction of specific proteins with lipopolysaccharide (LPS). Short tail fiber proteins (tail adhesin, gp12) mediate adsorption of T4-like bacteriophages to Escherichia coli, binding surface proteins or LPS. Produced as a recombinant protein, gp12 retains its ability to bind LPS. Since LPS is able to exert a major impact on the immune response in animals and in humans, we have tested LPS-binding phage protein gp12 as a potential modulator of the LPS-induced immune response. We have produced tail adhesin gp12 in a bacterial expression system and confirmed its ability to form trimers and to bind LPS in vitro by dynamic light scattering. This product had no negative effect on mammalian cell proliferation in vitro. Further, no harmful effects of this protein were observed in mice. Thus, gp12 was used in combination with LPS in a murine model, and it decreased the inflammatory response to LPS in vivo, as assessed by serum levels of cytokines IL-1 alpha and IL-6 and by histopathological analysis of spleen, liver, kidney and lungs. Thus, in future studies gp12 may be considered as a potential tool for modulating and specifically for counteracting LPS-related physiological effects in vivo. PMID:27471503

  15. T4 Phage Tail Adhesin Gp12 Counteracts LPS-Induced Inflammation In Vivo

    PubMed Central

    Miernikiewicz, Paulina; Kłopot, Anna; Soluch, Ryszard; Szkuta, Piotr; Kęska, Weronika; Hodyra-Stefaniak, Katarzyna; Konopka, Agnieszka; Nowak, Marcin; Lecion, Dorota; Kaźmierczak, Zuzanna; Majewska, Joanna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2016-01-01

    Bacteriophages that infect Gram-negative bacteria often bind to the bacterial surface by interaction of specific proteins with lipopolysaccharide (LPS). Short tail fiber proteins (tail adhesin, gp12) mediate adsorption of T4-like bacteriophages to Escherichia coli, binding surface proteins or LPS. Produced as a recombinant protein, gp12 retains its ability to bind LPS. Since LPS is able to exert a major impact on the immune response in animals and in humans, we have tested LPS-binding phage protein gp12 as a potential modulator of the LPS-induced immune response. We have produced tail adhesin gp12 in a bacterial expression system and confirmed its ability to form trimers and to bind LPS in vitro by dynamic light scattering. This product had no negative effect on mammalian cell proliferation in vitro. Further, no harmful effects of this protein were observed in mice. Thus, gp12 was used in combination with LPS in a murine model, and it decreased the inflammatory response to LPS in vivo, as assessed by serum levels of cytokines IL-1 alpha and IL-6 and by histopathological analysis of spleen, liver, kidney and lungs. Thus, in future studies gp12 may be considered as a potential tool for modulating and specifically for counteracting LPS-related physiological effects in vivo. PMID:27471503

  16. An overview of the structures of protein-DNA complexes

    PubMed Central

    Luscombe, Nicholas M; Austin, Susan E; Berman , Helen M; Thornton, Janet M

    2000-01-01

    On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes. PMID:11104519

  17. Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

    PubMed Central

    Edwards, Thomas E.; Phan, Isabelle; Abendroth, Jan; Dieterich, Shellie H.; Masoudi, Amir; Guo, Wenjin; Hewitt, Stephen N.; Kelley, Angela; Leibly, David; Brittnacher, Mitch J.; Staker, Bart L.; Miller, Samuel I.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.

    2010-01-01

    Background Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region. Methodology/Principal Findings Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 Å resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs. Conclusions/Significance The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics. PMID:20862217

  18. The multifaceted roles of intrinsic disorder in protein complexes.

    PubMed

    Uversky, Vladimir N

    2015-09-14

    Intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) are important constituents of many protein complexes, playing various structural, functional, and regulatory roles. In such disorder-based protein complexes, functional disorder is used both internally (for assembly, movement, and functional regulation of the different parts of a given complex) and externally (for interactions of a complex with its external regulators). In complex assembly, IDPs/IDPRs serve as the molecular glue that cements complexes or as highly flexible scaffolds. Disorder defines the order of complex assembly and the ability of a protein to be involved in polyvalent interactions. It is at the heart of various binding mechanisms and interaction modes ascribed to IDPs. Disorder in protein complexes is related to multifarious applications of induced folding and induced functional unfolding, or defines the entropic chain activities, such as stochastic machines and binding rheostats. This review opens a FEBS Letters Special Issue on Dynamics, Flexibility, and Intrinsic Disorder in protein assemblies and represents a brief overview of intricate roles played by IDPs and IDPRs in various aspects of protein complexes. PMID:26073257

  19. Chlorophyll-Protein Complexes of the Cyanophyte, Nostoc sp. 1

    PubMed Central

    Rusckowski, Mary; Zilinskas, Barbara A.

    1980-01-01

    Four chlorophyll-protein complexes have been resolved from the cyanophyte, Nostoc sp., by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis at 4 C. Complexes solubilized by SDS from Spinacia oleracea were run for comparison. As has been well documented, the P700-chlorophyll a-protein complex from the higher plant and blue-green algal samples are similar, and the light-harvesting pigment protein complex is present only in the former. Most noteworthy are two closely migrating chlorophyll proteins in Nostoc sp. which have approximately the same mobility as a single chlorophyll-protein band resolvable from spinach. The absorption maximum of the complex from spinach is at 667 nanometers, and those of the two complexes from Nostoc sp. are at 667 and 669 nanometers; the fluorescence emission maximum at −196 C is at 685 nanometers, and the 735 nanometer fluorescence peak, characteristic of the P700-chlorophyll a-protein complex, is absent. The apoproteins of these new complexes from Nostoc sp. and spinach are in the kilodalton range. It appears that at least one of these two chlorophyll-protein complexes from Nostoc sp. compares with those recently described by others from higher plants and green algae as likely photosystem II complexes, perhaps containing P680, although no photochemical data are yet available. Images PMID:16661198

  20. Hysteresis as a Marker for Complex, Overlapping Landscapes in Proteins

    PubMed Central

    Andrews, Benjamin T.; Capraro, Dominique T.; Sulkowska, Joanna I.; Onuchic, José N.; Jennings, Patricia A.

    2013-01-01

    Topologically complex proteins fold by multiple routes as a result of hard-to-fold regions of the proteins. Oftentimes these regions are introduced into the protein scaffold for function and increase frustration in the otherwise smooth-funneled landscape. Interestingly, while functional regions add complexity to folding landscapes, they may also contribute to a unique behavior referred to as hysteresis. While hysteresis is predicted to be rare, it is observed in various proteins, including proteins containing a unique peptide cyclization to form a fluorescent chromophore as well as proteins containing a knotted topology in their native fold. Here, hysteresis is demonstrated to be a consequence of the decoupling of unfolding events from the isomerization or hula-twist of a chromophore in one protein and the untying of the knot in a second protein system. The question now is- can hysteresis be a marker for the interplay of landscapes where complex folding and functional regions overlap? PMID:23525263

  1. Advances in protein complex analysis using mass spectrometry.

    PubMed

    Gingras, Anne-Claude; Aebersold, Ruedi; Raught, Brian

    2005-02-15

    Proteins often function as components of larger complexes to perform a specific function, and formation of these complexes may be regulated. For example, intracellular signalling events often require transient and/or regulated protein-protein interactions for propagation, and protein binding to a specific DNA sequence, RNA molecule or metabolite is often regulated to modulate a particular cellular function. Thus, characterizing protein complexes can offer important insights into protein function. This review describes recent important advances in mass spectrometry (MS)-based techniques for the analysis of protein complexes. Following brief descriptions of how proteins are identified using MS, and general protein complex purification approaches, we address two of the most important issues in these types of studies: specificity and background protein contaminants. Two basic strategies for increasing specificity and decreasing background are presented: whereas (1) tandem affinity purification (TAP) of tagged proteins of interest can dramatically improve the signal-to-noise ratio via the generation of cleaner samples, (2) stable isotopic labelling of proteins may be used to discriminate between contaminants and bona fide binding partners using quantitative MS techniques. Examples, as well as advantages and disadvantages of each approach, are presented. PMID:15611014

  2. Protein-protein binding affinities by pulse proteolysis: application to TEM-1/BLIP protein complexes.

    PubMed

    Hanes, Melinda S; Ratcliff, Kathleen; Marqusee, Susan; Handel, Tracy M

    2010-10-01

    Efficient methods for quantifying dissociation constants have become increasingly important for high-throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein-ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein-protein complex involving the β-lactamase TEM-1 and various β-lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C(m), of TEM-1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein-protein complexes. From a small set (n = 4) of TEM-1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔC(m) was observed. From this "calibration curve," accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis-derived ΔC(m) values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high-throughput mutagenesis binding studies. PMID:20669180

  3. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

    PubMed

    Krogan, Nevan J; Cagney, Gerard; Yu, Haiyuan; Zhong, Gouqing; Guo, Xinghua; Ignatchenko, Alexandr; Li, Joyce; Pu, Shuye; Datta, Nira; Tikuisis, Aaron P; Punna, Thanuja; Peregrín-Alvarez, José M; Shales, Michael; Zhang, Xin; Davey, Michael; Robinson, Mark D; Paccanaro, Alberto; Bray, James E; Sheung, Anthony; Beattie, Bryan; Richards, Dawn P; Canadien, Veronica; Lalev, Atanas; Mena, Frank; Wong, Peter; Starostine, Andrei; Canete, Myra M; Vlasblom, James; Wu, Samuel; Orsi, Chris; Collins, Sean R; Chandran, Shamanta; Haw, Robin; Rilstone, Jennifer J; Gandi, Kiran; Thompson, Natalie J; Musso, Gabe; St Onge, Peter; Ghanny, Shaun; Lam, Mandy H Y; Butland, Gareth; Altaf-Ul, Amin M; Kanaya, Shigehiko; Shilatifard, Ali; O'Shea, Erin; Weissman, Jonathan S; Ingles, C James; Hughes, Timothy R; Parkinson, John; Gerstein, Mark; Wodak, Shoshana J; Emili, Andrew; Greenblatt, Jack F

    2006-03-30

    Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology. PMID:16554755

  4. Antifreeze Proteins in Winter Rye Leaves Form Oligomeric Complexes1

    PubMed Central

    Yu, Xiao-Ming; Griffith, Marilyn

    1999-01-01

    Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides. PMID:10198095

  5. Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry*

    PubMed Central

    Kaake, Robyn M.; Wang, Xiaorong; Huang, Lan

    2010-01-01

    Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer. Recent evidence has suggested that protein interaction interfaces describe a new class of attractive targets for drug development. Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand how protein complexes work together in cells to maintain cell viability and normal homeostasis. Affinity purification coupled with quantitative mass spectrometry has become the primary method for studying in vivo protein interactions of protein complexes and whole organism proteomes. Recent developments in sample preparation and affinity purification strategies allow the capture, identification, and quantification of protein interactions of protein complexes that are stable, dynamic, transient, and/or weak. Current efforts have mainly focused on generating reliable, reproducible, and high confidence protein interaction data sets for functional characterization. The availability of increasing amounts of information on protein interactions in eukaryotic systems and new bioinformatics tools allow functional analysis of quantitative protein interaction data to unravel the biological significance of the identified protein interactions. Existing studies in this area have laid a solid foundation toward generating a complete map of in vivo protein interaction networks of protein complexes in cells or tissues. PMID:20445003

  6. Design and characterization of complex protein films

    NASA Astrophysics Data System (ADS)

    Bui, Holt P.

    Once a biomaterial is implanted into biological system, a layer of protein is immediately deposited on the surface of that material. The newly formed protein film will dictate how the implanted material will interact with the surrounding biological environment and lead to either the acceptance or rejection of the biomaterial. One method to enhance performance involves the activation the surface of the biomaterial with one or more proteins to direct specific interactions with the host environment. The focus of my dissertation was to develop and characterize model biomaterials surfaces that are activated with one or more proteins to help understand how the protein films may affect biological processes and a biomaterial's performance. One model system consisted of a patterned film of two proteins on a gold surface. Characterization of this protein pattern indicated that patterning protein films with a focused ion beam produced protein patterns with high biological contrast and high spatial control. The second model protein film involved the adsorption of fibronectin on surfaces with different surface energies. The characterization of the adsorbed fibronectin films suggest that fibronectin adsorbed on a hydrophilic surface is in an orientation that projects hydrophilic amino acid residues towards surface of the protein and dehydration causes reorientation to project hydrophobic amino acids towards the surface. In contrast, fibronectin is adsorbed onto a hydrophobic surface in a manner that resulted in dehydration and denaturation during the adsorption process. The last model protein film studied in this work consisted of fibronectin patterned in a manner so that the film consisted of spatially controlled domains of fibronectin adsorbed onto a hydrophilic surface as well as a hydrophobic surface. Lateral characterization of this pattern demonstrated a difference in secondary structure of fibronectin adsorbed on the two domains with varying surface energies.

  7. 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

  8. Multi-LZerD: Multiple protein docking for asymmetric complexes

    PubMed Central

    Esquivel-Rodríguez, Juan; Yang, Yifeng David; Kihara, Daisuke

    2012-01-01

    The tertiary structures of protein complexes provide a crucial insight about the molecular mechanisms that regulate their functions and assembly. However, solving protein complex structures by experimental methods is often more difficult than single protein structures. Here, we have developed a novel computational multiple protein docking algorithm, Multi-LZerD, that builds models of multimeric complexes by effectively reusing pairwise docking predictions of component proteins. A genetic algorithm is applied to explore the conformational space followed by a structure refinement procedure. Benchmark on eleven hetero-multimeric complexes resulted in near native conformations for all but one of them (a root mean square deviation smaller than 2.5Å). We also show that our method copes with unbound docking cases well, outperforming the methodology that can be directly compared to our approach. Multi-LZerD was able to predict near native structures for multimeric complexes of various topologies. PMID:22488467

  9. LINC complex proteins in cardiac structure, function, and disease

    PubMed Central

    Stroud, Matthew J; Banerjee, Indroneal; Lowe, Jennifer; Chen, Ju

    2014-01-01

    The LINC (LInker of Nucleoskeleton and Cytoskeleton) complex, composed of proteins within the inner and the outer nuclear membranes, connects the nuclear lamina to the cytoskeleton. The importance of this complex has been highlighted by the discovery of mutations in genes encoding LINC complex proteins, which are causative for skeletal or cardiac myopathies. Herein, this review summarizes structure, function, and interactions of major components of the LINC complex, highlights how mutations in these proteins may lead to cardiac disease, and outlines future challenges in the field. PMID:24481844

  10. Multiscale Model for the Assembly Kinetics of Protein Complexes.

    PubMed

    Xie, Zhong-Ru; Chen, Jiawen; Wu, Yinghao

    2016-02-01

    The assembly of proteins into high-order complexes is a general mechanism for these biomolecules to implement their versatile functions in cells. Natural evolution has developed various assembling pathways for specific protein complexes to maintain their stability and proper activities. Previous studies have provided numerous examples of the misassembly of protein complexes leading to severe biological consequences. Although the research focusing on protein complexes has started to move beyond the static representation of quaternary structures to the dynamic aspect of their assembly, the current understanding of the assembly mechanism of protein complexes is still largely limited. To tackle this problem, we developed a new multiscale modeling framework. This framework combines a lower-resolution rigid-body-based simulation with a higher-resolution Cα-based simulation method so that protein complexes can be assembled with both structural details and computational efficiency. We applied this model to a homotrimer and a heterotetramer as simple test systems. Consistent with experimental observations, our simulations indicated very different kinetics between protein oligomerization and dimerization. The formation of protein oligomers is a multistep process that is much slower than dimerization but thermodynamically more stable. Moreover, we showed that even the same protein quaternary structure can have very diverse assembly pathways under different binding constants between subunits, which is important for regulating the functions of protein complexes. Finally, we revealed that the binding between subunits in a complex can be synergistically strengthened during assembly without considering allosteric regulation or conformational changes. Therefore, our model provides a useful tool to understand the general principles of protein complex assembly. PMID:26738810

  11. SnapShot: SMC Protein Complexes Part II.

    PubMed

    Haering, Christian H; Gruber, Stephan

    2016-02-11

    This second of two SnapShots on SMC proteins depicts their roles at different stages of the eukaryotic cell cycle. The composition and architecture of SMC protein complexes and their regulators appear in SMC Protein Complexes Part I (available at http://www.cell.com/cell/pdf/S0092-8674%2815%2901690-6.pdf). To view this SnapShot, open or download the PDF. PMID:26871638

  12. Identifying protein complexes in protein-protein interaction networks by using clique seeds and graph entropy.

    PubMed

    Chen, Bolin; Shi, Jinhong; Zhang, Shenggui; Wu, Fang-Xiang

    2013-01-01

    The identification of protein complexes plays a key role in understanding major cellular processes and biological functions. Various computational algorithms have been proposed to identify protein complexes from protein-protein interaction (PPI) networks. In this paper, we first introduce a new seed-selection strategy for seed-growth style algorithms. Cliques rather than individual vertices are employed as initial seeds. After that, a result-modification approach is proposed based on this seed-selection strategy. Predictions generated by higher order clique seeds are employed to modify results that are generated by lower order ones. The performance of this seed-selection strategy and the result-modification approach are tested by using the entropy-based algorithm, which is currently the best seed-growth style algorithm to detect protein complexes from PPI networks. In addition, we investigate four pairs of strategies for this algorithm in order to improve its accuracy. The numerical experiments are conducted on a Saccharomyces cerevisiae PPI network. The group of best predictions consists of 1711 clusters, with the average f-score at 0.68 after removing all similar and redundant clusters. We conclude that higher order clique seeds can generate predictions with higher accuracy and that our improved entropy-based algorithm outputs more reasonable predictions than the original one. PMID:23112006

  13. Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects

    PubMed Central

    Arciola, Carla Renata; Campoccia, Davide; Ravaioli, Stefano; Montanaro, Lucio

    2015-01-01

    Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to ica locus are present in many coagulase-negative staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm production have been described. S. epidermidis and S. aureus undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection. PMID

  14. Bifunctional Role of the Treponema pallidum Extracellular Matrix Binding Adhesin Tp0751 ▿

    PubMed Central

    Houston, Simon; Hof, Rebecca; Francescutti, Teresa; Hawkes, Aaron; Boulanger, Martin J.; Cameron, Caroline E.

    2011-01-01

    Treponema pallidum, the causative agent of syphilis, is a highly invasive pathogenic spirochete capable of attaching to host cells, invading the tissue barrier, and undergoing rapid widespread dissemination via the circulatory system. The T. pallidum adhesin Tp0751 was previously shown to bind laminin, the most abundant component of the basement membrane, suggesting a role for this adhesin in host tissue colonization and bacterial dissemination. We hypothesized that similar to that of other invasive pathogens, the interaction of T. pallidum with host coagulation proteins, such as fibrinogen, may also be crucial for dissemination via the circulatory system. To test this prediction, we used enzyme-linked immunosorbent assay (ELISA) methodology to demonstrate specific binding of soluble recombinant Tp0751 to human fibrinogen. Click-chemistry-based palmitoylation profiling of heterologously expressed Tp0751 confirmed the presence of a lipid attachment site within this adhesin. Analysis of the Tp0751 primary sequence revealed the presence of a C-terminal putative HEXXH metalloprotease motif, and in vitro degradation assays confirmed that recombinant Tp0751 purified from both insect and Escherichia coli expression systems degrades human fibrinogen and laminin. The proteolytic activity of Tp0751 was abolished by the presence of the metalloprotease inhibitor 1,10-phenanthroline. Further, inductively coupled plasma-mass spectrometry showed that Tp0751 binds zinc and calcium. Collectively, these results indicate that Tp0751 is a zinc-dependent, membrane-associated protease that exhibits metalloprotease-like characteristics. However, site-directed mutagenesis of the HEXXH motif to HQXXH did not abolish the proteolytic activity of Tp0751, indicating that further mutagenesis studies are required to elucidate the critical active site residues associated with this protein. This study represents the first published description of a T. pallidum protease capable of degrading host

  15. Immersion freezing of ice nucleating active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Voigtländer, J.; Niedermeier, D.; Wex, H.; Stratmann, F.

    2012-08-01

    Biological particles, e.g. bacteria and their Ice Nucleating Active (INA) protein complexes, might play an important role for the ice formation in atmospheric mixed-phase clouds. Therefore, the immersion freezing behavior of INA protein complexes generated from a SnomaxTM solution/suspension was investigated as function of temperature in a range of -5 °C to -38 °C at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing of droplets containing small numbers of INA protein complexes occurs in a temperature range of -7 °C and -10 °C. The experiments performed in the lower temperature range, where all droplets freeze which contain at least one INA protein complex, are used to determine the average number of INA protein complexes present, assuming that the INA protein complexes are Poisson distributed over the droplet ensemble. Knowing the average number of INA protein complexes, the heterogeneous ice nucleation rate and rate coefficient of a single INA protein complex is determined by using the newly-developed CHESS model (stoCHastic model of idEntical poiSSon distributed ice nuclei). Therefore, we assume the ice nucleation process to be of stochastic nature, and a parameterization of the INA protein complex's nucleation rate. Analyzing the results of immersion freezing experiments from literature (SnomaxTM and Pseudomonas syringae bacteria), to results gained in this study, demonstrates that first, a similar temperature dependence of the heterogeneous ice nucleation rate for a single INA protein complex was found in all experiments, second, the shift of the ice fraction curves to higher temperatures can be explained consistently by a higher average number of INA protein complexes being present in the droplet ensemble, and finally the heterogeneous ice nucleation rate of one single INA protein complex might be also applicable for intact Pseudomonas syringae bacteria cells. The results obtained in this study allow a new perspective on the

  16. BigA is a novel adhesin of Brucella that mediates adhesion to epithelial cells.

    PubMed

    Czibener, Cecilia; Merwaiss, Fernando; Guaimas, Francisco; Del Giudice, Mariela Giselda; Serantes, Diego Armando Rey; Spera, Juan Manuel; Ugalde, Juan Esteban

    2016-04-01

    Adhesion to cells is the initial step in the infectious cycle of basically all pathogenic bacteria, and to do so, microorganisms have evolved surface molecules that target different cellular receptors. Brucella is an intracellular pathogen that infects a wide range of mammals whose virulence is completely dependent on the capacity to replicate in phagocytes. Although much has been done to elucidate how Brucella multiplies in macrophages, we still do not understand how bacteria invade epithelial cells to perform a replicative cycle or what adhesion molecules are involved in the process. We report the identification in Brucella abortus of a novel adhesin that harbours a bacterial immunoglobulin-like domain and demonstrate that this protein is involved in the adhesion to polarized epithelial cells such as the Caco-2 and Madin-Darby canine kidney models targeting the bacteria to the cell-cell interaction membrane. While deletion of the gene significantly reduced adhesion, over-expression dramatically increased it. Addition of the recombinant protein to cells induced cytoskeleton rearrangements and showed that this adhesin targets proteins of the cell-cell interaction membrane in confluent cultures. PMID:26400021

  17. Recording information on protein complexes in an information management system

    PubMed Central

    Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

    2011-01-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  18. Mass Spectrometry of Protein Complexes: From Origins to Applications

    NASA Astrophysics Data System (ADS)

    Mehmood, Shahid; Allison, Timothy M.; Robinson, Carol V.

    2015-04-01

    Now routine is the ability to investigate soluble and membrane protein complexes in the gas phase of a mass spectrometer while preserving folded structure and ligand-binding properties. Several recent transformative developments have occurred to arrive at this point. These include advances in mass spectrometry instrumentation, particularly with respect to resolution; the ability to study intact membrane protein complexes released from detergent micelles; and the use of protein unfolding in the gas phase to obtain stability parameters. Together, these discoveries are providing unprecedented information on the compositional heterogeneity of biomacromolecules, the unfolding trajectories of multidomain proteins, and the stability imparted by ligand binding to both soluble and membrane-embedded protein complexes. We review these recent breakthroughs, highlighting the challenges that had to be overcome and the physicochemical insight that can now be gained from studying proteins and their assemblies in the gas phase.

  19. Introduction of the mec Element (Methicillin Resistance) into Staphylococcus aureus Alters In Vitro Functional Activities of Fibrinogen and Fibronectin Adhesins

    PubMed Central

    Vaudaux, Pierre E.; Monzillo, Vincenza; Francois, Patrice; Lew, Daniel P.; Foster, Tim J.; Berger-Bächi, Brigitte

    1998-01-01

    Some methicillin-resistant strains of Staphylococcus aureus are defective in the production of major surface components such as protein A, clumping factor, or other important adhesins to extracellular matrix components which may play a role in bacterial colonization and infection. To evaluate the impact of methicillin resistance (mec) determinants on bacterial adhesion mediated by fibrinogen or fibronectin adhesins, we compared the in vitro attachment of two genetically distinct susceptible strains (NCTC8325 and Newman) to protein-coated surfaces with that of isogenic methicillin-resistant derivatives. All strains containing an intact mec element in their chromosomes were found to be defective in adhesion to fibrinogen and fibronectin immobilized on polymethylmethacrylate coverslips, regardless of the presence or absence of additional mutations in the femA, femB, or femC gene, known to decrease expression of methicillin resistance in S. aureus. Western ligand affinity blotting or immunoblotting of cell wall-associated adhesins revealed similar contents of fibrinogen- or fibronectin-binding proteins in methicillin-resistant strains compared to those of their methicillin-susceptible counterparts. In contrast to methicillin-resistant strains carrying a mec element in their genomes, methicillin-resistant strains constructed in vitro, by introducing the mecA gene on a plasmid, retained their adhesion phenotypes. In conclusion, the chromosomal insertion of the mec element into genetically defined strains of S. aureus impairs the in vitro functional activities of fibrinogen or fibronectin adhesins without altering their production. This effect is unrelated to the activity of the mecA gene. PMID:9517933

  20. Proteomic comparison of etioplast and chloroplast protein complexes.

    PubMed

    Plöscher, Matthias; Reisinger, Veronika; Eichacker, Lutz A

    2011-08-12

    Angiosperms grown in darkness develop etioplasts during skotomorphogenesis. It is well known that etioplasts accumulate large quantities of protochlorophyllideoxidoreductase, are devoid of chlorophyll and are the site to assemble the photosynthetic machinery during photomorphogenesis. Proteomic investigation of the membrane protein complexes by Native PAGE, in combination with CyDye labelling and mass spectrometric analysis revealed that etioplasts and chloroplasts share a number of membrane protein complexes characteristic for electron transport, chlorophyll and protein synthesis as well as fatty acid biosynthesis. The complex regulatory function in both developmental states is discussed. PMID:21440687

  1. Protein camouflage in cytochrome c-calixarene complexes

    NASA Astrophysics Data System (ADS)

    McGovern, Róise E.; Fernandes, Humberto; Khan, Amir R.; Power, Nicholas P.; Crowley, Peter B.

    2012-07-01

    Small molecules that recognize protein surfaces are important tools for modifying protein interaction properties. Since the 1980s, several thousand studies concerning calixarenes and host-guest interactions have been published. Although there is growing interest in protein-calixarene interactions, only limited structural information has been available to date. We now report the crystal structure of a protein-calixarene complex. The water-soluble p-sulfonatocalix[4]arene is shown to bind the lysine-rich cytochrome c at three different sites. Binding curves obtained from NMR titrations reveal an interaction process that involves two or more binding sites. Together, the data indicate a dynamic complex in which the calixarene explores the surface of cytochrome c. In addition to providing valuable information on protein recognition, the data also indicate that the calixarene is a mediator of protein-protein interactions, with potential applications in generating assemblies and promoting crystallization.

  2. Sizing Large Proteins and Protein Complexes by Electrospray Ionization Mass Spectrometry and Ion Mobility

    PubMed Central

    Kaddis, Catherine S.; Lomeli, Shirley H.; Yin, Sheng; Berhane, Beniam; Apostol, Marcin I.; Kickhoefer, Valerie A.; Rome, Leonard H.; Loo, Joseph A.

    2009-01-01

    Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method termed GEMMA (Gas-Phase Electrophoretic Mobility Molecular Analysis), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared to other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and x-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm3. Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water. PMID:17434746

  3. Crystallization and preliminary X-ray data of the FadA adhesin from Fusobacterium nucleatum

    SciTech Connect

    Nithianantham, Stanley; Xu, Minghua; Wu, Nan; Han, Yiping W.; Shoham, Menachem

    2006-12-01

    The FadA adhesin from F. nucleatum, which is involved in bacterial attachment and invasion of human oral epithelial cells, has been crystallized in space group P6{sub 1} or P6{sub 5}, and X-ray data have been collected to 1.9 Å resolution. Fusobacterium nucleatum is a Gram-negative anaerobe prevalent in the oral cavity that is associated with periodontal disease, preterm birth and infections in other parts of the human body. The bacteria attach to and invade epithelial and endothelial cells in the gum tissue and elsewhere via a 13.7 kDa adhesin protein FadA (Fusobacterium adhesin A). FadA exists in two forms: the intact form (pre-FadA), consisting of 129 amino acids, and the mature form (mFadA), which lacks an 18-residue signal sequence. Both forms have been expressed in Escherichia coli and purified. mFadA has been crystallized. The crystals belong to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 59.3, c = 125.7 Å and one molecule per asymmetric unit. The crystals exhibit an unusually high solvent content of 74%. Synchrotron X-ray data have been collected to 1.9 Å. The crystals are suitable for X-ray structure determination. The crystal structure of FadA may provide a basis for the development of therapeutic agents to combat periodontal disease and other infections associated with F. nucleatum.

  4. Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

    SciTech Connect

    Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N.; Auberry, Deanna L.; Hill, Eric A.; Hooker, Brian S.; Moore, Priscilla A.; Moore, Ronald J.; Shi, Liang; Wiley, H. S.; Kery, Vladimir

    2005-04-11

    Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.

  5. Protein structures in SDS micelle-protein complexes.

    PubMed Central

    Parker, W; Song, P S

    1992-01-01

    Sodium dodecyl sulfate (SDS) is used more often than any other detergent as an excellent denaturing or "unfolding" detergent. However, formation of ordered structure (alpha-helix or beta-sheet) in certain peptides is known to be induced by interaction with SDS micelles. The SDS-induced structures formed by these peptides are amphiphilic, having both a hydrophobic and a hydrophilic face. Previous work in this area has revealed that SDS induces helical folding in a wide variety of non-helical proteins. Here, we describe the interaction of several structurally unrelated proteins with SDS micelles and the correlation of these structures to helical amphiphilic regions present in the primary sequence. It is likely that the ability of native nonordered protein structures to form induced amphiphilic ordered structures is rather common. PMID:1600087

  6. Embracing proteins: structural themes in aptamer-protein complexes.

    PubMed

    Gelinas, Amy D; Davies, Douglas R; Janjic, Nebojsa

    2016-02-01

    Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer-protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer-protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity. PMID:26919170

  7. Structure of the Head of the Bartonella Adhesin BadA

    PubMed Central

    Szczesny, Pawel; Linke, Dirk; Ursinus, Astrid; Bär, Kerstin; Schwarz, Heinz; Riess, Tanja M.; Kempf, Volkhard A. J.; Lupas, Andrei N.; Martin, Jörg; Zeth, Kornelius

    2008-01-01

    Trimeric autotransporter adhesins (TAAs) are a major class of proteins by which pathogenic proteobacteria adhere to their hosts. Prominent examples include Yersinia YadA, Haemophilus Hia and Hsf, Moraxella UspA1 and A2, and Neisseria NadA. TAAs also occur in symbiotic and environmental species and presumably represent a general solution to the problem of adhesion in proteobacteria. The general structure of TAAs follows a head-stalk-anchor architecture, where the heads are the primary mediators of attachment and autoagglutination. In the major adhesin of Bartonella henselae, BadA, the head consists of three domains, the N-terminal of which shows strong sequence similarity to the head of Yersinia YadA. The two other domains were not recognizably similar to any protein of known structure. We therefore determined their crystal structure to a resolution of 1.1 Å. Both domains are β-prisms, the N-terminal one formed by interleaved, five-stranded β-meanders parallel to the trimer axis and the C-terminal one by five-stranded β-meanders orthogonal to the axis. Despite the absence of statistically significant sequence similarity, the two domains are structurally similar to domains from Haemophilus Hia, albeit in permuted order. Thus, the BadA head appears to be a chimera of domains seen in two other TAAs, YadA and Hia, highlighting the combinatorial evolutionary strategy taken by pathogens. PMID:18688279

  8. PEGylated Albumin-Based Polyion Complex Micelles for Protein Delivery.

    PubMed

    Jiang, Yanyan; Lu, Hongxu; Chen, Fan; Callari, Manuela; Pourgholami, Mohammad; Morris, David L; Stenzel, Martina H

    2016-03-14

    An increasing amount of therapeutic agents are based on proteins. However, proteins as drug have intrinsic problems such as their low hydrolytic stability. Delivery of proteins using nanoparticles has increasingly been the focus of interest with polyion complex micelles, prepared from charged block copolymer and the oppositely charged protein, as an example of an attractive carrier for proteins. Inspired by this approach, a more biocompatible pathway has been developed here, which replaces the charged synthetic polymer with an abundant protein, such as albumin. Although bovine serum albumin (BSA) was observed to form complexes with positively charged proteins directly, the resulting protein nanoparticle were not stable and aggregated to large precipitates over the course of a day. Therefore, maleimide functionalized poly(oligo (ethylene glycol) methyl ether methacrylate) (MI-POEGMEMA) (Mn = 26000 g/mol) was synthesized to generate a polymer-albumin conjugate, which was able to condense positively charged proteins, here lysozyme (Lyz) as a model. The PEGylated albumin polyion complex micelle with lysozyme led to nanoparticles between 15 and 25 nm in size depending on the BSA to Lyz ratio. The activity of the encapsulated protein was tested using Sprouty 1 (C-12; Spry1) proteins, which can act as an endogenous angiogenesis inhibitor. Condensation of Spry1 with the PEGylated albumin could improve the anticancer efficacy of Spry1 against the breast cancer cells lowering the IC50 value of the protein. Furthermore, the high anticancer efficacy of the POEGMEMA-BSA/Spry1 complex micelle was verified by effectively inhibiting the growth of three-dimensional MCF-7 multicellular tumor spheroids. The PEGylated albumin complex micelle has great potential as a drug delivery vehicle for a new generation of cancer pharmaceuticals. PMID:26809948

  9. Protein Connectivity in Chemotaxis Receptor Complexes.

    PubMed

    Eismann, Stephan; Endres, Robert G

    2015-12-01

    The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET) measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures. PMID:26646441

  10. Protein Connectivity in Chemotaxis Receptor Complexes

    PubMed Central

    Eismann, Stephan; Endres, Robert G.

    2015-01-01

    The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET) measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures. PMID:26646441

  11. Identification of Post-translational Modifications of Plant Protein Complexes

    PubMed Central

    Piquerez, Sophie J. M.; Balmuth, Alexi L.; Sklenář, Jan; Jones, Alexandra M.E.; Rathjen, John P.; Ntoukakis, Vardis

    2014-01-01

    Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein. PMID:24637539

  12. Coupling protein complex analysis to peptide based proteomics.

    PubMed

    Gao, Qiang; Madian, Ashraf G; Liu, Xiuping; Adamec, Jiri; Regnier, Fred E

    2010-12-01

    Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20°C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80°C while lowest recovery was achieved at 4°C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes. PMID:21036361

  13. Building a Hierarchical Organization of Protein Complexes Out of Protein Association Data

    PubMed Central

    Stojmirović, Aleksandar; Yu, Yi-Kuo

    2014-01-01

    Organizing experimentally determined protein associations as a hierarchy can be a good approach to elucidating the content of protein complexes and the modularity of subcomplexes. Several challenges exist. First, intrinsically sticky proteins, such as chaperones, are often falsely assigned to many functionally unrelated complexes. Second, the reported collections of proteins may not be true “complexes” in the sense that they bind together and perform a joint cellular function. Third, due to imperfect sensitivity of protein detection methods, both false positive and false negative assignments of a protein to complexes may occur. We mitigate the first issue by down-weighting sticky proteins by their occurrence frequencies. We approach the other two problems by merging nearly identical complexes and by constructing a directed acyclic graph (DAG) based on the relationship of partial inclusion. The constructed DAG, within which smaller complexes form parts of the larger, can reveal how different complexes are joined. By merging almost identical complexes one can deemphasize the influence of false positives, while allowing false negatives to be rescued by other nearly identical association data. We investigate several protein weighting schemes and compare their corresponding DAGs using yeast and human complexes. We find that the scheme incorporating weights based on information flow in the network of direct protein–protein interactions produces biologically most meaningful DAGs. In either yeast or human, isolated nodes form a large proportion of the final hierarchy. While most connected components encompass very few nodes, the largest one for each species contains a sizable portion of all nodes. By considering examples of subgraphs composed of nodes containing a specified protein, we illustrate that the graphs' topological features can correctly suggest the biological roles of protein complexes. The input data, final results and the source code are available at ftp

  14. Cross-linking Measurements of In Vivo Protein Complex Topologies*

    PubMed Central

    Zheng, Chunxiang; Yang, Li; Hoopmann, Michael R.; Eng, Jimmy K.; Tang, Xiaoting; Weisbrod, Chad R.; Bruce, James E.

    2011-01-01

    Identification and measurement of in vivo protein interactions pose critical challenges in the goal to understand biological systems. The measurement of structures and topologies of proteins and protein complexes as they exist in cells is particularly challenging, yet critically important to improve understanding of biological function because proteins exert their intended function only through the structures and interactions they exhibit in vivo. In the present study, protein interactions in E. coli cells were identified in our unbiased cross-linking approach, yielding the first in vivo topological data on many interactions and the largest set of identified in vivo cross-linked peptides produced to date. These data show excellent agreement with protein and complex crystal structures where available. Furthermore, our unbiased data provide novel in vivo topological information that can impact understanding of biological function, even for cases where high resolution structures are not yet available. PMID:21697552

  15. Graph theory and stability analysis of protein complex interaction networks.

    PubMed

    Huang, Chien-Hung; Chen, Teng-Hung; Ng, Ka-Lok

    2016-04-01

    Protein complexes play an essential role in many biological processes. Complexes can interact with other complexes to form protein complex interaction network (PCIN) that involves in important cellular processes. There are relatively few studies on examining the interaction topology among protein complexes; and little is known about the stability of PCIN under perturbations. We employed graph theoretical approach to reveal hidden properties and features of four species PCINs. Two main issues are addressed, (i) the global and local network topological properties, and (ii) the stability of the networks under 12 types of perturbations. According to the topological parameter classification, we identified some critical protein complexes and validated that the topological analysis approach could provide meaningful biological interpretations of the protein complex systems. Through the Kolmogorov-Smimov test, we showed that local topological parameters are good indicators to characterise the structure of PCINs. We further demonstrated the effectiveness of the current approach by performing the scalability and data normalization tests. To measure the robustness of PCINs, we proposed to consider eight topological-based perturbations, which are specifically applicable in scenarios of targeted, sustained attacks. We found that the degree-based, betweenness-based and brokering-coefficient-based perturbations have the largest effect on network stability. PMID:26997661

  16. Biochemical isolation of Argonaute protein complexes by Ago-APP

    PubMed Central

    Hauptmann, Judith; Schraivogel, Daniel; Bruckmann, Astrid; Manickavel, Sudhir; Jakob, Leonhard; Eichner, Norbert; Pfaff, Janina; Urban, Marc; Sprunck, Stefanie; Hafner, Markus; Tuschl, Thomas; Deutzmann, Rainer; Meister, Gunter

    2015-01-01

    During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells. PMID:26351695

  17. SnapShot: SMC Protein Complexes Part I.

    PubMed

    Haering, Christian H; Gruber, Stephan

    2016-01-14

    This first of two SnapShots on SMC proteins depicts the composition and architecture of SMC protein complexes and their regulators. Their roles at different stages of the cell cycle will appear in Part II. To view this SnapShot, open or download the PDF. PMID:26771499

  18. Linking structural features of protein complexes and biological function.

    PubMed

    Sowmya, Gopichandran; Breen, Edmond J; Ranganathan, Shoba

    2015-09-01

    Protein-protein interaction (PPI) establishes the central basis for complex cellular networks in a biological cell. Association of proteins with other proteins occurs at varying affinities, yet with a high degree of specificity. PPIs lead to diverse functionality such as catalysis, regulation, signaling, immunity, and inhibition, playing a crucial role in functional genomics. The molecular principle of such interactions is often elusive in nature. Therefore, a comprehensive analysis of known protein complexes from the Protein Data Bank (PDB) is essential for the characterization of structural interface features to determine structure-function relationship. Thus, we analyzed a nonredundant dataset of 278 heterodimer protein complexes, categorized into major functional classes, for distinguishing features. Interestingly, our analysis has identified five key features (interface area, interface polar residue abundance, hydrogen bonds, solvation free energy gain from interface formation, and binding energy) that are discriminatory among the functional classes using Kruskal-Wallis rank sum test. Significant correlations between these PPI interface features amongst functional categories are also documented. Salt bridges correlate with interface area in regulator-inhibitors (r = 0.75). These representative features have implications for the prediction of potential function of novel protein complexes. The results provide molecular insights for better understanding of PPIs and their relation to biological functions. PMID:26131659

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

    PubMed Central

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

    2013-01-01

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

  20. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  1. Optimized Affinity Capture of Yeast Protein Complexes.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented. PMID:27371596

  2. Bringing single-molecule spectroscopy to macromolecular protein complexes

    PubMed Central

    Joo, Chirlmin; Fareh, Mohamed; Kim, V. Narry

    2013-01-01

    Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. PMID:23200186

  3. Adhesins of Leptospira interrogans Mediate the Interaction to Fibrinogen and Inhibit Fibrin Clot Formation In Vitro

    PubMed Central

    Oliveira, Rosane; Domingos, Renan F.; Siqueira, Gabriela H.; Fernandes, Luis G.; Souza, Natalie M.; Vieira, Monica L.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Nascimento, Ana L. T. O.

    2013-01-01

    We report in this work that Leptospira strains, virulent L. interrogans serovar Copenhageni, attenuated L. interrogans serovar Copenhageni and saprophytic L. biflexa serovar Patoc are capable of binding fibrinogen (Fg). The interaction of leptospires with Fg inhibits thrombin- induced fibrin clot formation that may affect the haemostatic equilibrium. Additionally, we show that plasminogen (PLG)/plasmin (PLA) generation on the surface of Leptospira causes degradation of human Fg. The data suggest that PLA-coated leptospires were capable to employ their proteolytic activity to decrease one substrate of the coagulation cascade. We also present six leptospiral adhesins and PLG- interacting proteins, rLIC12238, Lsa33, Lsa30, OmpL1, rLIC11360 and rLIC11975, as novel Fg-binding proteins. The recombinant proteins interact with Fg in a dose-dependent and saturable fashion when increasing protein concentration was set to react to a fix human Fg concentration. The calculated dissociation equilibrium constants (KD) of these reactions ranged from 733.3±276.8 to 128±89.9 nM for rLIC12238 and Lsa33, respectively. The interaction of recombinant proteins with human Fg resulted in inhibition of fibrin clot by thrombin-catalyzed reaction, suggesting that these versatile proteins could mediate Fg interaction in Leptospira. Our data reveal for the first time the inhibition of fibrin clot by Leptospira spp. and presents adhesins that could mediate these interactions. Decreasing fibrin clot would cause an imbalance of the coagulation cascade that may facilitate bleeding and help bacteria dissemination PMID:24009788

  4. Electrophoresis of proteins and protein-protein complexes in native polyacrylamide gels using a horizontal gel apparatus.

    PubMed

    Su, C; Wang, F; Ciolek, D; Pan, Y C

    1994-11-15

    Electrophoresis of proteins and protein-protein complexes in polyacrylamide gels under native conditions using a horizontal gel apparatus is described. The advantage of this system is that it permits the detection of both negatively and positively charged proteins as well as protein-protein complexes in the same gel. During electrophoresis, a continuous gel sandwiched between two glass plates is placed horizontally on the platform and submerged in a reservoir buffer. The sample wells are made along the center of the gel, allowing positively and negatively charged proteins to migrate toward the cathode and anode, respectively. Several proteins with varying molecular weights and isoelectric point (pI) values and pairs of proteins capable of forming protein-protein complexes were chosen as model systems to illustrate the methodology. The effects of several parameters on the performance of the gel system including protein molecular weight, pI, and gel concentration were also examined and the results obtained by this method are comparable to those obtained by the vertical system. Following electrophoresis, both negatively and positively charged proteins as well as protein-protein complexes can be transferred by electroblotting onto polyvinylidene difluoride membranes for further analyses. PMID:7695108

  5. Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

    PubMed Central

    Miczak, A; Kaberdin, V R; Wei, C L; Lin-Chao, S

    1996-01-01

    The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed. Images Fig. 1 Fig. 2 PMID:8632981

  6. Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes

    SciTech Connect

    Martinovic, Suzana; Pasa-Tolic, Liljiana; Smith, Richard D.

    2004-10-01

    Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of non-covalent complexes on the intact complex level.

  7. The dynamics and pH-dependence of Ag43 adhesins' self-association probed by atomic force spectroscopy

    NASA Astrophysics Data System (ADS)

    Jacquot, Adrien; Sakamoto, Chizuko; Razafitianamarahavo, Angelina; Caillet, Céline; Merlin, Jenny; Fahs, Ahmad; Ghigo, Jean-Marc; Duval, Jérôme F. L.; Beloin, Christophe; Francius, Grégory

    2014-10-01

    Self-associating auto-transporter (SAAT) adhesins are two-domain cell surface proteins involved in bacteria auto-aggregation and biofilm formation. Antigen 43 (Ag43) is a SAAT adhesin commonly found in Escherichia coli whose variant Ag43a has been shown to promote persistence of uropathogenic E. coli within the bladder. The recent resolution of the tri-dimensional structure of the 499 amino-acids' β-domain in Ag43a has shed light on the possible mechanism governing the self-recognition of SAAT adhesins, in particular the importance of trans-interactions between the L shaped β-helical scaffold of two α-domains of neighboring adhesins. In this study, we use single-molecule force spectroscopy (SMFS) and dynamic force spectroscopy (DFS) to unravel the dynamics of Ag43-self association under various pH and molecular elongation rate conditions that mimic the situations encountered by E. coli in its natural environment. Results evidenced an important stretchability of Ag43α with unfolding of sub-domains leading to molecular extension as long as 150 nm. Nanomechanical analysis of molecular stretching data suggested that self-association of Ag43 can lead to the formation of dimers and tetramers driven by rapid and weak cis- as well as slow but strong trans-interaction forces with a magnitude as large as 100-250 pN. The dynamics of cis- and trans-interactions were demonstrated to be strongly influenced by pH and applied shear force, thus suggesting that environmental conditions can modulate Ag43-mediated aggregation of bacteria at the molecular level.Self-associating auto-transporter (SAAT) adhesins are two-domain cell surface proteins involved in bacteria auto-aggregation and biofilm formation. Antigen 43 (Ag43) is a SAAT adhesin commonly found in Escherichia coli whose variant Ag43a has been shown to promote persistence of uropathogenic E. coli within the bladder. The recent resolution of the tri-dimensional structure of the 499 amino-acids' β-domain in Ag43a has shed

  8. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  9. Negative Ions Enhance Survival of Membrane Protein Complexes.

    PubMed

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

    2016-06-01

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

  10. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  11. A-Kinase Anchoring Proteins: From protein complexes to physiology and disease

    PubMed Central

    Carnegie, Graeme K.; Means, Christopher K.; Scott, John D.

    2009-01-01

    Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review we focus on recent advances in the elucidation of AKAP function. PMID:19319965

  12. Molecular dynamics simulation strategies for protein-micelle complexes.

    PubMed

    Cheng, Xi; Kim, Jin-Kyoung; Kim, Yangmee; Bowie, James U; Im, Wonpil

    2016-07-01

    The structure and stability of membrane proteins can vary widely in different detergents and this variability has great practical consequences for working with membrane proteins. Nevertheless, the mechanisms that operate to alter the behavior of proteins in micelles are poorly understood and not predictable. Atomic simulations could provide considerable insight into these mechanisms. Building protein-micelle complexes for simulation is fraught with uncertainty, however, in part because it is often unknown how many detergent molecules are present in the complex. Here, we describe several convenient ways to employ Micelle Builder in CHARMM-GUI to rapidly construct protein-micelle complexes and performed simulations of the isolated voltage-sensor domain of voltage-dependent potassium-selective channel and an antimicrobial peptide papiliocin with varying numbers of detergents. We found that once the detergent number exceeds a threshold, protein-detergent interactions change very little and remain very consistent with experimental observations. Our results provide a platform for future studies of the interplays between protein structure and detergent properties at the atomic level. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26679426

  13. Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings

    PubMed Central

    Brzozowska, Ewa; Koba, Marcin; Śmietana, Mateusz; Górska, Sabina; Janik, Monika; Gamian, Andrzej; Bock, Wojtek J.

    2016-01-01

    This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests – ELISA and BIAcore – the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria. PMID:27231592

  14. Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings.

    PubMed

    Brzozowska, Ewa; Koba, Marcin; Śmietana, Mateusz; Górska, Sabina; Janik, Monika; Gamian, Andrzej; Bock, Wojtek J

    2016-03-01

    This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests - ELISA and BIAcore - the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria. PMID:27231592

  15. Identifying hierarchical and overlapping protein complexes based on essential protein-protein interactions and "seed-expanding" method.

    PubMed

    Ren, Jun; Zhou, Wei; Wang, Jianxin

    2014-01-01

    Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and "seed-expanding." First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes. PMID:25143945

  16. Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

    PubMed Central

    Ren, Jun; Zhou, Wei; Wang, Jianxin

    2014-01-01

    Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes. PMID:25143945

  17. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters

    PubMed Central

    Hanna, Eileen Marie; Zaki, Nazar; Amin, Amr

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present “DyCluster”, a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster. PMID:26641660

  18. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters.

    PubMed

    Hanna, Eileen Marie; Zaki, Nazar; Amin, Amr

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present "DyCluster", a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster. PMID:26641660

  19. Detecting protein complexes in protein interaction networks using a ranking algorithm with a refined merging procedure

    PubMed Central

    2014-01-01

    Background Developing suitable methods for the identification of protein complexes remains an active research area. It is important since it allows better understanding of cellular functions as well as malfunctions and it consequently leads to producing more effective cures for diseases. In this context, various computational approaches were introduced to complement high-throughput experimental methods which typically involve large datasets, are expensive in terms of time and cost, and are usually subject to spurious interactions. Results In this paper, we propose ProRank+, a method which detects protein complexes in protein interaction networks. The presented approach is mainly based on a ranking algorithm which sorts proteins according to their importance in the interaction network, and a merging procedure which refines the detected complexes in terms of their protein members. ProRank + was compared to several state-of-the-art approaches in order to show its effectiveness. It was able to detect more protein complexes with higher quality scores. Conclusions The experimental results achieved by ProRank + show its ability to detect protein complexes in protein interaction networks. Eventually, the method could potentially identify previously-undiscovered protein complexes. The datasets and source codes are freely available for academic purposes at http://faculty.uaeu.ac.ae/nzaki/Research.htm. PMID:24944073

  20. Rapid purification of protein complexes from mammalian cells

    PubMed Central

    Medina, Dan; Moskowitz, Neal; Khan, Subarna; Christopher, Scott; Germino, Joseph

    2000-01-01

    The evaluation of the protein binding partner(s) of biologically important proteins is currently an area of intense research, especially since the development of the yeast two-hybrid assay. However, not all protein–protein interactions uncovered by this assay are biologically relevant and another confirmatory assay must be performed. Ideally, this assay should be rapid, versatile and performed under conditions which mimic the ‘normal’ physiological state as closely as possible. Towards this goal, we have constructed two eukaryotic expression vectors that facilitate the purification of a protein of interest, along with any associated proteins, from mammalian cells. These vectors incorporate the following features: (i) a tetracycline-responsive promoter so that the level of protein production can be regulated; (ii) an N-terminal glutathione S-transferase tag or a triple repeat of the HA1 epitope, to facilitate purification of the protein either by glutathione affinity chromatography or immunoprecipitation, respectively, followed by a multiple cloning site; (iii) the gene for the enhanced green fluorescent protein (for detection of the presence of the fusion protein and subcellular localization); (iv) a puromycin marker for the selection of stable transformants; (v) a truncated EBNA protein and oriP sequence for episomal replication of the vector. These latter two features permit expansion of small cultures of transfected cells under puromycin selection, thereby increasing the amount of tagged protein that can be purified. We show that these vectors can be used to direct the doxycycline-inducible expresssion of tagged proteins and to recover tagged CIP1–p21 protein complexes from HeLa cells. Furthermore, we show that these tagged p21-purified complexes contain both cyclin A and Cdk2, which are known to interact with p21, but not β-actin. PMID:10871384

  1. Adhesin degradation accelerates delivery of heat-labile toxin by enterotoxigenic Escherichia coli.

    PubMed

    Roy, Koushik; Kansal, Rita; Bartels, Scott R; Hamilton, David J; Shaaban, Salwa; Fleckenstein, James M

    2011-08-26

    Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development. PMID:21757737

  2. Analyzing Large Protein Complexes by Structural Mass Spectrometry

    PubMed Central

    Kirshenbaum, Noam; Michaelevski, Izhak; Sharon, Michal

    2010-01-01

    Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization. One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol

  3. That's my story, and I'm sticking to it—an update on B. burgdorferi adhesins

    PubMed Central

    Brissette, Catherine A.; Gaultney, Robert A.

    2014-01-01

    Adhesion is the initial event in the establishment of any infection. Borrelia burgdorferi, the etiological agent of Lyme disease, possesses myriad proteins termed adhesins that facilitate contact with its vertebrate hosts. B. burgdorferi adheres to host tissues through interactions with host cells and extracellular matrix, as well as other molecules present in serum and extracellular fluids. These interactions, both general and specific, are critical in the establishment of infection. Modulation of borrelial adhesion to host tissues affects the microorganisms's ability to colonize, disseminate, and persist. In this review, we update the current knowledge on structure, function, and role in pathogenesis of these “sticky” B. burgdorferi infection-associated proteins. PMID:24772392

  4. Emergence of Complexity in Protein Functions and Metabolic Networks

    NASA Technical Reports Server (NTRS)

    Pohorille, Andzej

    2009-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis of very large libraries of random amino acid sequences and subsequently subjecting them to in vitro evolution. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions, important clues have been uncovered. Considerable progress has been also achieved in understanding the origins of membrane proteins. We will address this issue in the example of ion channels - proteins that mediate transport of ions across cell walls. Remarkably, despite overall complexity of these proteins in contemporary cells, their structural motifs are quite simple, with -helices being most common. By combining results of experimental and computer simulation studies on synthetic models and simple, natural channels, I will show that, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during

  5. Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

    PubMed Central

    Hyung, Suk-Joon; Ruotolo, Brandon T.

    2013-01-01

    Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

  6. Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

    PubMed Central

    Zweers, Jessica C; Barák, Imrich; Becher, Dörte; Driessen, Arnold JM; Hecker, Michael; Kontinen, Vesa P; Saller, Manfred J; Vavrová, L'udmila; van Dijl, Jan Maarten

    2008-01-01

    Background The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations. Conclusion While inactivation of quality control systems has the potential to improve protein production yields, this could be achieved at the expense of product quality. Mechanisms underlying degradation of secretory proteins are nowadays well understood and often controllable. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins. PMID:18394159

  7. A secretory kinase complex regulates extracellular protein phosphorylation.

    PubMed

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. PMID:25789606

  8. Solid-State NMR Spectroscopy of Protein Complexes

    PubMed Central

    Sun, Shangjin; Han, Yun; Paramasivam, Sivakumar; Yan, Si; Siglin, Amanda E.; Williams, John C.; Byeon, In-Ja L.; Ahn, Jinwoo; Gronenborn, Angela M.; Polenova, Tatyana

    2016-01-01

    Protein-protein interactions are vital for many biological processes. These interactions often result in the formation of protein assemblies that are large in size, insoluble and difficult to crystallize, and therefore are challenging to study by structure biology techniques, such as single crystal X-ray diffraction and solution NMR spectroscopy. Solid-state NMR (SSNMR) spectroscopy is emerging as a promising technique for studies of such protein assemblies because it is not limited by molecular size, solubility or lack of long-range order. In the past several years, we have applied magic angle spinning SSNMR based methods to study several protein complexes. In this chapter, we discuss the general solid-state NMR methodologies employed for structural and dynamics analyses of protein complexes with specific examples from our work on thioredoxin reassemblies, HIV-1 capsid protein assemblies and microtubule-associated protein assemblies. We present protocols for sample preparation and characterization, pulse sequences, SSNMR spectra collection and data analysis. PMID:22167681

  9. Displacement affinity chromatography of protein phosphatase one (PP1) complexes

    PubMed Central

    Moorhead, Greg BG; Trinkle-Mulcahy, Laura; Nimick, Mhairi; De Wever, Veerle; Campbell, David G; Gourlay, Robert; Lam, Yun Wah; Lamond, Angus I

    2008-01-01

    Background Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes. PMID:19000314

  10. 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

  11. The Three-dimensional Structure of the Extracellular Adhesion Domain of the Sialic Acid-binding Adhesin SabA from Helicobacter pylori

    PubMed Central

    Pang, Siew Siew; Nguyen, Stanley Thai Son; Perry, Andrew J.; Day, Christopher J.; Panjikar, Santosh; Tiralongo, Joe; Whisstock, James C.; Kwok, Terry

    2014-01-01

    The gastric pathogen Helicobacter pylori is a major cause of acute chronic gastritis and the development of stomach and duodenal ulcers. Chronic infection furthermore predisposes to the development of gastric cancer. Crucial to H. pylori survival within the hostile environment of the digestive system are the adhesins SabA and BabA; these molecules belong to the same protein family and permit the bacteria to bind tightly to sugar moieties LewisB and sialyl-LewisX, respectively, on the surface of epithelial cells lining the stomach and duodenum. To date, no representative SabA/BabA structure has been determined, hampering the development of strategies to eliminate persistent H. pylori infections that fail to respond to conventional therapy. Here, using x-ray crystallography, we show that the soluble extracellular adhesin domain of SabA shares distant similarity to the tetratricopeptide repeat fold family. The molecule broadly resembles a golf putter in shape, with the head region featuring a large cavity surrounded by loops that vary in sequence between different H. pylori strains. The N-terminal and C-terminal helices protrude at right angles from the head domain and together form a shaft that connects to a predicted outer membrane protein-like β-barrel trans-membrane domain. Using surface plasmon resonance, we were able to detect binding of the SabA adhesin domain to sialyl-LewisX and LewisX but not to LewisA, LewisB, or LewisY. Substitution of the highly conserved glutamine residue 159 in the predicted ligand-binding pocket abrogates the binding of the SabA adhesin domain to sialyl-LewisX and LewisX. Taken together, these data suggest that the adhesin domain of SabA is sufficient in isolation for specific ligand binding. PMID:24375407

  12. Biclustering Protein Complex Interactions with a Biclique FindingAlgorithm

    SciTech Connect

    Ding, Chris; Zhang, Anne Ya; Holbrook, Stephen

    2006-12-01

    Biclustering has many applications in text mining, web clickstream mining, and bioinformatics. When data entries are binary, the tightest biclusters become bicliques. We propose a flexible and highly efficient algorithm to compute bicliques. We first generalize the Motzkin-Straus formalism for computing the maximal clique from L{sub 1} constraint to L{sub p} constraint, which enables us to provide a generalized Motzkin-Straus formalism for computing maximal-edge bicliques. By adjusting parameters, the algorithm can favor biclusters with more rows less columns, or vice verse, thus increasing the flexibility of the targeted biclusters. We then propose an algorithm to solve the generalized Motzkin-Straus optimization problem. The algorithm is provably convergent and has a computational complexity of O(|E|) where |E| is the number of edges. It relies on a matrix vector multiplication and runs efficiently on most current computer architectures. Using this algorithm, we bicluster the yeast protein complex interaction network. We find that biclustering protein complexes at the protein level does not clearly reflect the functional linkage among protein complexes in many cases, while biclustering at protein domain level can reveal many underlying linkages. We show several new biologically significant results.

  13. Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

    PubMed Central

    Fabre, Bertrand; Lambour, Thomas; Garrigues, Luc; Amalric, François; Vigneron, Nathalie; Menneteau, Thomas; Stella, Alexandre; Monsarrat, Bernard; Van den Eynde, Benoît; Burlet-Schiltz, Odile; Bousquet-Dubouch, Marie-Pierre

    2015-01-01

    In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin–proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high-resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes. PMID:25561571

  14. Architecture and function of IFT complex proteins in ciliogenesis

    PubMed Central

    Taschner, Michael; Bhogaraju, Sagar; Lorentzen, Esben

    2014-01-01

    Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT. PMID:22118932

  15. Economy of operon formation: cotranscription minimizes shortfall in protein complexes.

    PubMed

    Sneppen, Kim; Pedersen, Steen; Krishna, Sandeep; Dodd, Ian; Semsey, Szabolcs

    2010-01-01

    Genes of prokaryotes and Archaea are often organized in cotranscribed groups, or operons. In contrast, eukaryotic genes are generally transcribed independently. Here we show that there is a substantial economic gain for the cell to cotranscribe genes encoding protein complexes because it synchronizes the fluctuations, or noise, in the levels of the different components. This correlation substantially reduces the shortfall in production of the complex. This benefit is relatively large in small cells such as bacterial cells, in which there are few mRNAs and proteins per cell, and is diminished in larger cells such as eukaryotic cells. PMID:20877578

  16. Discovery of host-viral protein complexes during infection

    PubMed Central

    Rowles, Daniell L.; Terhune, Scott S.; Cristea, Ileana M.

    2014-01-01

    Summary Viruses have co-evolved with their hosts, developing effective approaches for hijacking and manipulating host cellular processes. Therefore, for their efficient replication and spread, viruses depend on dynamic and temporally-regulated interactions with host proteins. The rapid identification of host proteins targeted by viral proteins during infection provides significant insights into mechanisms of viral protein function. The resulting discoveries often lead to unique and innovative hypotheses on viral protein function. Here, we describe a robust method for identifying virus-host protein interactions and protein complexes, which we have successfully utilized to characterize spatial-temporal protein interactions during infections with either DNA or RNA viruses, including human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), human immunodeficiency virus (HIV-1), Sindbis, and West Nile virus (WNV). This approach involves cryogenic cell lysis, rapid immunoaffinity purification targeting a virus or host protein, followed by identification of associated proteins using mass spectrometry. Like most proteomic approaches, this methodology has evolved over the past few years and continues to evolve. We are presenting here the updated approaches for each step, and discuss alternative strategies allowing for the protocol to be optimized for different biological systems. PMID:23996249

  17. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  18. Purification of a mycobacterial adhesin for fibronectin.

    PubMed Central

    Ratliff, T L; McCarthy, R; Telle, W B; Brown, E J

    1993-01-01

    Previous studies have demonstrated that mycobacteria attach to fibronectin (FN). The attachment of mycobacteria to FN is considered to be biologically important in Mycobacterium bovis BCG therapy for superficial bladder cancer, initiation of delayed hypersensitivity to mycobacterial antigens, and the phagocytosis of mycobacteria by epithelial cells. Therefore, we purified the mycobacterial receptor for FN. Culture supernatants from 3-week cultures of Mycobacterium vaccae, which contained proteins that bound FN and inhibited the attachment of both M. vaccae and BCG to FN, were used as a source of receptor. Lyophilized M. vaccae supernatants were reconstituted in 0.02 M bis-Tris (pH 6.0) and applied sequentially to an ACA 54 gel filtration column and a DEAE-Sephacel anion-exchange column. A purified inhibitory protein of 55 kDa (p55) was obtained. The purified p55 protein was observed to bind to FN and to inhibit 125I-FN binding to viable BCG in a dose-dependent manner. Polyclonal and monoclonal antibodies to the protein were generated. The resulting polyclonal antiserum blotted a single protein band at 55 kDa in crude M. vaccae supernatants, cross-reacted with a 55-kDa BCG protein by Western blot (immunoblot), and recognized a 55-kDa band that was associated with the BCG cell wall, which is consistent with its function as a FN receptor. A monoclonal immunoglobulin M(lambda) was isolated from mice immunized with purified M. vaccae p55 protein that was not functional in Western blots but inhibited the attachment of viable BCG to FN. These studies demonstrate that a protein or antigenically related proteins with M(r)s of 55,000 function as FN receptors for at least two distinct mycobacteria. Images PMID:8478078

  19. Characterization of invasive Neisseria meningitidis from Atlantic Canada, 2009 to 2013: With special reference to the nonpolysaccharide vaccine targets (PorA, factor H binding protein, Neisseria heparin-binding antigen and Neisseria adhesin A)

    PubMed Central

    Tsang, Raymond SW; Law, Dennis KS; Gad, Rita R; Mailman, Tim; German, Gregory; Needle, Robert

    2015-01-01

    BACKGROUND: Serogroup B Neisseria meningitidis (MenB) has always been a major cause of invasive meningococcal disease (IMD) in Canada. With the successful implementation of a meningitis C conjugate vaccine, the majority of IMD in Canada is now caused by MenB. OBJECTIVE: To investigate IMD case isolates in Atlantic Canada from 2009 to 2013. Data were analyzed to determine the potential coverage of the newly licensed MenB vaccine. METHODS: Serogroup, serotype and serosubtype antigens were determined from IMD case isolates. Clonal analysis was performed using multilocus sequence typing. The protein-based vaccine antigen genes were sequenced and the predicted peptides were investigated. RESULTS: The majority of the IMD isolates were MenB (82.5%, 33 of 40) and, in particular, sequence type (ST)-154 B:4:P1.4 was responsible for 47.5% (19 of 40) of all IMD case isolates in Atlantic Canada. Isolates of this clone expressed the PorA antigen P1.4 and possessed the nhba genes encoding for Neisseria heparin-binding antigen peptide 2, which together matched exactly with two of the four components of the new four-component meningococcal B vaccine. Nineteen MenB isolates had two antigenic matches, another five MenB and one meningitis Y isolate had one antigenic match. This provided 75.8% (25 of 33) potential coverage for MenB, or a 62.5% (25 of 40) overall potential coverage for IMD. CONCLUSION: From 2009 to 2013, IMD in Atlantic Canada was mainly caused by MenB and, in particular, the B:4:P1.4 ST-154 clone, which accounted for 47.5% of all IMD case isolates. The new four-component meningococcal B vaccine appeared to offer adequate coverage against MenB in Atlantic Canada. PMID:26744586

  20. Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes.

    PubMed

    Petrotchenko, Evgeniy V; Olkhovik, Vyacheslav K; Borchers, Christoph H

    2005-08-01

    An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative "cross-linked peptides" (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H12 form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the

  1. Information-driven modeling of protein-peptide complexes.

    PubMed

    Trellet, Mikael; Melquiond, Adrien S J; Bonvin, Alexandre M J J

    2015-01-01

    Despite their biological importance in many regulatory processes, protein-peptide recognition mechanisms are difficult to study experimentally at the structural level because of the inherent flexibility of peptides and the often transient interactions on which they rely. Complementary methods like biomolecular docking are therefore required. The prediction of the three-dimensional structure of protein-peptide complexes raises unique challenges for computational algorithms, as exemplified by the recent introduction of protein-peptide targets in the blind international experiment CAPRI (Critical Assessment of PRedicted Interactions). Conventional protein-protein docking approaches are often struggling with the high flexibility of peptides whose short sizes impede protocols and scoring functions developed for larger interfaces. On the other side, protein-small ligand docking methods are unable to cope with the larger number of degrees of freedom in peptides compared to small molecules and the typically reduced available information to define the binding site. In this chapter, we describe a protocol to model protein-peptide complexes using the HADDOCK web server, working through a test case to illustrate every steps. The flexibility challenge that peptides represent is dealt with by combining elements of conformational selection and induced fit molecular recognition theories. PMID:25555727

  2. Characterization of the Modular Design of the Autolysin/Adhesin Aaa from Staphylococcus Aureus

    PubMed Central

    Hirschhausen, Nina; Schlesier, Tim; Peters, Georg; Heilmann, Christine

    2012-01-01

    Background Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. Methodology/Principal Findings Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. Conclusions/Significance We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus

  3. Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA

    PubMed Central

    Fusco, William G.; Choudhary, Neelima R.; Stewart, Shelley M.; Alam, S. Munir; Sempowski, Gregory D.; Elkins, Christopher

    2015-01-01

    Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrAI) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine. PMID:25897604

  4. Protein corona – from molecular adsorption to physiological complexity

    PubMed Central

    Docter, Dominic; Maskos, Michael

    2015-01-01

    Summary In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP–protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs. PMID:25977856

  5. Protein Ligand Complex Guided Approach for Virtual Screening.

    PubMed

    Karthikeyan, Muthukumarasamy; Pandit, Deepak; Vyas, Renu

    2015-01-01

    The target ligand association data is a rich source of information which is not exploited enough for drug design efforts in virtual screening. A java based open-source toolkit for Protein Ligand Network Extraction (J-ProLiNE) focused on protein-ligand complex analysis with several features integrated in a distributed computing network has been developed. Sequence alignment and similarity search components have been automated to yield local, global alignment scores along with similarity and distance scores. 10000 proteins with co-crystallized ligands from pdb and MOAD databases were extracted and analyzed for revealing relationships between targets, ligands and scaffolds. Through this analysis, we could generate a protein ligand network to identify the promiscuous and selective scaffolds for multiple classes of proteins targets. Using J-ProLiNE we created a 507 x 507 matrix of protein targets and native ligands belonging to six enzyme classes and analyzed the results to elucidate the protein-protein, protein-ligand and ligand-ligand interactions. In yet another application of the J-ProLiNE software, we were able to process kinase related information stored in US patents to construct disease-gene-ligand-scaffold networks. It is hoped that the studies presented here will enable target ligand knowledge based virtual screening for inhibitor design. PMID:26138572

  6. Pigment Analysis of Chloroplast Pigment-Protein Complexes in Wheat

    PubMed Central

    Eskins, Kenneth; Duysen, Murray E.; Olson, Linda

    1983-01-01

    Pigment-protein complexes separated from wheat (Triticum aestivum L. selection ND96-25 by two gel electrophoresis techniques were analyzed by high-performance liquid chromatography for chlorophylls and carotenoids. The two techniques are compared, and pigment analyses are given for the major reaction centers and light-harvesting complexes. Reaction centers contain mostly chlorophyll a, carotene, and lutein, whereas light-harvesting complexes contain chlorophyll a, chlorophyll b, lutein, and neoxanthin. The amounts of violaxanthin are variable. Images Fig. 1 PMID:16662906

  7. Cardiac mitochondrial matrix and respiratory complex protein phosphorylation

    PubMed Central

    Covian, Raul

    2012-01-01

    It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain α-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on 32P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the

  8. Study of protein complexes via homology modeling, applied to cysteine proteases and their protein inhibitors.

    PubMed

    Tastan Bishop, Ozlem; Kroon, Matthys

    2011-12-01

    This paper develops and evaluates large-scale calculation of 3D structures of protein complexes by homology modeling as a promising new approach for protein docking. The complexes investigated were papain-like cysteine proteases and their protein inhibitors, which play numerous roles in human and parasitic metabolisms. The structural modeling was performed in two parts. For the first part (evaluation set), nine crystal structure complexes were selected, 1325 homology models of known complexes were rebuilt by various templates including hybrids, allowing an analysis of the factors influencing the accuracy of the models. The important considerations for modeling the interface were protease coverage and inhibitor sequence identity. In the second part (study set), the findings of the evaluation set were used to select appropriate templates to model novel cysteine protease-inhibitor complexes from human and malaria parasites Plasmodium falciparum and Plasmodium vivax. The energy scores, considering the evaluation set, indicate that the models are of high accuracy. PMID:21365221

  9. Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

    ERIC Educational Resources Information Center

    Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

    2005-01-01

    In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

  10. Protein import and the origin of red complex plastids.

    PubMed

    Gould, Sven B; Maier, Uwe-G; Martin, William F

    2015-06-15

    The number and nature of endosymbioses involving red algal endosymbionts are debated. Gene phylogenies have become the most popular tool to untangle this issue, but they deliver conflicting results. As gene and lineage sampling has increased, so have both the number of conflicting trees and the number of suggestions in the literature for multiple tertiary, and even quaternary, symbioses that might reconcile the tree conflicts. Independent lines of evidence that can address the issue are needed. Here we summarize the mechanism and machinery of protein import into complex red plastids. The process involves protein translocation machinery, known as SELMA, that arose once in evolution, that facilitates protein import across the second outermost of the four plastid membranes, and that is always targeted specifically to that membrane, regardless of where it is encoded today. It is widely accepted that the unity of protein import across the two membranes of primary plastids is strong evidence for their single cyanobacterial origin. Similarly, the unity of SELMA-dependent protein import across the second outermost plastid membrane constitutes strong evidence for the existence of a single red secondary endosymbiotic event at the common origin of all red complex plastids. We furthermore propose that the two outer membranes of red complex plastids are derived from host endoplasmic reticulum in the initial red secondary endosymbiotic event. PMID:26079086

  11. Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte Complex Micelle.

    PubMed

    Wu, Fu-Gen; Jiang, Yao-Wen; Chen, Zhan; Yu, Zhi-Wu

    2016-04-19

    The folding/unfolding behavior of proteins (enzymes) in confined space is important for their properties and functions, but such a behavior remains largely unexplored. In this article, we reported our finding that lysozyme and a double hydrophilic block copolymer, methoxypoly(ethylene glycol)5K-block-poly(l-aspartic acid sodium salt)10 (mPEG(5K)-b-PLD10), can form a polyelectrolyte complex micelle with a particle size of ∼30 nm, as verified by dynamic light scattering and transmission electron microscopy. The unfolding and refolding behaviors of lysozyme molecules in the presence of the copolymer were studied by microcalorimetry and circular dichroism spectroscopy. Upon complex formation with mPEG(5K)-b-PLD10, lysozyme changed from its initial native state to a new partially unfolded state. Compared with its native state, this copolymer-complexed new folding state of lysozyme has different secondary and tertiary structures, a decreased thermostability, and significantly altered unfolding/refolding behaviors. It was found that the native lysozyme exhibited reversible unfolding and refolding upon heating and subsequent cooling, while lysozyme in the new folding state (complexed with the oppositely charged PLD segments of the polymer) could unfold upon heating but could not refold upon subsequent cooling. By employing the heating-cooling-reheating procedure, the prevention of complex formation between lysozyme and polymer due to the salt screening effect was observed, and the resulting uncomplexed lysozyme regained its proper unfolding and refolding abilities upon heating and subsequent cooling. Besides, we also pointed out the important role the length of the PLD segment played during the formation of micelles and the monodispersity of the formed micelles. Furthermore, the lysozyme-mPEG(5K)-b-PLD10 mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates in solution as frequently observed in other protein

  12. Atomic force and super-resolution microscopy support a role for LapA as a cell-surface biofilm adhesin of Pseudomonas fluorescens

    PubMed Central

    Ivanov, Ivan E.; Boyd, Chelsea D.; Newell, Peter D.; Schwartz, Mary E.; Turnbull, Lynne; Johnson, Michael S.; Whitchurch, Cynthia B.; O’Toole, George A.; Camesano, Terri A.

    2012-01-01

    Pseudomonas fluorescence Pf0-1 requires the large repeat protein LapA for stable surface attachment. This study presents direct evidence that LapA is a cell-surface-localized adhesin. Atomic force microscopy (AFM) revealed a significant twofold reduction in adhesion force for mutants lacking the LapA protein on the cell surface compared to the wild-type strain. Deletion of lapG, a gene encoding a periplasmic cysteine protease that functions to release LapA from the cell surface, resulted in a twofold increase in the force of adhesion. Three-dimensional structured illumination microscopy (3D-SIM) revealed the presence of the LapA protein on the cell surface, consistent with its role as an adhesin. The protein is only visualized in the cytoplasm for a mutant of the ABC transporter responsible for translocating LapA to the cell surface. Together, these data highlight the power of combining the use of AFM and 3D-SIM with genetic studies to demonstrate that LapA, a member of a large group of RTX-like repeat proteins, is a cell-surface adhesin. PMID:23064158

  13. Oral Streptococci Utilize a Siglec-Like Domain of Serine-Rich Repeat Adhesins to Preferentially Target Platelet Sialoglycans in Human Blood

    PubMed Central

    Deng, Lingquan; Bensing, Barbara A.; Thamadilok, Supaporn; Yu, Hai; Lau, Kam; Chen, Xi; Ruhl, Stefan; Sullam, Paul M.; Varki, Ajit

    2014-01-01

    Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group streptococci have a

  14. Exploration of the dynamic properties of protein complexes predicted from spatially constrained protein-protein interaction networks.

    PubMed

    Yen, Eric A; Tsay, Aaron; Waldispuhl, Jerome; Vogel, Jackie

    2014-05-01

    Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and visualization of the hidden

  15. Structural Reconstruction of Protein-Protein Complexes Involved in Intracellular Signaling.

    PubMed

    Kirsch, Klára; Sok, Péter; Reményi, Attila

    2016-01-01

    Signaling complexes within the cell convert extracellular cues into physiological outcomes. Their assembly involves signaling enzymes, allosteric regulators and scaffold proteins that often contain long stretches of disordered protein regions, display multi-domain architectures, and binding affinity between individual components is low. These features are indispensable for their central roles as dynamic information processing hubs, on the other hand they also make reconstruction of structurally homogeneous complex samples highly challenging. In this present chapter we discuss protein machinery which influences extracellular signal reception, intracellular pathway activity, and cytoskeletal or transcriptional activity. PMID:27165334

  16. Modeling of protein binary complexes using structural mass spectrometry data

    PubMed Central

    Kamal, J.K. Amisha; Chance, Mark R.

    2008-01-01

    In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints—positive and/or negative—in the docking step and are also used to decide the type of energy filter—electrostatics or desolvation—in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure. PMID:18042684

  17. From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

    PubMed Central

    Gao, Mu; Skolnick, Jeffrey

    2009-01-01

    DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Cα deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein. PMID:19343221

  18. Two Clathrin Adaptor Protein Complexes Instruct Axon-Dendrite Polarity.

    PubMed

    Li, Pengpeng; Merrill, Sean A; Jorgensen, Erik M; Shen, Kang

    2016-05-01

    The cardinal feature of neuronal polarization is the establishment and maintenance of axons and dendrites. How axonal and dendritic proteins are sorted and targeted to different compartments is poorly understood. Here, we identified distinct dileucine motifs that are necessary and sufficient to target transmembrane proteins to either the axon or the dendrite through direct interactions with the clathrin-associated adaptor protein complexes (APs) in C. elegans. Axonal targeting requires AP-3, while dendritic targeting is mediated by AP-1. The axonal dileucine motif binds to AP-3 with higher efficiency than to AP-1. Both AP-3 and AP-1 are localized to the Golgi but occupy adjacent domains. We propose that AP-3 and AP-1 directly select transmembrane proteins and target them to axon and dendrite, respectively, by sorting them into distinct vesicle pools. PMID:27151641

  19. Ca2+-stabilized adhesin helps an Antarctic bacterium reach out and bind ice

    PubMed Central

    Vance, Tyler D. R.; Olijve, Luuk L. C.; Campbell, Robert L.; Voets, Ilja K.; Davies, Peter L.; Guo, Shuaiqi

    2014-01-01

    The large size of a 1.5-MDa ice-binding adhesin [MpAFP (Marinomonas primoryensis antifreeze protein)] from an Antarctic Gram-negative bacterium, M. primoryensis, is mainly due to its highly repetitive RII (Region II). MpAFP_RII contains roughly 120 tandem copies of an identical 104-residue repeat. We have previously determined that a single RII repeat folds as a Ca2+-dependent immunoglobulin-like domain. Here, we solved the crystal structure of RII tetra-tandemer (four tandem RII repeats) to a resolution of 1.8 Å. The RII tetra-tandemer reveals an extended (~190-Å × ~25-Å), rod-like structure with four RII-repeats aligned in series with each other. The inter-repeat regions of the RII tetra-tandemer are strengthened by Ca2+ bound to acidic residues. SAXS (small-angle X-ray scattering) profiles indicate the RII tetra-tandemer is significantly rigidified upon Ca2+ binding, and that the protein's solution structure is in excellent agreement with its crystal structure. We hypothesize that >600 Ca2+ help rigidify the chain of ~120 104-residue repeats to form a ~0.6 μm rod-like structure in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed extender role of RII can help the strictly aerobic, motile bacterium bind ice in the upper reaches of the Antarctic lake where oxygen and nutrients are most abundant. Ca2+-induced rigidity of tandem Ig-like repeats in large adhesins might be a general mechanism used by bacteria to bind to their substrates and help colonize specific niches. PMID:24892750

  20. Over-producing soluble protein complex and validating protein-protein interaction through a new bacterial co-expression system.

    PubMed

    Zeng, Jumei; Zhang, Lei; Li, Yuqing; Wang, Yi; Wang, Mingchao; Duan, Xin; He, Zheng-Guo

    2010-01-01

    Many proteins exert their functions through a protein complex and protein-protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein-protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein-protein interaction of interest in a wide range of biological organisms. PMID:19747546

  1. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy.

    PubMed

    Turk, Rolf; Hsiao, Jordy J; Smits, Melinda M; Ng, Brandon H; Pospisil, Tyler C; Jones, Kayla S; Campbell, Kevin P; Wright, Michael E

    2016-06-01

    Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research. PMID:27099343

  2. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data. PMID:27165321

  3. Plasmid-protein relaxation complexes in Staphylococcus aureus.

    PubMed

    Novick, R

    1976-09-01

    Protein-deoxyribonucleic acid relaxation complexes have been demonstrated for six Staphylococcus aureus plasmids out of sixteen examined. Four of these encode stretomycin resistence, have molecular weights of about 2.7 x 10(6), and are isolated as supercoiled molecules that are virtally 100% relaxable by treatment with sodium dodecyl sulfate. It is probable that these four isolates represent a single widely disseminated plasmid species. The other two plasmids showing relaxation complexes have molecular weights of about 3 x 10(6) and encode chloramphenicol resistance. The complexes in these cases are unstable, and it has not been possible to induce more than 50% relaxation by any of the standard treatments. Ten other plasmids do not show detectable complexes. These include three penicillinase plasmids, four tetracycline-resistance plasmids, one plasmid carrying kanamycin-neomycin resistance, and finally, two chloramphenicol-resistance plasmids. PMID:956124

  4. Efficient Prediction of Co-Complexed Proteins Based on Coevolution

    PubMed Central

    de Vienne, Damien M.; Azé, Jérôme

    2012-01-01

    The prediction of the network of protein-protein interactions (PPI) of an organism is crucial for the understanding of biological processes and for the development of new drugs. Machine learning methods have been successfully applied to the prediction of PPI in yeast by the integration of multiple direct and indirect biological data sources. However, experimental data are not available for most organisms. We propose here an ensemble machine learning approach for the prediction of PPI that depends solely on features independent from experimental data. We developed new estimators of the coevolution between proteins and combined them in an ensemble learning procedure. We applied this method to a dataset of known co-complexed proteins in Escherichia coli and compared it to previously published methods. We show that our method allows prediction of PPI with an unprecedented precision of 95.5% for the first 200 sorted pairs of proteins compared to 28.5% on the same dataset with the previous best method. A close inspection of the best predicted pairs allowed us to detect new or recently discovered interactions between chemotactic components, the flagellar apparatus and RNA polymerase complexes in E. coli. PMID:23152796

  5. Characterization of FimH adhesins expressed by Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum: reconstitution of mannose-binding properties by single amino acid substitution.

    PubMed

    Kisiela, Dagmara; Sapeta, Anna; Kuczkowski, Maciej; Stefaniak, Tadeusz; Wieliczko, Alina; Ugorski, Maciej

    2005-09-01

    Recombinant FimH adhesins of type 1 fimbriae from Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, in contrast to those of Salmonella enterica serovar Typhimurium, did not bind to high-mannose oligosaccharides or to human colon carcinoma HT-29 cells. However, mutated FimH proteins from biovar Gallinarum and biovar Pullorum, in which the isoleucine at position 78 was replaced by the threonine found in S. enterica serovar Typhimurium, bound well to glycoproteins carrying high-mannose oligosaccharides and colon carcinoma cells. The loss of sugar-binding properties by biovar Gallinarum and biovar Pullorum FimH adhesins, which are a part of the type 1 fimbriae, is most probably the result of a single T78I mutation, as was proven by site-directed mutagenesis of FimH proteins. PMID:16113346

  6. Erythrocyte gangliosides act as receptors for Neisseria subflava: identification of the Sia-1 adhesin.

    PubMed Central

    Nyberg, G; Strömberg, N; Jonsson, A; Karlsson, K A; Normark, S

    1990-01-01

    Neisseria gonorrhoeae was recently shown to bind to a subset of lactose-containing glycolipids (N. Strömberg, C. Deal, G. Nyberg, S. Normark, M. So, and K.-A. Karlsson, Proc. Natl. Acad. Sci. USA 85:4902-4906, 1988). A number of commensal Neisseria strains were also shown to be lactose binders. In addition, Neisseria subflava bound to immobilized gangliosides, such as hematoside and sialosyl paragloboside, carrying the NeuAc alpha 2-3Gal beta 1-4Glc sequence. To a lesser extent, N. gonorrhoeae also bound to this receptor in vitro. In N. subflava GN01, this binding property mediated agglutination of human erythrocytes in a neuraminidase-sensitive fashion. Nitrosoguanidine-induced nonhemagglutinative mutants of N. subflava GN01 had lost the ability to bind hematoside and sialosylparagloboside but remained able to bind lactosylceramide and gangliotetraosylceramide. These mutants fell into three classes with respect to their outer membrane protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Class 1 mutants were identical to the parent strain save for the loss of a 27-kilodalton (kDa) protein. Class 2 mutants showed an outer membrane protein profile identical to that of the wild type, whereas mutants belonging to class 3 showed a number of changes, including the apparent absence of the 27-kDa protein. The 27-kDa protein from N. subflava GN01 was purified from the supernatant. A polyclonal antiserum to the purified Sia-1 protein as well as a Sia-1-specific monoclonal antibody inhibited hemagglutination by strain GN01. The purified Sia-1 protein in the presence of diluted anti-Sia-1 antiserum mediated a neuraminidase-sensitive hemagglutination. The purified Sia protein from a class 2 mutant was not able to hemagglutinate when cross-linked with antibodies, suggesting that it is a mutant form of Sia-1 affected in the receptor-binding site. Immunoelectron microscopy with a Sia-1-specific monoclonal antibody revealed that the adhesin was

  7. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    NASA Astrophysics Data System (ADS)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  8. The mammalian autophagy initiator complex contains 2 HORMA domain proteins

    PubMed Central

    Michel, Max; Schwarten, Melanie; Decker, Christina; Nagel-Steger, Luitgard; Willbold, Dieter; Weiergräber, Oliver H

    2015-01-01

    ATG101 is an essential component of the ULK complex responsible for initiating cellular autophagy in mammalian cells; its 3-dimensional structure and molecular function, however, are currently unclear. Here we present the X-ray structure of human ATG101. The protein displays an open HORMA domain fold. Both structural properties and biophysical evidence indicate that ATG101 is locked in this conformation, in contrast to the prototypical HORMA domain protein MAD2. Moreover, we discuss a potential mode of dimerization with ATG13 as a fundamental aspect of ATG101 function. PMID:26236954

  9. Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations.

    PubMed

    Waadt, Rainer; Schlücking, Kathrin; Schroeder, Julian I; Kudla, Jörg

    2014-01-01

    The analyses of protein-protein interactions are crucial for understanding cellular processes including signal transduction, protein trafficking, and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further, we discuss different BiFC applications and provide examples for proper BiFC analyses in planta. PMID:24057390

  10. A sialoreceptor binding motif in the Mycoplasma synoviae adhesin VlhA.

    PubMed

    May, Meghan; Dunne, Dylan W; Brown, Daniel R

    2014-01-01

    Mycoplasma synoviae depends on its adhesin VlhA to mediate cytadherence to sialylated host cell receptors. Allelic variants of VlhA arise through recombination between an assemblage of promoterless vlhA pseudogenes and a single transcription promoter site, creating lineages of M. synoviae that each express a different vlhA allele. The predicted full-length VlhA sequences adjacent to the promoter of nine lineages of M. synoviae varying in avidity of cytadherence were aligned with that of the reference strain MS53 and with a 60-a.a. hemagglutinating VlhA C-terminal fragment from a Tunisian lineage of strain WVU1853(T). Seven different sequence variants of an imperfectly conserved, single-copy, 12-a.a. candidate cytadherence motif were evident amid the flanking variable residues of the 11 total sequences examined. The motif was predicted to adopt a short hairpin structure in a low-complexity region near the C-terminus of VlhA. Biotinylated synthetic oligopeptides representing four selected variants of the 12-a.a. motif, with the whole synthesized 60-a.a. fragment as a positive control, differed (P<0.01) in the extent they bound to chicken erythrocyte membranes. All bound to a greater extent (P<0.01) than scrambled or irrelevant VlhA domain negative control peptides did. Experimentally introduced branched-chain amino acid (BCAA) substitutions Val3Ile and Leu7Ile did not significantly alter binding, whereas fold-destabilizing substitutions Thr4Gly and Ala9Gly tended to reduce it (P<0.05). Binding was also reduced to background levels (P<0.01) when the peptides were exposed to desialylated membranes, or were pre-saturated with free sialic acid before exposure to untreated membranes. From this evidence we conclude that the motif P-X-(BCAA)-X-F-X-(BCAA)-X-A-K-X-G binds sialic acid and likely mediates VlhA-dependent M. synoviae attachment to host cells. This conserved mechanism retains the potential for fine-scale rheostasis in binding avidity, which could be a general

  11. A Sialoreceptor Binding Motif in the Mycoplasma synoviae Adhesin VlhA

    PubMed Central

    May, Meghan; Dunne, Dylan W.; Brown, Daniel R.

    2014-01-01

    Mycoplasma synoviae depends on its adhesin VlhA to mediate cytadherence to sialylated host cell receptors. Allelic variants of VlhA arise through recombination between an assemblage of promoterless vlhA pseudogenes and a single transcription promoter site, creating lineages of M. synoviae that each express a different vlhA allele. The predicted full-length VlhA sequences adjacent to the promoter of nine lineages of M. synoviae varying in avidity of cytadherence were aligned with that of the reference strain MS53 and with a 60-a.a. hemagglutinating VlhA C-terminal fragment from a Tunisian lineage of strain WVU1853T. Seven different sequence variants of an imperfectly conserved, single-copy, 12-a.a. candidate cytadherence motif were evident amid the flanking variable residues of the 11 total sequences examined. The motif was predicted to adopt a short hairpin structure in a low-complexity region near the C-terminus of VlhA. Biotinylated synthetic oligopeptides representing four selected variants of the 12-a.a. motif, with the whole synthesized 60-a.a. fragment as a positive control, differed (P<0.01) in the extent they bound to chicken erythrocyte membranes. All bound to a greater extent (P<0.01) than scrambled or irrelevant VlhA domain negative control peptides did. Experimentally introduced branched-chain amino acid (BCAA) substitutions Val3Ile and Leu7Ile did not significantly alter binding, whereas fold-destabilizing substitutions Thr4Gly and Ala9Gly tended to reduce it (P<0.05). Binding was also reduced to background levels (P<0.01) when the peptides were exposed to desialylated membranes, or were pre-saturated with free sialic acid before exposure to untreated membranes. From this evidence we conclude that the motif P-X-(BCAA)-X-F-X-(BCAA)-X-A-K-X-G binds sialic acid and likely mediates VlhA-dependent M. synoviae attachment to host cells. This conserved mechanism retains the potential for fine-scale rheostasis in binding avidity, which could be a general

  12. Comparison of Surface Proteomes of Adherence Variants of Listeria Monocytogenes Using LC-MS/MS for Identification of Potential Surface Adhesins

    PubMed Central

    Tiong, Hung King; Hartson, Steven D.; Muriana, Peter M.

    2016-01-01

    The ability of Listeria monocytogenes to adhere and form biofilms leads to persistence in food processing plants and food-associated listeriosis. The role of specific surface proteins as adhesins to attach Listeria cells to various contact surfaces has not been well characterized to date. In prior research comparing different methods for surface protein extraction, the Ghost urea method revealed cleaner protein content as verified by the least cytoplasmic protein detected in surface extracts using LC-MS/MS. The same technique was utilized to extract and detect surface proteins among two surface-adherent phenotypic strains of L. monocytogenes (i.e., strongly and weakly adherent). Of 640 total proteins detected among planktonic and sessile cells, 21 protein members were exclusively detected in the sessile cells. Relative LC-MS/MS detection and quantification of surface-extracted proteins from the planktonic weakly adherent (CW35) and strongly adherent strains (99-38) were examined by protein mass normalization of proteins. We found that L. monocytogenes 99-38 exhibited a total of 22 surface proteins that were over-expressed: 11 proteins were detected in surface extracts of both sessile and planktonic 99-38 that were ≥5-fold over-expressed while another 11 proteins were detected only in planktonic 99-38 cells that were ≥10-fold over-expressed. Our results suggest that these protein members are worthy of further investigation for their involvement as surface adhesins. PMID:27196934

  13. Comparison of Surface Proteomes of Adherence Variants of Listeria Monocytogenes Using LC-MS/MS for Identification of Potential Surface Adhesins.

    PubMed

    Tiong, Hung King; Hartson, Steven D; Muriana, Peter M

    2016-01-01

    The ability of Listeria monocytogenes to adhere and form biofilms leads to persistence in food processing plants and food-associated listeriosis. The role of specific surface proteins as adhesins to attach Listeria cells to various contact surfaces has not been well characterized to date. In prior research comparing different methods for surface protein extraction, the Ghost urea method revealed cleaner protein content as verified by the least cytoplasmic protein detected in surface extracts using LC-MS/MS. The same technique was utilized to extract and detect surface proteins among two surface-adherent phenotypic strains of L. monocytogenes (i.e., strongly and weakly adherent). Of 640 total proteins detected among planktonic and sessile cells, 21 protein members were exclusively detected in the sessile cells. Relative LC-MS/MS detection and quantification of surface-extracted proteins from the planktonic weakly adherent (CW35) and strongly adherent strains (99-38) were examined by protein mass normalization of proteins. We found that L. monocytogenes 99-38 exhibited a total of 22 surface proteins that were over-expressed: 11 proteins were detected in surface extracts of both sessile and planktonic 99-38 that were ≥5-fold over-expressed while another 11 proteins were detected only in planktonic 99-38 cells that were ≥10-fold over-expressed. Our results suggest that these protein members are worthy of further investigation for their involvement as surface adhesins. PMID:27196934

  14. Characterization of known protein complexes using k-connectivity and other topological measures

    PubMed Central

    Gallagher, Suzanne R; Goldberg, Debra S

    2015-01-01

    Many protein complexes are densely packed, so proteins within complexes often interact with several other proteins in the complex. Steric constraints prevent most proteins from simultaneously binding more than a handful of other proteins, regardless of the number of proteins in the complex. Because of this, as complex size increases, several measures of the complex decrease within protein-protein interaction networks. However, k-connectivity, the number of vertices or edges that need to be removed in order to disconnect a graph, may be consistently high for protein complexes. The property of k-connectivity has been little used previously in the investigation of protein-protein interactions. To understand the discriminative power of k-connectivity and other topological measures for identifying unknown protein complexes, we characterized these properties in known Saccharomyces cerevisiae protein complexes in networks generated both from highly accurate X-ray crystallography experiments which give an accurate model of each complex, and also as the complexes appear in high-throughput yeast 2-hybrid studies in which new complexes may be discovered. We also computed these properties for appropriate random subgraphs.We found that clustering coefficient, mutual clustering coefficient, and k-connectivity are better indicators of known protein complexes than edge density, degree, or betweenness. This suggests new directions for future protein complex-finding algorithms. PMID:26913183

  15. A profile of protein-protein interaction: Crystal structure of a lectin-lectin complex.

    PubMed

    Surya, Sukumaran; Abhilash, Joseph; Geethanandan, Krishnan; Sadasivan, Chittalakkottu; Haridas, Madhathilkovilakathu

    2016-06-01

    Proteins may utilize complex networks of interactions to create/proceed signaling pathways of highly adaptive responses such as programmed cell death. Direct binary interactions study of proteins may help propose models for protein-protein interaction. Towards this goal we applied a combination of thermodynamic kinetics and crystal structure analyses to elucidate the complexity and diversity in such interactions. By determining the heat change on the association of two galactose-specific legume lectins from Butea monosperma (BML) and Spatholobus parviflorus (SPL) belonging to Fabaceae family helped to compute the binding equilibrium. It was extended further by X-ray structural analysis of BML-SPL binary complex. In order to chart the proteins interacting mainly through their interfaces, identification of the nature of forces which stabilized the association of the lectin-lectin complex was examined. Comprehensive analysis of the BMLSPL complex by isothermal titration calorimetry and X-ray crystal structure threw new light on the lectin-lectin interactions suggesting of their use in diverse areas of glycobiology. PMID:26945504

  16. Analysis of secondary structural and physicochemical changes in protein-protein complexes.

    PubMed

    Saranya, N; Saravanan, K M; Michael Gromiha, M; Selvaraj, S

    2016-03-01

    Conformation switching in protein-protein complexes is considered important for the molecular recognition process. Overall analysis of 123 protein-protein complexes in a benchmark data-set showed that 6.8% of residues switched over their secondary structure conformation upon complex formation. Amino acid residue-wise preference for conformation change has been analyzed in binding and non-binding site residues separately. In this analysis, residues such as Ser, Leu, Glu, and Lys had higher frequency of secondary structural conformation change. The change of helix to coil and sheet to coil conformation and vice versa has been observed frequently, whereas the conformation change of helix to extended sheet occurred rarely in the studied complexes. Influence of conformation change toward the N and C terminal on either side of the binding site residues has been analyzed. Further, analysis on φ and ψ angle variation, conservation, stability, and solvent accessibility have been performed on binding site residues. Knowledge obtained from the present study could be effectively employed in the protein-protein modeling and docking studies. PMID:25990569

  17. Complex architecture of major histocompatibility complex class II promoters: reiterated motifs and conserved protein-protein interactions.

    PubMed Central

    Jabrane-Ferrat, N; Fontes, J D; Boss, J M; Peterlin, B M

    1996-01-01

    The S box (also known as at the H, W, or Z box) is the 5'-most element of the conserved upstream sequences in promoters of major histocompatibility complex class II genes. It is important for their B-cell-specific and interferon gamma-inducible expression. In this study, we demonstrate that the S box represents a duplication of the downstream X box. First, RFX, which is composed of the RFX5-p36 heterodimer that binds to the X box, also binds to the S box and its 5'-flanking sequence. Second, NF-Y, which binds to the Y box and increases interactions between RFX and the X box, also increases the binding of RFX to the S box. Third, RFXs bound to S and X boxes interact with each other in a spatially constrained manner. Finally, we confirmed these protein-protein and protein-DNA interactions by expressing a hybrid RFX5-VP16 protein in cells. We conclude that RFX binds to S and X boxes and that complex interactions between RFX and NF-Y direct B-cell-specific and interferon gamma-inducible expression or major histocompatibility complex class II genes. PMID:8756625

  18. Size dependent complexity of sequences in protein families

    NASA Astrophysics Data System (ADS)

    Li, J.; Wang, J.; Wang, W.

    2005-10-01

    The size dependent complexity of protein sequences in various families in the FSSP database is characterized by sequence entropy, sequence similarity and sequence identity. As the average length Lf of sequences in the family increases, an increasing trend of the sequence entropy and a decreasing trend of the sequence similarity and sequence identity are found. As Lf increases beyond 250, a saturation of the sequence entropy, the sequence similarity and the sequence identity is observed. Such a saturated behavior of complexity is attributed to the saturation of the probability Pg of global (long-range) interactions in protein structures when Lf >250. It is also found that the alphabet size of residue types describing the sequence diversity depends on the value of Lf, and becomes saturated at 12.

  19. Immersion freezing of ice nucleation active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Wex, H.; Šantl-Temkiv, T.; Voigtländer, J.; Niedermeier, D.; Stratmann, F.

    2013-06-01

    Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between -5 °C to -38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about -6 °C to about -10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached

  20. Protein-protein complex structure predictions by multimeric threading and template recombination

    PubMed Central

    Mukherjee, Srayanta; Zhang, Yang

    2011-01-01

    Summary The number of protein-protein complex structures is nearly 6-times smaller than that of tertiary structures in PDB which limits the power of homology-based approaches to complex structure modeling. We present a new threading-recombination approach, COTH, to boost the protein complex structure library by combining tertiary structure templates with complex alignments. The query sequences are first aligned to complex templates using a modified dynamic programming algorithm, guided by ab initio binding-site predictions. The monomer alignments are then shifted to the multimeric template framework by structural alignments. COTH was tested on 500 non-homologous dimeric proteins, which can successfully detect correct templates for half of the cases after homologous templates are excluded, which significantly outperforms conventional homology modeling algorithms. It also shows a higher accuracy in interface modeling than rigid-body docking of unbound structures from ZDOCK although with lower coverage. These data demonstrate new avenues to model complex structures from non-homologous templates. PMID:21742262

  1. Supercharging Protein Complexes from Aqueous Solution Disrupts their Native Conformations

    NASA Astrophysics Data System (ADS)

    Sterling, Harry J.; Kintzer, Alexander F.; Feld, Geoffrey K.; Cassou, Catherine A.; Krantz, Bryan A.; Williams, Evan R.

    2012-02-01

    The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol ( m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared with the "native" complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a prechannel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of Coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets, where enrichment of the supercharging reagent during droplet evaporation occurs.

  2. How to Build a Complex, Functional Propeller Protein, From Parts.

    PubMed

    Clark, Patricia L

    2016-04-01

    By combining ancestral sequence reconstruction and in vitro evolution, Smock et al. identified single motifs that assemble into a functional five-bladed β-propeller, and a likely route for conversion into the more complex, extant single chain fusion. Interestingly, although sequence diversification destabilized five-motif fusions, it also destabilized aggregation-prone intermediates, increasing the level of functional protein in vivo. PMID:26971075

  3. Rational stabilization of complex proteins: a divide and combine approach

    PubMed Central

    Lamazares, Emilio; Clemente, Isabel; Bueno, Marta; Velázquez-Campoy, Adrián; Sancho, Javier

    2015-01-01

    Increasing the thermostability of proteins is often crucial for their successful use as analytic, synthetic or therapeutic tools. Most rational thermostabilization strategies were developed on small two-state proteins and, unsurprisingly, they tend to fail when applied to the much more abundant, larger, non-fully cooperative proteins. We show that the key to stabilize the latter is to know the regions of lower stability. To prove it, we have engineered apoflavodoxin, a non-fully cooperative protein on which previous thermostabilizing attempts had failed. We use a step-wise combination of structure-based, rationally-designed, stabilizing mutations confined to the less stable structural region, and obtain variants that, according to their van't Hoff to calorimetric enthalpy ratios, exhibit fully-cooperative thermal unfolding with a melting temperature of 75°C, 32 degrees above the lower melting temperature of the non-cooperative wild type protein. The ideas introduced here may also be useful for the thermostabilization of complex proteins through formulation or using specific stabilizing ligands (e.g. pharmacological chaperones). PMID:25774740

  4. Development of a Split SNAP-CLIP Double Labeling System for Tracking Proteins Following Dissociation from Protein-Protein Complexes in Living Cells.

    PubMed

    Mie, Masayasu; Naoki, Tatsuhiko; Kobatake, Eiry

    2016-08-16

    The split SNAP-tag protein-fragment complementation assay (PCA) is a useful tool for imaging protein-protein interactions (PPIs) in living cells. In contrast to conventional methods employed for imaging PPIs, the split SNAP-tag PCA enables tracking of proteins following dissociation from protein-protein complexes. A limitation of this system, however, is that it only allows for labeling and tracking of one of the proteins forming the protein-protein complex. To track both proteins forming a protein-protein complex, each protein needs to be appropriately labeled. In this study, a split SNAP-CLIP double labeling system is developed and applied for tracking of each protein forming a protein-protein complex. As a proof-of concept, FM protein for PPIs and protein kinase C alpha (PKCα) for translocation are introduced to a split SNAP-CLIP double labeling system. The results show a split SNAP-CLIP double labeling system enables labeling of both proteins in a protein-protein complex and subsequent tracking of each of the proteins following dissociation from the protein-protein complexes in living cells. PMID:27448142

  5. Transformation of a fragment of beta-structural bacteriophage T4 adhesin to stable alpha-helical trimer.

    PubMed

    Miroshnikov, K A; Sernova, N V; Shneider, M M; Mesyanzhinov, V V

    2000-12-01

    Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells. Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a beta-helix, is supposed to be a major structural feature of this protein. We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively. When expressed in E. coli cells, these gp12 fragments formed labile beta-structural trimers. Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant alpha-helical structure. This structure is probably similar to that of fibritin, which has a triple alpha-helical coiled-coil structure. Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization. PMID:11173503

  6. A secretory kinase complex regulates extracellular protein phosphorylation

    PubMed Central

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001 PMID:25789606

  7. Small-Angle X-Ray Scattering From RNA, Proteins, And Protein Complexes

    SciTech Connect

    Lipfert, Jan; Doniach, Sebastian; /Stanford U., Phys. Dept. /Stanford U., Appl. Phys. Dept. /SLAC, SSRL

    2007-09-18

    Small-angle X-ray scattering (SAXS) is increasingly used to characterize the structure and interactions of biological macromolecules and their complexes in solution. Although still a low-resolution technique, the advent of high-flux synchrotron sources and the development of algorithms for the reconstruction of 3-D electron density maps from 1-D scattering profiles have made possible the generation of useful low-resolution molecular models from SAXS data. Furthermore, SAXS is well suited for the study of unfolded or partially folded conformational ensembles as a function of time or solution conditions. Here, we review recently developed algorithms for 3-D structure modeling and applications to protein complexes. Furthermore, we discuss the emerging use of SAXS as a tool to study membrane protein-detergent complexes. SAXS is proving useful to study the folding of functional RNA molecules, and finally we discuss uses of SAXS to study ensembles of denatured proteins.

  8. Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

    PubMed Central

    Han, Yiping W.; Ikegami, Akihiko; Rajanna, Chythanya; Kawsar, Hameem I.; Zhou, Yun; Li, Mei; Sojar, Hakimuddin T.; Genco, Robert J.; Kuramitsu, Howard K.; Deng, Cheri X.

    2005-01-01

    Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity. PMID:16030227

  9. Cloning of a Streptococcus sanguis adhesin which mediates binding to saliva-coated hydroxyapatite.

    PubMed Central

    Ganeshkumar, N; Song, M; McBride, B C

    1988-01-01

    Chromosomal DNA from a salivary aggregating strain of Streptococcus sanguis 12 was partially digested with PstI and ligated into the plasmid vector pUC18 and transformed into Escherichia coli JM83. A total of 1,700 recombinant clones of E. coli were examined by a colony immunoassay with antisera raised against either S. sanguis 12 whole cells or S. sanguis 12 surface fibrils. Five clones which reacted with one or the other antiserum were shown to be unique by Western blotting (immunoblotting) and restriction endonuclease digestion. One recombinant plasmid pSA2 expressed two proteins with Mrs of 20,000 and 36,000. The 36,000-Mr protein has been designated SsaB. Both proteins were purified to homogeneity by Sephadex G-75 and ion-exchange chromatography. The proteins were present in mutanolysin digests of whole-cell lysates of S. sanguis 12 and in the non-saliva-aggregating variant 12na and the hydrophilic variant 12L. Polyclonal antiserum raised against the SsaB protein reacted strongly with the cell surfaces of S. sanguis 12 and 12na but not with that of 12L. SsaB inhibited the adhesion of S. sanguis 12na to saliva-coated hydroxyapatite, indicating that the adhesin mediates the binding to the pH-sensitive receptor. Images PMID:3356463

  10. The Streptococcus pneumoniae adhesin PsrP binds to Keratin 10 on lung cells

    PubMed Central

    Shivshankar, Pooja; Sanchez, Carlos; Rose, Lloyd F.; Orihuela, Carlos J.

    2009-01-01

    Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island encoded adhesin that mediates attachment to lung cells. It is a member of the Serine-rich repeat protein (SRRP) family and the largest bacterial protein known. PsrP production by S. pneumoniae was confirmed by immunoblotting and a truncated version of the protein was determined to be glycosylated. Using isogenic psrP mutants complemented with various PsrP constructs and competitive inhibition assays with recombinant proteins, we determined that PsrP requires an extended SRR2 domain for function and that adhesion is mediated through amino acids 273-341 of its Basic Region (BR) domain. Affinity chromatography, immunoprecipitation, ELISA, FACS, and immunofluorescent co-localization studies determined that PsrP binds to Keratin 10 (K10) on the surface of lung but not nasopharyngeal epithelial cells. Unglycosylated K10 bound to wild type but not psrP deficient pneumococci; suggesting that unlike other SRRPs, PsrP-mediated adhesion was independent of lectin activity. Finally, mice immunized with recombinant (r)PsrPBR had significantly less bacteria in their blood and improved survival versus controls following intranasal challenge. We conclude that the BR domain of PsrP binds to K10 in a lectin-independent manner; that K10 is expressed on lung cells; and that vaccination with rPsrPBR is protective against pneumococcal disease. PMID:19627498

  11. Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin.

    PubMed Central

    Paruchuri, D K; Seifert, H S; Ajioka, R S; Karlsson, K A; So, M

    1990-01-01

    We recently identified a set of mammalian cell receptors for Neisseria gonorrhoeae that are glycolipids. These receptors, lactosylceramide [Gal(beta 1-4)Glc(beta 1-1)Cer], gangliotriosylceramide [GalNAc( beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], and gangliotetraosylceramide [Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], were shown to be specifically bound by a gonococcal outer membrane protein distinct from pilin and protein II. Here we report the isolation of the gene encoding the gangliotetraosylceramide-binding adhesin from a N. gonorrhoeae MS11 gene bank in Escherichia coli. Transposon mutagenesis studies in E. coli indicate that the adhesion is a protein with a molecular mass of 36,000 Da. The gene encoding the 36-kDa protein is duplicated in MS11 since two transposon insertions were required to abolish expression of the gene in this bacterium. This protein is present on the surface of the gonococcus and is not associated with the pilus. Images PMID:2153292

  12. Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin.

    PubMed

    Paruchuri, D K; Seifert, H S; Ajioka, R S; Karlsson, K A; So, M

    1990-01-01

    We recently identified a set of mammalian cell receptors for Neisseria gonorrhoeae that are glycolipids. These receptors, lactosylceramide [Gal(beta 1-4)Glc(beta 1-1)Cer], gangliotriosylceramide [GalNAc( beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], and gangliotetraosylceramide [Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], were shown to be specifically bound by a gonococcal outer membrane protein distinct from pilin and protein II. Here we report the isolation of the gene encoding the gangliotetraosylceramide-binding adhesin from a N. gonorrhoeae MS11 gene bank in Escherichia coli. Transposon mutagenesis studies in E. coli indicate that the adhesion is a protein with a molecular mass of 36,000 Da. The gene encoding the 36-kDa protein is duplicated in MS11 since two transposon insertions were required to abolish expression of the gene in this bacterium. This protein is present on the surface of the gonococcus and is not associated with the pilus. PMID:2153292

  13. Radioprotection by polyethylene glycol-protein complexes in mice

    SciTech Connect

    Gray, B.H.; Stull, R.W.

    1983-03-01

    Polyethylene glycol of about 5000 D was activated with cyanuric chloride, and the activated compound was complexed to each of three proteins. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase were each radioprotectants when administered prophylactically to female B6CBF1 mice before irradiation. The dose reduction factor for these mice was 1.2 when 5000 units of polyethylene glycol-catalase was administered before /sup 60/Co irradiation. Female B6CBF1 mice administered prophylactic intravenous injections of catalase, polyethylene glycol-albumin, or heat-denatured polyethylene glycol-catalase had survival rates similar to phosphate-buffered saline-injected control mice following /sup 60/Co irradiation. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase have radioprotective activity in B6CBF1 mice, which appears to depend in part on enzymatic activities of the complex. However, no radioprotective effect was observed in male C57BL/6 mice injected with each polyethylene glycol-protein complex at either 3 or 24 hr before irradiation. The mechanism for radioprotection by these complexes may depend in part on other factors.

  14. Taking advantage of local structure descriptors to analyze interresidue contacts in protein structures and protein complexes.

    PubMed

    Martin, Juliette; Regad, Leslie; Etchebest, Catherine; Camproux, Anne-Claude

    2008-11-15

    Interresidue protein contacts in proteins structures and at protein-protein interface are classically described by the amino acid types of interacting residues and the local structural context of the contact, if any, is described using secondary structures. In this study, we present an alternate analysis of interresidue contact using local structures defined by the structural alphabet introduced by Camproux et al. This structural alphabet allows to describe a 3D structure as a sequence of prototype fragments called structural letters, of 27 different types. Each residue can then be assigned to a particular local structure, even in loop regions. The analysis of interresidue contacts within protein structures defined using Voronoï tessellations reveals that pairwise contact specificity is greater in terms of structural letters than amino acids. Using a simple heuristic based on specificity score comparison, we find that 74% of the long-range contacts within protein structures are better described using structural letters than amino acid types. The investigation is extended to a set of protein-protein complexes, showing that the similar global rules apply as for intraprotein contacts, with 64% of the interprotein contacts best described by local structures. We then present an evaluation of pairing functions integrating structural letters to decoy scoring and show that some complexes could benefit from the use of structural letter-based pairing functions. PMID:18491388

  15. pyDockSAXS: protein-protein complex structure by SAXS and computational docking.

    PubMed

    Jiménez-García, Brian; Pons, Carles; Svergun, Dmitri I; Bernadó, Pau; Fernández-Recio, Juan

    2015-07-01

    Structural characterization of protein-protein interactions at molecular level is essential to understand biological processes and identify new therapeutic opportunities. However, atomic resolution structural techniques cannot keep pace with current advances in interactomics. Low-resolution structural techniques, such as small-angle X-ray scattering (SAXS), can be applied at larger scale, but they miss atomic details. For efficient application to protein-protein complexes, low-resolution information can be combined with theoretical methods that provide energetic description and atomic details of the interactions. Here we present the pyDockSAXS web server (http://life.bsc.es/pid/pydocksaxs) that provides an automatic pipeline for modeling the structure of a protein-protein complex from SAXS data. The method uses FTDOCK to generate rigid-body docking models that are subsequently evaluated by a combination of pyDock energy-based scoring function and their capacity to describe SAXS data. The only required input files are structural models for the interacting partners and a SAXS curve. The server automatically provides a series of structural models for the complex, sorted by the pyDockSAXS scoring function. The user can also upload a previously computed set of docking poses, which opens the possibility to filter the docking solutions by potential interface residues or symmetry restraints. The server is freely available to all users without restriction. PMID:25897115

  16. Retinal Cone Photoreceptors Require Phosducin-Like Protein 1 for G Protein Complex Assembly and Signaling

    PubMed Central

    Tracy, Christopher M.; Kolesnikov, Alexander V.; Blake, Devon R.; Chen, Ching-Kang; Baehr, Wolfgang; Kefalov, Vladimir J.; Willardson, Barry M.

    2015-01-01

    G protein β subunits (Gβ) play essential roles in phototransduction as part of G protein βγ (Gβγ) and regulator of G protein signaling 9 (RGS9)-Gβ5 heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (Gt2) and RGS9-Gβ5 subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gβ complex formation in G protein signaling. PMID:25659125

  17. Encounter complexes and dimensionality reduction in protein–protein association

    PubMed Central

    Kozakov, Dima; Li, Keyong; Hall, David R; Beglov, Dmitri; Zheng, Jiefu; Vakili, Pirooz; Schueler-Furman, Ora; Paschalidis, Ioannis Ch; Clore, G Marius; Vajda, Sandor

    2014-01-01

    An outstanding challenge has been to understand the mechanism whereby proteins associate. We report here the results of exhaustively sampling the conformational space in protein–protein association using a physics-based energy function. The agreement between experimental intermolecular paramagnetic relaxation enhancement (PRE) data and the PRE profiles calculated from the docked structures shows that the method captures both specific and non-specific encounter complexes. To explore the energy landscape in the vicinity of the native structure, the nonlinear manifold describing the relative orientation of two solid bodies is projected onto a Euclidean space in which the shape of low energy regions is studied by principal component analysis. Results show that the energy surface is canyon-like, with a smooth funnel within a two dimensional subspace capturing over 75% of the total motion. Thus, proteins tend to associate along preferred pathways, similar to sliding of a protein along DNA in the process of protein-DNA recognition. DOI: http://dx.doi.org/10.7554/eLife.01370.001 PMID:24714491

  18. Biodegradation of the chitin-protein complex in crustacean cuticle

    USGS Publications Warehouse

    Artur, Stankiewicz B.; Mastalerz, Maria; Hof, C.H.J.; Bierstedt, A.; Flannery, M.B.; Briggs, D.E.G.; Evershed, R.P.

    1998-01-01

    Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative analysis of amino acids (by HPLC) and chitin showed that the major loss of proteins and chitin occurred between weeks 1 and 2. After 8 weeks tyrosine, tryptophan and valine are the most prominent amino acid moieties, showing their resistance to degradation. The presence of cyclic ketones in the pyrolysates indicates that mucopolysaccharides or other bound non-chitinous carbohydrates are also resistant to decay. There is no evidence of structural degradation of chitin prior to 8 weeks when FTIR revealed a reduction in chitin-specific bands. The chemical changes are paralleled by structural changes in the cuticle, which becomes an increasingly open structure consisting of loose chitinous fibres. The rapid rate of decay in the experiments suggests that where chitin and protein are preserved in fossil cuticles degradation must have been inhibited.Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative

  19. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    SciTech Connect

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S. E-mail: mkukrishna@rediffmail.com

    2005-08-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V{sub M}) of 3.3 Å{sup 3} Da{sup −1}, corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

  20. Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

    PubMed

    Paquet, Françoise; Delalande, Olivier; Goffinont, Stephane; Culard, Françoise; Loth, Karine; Asseline, Ulysse; Castaing, Bertrand; Landon, Celine

    2014-01-01

    In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA. PMID:24558431

  1. Electrostatic Rate Enhancement and Transient Complex of Protein-Protein Association

    PubMed Central

    Alsallaq, Ramzi; Zhou, Huan-Xiang

    2012-01-01

    The association of two proteins is bounded by the rate at which they, via diffusion, find each other while in appropriate relative orientations. Orientational constraints restrict this rate to ~105 – 106 M−1s−1. Proteins with higher association rates generally have complementary electrostatic surfaces; proteins with lower association rates generally are slowed down by conformational changes upon complex formation. Previous studies (Zhou, Biophys. J. 1997;73:2441–2445) have shown that electrostatic enhancement of the diffusion-limited association rate can be accurately modeled by kD = kD0 exp(−*/ kBT), where kD and kD0 are the rates in the presence and absence of electrostatic interactions, respectively, * is the average electrostatic interaction energy in a “transient-complex” ensemble, and kBT is thermal energy. The transient-complex ensemble separates the bound state from the unbound state. Predictions of the transient-complex theory on four protein complexes were found to agree well with experiment when the electrostatic interaction energy was calculated with the linearized Poisson-Boltzmann (PB) equation (Alsallaq and Zhou, Structure 2007, 15:215–224). Here we show that the agreement is further improved when the nonlinear PB equation is used. These predictions are obtained with the dielectric boundary defined as the protein van der Waals surface. When the dielectric boundary is instead specified as the molecular surface, electrostatic interactions in the transient complex become repulsive and are thus predicted to retard association. Together these results demonstrate that the transient-complex theory is predictive of electrostatic rate enhancement and can help parameterize PB calculations. PMID:17932929

  2. Contractile vacuole complex--its expanding protein inventory.

    PubMed

    Plattner, Helmut

    2013-01-01

    The contractile vacuole complex (CVC) of some protists serves for the osmotic equilibration of water and ions, notably Ca(2+), by chemiosmotic exploitation of a H(+) gradient generated by the organelle-resident V-type H(+)-ATPase. Ca(2+) is mostly extruded, but there is also some reflux into the cytosol via Ca(2+)-release channels. Most data available are from Dictyostelium and Paramecium. In Paramecium, the major parts of CVC contain several v-/R-SNARE (synaptobrevins) and t-/Q-SNARE (syntaxins) proteins. This is complemented by Rab-type GTPases (shown in Tetrahymena) and exocyst components (Chlamydomonas). All this reflects a multitude of membrane interactions and fusion processes. Ca(2+)/H(+) and other exchangers are to be postulated, as are aquaporins and mechanosensitive Ca(2+) channels. From the complexity of the organelle, many more proteins may be expected. For instance, the pore is endowed with its own set of proteins. We may now envisage the regulation of membrane dynamics (reversible tubulation) and the epigenetic control of organelle shape, size and positioning. New aspects about organelle function and biogenesis are sketched in Section 7. The manifold regulators currently known from CVC suggest the cooperation of widely different mechanisms to maintain its dynamic function and to drive its biogenesis. PMID:24016530

  3. Assembly and solution structure of the core retromer protein complex.

    PubMed

    Norwood, Suzanne J; Shaw, Daniel J; Cowieson, Nathan P; Owen, David J; Teasdale, Rohan D; Collins, Brett M

    2011-01-01

    Retromer is a peripheral membrane protein complex that has pleiotropic roles in endosomal membrane trafficking. The core of retromer possesses three subunits, VPS35, VPS29 and VPS26, that play different roles in binding to cargo, regulatory proteins and complex stabilization. We have performed an investigation of the thermodynamics of core retromer assembly using isothermal titration calorimetry (ITC) demonstrating that VPS35 acts as the central subunit to which VPS29 and VPS26 bind independently. Furthermore, we confirm that the conserved PRLYL motif of the large VPS35 subunit is critical for direct VPS26 interaction. Heat capacity measurements of VPS29 and VPS26 binding to VPS35 indicate extensive binding interfaces and suggest conformational alterations in VPS29 or VPS35 upon complex formation. Solution studies of the retromer core using small-angle X-ray scattering allow us to propose a model whereby VPS35 forms an extended platform with VPS29 and VPS26 bound at distal ends, with the potential for forming dimeric assemblies. PMID:20875039

  4. Inferring drug-disease associations based on known protein complexes

    PubMed Central

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html. PMID:26044949

  5. Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

    PubMed

    Sudhir, Putty-Reddy; Chen, Chung-Hsuan

    2016-01-01

    A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

  6. Modeling and fitting protein-protein complexes to predict change of binding energy

    PubMed Central

    Dourado, Daniel F.A.R.; Flores, Samuel Coulbourn

    2016-01-01

    It is possible to accurately and economically predict change in protein-protein interaction energy upon mutation (ΔΔG), when a high-resolution structure of the complex is available. This is of growing usefulness for design of high-affinity or otherwise modified binding proteins for therapeutic, diagnostic, industrial, and basic science applications. Recently the field has begun to pursue ΔΔG prediction for homology modeled complexes, but so far this has worked mostly for cases of high sequence identity. If the interacting proteins have been crystallized in free (uncomplexed) form, in a majority of cases it is possible to find a structurally similar complex which can be used as the basis for template-based modeling. We describe how to use MMB to create such models, and then use them to predict ΔΔG, using a dataset consisting of free target structures, co-crystallized template complexes with sequence identify with respect to the targets as low as 44%, and experimental ΔΔG measurements. We obtain similar results by fitting to a low-resolution Cryo-EM density map. Results suggest that other structural constraints may lead to a similar outcome, making the method even more broadly applicable. PMID:27173910

  7. Analysis of the interface variability in NMR structure ensembles of protein-protein complexes.

    PubMed

    Calvanese, Luisa; D'Auria, Gabriella; Vangone, Anna; Falcigno, Lucia; Oliva, Romina

    2016-06-01

    NMR structures consist in ensembles of conformers, all satisfying the experimental restraints, which exhibit a certain degree of structural variability. We analyzed here the interface in NMR ensembles of protein-protein heterodimeric complexes and found it to span a wide range of different conservations. The different exhibited conservations do not simply correlate with the size of the systems/interfaces, and are most probably the result of an interplay between different factors, including the quality of experimental data and the intrinsic complex flexibility. In any case, this information is not to be missed when NMR structures of protein-protein complexes are analyzed; especially considering that, as we also show here, the first NMR conformer is usually not the one which best reflects the overall interface. To quantify the interface conservation and to analyze it, we used an approach originally conceived for the analysis and ranking of ensembles of docking models, which has now been extended to directly deal with NMR ensembles. We propose this approach, based on the conservation of the inter-residue contacts at the interface, both for the analysis of the interface in whole ensembles of NMR complexes and for the possible selection of a single conformer as the best representative of the overall interface. In order to make the analyses automatic and fast, we made the protocol available as a web tool at: https://www.molnac.unisa.it/BioTools/consrank/consrank-nmr.html. PMID:26968364

  8. Protein complexes and functional modules in molecular networks

    NASA Astrophysics Data System (ADS)

    Spirin, Victor; Mirny, Leonid A.

    2003-10-01

    Proteins, nucleic acids, and small molecules form a dense network of molecular interactions in a cell. Molecules are nodes of this network, and the interactions between them are edges. The architecture of molecular networks can reveal important principles of cellular organization and function, similarly to the way that protein structure tells us about the function and organization of a protein. Computational analysis of molecular networks has been primarily concerned with node degree [Wagner, A. & Fell, D. A. (2001) Proc. R. Soc. London Ser. B 268, 1803-1810; Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabasi, A. L. (2000) Nature 407, 651-654] or degree correlation [Maslov, S. & Sneppen, K. (2002) Science 296, 910-913], and hence focused on single/two-body properties of these networks. Here, by analyzing the multibody structure of the network of protein-protein interactions, we discovered molecular modules that are densely connected within themselves but sparsely connected with the rest of the network. Comparison with experimental data and functional annotation of genes showed two types of modules: (i) protein complexes (splicing machinery, transcription factors, etc.) and (ii) dynamic functional units (signaling cascades, cell-cycle regulation, etc.). Discovered modules are highly statistically significant, as is evident from comparison with random graphs, and are robust to noise in the data. Our results provide strong support for the network modularity principle introduced by Hartwell et al. [Hartwell, L. H., Hopfield, J. J., Leibler, S. & Murray, A. W. (1999) Nature 402, C47-C52], suggesting that found modules constitute the "building blocks" of molecular networks.

  9. Identification of short peptide sequences in complex milk protein hydrolysates.

    PubMed

    O'Keeffe, Martina B; FitzGerald, Richard J

    2015-10-01

    Numerous low molecular mass bioactive peptides (BAPs) can be generated during the hydrolysis of bovine milk proteins. Low molecular mass BAP sequences are less likely to be broken down by digestive enzymes and are thus more likely to be active in vivo. However, the identification of short peptides remains a challenge during mass spectrometry (MS) analysis due to issues with the transfer and over-fragmentation of low molecular mass ions. A method is described herein using time-of-flight ESI-MS/MS to effectively fragment and identify short peptides. This includes (a) short synthetic peptides, (b) short peptides within a defined hydrolysate sample, i.e. a prolyl endoproteinase hydrolysate of β-casein and (c) short peptides within a complex hydrolysate, i.e. a Corolase PP digest of sodium caseinate. The methodology may find widespread utilisation in the efficient identification of low molecular mass peptide sequences in food protein hydrolysates. PMID:25872436

  10. GeLC-MS/MS Analysis of Complex Protein Mixtures

    PubMed Central

    Dzieciatkowska, Monika; Hill, Ryan; Hansen, Kirk C.

    2015-01-01

    Discovery-based proteomics has found its place in nearly every facet of biological research. A key objective of this approach is to maximize sequence coverage for proteins across a wide concentration range. Fractionating samples at the protein level is one of the most common ways to circumvent challenges due to sample complexity and improve proteome coverage. Of the available methods, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) is a robust and reproducible method for qualitative and quantitative proteomic analysis. Here we describe a general GeLC-MS/MS protocol and include technical advice and outline caveats to increase the probability of a successful analysis. PMID:24791981

  11. Force-induced remodelling of proteins and their complexes

    PubMed Central

    Chen, Yun; Radford, Sheena E; Brockwell, David J

    2015-01-01

    Force can drive conformational changes in proteins, as well as modulate their stability and the affinity of their complexes, allowing a mechanical input to be converted into a biochemical output. These properties have been utilised by nature and force is now recognised to be widely used at the cellular level. The effects of force on the biophysical properties of biological systems can be large and varied. As these effects are only apparent in the presence of force, studies on the same proteins using traditional ensemble biophysical methods can yield apparently conflicting results. Where appropriate, therefore, force measurements should be integrated with other experimental approaches to understand the physiological context of the system under study. PMID:25710390

  12. Purification and characterization of HIV–human protein complexes

    PubMed Central

    Jäger, Stefanie; Gulbahce, Natali; Cimermancic, Peter; Kane, Joshua; He, Nanhai; Chou, Seemay; D’Orso, Iván; Fernandes, Jason; Jang, Gwendolyn; Frankel, Alan D.; Alber, Tom; Zhou, Qiang; Krogan, Nevan J.

    2011-01-01

    To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen–host protein–protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10–100 proteins), generation of comprehensive host–virus PPI maps using different experimental platforms, including affinity tag purification-mass spectrometry (AP-MS) and yeast two-hybrid (Y2H) approaches, can be achieved. Global maps such as these provide unbiased insight into the molecular mechanisms of viral entry, replication and assembly. However, to date, only two-hybrid methodology has been used in a systematic fashion to characterize viral–host protein–protein interactions, although a deluge of data exists in databases that manually curate from the literature individual host–pathogen PPIs. We will summarize this work and also describe an AP-MS platform that can be used to characterize viral-human protein complexes and discuss its application for the HIV genome. PMID:20708689

  13. Simulating evolution of protein complexes through gene duplication and co-option.

    PubMed

    Haarsma, Loren; Nelesen, Serita; VanAndel, Ethan; Lamine, James; VandeHaar, Peter

    2016-06-21

    We present a model of the evolution of protein complexes with novel functions through gene duplication, mutation, and co-option. Under a wide variety of input parameters, digital organisms evolve complexes of 2-5 bound proteins which have novel functions but whose component proteins are not independently functional. Evolution of complexes with novel functions happens more quickly as gene duplication rates increase, point mutation rates increase, protein complex functional probability increases, protein complex functional strength increases, and protein family size decreases. Evolution of complexity is inhibited when the metabolic costs of making proteins exceeds the fitness gain of having functional proteins, or when point mutation rates get so large the functional proteins undergo deleterious mutations faster than new functional complexes can evolve. PMID:27038665

  14. The Complex Energy Landscape of the Protein IscU

    PubMed Central

    Bothe, Jameson R.; Tonelli, Marco; Ali, Ibrahim K.; Dai, Ziqi; Frederick, Ronnie O.; Westler, William M.; Markley, John L.

    2015-01-01

    IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural information from NMR and SAXS suggests that the structures of the cold-induced and heat-induced D states are similar. Both states exhibit similar 1H-15N HSQC spectra and the same pattern of peptidyl-prolyl peptide bond configurations by NMR, and both appear to be similarly expanded compared with the S state based on analysis of SAXS data. Whereas in other proteins the cold-denatured states have been found to be slightly more compact than the heat-denatured states, these two states occupy similar volumes in IscU. PMID:26331259

  15. Mapping energy transfer channels in fucoxanthin-chlorophyll protein complex.

    PubMed

    Gelzinis, Andrius; Butkus, Vytautas; Songaila, Egidijus; Augulis, Ramūnas; Gall, Andrew; Büchel, Claudia; Robert, Bruno; Abramavicius, Darius; Zigmantas, Donatas; Valkunas, Leonas

    2015-02-01

    Fucoxanthin-chlorophyll protein (FCP) is the key molecular complex performing the light-harvesting function in diatoms, which, being a major group of algae, are responsible for up to one quarter of the total primary production on Earth. These photosynthetic organisms contain an unusually large amount of the carotenoid fucoxanthin, which absorbs the light in the blue-green spectral region and transfers the captured excitation energy to the FCP-bound chlorophylls. Due to the large number of fucoxanthins, the excitation energy transfer cascades in these complexes are particularly tangled. In this work we present the two-color two-dimensional electronic spectroscopy experiments on FCP. Analysis of the data using the modified decay associated spectra permits a detailed mapping of the excitation frequency dependent energy transfer flow with a femtosecond time resolution. PMID:25445318

  16. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    SciTech Connect

    Nielsen, Anders Lade

    2009-10-23

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  17. Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

    PubMed Central

    Sudhir, Putty-Reddy; Chen, Chung-Hsuan

    2016-01-01

    A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

  18. STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

    PubMed Central

    Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

    2009-01-01

    Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

  19. Small Cofactors May Assist Protein Emergence from RNA World: Clues from RNA-Protein Complexes

    PubMed Central

    Shen, Liang; Ji, Hong-Fang

    2011-01-01

    It is now widely accepted that at an early stage in the evolution of life an RNA world arose, in which RNAs both served as the genetic material and catalyzed diverse biochemical reactions. Then, proteins have gradually replaced RNAs because of their superior catalytic properties in catalysis over time. Therefore, it is important to investigate how primitive functional proteins emerged from RNA world, which can shed light on the evolutionary pathway of life from RNA world to the modern world. In this work, we proposed that the emergence of most primitive functional proteins are assisted by the early primitive nucleotide cofactors, while only a minority are induced directly by RNAs based on the analysis of RNA-protein complexes. Furthermore, the present findings have significant implication for exploring the composition of primitive RNA, i.e., adenine base as principal building blocks. PMID:21789260

  20. Insertional inactivation of an intrageneric coaggregation-relevant adhesin locus from Streptococcus gordonii DL1 (Challis).

    PubMed Central

    Whittaker, C J; Clemans, D L; Kolenbrander, P E

    1996-01-01

    Transposon Tn916 was used to insertionally inactivate a coaggregation-relevant locus of Streptococcus gordonii DL1 (Challis). One mutant (F11) was isolated that lost the ability to coaggregate with the streptococcal partners of DL1 but retained the ability to coaggregate with partners belonging to other genera. A probe specific for the region flanking the Tn916 insertion was used to isolate a locus-specific fragment from a chromosomal lambda library. Southern analysis of the resulting phagemids revealed that a 0.5-kb EcoRI fragment hybridized with the F11 probe. Cloning of the 0.5-kb EcoRI fragment into the E. coli-streptococcal insertion vector p(omega) yielded pCW4, which was used to insertionally inactivate the putative coaggregation-relevant gene in DL1. Insertion mutants showed altered coaggregation with streptococci but retained wild-type coaggregation properties with other genera of bacteria. Comparison of immunoblots of cell surface proteins showed a 100-kDa protein in DL1 which was not detected in the Tn916 and pCW4 insertion mutants. These results indicate that the 0.5-kb EcoRI fragment is part of an adhesin-relevant locus that is involved in the production of a 100-kDa protein at the cell surface. PMID:8926080

  1. Adhesins involved in attachment to abiotic surfaces by Gram-negative bacteria

    PubMed Central

    Berne, Cécile; Ducret, Adrien; Hardy, Gail G; Brun, Yves V.

    2015-01-01

    During the first step of biofilm formation, initial attachment is dictated by physicochemical and electrostatic interactions between the surface and the bacterial envelope. Depending upon the nature of these interactions, attachment can be transient or permanent. To achieve irreversible attachment, bacterial cells have developed a series of surface adhesins promoting specific or non-specific adhesion under various environmental conditions. This chapter will review the recent advances in our understanding of the secretion, assembly and regulation of the bacterial adhesins during biofilm formation with a particular emphasis on the fimbrial, non-fimbrial and discrete polysaccharide adhesins in Gram-negative bacteria. PMID:26350310

  2. Structural Assembly of Multidomain Proteins and Protein Complexes Guided by the Overall Rotational Diffusion Tensor

    PubMed Central

    Ryabov, Yaroslav; Fushman, David

    2008-01-01

    We present a simple and robust approach that uses the overall rotational diffusion tensor as a structural constraint for domain positioning in multidomain proteins and protein-protein complexes. This method offers the possibility to use NMR relaxation data for detailed structure characterization of such systems provided the structures of individual domains are available. The proposed approach extends the concept of using long-range information contained in the overall rotational diffusion tensor. In contrast to the existing approaches, we use both the principal axes and principal values of protein’s rotational diffusion tensor to determine not only the orientation but also the relative positioning of the individual domains in a protein. This is achieved by finding the domain arrangement in a molecule that provides the best possible agreement with all components of the overall rotational diffusion tensor derived from experimental data. The accuracy of the proposed approach is demonstrated for two protein systems with known domain arrangement and parameters of the overall tumbling: the HIV-1 protease homodimer and Maltose Binding Protein. The accuracy of the method and its sensitivity to domain positioning is also tested using computer-generated data for three protein complexes, for which the experimental diffusion tensors are not available. In addition, the proposed method is applied here to determine, for the first time, the structure of both open and closed conformations of Lys48-linked di-ubiquitin chain, where domain motions render impossible accurate structure determination by other methods. The proposed method opens new avenues for improving structure characterization of proteins in solution. PMID:17550252

  3. Partner-Aware Prediction of Interacting Residues in Protein-Protein Complexes from Sequence Data

    PubMed Central

    Ahmad, Shandar; Mizuguchi, Kenji

    2011-01-01

    Computational prediction of residues that participate in protein-protein interactions is a difficult task, and state of the art methods have shown only limited success in this arena. One possible problem with these methods is that they try to predict interacting residues without incorporating information about the partner protein, although it is unclear how much partner information could enhance prediction performance. To address this issue, the two following comparisons are of crucial significance: (a) comparison between the predictability of inter-protein residue pairs, i.e., predicting exactly which residue pairs interact with each other given two protein sequences; this can be achieved by either combining conventional single-protein predictions or making predictions using a new model trained directly on the residue pairs, and the performance of these two approaches may be compared: (b) comparison between the predictability of the interacting residues in a single protein (irrespective of the partner residue or protein) from conventional methods and predictions converted from the pair-wise trained model. Using these two streams of training and validation procedures and employing similar two-stage neural networks, we showed that the models trained on pair-wise contacts outperformed the partner-unaware models in predicting both interacting pairs and interacting single-protein residues. Prediction performance decreased with the size of the conformational change upon complex formation; this trend is similar to docking, even though no structural information was used in our prediction. An example application that predicts two partner-specific interfaces of a protein was shown to be effective, highlighting the potential of the proposed approach. Finally, a preliminary attempt was made to score docking decoy poses using prediction of interacting residue pairs; this analysis produced an encouraging result. PMID:22194998

  4. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs.

    PubMed

    Schneider, Tim; Hung, Lee-Hsueh; Schreiner, Silke; Starke, Stefan; Eckhof, Heinrich; Rossbach, Oliver; Reich, Stefan; Medenbach, Jan; Bindereif, Albrecht

    2016-01-01

    Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component. PMID:27510448

  5. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs

    PubMed Central

    Schneider, Tim; Hung, Lee-Hsueh; Schreiner, Silke; Starke, Stefan; Eckhof , Heinrich; Rossbach, Oliver; Reich, Stefan; Medenbach, Jan; Bindereif , Albrecht

    2016-01-01

    Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component. PMID:27510448

  6. A novel algorithm for detecting protein complexes with the breadth first search.

    PubMed

    Tang, Xiwei; Wang, Jianxin; Li, Min; He, Yiming; Pan, Yi

    2014-01-01

    Most biological processes are carried out by protein complexes. A substantial number of false positives of the protein-protein interaction (PPI) data can compromise the utility of the datasets for complexes reconstruction. In order to reduce the impact of such discrepancies, a number of data integration and affinity scoring schemes have been devised. The methods encode the reliabilities (confidence) of physical interactions between pairs of proteins. The challenge now is to identify novel and meaningful protein complexes from the weighted PPI network. To address this problem, a novel protein complex mining algorithm ClusterBFS (Cluster with Breadth-First Search) is proposed. Based on the weighted density, ClusterBFS detects protein complexes of the weighted network by the breadth first search algorithm, which originates from a given seed protein used as starting-point. The experimental results show that ClusterBFS performs significantly better than the other computational approaches in terms of the identification of protein complexes. PMID:24818139

  7. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins

    PubMed Central

    Ju Shin, Yeong; Kyun Park, Seung; Jung Jung, Yoo; Na Kim, Ye; Sung Kim, Ki; Kyu Park, Ok; Kwon, Seung-Hae; Ho Jeon, Sung; Trinh, Le A.; Fraser, Scott E.; Kee, Yun; Joon Hwang, Byung

    2015-01-01

    Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein. PMID:26373678

  8. Polysaccharide-Protein Complexes in a Coarse-Grained Model.

    PubMed

    Poma, Adolfo B; Chwastyk, Mateusz; Cieplak, Marek

    2015-09-10

    We construct two variants of coarse-grained models of three hexaoses: one based on the centers of mass of the monomers and the other associated with the C4 atoms. The latter is found to be better defined and more suitable for studying interactions with proteins described within α-C based models. We determine the corresponding effective stiffness constants through all-atom simulations and two statistical methods. One method is the Boltzmann inversion (BI) and the other, named energy-based (EB), involves direct monitoring of energies as a function of the variables that define the stiffness potentials. The two methods are generally consistent in their account of the stiffness. We find that the elastic constants differ between the hexaoses and are noticeably different from those determined for the crystalline cellulose Iβ. The nonbonded couplings through hydrogen bonds between different sugar molecules are modeled by the Lennard-Jones potentials and are found to be stronger than the hydrogen bonds in proteins. We observe that the EB method agrees with other theoretical and experimental determinations of the nonbonded parameters much better than BI. We then consider the hexaose-Man5B catalytic complexes and determine the contact energies between their the C4-α-C atoms. These interactions are found to be stronger than the proteinic hydrogen bonds: about four times as strong for cellohexaose and two times for mannohexaose. The fluctuational dynamics of the coarse-grained complexes are found to be compatible with previous all-atom studies by Bernardi et al. PMID:26291477

  9. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis

    PubMed Central

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S.; Pérez, Amparo Cuéllar; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-01-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  10. Nucleocytoplasmic Transport of RNAs and RNA-Protein Complexes.

    PubMed

    Sloan, Katherine E; Gleizes, Pierre-Emmanuel; Bohnsack, Markus T

    2016-05-22

    RNAs and ribonucleoprotein complexes (RNPs) play key roles in mediating and regulating gene expression. In eukaryotes, most RNAs are transcribed, processed and assembled with proteins in the nucleus and then either function in the cytoplasm or also undergo a cytoplasmic phase in their biogenesis. This compartmentalization ensures that sequential steps in gene expression and RNP production are performed in the correct order and it allows important quality control mechanisms that prevent the involvement of aberrant RNAs/RNPs in these cellular pathways. The selective exchange of RNAs/RNPs between the nucleus and cytoplasm is enabled by nuclear pore complexes, which function as gateways between these compartments. RNA/RNP transport is facilitated by a range of nuclear transport receptors and adaptors, which are specifically recruited to their cargos and mediate interactions with nucleoporins to allow directional translocation through nuclear pore complexes. While some transport factors are only responsible for the export/import of a certain class of RNA/RNP, others are multifunctional and, in the case of large RNPs, several export factors appear to work together to bring about export. Recent structural studies have revealed aspects of the mechanisms employed by transport receptors to enable specific cargo recognition, and genome-wide approaches have provided the first insights into the diverse composition of pre-mRNPs during export. Furthermore, the regulation of RNA/RNP export is emerging as an important means to modulate gene expression under stress conditions and in disease. PMID:26434509

  11. [Role of Bacterial Adhesin RAPA1 in Formation of Efficient Symbiosis of Rhizobium leguminosarum with Bean Plants].

    PubMed

    Nigmatullina, L R; Lavina, A M; Vershinina, Z R; Baimiev, Al Kh

    2015-01-01

    Bacterial adhesins, the proteins responsible for attachment of plant growth-promoting rhizobacteria to plant roots, are involved in formation of stable associative symbioses. In the present work enhanced expression of the rapA1 adhesin gene in Rhizobium leguminosarum PVu5 was shown to improve the efficiency of nodulation on bean roots inoculated with the modified strain. The rapA1 gene was cloned into the pJN105Turbo plasmid, this construct was used for transformation of R. leguminosarum PVu5, bean plants were inoculated by this transgenic strain, and efficiency of root nodule formation was determined. In the plants treated with rapA1-transgenic rhizobia, the number of root nodules was on average two times higher than in the plants inoculated with the original strain. Aggregation of R. leguminosarum was achieved when the rapA1 gene expression was enhanced either in rhizobia or in the co-cultured modified strain E. coli pJN105TurboRapA1. PMID:26964360

  12. The pgaABCD Locus of Escherichia coli Promotes the Synthesis of a Polysaccharide Adhesin Required for Biofilm Formation

    PubMed Central

    Wang, Xin; Preston, James F.; Romeo, Tony

    2004-01-01

    Production of a polysaccharide matrix is a hallmark of bacterial biofilms, but the composition of matrix polysaccharides and their functions are not widely understood. Previous studies of the regulation of Escherichia coli biofilm formation suggested the involvement of an unknown adhesin. We now establish that the pgaABCD (formerly ycdSRQP) locus affects biofilm development by promoting abiotic surface binding and intercellular adhesion. All of the pga genes are required for optimal biofilm formation under a variety of growth conditions. A pga-dependent cell-bound polysaccharide was isolated and determined by nuclear magnetic resonance analyses to consist of unbranched β-1,6-N-acetyl-d-glucosamine, a polymer previously unknown from the gram-negative bacteria but involved in adhesion by staphylococci. The pga genes are predicted to encode envelope proteins involved in synthesis, translocation, and possibly surface docking of this polysaccharide. As predicted, if poly-β-1,6-GlcNAc (PGA) mediates cohesion, metaperiodate caused biofilm dispersal and the release of intact cells, whereas treatment with protease or other lytic enzymes had no effect. The pgaABCD operon exhibits features of a horizontally transferred locus and is present in a variety of eubacteria. Therefore, we propose that PGA serves as an adhesin that stabilizes biofilms of E. coli and other bacteria. PMID:15090514

  13. In vitro effect of temperature on the conformational structure and collagen binding of SdrF, a Staphylococcus epidermidis adhesin.

    PubMed

    Di Poto, Antonella; Papi, Massimiliano; Trivedi, Sheetal; Maiorana, Alessandro; Gavazzo, Paola; Vassalli, Massimo; Lowy, Franklin D; De Spirito, Marco; Montanaro, Lucio; Imbriani, Marcello; Arciola, Carla Renata; Visai, Livia

    2015-07-01

    Staphylococcus epidermidis is the leading etiologic agent of device-related infections. S. epidermidis is able to bind, by means of the adhesins of its cell wall, the host matrix proteins filming the artificial surfaces. Thence, bacteria cling to biomaterials and infection develops. The effect of temperature on integrity, structure, and biological activity of the collagen-binding adhesin (SdrF) of S. epidermidis has been here investigated. By cloning in E. coli XL1-Blue, a recombinant of the SdrF binding domain B (rSdrFB), carrying an N-terminal polyhistidine, was obtained. Purification was by HiTrap(TM) Chelating HP columns. Assessment of purity, molecular weight, and integrity was by SDS-PAGE. The rSdrFB-collagen binding was investigated by ELISA. A full three-dimensional reconstruction of rSdrFB was achieved by small-angle X-ray scattering (SAXS). At 25 °C, rSdrFB bound to type I collagen in a dose-dependent, saturable manner, with a Kd of 2.48 × 10(-7) M. When temperature increased from 25 to 37 °C, a strong conformational change occurred, together with the abolition of the rSdrFB-collagen binding. The rSdrFB integrity was not affected by temperature variation. SdrFB-collagen binding is switched on/off depending on the temperature. Implications with the infection pathogenesis are enlightened. PMID:25683665

  14. Determination of adhesin gene sequences in, and biofilm formation by, O157 and non-O157 Shiga toxin-producing Escherichia coli strains isolated from different sources.

    PubMed

    Biscola, Franciele Tafarello; Abe, Cecilia Mari; Guth, Beatriz Ernestina Cabilio

    2011-04-01

    Biofilm formation by Shiga toxin-producing Escherichia coli (STEC) has been associated with the expression of different adhesins (type 1 fimbria, curli, Ag43, Cah, and EhaA). In this study, biofilm formation and the presence of adhesin-related gene sequences were determined by PCR in 18 O157 strains and 33 non-O157 strains isolated from different sources (human, animal, food, and water). The expression of different adhesins was also assessed by reverse transcription-PCR (RT-PCR), Congo red agar plates, and mannose-sensitive hemagglutination (MSHA) assay. Biofilm formation occurred in 5/18 (28%) O157 STEC strains and 17/33 (51%) non-O157 STEC strains from different serotypes and sources, when the assays were performed at 28°C for 48 h. Among the non-O157 biofilm-producing isolates, 12/17 (71%) expressed type 1 fimbriae and 11/17 (65%) expressed curli and produced cellulose, while 8/17 (47%) were considered to be Ag43(+) by RT-PCR. Among O157 strains, a close correlation was observed between biofilm formation and expression of curli and cellulose. In non-O157 strains, it seems that, in addition to the presence of curli, the ability to form biofilm is associated with the presence of other factors such as type 1 fimbriae and autotransporter proteins, which may contribute to the persistence of these organisms in the environment. PMID:21317257

  15. Detection of an endothelin-1-binding protein complex by low temperature SDS-PAGE

    SciTech Connect

    Takasuka, T.; Horii, I.; Furuichi, Y.; Watanabe, T. )

    1991-04-15

    We found that the complex of ET-1 and its binding protein was stable enough to be separated by SDS-PAGE when electrophoresis was run at a low temperature. Cross-linking was not necessary for the detection of {sup 125}I-ET-1 and its binding protein complex by autoradiography. This simple method could be used in qualitative (estimation of apparent molecular weight of ET-1 binding protein) and quantitative (determination of relative content of ET-binding protein) analysis of the ET-binding protein complex. ET-binding protein complexes of various animal species and organs were investigated by this method.

  16. Structures of C-mannosylated anti-adhesives bound to the type 1 fimbrial FimH adhesin

    PubMed Central

    de Ruyck, Jerome; Lensink, Marc F.; Bouckaert, Julie

    2016-01-01

    Selective inhibitors of the type 1 fimbrial adhesin FimH are recognized as attractive alternatives for antibiotic therapies and prophylaxes against Escherichia coli infections such as urinary-tract infections. To construct these inhibitors, the α-d-mannopyranoside of high-mannose N-glycans, recognized with exclusive specificity on glycoprotein receptors by FimH, forms the basal structure. A hydrophobic aglycon is then linked to the mannose by the O1 oxygen inherently present in the α-anomeric configuration. Substitution of this O atom by a carbon introduces a C-glycosidic bond, which may enhance the therapeutic potential of such compounds owing to the inability of enzymes to degrade C-glycosidic bonds. Here, the first crystal structures of the E. coli FimH adhesin in complex with C-glycosidically linked mannopyranosides are presented. These findings explain the role of the spacer in positioning biphenyl ligands for interactions by means of aromatic stacking in the tyrosine gate of FimH and how the normally hydrated C-glycosidic link is tolerated. As these new compounds can bind FimH, it can be assumed that they have the potential to serve as potent new antagonists of FimH, paving the way for the design of a new family of anti-adhesive compounds against urinary-tract infections. PMID:27158502

  17. Structures of C-mannosylated anti-adhesives bound to the type 1 fimbrial FimH adhesin.

    PubMed

    de Ruyck, Jerome; Lensink, Marc F; Bouckaert, Julie

    2016-05-01

    Selective inhibitors of the type 1 fimbrial adhesin FimH are recognized as attractive alternatives for antibiotic therapies and prophylaxes against Escherichia coli infections such as urinary-tract infections. To construct these inhibitors, the α-d-mannopyranoside of high-mannose N-glycans, recognized with exclusive specificity on glycoprotein receptors by FimH, forms the basal structure. A hydrophobic aglycon is then linked to the mannose by the O1 oxygen inherently present in the α-anomeric configuration. Substitution of this O atom by a carbon introduces a C-glycosidic bond, which may enhance the therapeutic potential of such compounds owing to the inability of enzymes to degrade C-glycosidic bonds. Here, the first crystal structures of the E. coli FimH adhesin in complex with C-glycosidically linked mannopyranosides are presented. These findings explain the role of the spacer in positioning biphenyl ligands for interactions by means of aromatic stacking in the tyrosine gate of FimH and how the normally hydrated C-glycosidic link is tolerated. As these new compounds can bind FimH, it can be assumed that they have the potential to serve as potent new antagonists of FimH, paving the way for the design of a new family of anti-adhesive compounds against urinary-tract infections. PMID:27158502

  18. Heterodimeric protein complex identification by naïve Bayes classifiers

    PubMed Central

    2013-01-01

    Background Protein complexes are basic cellular entities that carry out the functions of their components. It can be found that in databases of protein complexes of yeast like CYC2008, the major type of known protein complexes is heterodimeric complexes. Although a number of methods for trying to predict sets of proteins that form arbitrary types of protein complexes simultaneously have been proposed, it can be found that they often fail to predict heterodimeric complexes. Results In this paper, we have designed several features characterizing heterodimeric protein complexes based on genomic data sets, and proposed a supervised-learning method for the prediction of heterodimeric protein complexes. This method learns the parameters of the features, which are embedded in the naïve Bayes classifier. The log-likelihood ratio derived from the naïve Bayes classifier with the parameter values obtained by maximum likelihood estimation gives the score of a given pair of proteins to predict whether the pair is a heterodimeric complex or not. A five-fold cross-validation shows good performance on yeast. The trained classifiers also show higher predictability than various existing algorithms on yeast data sets with approximate and exact matching criteria. Conclusions Heterodimeric protein complex prediction is a rather harder problem than heteromeric protein complex prediction because heterodimeric protein complex is topologically simpler. However, it turns out that by designing features specialized for heterodimeric protein complexes, predictability of them can be improved. Thus, the design of more sophisticate features for heterodimeric protein complexes as well as the accumulation of more accurate and useful genome-wide data sets will lead to higher predictability of heterodimeric protein complexes. Our tool can be downloaded from http://imi.kyushu-u.ac.jp/~om/. PMID:24299017

  19. Excitation energy transfer in photosynthetic protein-pigment complexes

    NASA Astrophysics Data System (ADS)

    Yeh, Shu-Hao

    Quantum biology is a relatively new research area which investigates the rules that quantum mechanics plays in biology. One of the most intriguing systems in this field is the coherent excitation energy transport (EET) in photosynthesis. In this document I will discuss the theories that are suitable for describing the photosynthetic EET process and the corresponding numerical results on several photosynthetic protein-pigment complexes (PPCs). In some photosynthetic EET processes, because of the electronic coupling between the chromophores within the system is about the same order of magnitude as system-bath coupling (electron-phonon coupling), a non-perturbative method called hierarchy equation of motion (HEOM) is applied to study the EET dynamics. The first part of this thesis includes brief introduction and derivation to the HEOM approach. The second part of this thesis the HEOM method will be applied to investigate the EET process within the B850 ring of the light harvesting complex 2 (LH2) from purple bacteria, Rhodopseudomonas acidophila. The dynamics of the exciton population and coherence will be analyzed under different initial excitation configurations and temperatures. Finally, how HEOM can be implemented to simulate the two-dimensional electronic spectra of photosynthetic PPCs will be discussed. Two-dimensional electronic spectroscopy is a crucial experimental technique to probe EET dynamics in multi-chromophoric systems. The system we are interested in is the 7-chromophore Fenna-Matthews-Olson (FMO) complex from green sulfur bacteria, Prosthecochloris aestuarii. Recent crystallographic studies report the existence of an additional (eighth) chromophore in some of the FMO monomers. By applying HEOM we are able to calculate the two-dimensional electronic spectra of the 7-site and 8-site FMO complexes and investigate the functionality of the eighth chromophore.

  20. Impact of natural variation in bacterial F17G adhesins on crystallization behaviour.

    PubMed

    Buts, Lieven; Wellens, Adinda; Van Molle, Inge; Wyns, Lode; Loris, Remy; Lahmann, Martina; Oscarson, Stefan; De Greve, Henri; Bouckaert, Julie

    2005-08-01

    Since the introduction of structural genomics, the protein has been recognized as the most important variable in crystallization. Recent strategies to modify a protein to improve crystal quality have included rationally engineered point mutations, truncations, deletions and fusions. Five naturally occurring variants, differing in 1-18 amino acids, of the 177-residue lectin domain of the F17G fimbrial adhesin were expressed and purified in identical ways. For four out of the five variants crystals were obtained, mostly in non-isomorphous space groups, with diffraction limits ranging between 2.4 and 1.1 A resolution. A comparative analysis of the crystal-packing contacts revealed that the variable amino acids are often involved in lattice contacts and a single amino-acid substitution can suffice to radically change crystal packing. A statistical approach proved reliable to estimate the compatibilities of the variant sequences with the observed crystal forms. In conclusion, natural variation, universally present within prokaryotic species, is a valuable genetic resource that can be favourably employed to enhance the crystallization success rate with considerably less effort than other strategies. PMID:16041081

  1. Comparison of tertiary structures of proteins in protein-protein complexes with unbound forms suggests prevalence of allostery in signalling proteins

    PubMed Central

    2012-01-01

    Background Most signalling and regulatory proteins participate in transient protein-protein interactions during biological processes. They usually serve as key regulators of various cellular processes and are often stable in both protein-bound and unbound forms. Availability of high-resolution structures of their unbound and bound forms provides an opportunity to understand the molecular mechanisms involved. In this work, we have addressed the question “What is the nature, extent, location and functional significance of structural changes which are associated with formation of protein-protein complexes?” Results A database of 76 non-redundant sets of high resolution 3-D structures of protein-protein complexes, representing diverse functions, and corresponding unbound forms, has been used in this analysis. Structural changes associated with protein-protein complexation have been investigated using structural measures and Protein Blocks description. Our study highlights that significant structural rearrangement occurs on binding at the interface as well as at regions away from the interface to form a highly specific, stable and functional complex. Notably, predominantly unaltered interfaces interact mainly with interfaces undergoing substantial structural alterations, revealing the presence of at least one structural regulatory component in every complex. Interestingly, about one-half of the number of complexes, comprising largely of signalling proteins, show substantial localized structural change at surfaces away from the interface. Normal mode analysis and available information on functions on some of these complexes suggests that many of these changes are allosteric. This change is largely manifest in the proteins whose interfaces are altered upon binding, implicating structural change as the possible trigger of allosteric effect. Although large-scale studies of allostery induced by small-molecule effectors are available in literature, this is, to our

  2. Atomistic Simulation of Lignocellulosic Biomass and Associated Cellulosomal Protein Complexes

    SciTech Connect

    Petridis, Loukas; Crowley, Michael F; Smith, Jeremy C

    2010-01-01

    Computer simulations have been performed to obtain an atomic-level understanding of lignocellulose structure and the assembly of its associated cellulosomal protein complexes. First, a CHARMM molecular mechanics force field for lignin is derived and validated by performing a molecular dynamics simulation of a crystal of a lignin fragment molecule and comparing simulation-derived structural features with experimental results. Together with the existing force field for polysaccharides, this work provides the basis for full simulations of lignocellulose. Second, the underlying molecular mechanism governing the assembly of various cellulosomal modules is investigated by performing a novel free-energy calculation of the cohesin-dockerin dissociation. Our calculation indicates a free-energy barrier of ~17 kcal/mol and further reveals a stepwise dissociation pathway involving both the central -sheet interface and its adjacent solvent-exposed loop/turn regions clustered at both ends of the -barrel structure.

  3. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

    PubMed

    Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

    2016-09-01

    The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  4. RNA and protein complexes of trp RNA-binding attenuation protein characterized by mass spectrometry.

    PubMed

    Akashi, Satoko; Watanabe, Masahiro; Heddle, Jonathan G; Unzai, Satoru; Park, Sam-Yong; Tame, Jeremy R H

    2009-03-15

    We have characterized both wild-type and mutant TRAP (trp RNA-binding attenuation protein) from Bacillus stearothermophilus , and their complexes with RNA or its regulator anti-TRAP protein (AT), by electrospray ionization mass spectrometry (ESI-MS). Wild-type TRAP mainly forms homo-11mer rings. The mutant used carries three copies of the TRAP monomer on a single polypeptide chain so that it associates to form a 12mer ring with four polypeptide molecules. Mass spectra showed that both the wild-type TRAP 11mer and the mutant TRAP 12mer can bind a cognate single-stranded RNA molecule with a molar ratio of 1:1. The crystal structure of wild-type TRAP complexed with AT shows a TRAP 12mer ring surrounded by six AT trimers. However, nanoESI-MS of wild-type TRAP mixed with AT shows four species with different binding stoichiometries, and the complex observed by crystallography represents only a minor species in solution; most of the TRAP remains in an 11mer ring form. Mass spectra of mutant TRAP showed only a single species, TRAP 12mer + six copies of AT trimer, which is observed by crystallography. These results suggest that crystallization selects only the most symmetrical TRAP-AT complex from the solution, whereas ESI-MS can take a "snapshot" of all the species in solution. PMID:19219981

  5. Uropathogenic E. coli adhesin-induced host cell receptor conformational changes: implications in transmembrane signaling transduction

    PubMed Central

    Wang, Huaibin; Min, Guangwei; Glockshuber, Rudi; Sun, Tung-Tien; Kong, Xiang-Peng

    2009-01-01

    Urinary tract infection (UTI) is the second most common infectious disease, and is caused predominantly by type 1-fimbriated uropathogenic E. coli (UPEC). UPEC initiates infection by attaching to uroplakin Ia, its urothelial surface receptor, via the FimH adhesins capping the distal end of its fimbriae. Uroplakin Ia, together with uroplakins Ib, II and IIIa, forms a 16 nm receptor complex that is assembled into hexagonally packed two-dimensional crystals (urothelial plaques) covering >90% of the urothelial apical surface. Recent studies indicate that FimH is the invasin of UPEC as its attachment to the urothelial surface can induce cellular signaling events including calcium elevation and the phosphorylation of the uroplakin IIIa cytoplasmic tail, leading to cytoskeletal rearrangements and bacterial invasion. However, it remains unknown how the binding of FimH to the uroplakin receptor triggers a signal that can be transmitted through the highly impermeable urothelial apical membrane. We show here by cryo-electron microscopy that FimH-binding to the extracellular domain of UPIa induces global conformational changes in the entire uroplakin receptor complex, including a coordinated movement of the tightly bundled transmembrane helices. This movement of the transmembrane helix bundles can cause a corresponding lateral translocation of the uroplakin cytoplasmic tails, which can be sufficient to trigger downstream signaling events. Our results suggest a novel pathogen-induced transmembrane signal transduction mechanism that plays a key role in the initial stages of UPEC invasion and receptor-mediated bacterial invasion in general. PMID:19577575

  6. Protein biotinylation visualized by a complex structure of biotin protein ligase with a substrate.

    PubMed

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2008-05-23

    Biotin protein ligase (BPL) catalyzes the biotinylation of the biotin carboxyl carrier protein (BCCP) only at a special lysine residue. Here we report the first structure of BPL.BCCP complex crystals, which are prepared using two BPL mutants: R48A and R48A/K111A. From a detailed structural characterization, it is likely that the mutants retain functionality as enzymes but have a reduced activity to produce the reaction intermediate biotinyl-5'-AMP. The observed biotin and partly disordered ATP in the mutant structures may act as a non-reactive analog of the substrates or biotinyl-5'-AMP, thereby providing the complex crystals. The four crystallographically independent BPL.BCCP complexes obtained can be classified structurally into three groups: the formation stages 1 and 2 with apo-BCCP and the product stage with biotinylated holo-BCCP. Residues responsible for the complex formation as well as for the biotinylation reaction have been identified. The C-terminal domain of BPL shows especially large conformational changes to accommodate BCCP, suggesting its functional importance. The formation stage 1 complex shows the closest distance between the carboxyl carbon of biotin and the special lysine of BCCP, suggesting its relevance to the unobserved reaction stage. Interestingly, bound ATP and biotin are also seen in the product stage, indicating that the substrates may be recruited into the product stage complex before the release of holo-BCCP, probably for the next reaction cycle. The existence of formation and product stages before and after the reaction stage would be favorable to ensure both the reaction efficiency and the extreme substrate specificity of the biotinylation reaction. PMID:18372281

  7. The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis.

    PubMed Central

    Patti, J M; Bremell, T; Krajewska-Pietrasik, D; Abdelnour, A; Tarkowski, A; Rydén, C; Höök, M

    1994-01-01

    The importance of a collagen-binding adhesin in the pathogenesis of septic arthritis has been examined by comparing the virulence of two sets of Staphylococcus aureus mutants in an animal model. Collagen adhesin-negative mutant PH100 was constructed by replacing the chromosomal collagen adhesin gene (cna) in a clinical strain, Phillips, with an inactivated copy of the gene. Collagen adhesin-positive mutant S. aureus CYL574 was generated by introducing the cna gene into CYL316, a strain that normally lacks the cna gene. Biochemical, immunological, and functional analyses of the generated mutants and their respective parent strains showed that binding of 125I-labeled collagen, expression of an immunoreactive collagen adhesin, and bacterial adherence to cartilage were directly correlated with the presence of a functional cna gene. Greater than 70% of the mice injected with the Cna+ strains developed clinical signs of arthritis, whereas less than 27% of the animals injected with Cna- strains showed symptoms of disease. Furthermore, mice injected with the Cna+ strain Phillips had remarkably elevated levels of immunoglobulin G1 and interleukin-6 compared with mice injected with the Cna- mutant PH100. Taken together, these results demonstrate that collagen adhesin plays an important role in the pathogenesis of septic arthritis induced by S. aureus. Images PMID:8262622

  8. Mitogen activated protein kinase at the nuclear pore complex

    PubMed Central

    Faustino, Randolph S; Maddaford, Thane G; Pierce, Grant N

    2011-01-01

    Abstract Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function. PMID:20497490

  9. Arabidopsis flower development-of protein complexes, targets, and transport.

    PubMed

    Becker, Annette; Ehlers, Katrin

    2016-03-01

    Tremendous progress has been achieved over the past 25 years or more of research on the molecular mechanisms of floral organ identity, patterning, and development. While collections of floral homeotic mutants of Antirrhinum majus laid the foundation already at the beginning of the previous century, it was the genetic analysis of these mutants in A. majus and Arabidopsis thaliana that led to the development of the ABC model of floral organ identity more than 20 years ago. This intuitive model kick-started research focused on the genetic mechanisms regulating flower development, using mainly A. thaliana as a model plant. In recent years, interactions among floral homeotic proteins have been elucidated, and their direct and indirect target genes are known to a large extent. Here, we provide an overview over the advances in understanding the molecular mechanism orchestrating A. thaliana flower development. We focus on floral homeotic protein complexes, their target genes, evidence for their transport in floral primordia, and how these new results advance our view on the processes downstream of floral organ identity, such as organ boundary formation or floral organ patterning. PMID:25845756

  10. MOEPGA: A novel method to detect protein complexes in yeast protein-protein interaction networks based on MultiObjective Evolutionary Programming Genetic Algorithm.

    PubMed

    Cao, Buwen; Luo, Jiawei; Liang, Cheng; Wang, Shulin; Song, Dan

    2015-10-01

    The identification of protein complexes in protein-protein interaction (PPI) networks has greatly advanced our understanding of biological organisms. Existing computational methods to detect protein complexes are usually based on specific network topological properties of PPI networks. However, due to the inherent complexity of the network structures, the identification of protein complexes may not be fully addressed by using single network topological property. In this study, we propose a novel MultiObjective Evolutionary Programming Genetic Algorithm (MOEPGA) which integrates multiple network topological features to detect biologically meaningful protein complexes. Our approach first systematically analyzes the multiobjective problem in terms of identifying protein complexes from PPI networks, and then constructs the objective function of the iterative algorithm based on three common topological properties of protein complexes from the benchmark dataset, finally we describe our algorithm, which mainly consists of three steps, population initialization, subgraph mutation and subgraph selection operation. To show the utility of our method, we compared MOEPGA with several state-of-the-art algorithms on two yeast PPI datasets. The experiment results demonstrate that the proposed method can not only find more protein complexes but also achieve higher accuracy in terms of fscore. Moreover, our approach can cover a certain number of proteins in the input PPI network in terms of the normalized clustering score. Taken together, our method can serve as a powerful framework to detect protein complexes in yeast PPI networks, thereby facilitating the identification of the underlying biological functions. PMID:26298638

  11. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes.

    PubMed

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  12. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes

    PubMed Central

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  13. Functional Mapping of Protein-Protein Interactions in an Enzyme Complex by Directed Evolution

    PubMed Central

    Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; Sasso, Severin; Gamper, Marianne; Kast, Peter

    2014-01-01

    The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes. PMID:25551646

  14. Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes

    PubMed Central

    Kaiser, Peter; Meierhofer, David; Wang, Xiaorong; Huang, Lan

    2011-01-01

    Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry–based proteomics combined with affinity-tag-based protein purification is one of the most effective strategies to isolate and identify protein complexes. The development of tandem-affinity purification approaches has revolutionized proteomics experiments. These two-step affinity purification strategies allow rapid, effective purification of protein complexes and, at the same time, minimize background. Identification of even very low-abundant protein complexes with modern sensitive mass spectrometers has become routine. Here, we describe two general strategies for tandem-affinity purification followed by mass spectrometric identification of protein complexes. PMID:18370112

  15. Assembly and export of a Toxoplasma microneme complex in Giardia lamblia.

    PubMed

    Gaechter, Verena; Hehl, Adrian B

    2005-11-01

    The microneme proteins of Toxoplasma gondii belong to a large family of adhesins of apicomplexan parasites involved in motility and host cell invasion. During secretory transport, soluble micronemes associate with membrane-bound carriers/escorters and become exposed on the parasite surface as complexes with an array of adhesive domains. Previously, we have exploited the intestinal protozoan Giardia lamblia as an expression system to produce correctly folded and unglycosylated monomeric surface proteins of T. gondii. Here, we report assembly and export of a trimeric microneme (MIC1/4/6) adhesin complex from Toxoplasma. Co-expressed, recombinant microneme proteins were used to investigate structural requirements for microneme complex formation. In addition, export of a microneme subunit induced development of novel Golgi-like compartments demonstrating the existence of post endoplasmic reticulum structures involved in constitutive secretion in this 'Golgi-less' cell. Recreation of the trimeric microneme escorter-cargo system in Giardia is a versatile tool to analyse universal requirements for complex assembly, receptor-ligand interactions and Golgi neogenesis in the basal Giardia secretory system. PMID:16188260

  16. Functional mammalian spliceosomal complex E contains SMN complex proteins in addition to U1 and U2 snRNPs

    PubMed Central

    Makarov, Evgeny M.; Owen, Nicholas; Bottrill, Andrew; Makarova, Olga V.

    2012-01-01

    Spliceosomes remove introns from primary gene transcripts. They assemble de novo on each intron through a series of steps that involve the incorporation of five snRNP particles and multiple non-snRNP proteins. In mammals, all the intermediate complexes have been characterized on one transcript (MINX), with the exception of the very first, complex E. We have purified this complex by two independent procedures using antibodies to either U1-A or PRPF40A proteins, which are known to associate at an early stage of assembly. We demonstrate that the purified complexes are functional in splicing using commitment assays. These complexes contain components expected to be in the E complex and a number of previously unrecognized factors, including survival of motor neurons (SMN) and proteins of the SMN-associated complex. Depletion of the SMN complex proteins from nuclear extracts inhibits formation of the E complex and causes non-productive complexes to accumulate. This suggests that the SMN complex stabilizes the association of U1 and U2 snRNPs with pre-mRNA. In addition, the antibody to PRPF40A precipitated U2 snRNPs from nuclear extracts, indicating that PRPF40A associates with U2 snRNPs. PMID:22110043

  17. Using contrast patterns between true complexes and random subgraphs in PPI networks to predict unknown protein complexes

    PubMed Central

    Liu, Quanzhong; Song, Jiangning; Li, Jinyan

    2016-01-01

    Most protein complex detection methods utilize unsupervised techniques to cluster densely connected nodes in a protein-protein interaction (PPI) network, in spite of the fact that many true complexes are not dense subgraphs. Supervised methods have been proposed recently, but they do not answer why a group of proteins are predicted as a complex, and they have not investigated how to detect new complexes of one species by training the model on the PPI data of another species. We propose a novel supervised method to address these issues. The key idea is to discover emerging patterns (EPs), a type of contrast pattern, which can clearly distinguish true complexes from random subgraphs in a PPI network. An integrative score of EPs is defined to measure how likely a subgraph of proteins can form a complex. New complexes thus can grow from our seed proteins by iteratively updating this score. The performance of our method is tested on eight benchmark PPI datasets and compared with seven unsupervised methods, two supervised and one semi-supervised methods under five standards to assess the quality of the predicted complexes. The results show that in most cases our method achieved a better performance, sometimes significantly. PMID:26868667

  18. Repetitive Sequence Variations in the Promoter Region of the Adhesin-Encoding Gene sabA of Helicobacter pylori Affect Transcription

    PubMed Central

    Harvey, Vivian C.; Acio, Catherine R.; Bredehoft, Amy K.; Zhu, Laurence; Hallinger, Daniel R.; Quinlivan-Repasi, Vanessa; Harvey, Samuel E.

    2014-01-01

    The pathogenesis of diseases elicited by the gastric pathogen Helicobacter pylori is partially determined by the effectiveness of adaptation to the variably acidic environment of the host stomach. Adaptation includes appropriate adherence to the gastric epithelium via outer membrane protein adhesins such as SabA. The expression of sabA is subject to regulation via phase variation in the promoter and coding regions as well as repression by the two-component system ArsRS. In this study, we investigated the role of a homopolymeric thymine [poly(T)] tract −50 to −33 relative to the sabA transcriptional start site in H. pylori strain J99. We quantified sabA expression in H. pylori J99 by quantitative reverse transcription-PCR (RT-PCR), demonstrating significant changes in sabA expression associated with experimental manipulations of poly(T) tract length. Mimicking the length increase of this tract by adding adenines instead of thymines had similar effects, while the addition of other nucleotides failed to affect sabA expression in the same manner. We hypothesize that modification of the poly(T) tract changes DNA topology, affecting regulatory protein interaction(s) or RNA polymerase binding efficiency. Additionally, we characterized the interaction between the sabA promoter region and ArsR, a response regulator affecting sabA expression. Using recombinant ArsR in electrophoretic mobility shift assays (EMSA), we localized binding to a sequence with partial dyad symmetry −20 and +38 relative to the sabA +1 site. The control of sabA expression by both ArsRS and phase variation at two distinct repeat regions suggests the control of sabA expression is both complex and vital to H. pylori infection. PMID:25022855

  19. Sampling small-scale and large-scale conformational changes in proteins and molecular complexes

    NASA Astrophysics Data System (ADS)

    Yun, Mi-Ran; Mousseau, N.; Derreumaux, P.

    2007-03-01

    Sampling of small-scale and large-scale motions is important in various computational tasks, such as protein-protein docking and ligand binding. Here, we report further development and applications of the activation-relaxation technique for internal coordinate space trajectories (ARTIST). This method generates conformational moves of any complexity and size by identifying and crossing well-defined saddle points connecting energy minima. Simulations on two all-atom proteins and three protein complexes containing between 70 and 300 amino acids indicate that ARTIST opens the door to the full treatment of all degrees of freedom in dense systems such as protein-protein complexes.

  20. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.

    PubMed

    Leitner, Alexander; Joachimiak, Lukasz A; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-07-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  1. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

    PubMed Central

    Leitner, Alexander; Joachimiak, Lukasz A.; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-01-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  2. Assessment of Adhesins as an Indicator of Pathovar-Associated Virulence Factors in Bovine Escherichia coli.

    PubMed

    Valat, Charlotte; Forest, Karine; Auvray, Frédéric; Métayer, Véronique; Méheut, Thomas; Polizzi, Charlène; Gay, Emilie; Haenni, Marisa; Oswald, Eric; Madec, Jean-Yves

    2014-12-01

    The CS31A, F17, and F5 adhesins are usually targeted by serology-based methods to detect pathogenic Escherichia coli associated with calf enteritis. However, the virulence traits of the selected isolates are still poorly described. Here, from a set of 349 diarrheagenic E. coli isolates from cattle, we demonstrated a 70.8% concordance rate (Cohen's kappa, 0.599) between serology- and PCR-based approaches for the detection of adhesins under field conditions. A 79% to 82.4% correspondence between the two methods was found for fimbrial adhesins, whereas major discrepancies (33%) were observed for CS31A-type antigens. Various F17A variants were found, such as F17Ac (20K) (50%), F17Aa (FY) (18.9%), F17Ab (8.1%), and F17Ad (111K) (5.4%), including a high proportion (17.6%) of new F17A internal combinations (F17Aab, F17Aac, and F17Abc) or untypeable variants. In addition, the highest proportion of pathovar-associated virulence factor (VF) genes was observed among E. coli isolates that produced F5/F41 adhesins. A specific link between the heat-stable toxins related to the enterotoxigenic E. coli (ETEC) pathovar and adhesins was identified. STa was significantly linked to F5/F41 and EAST1 to CS31A adhesins (P < 0.001), respectively, whereas NTEC was associated with F17 adhesin (P = 0.001). Clustering between phylogroups according to the adhesin types was also observed. Also, few Shiga toxin-producing E. coli (STEC) or enteropathogenic E. coli (EPEC) pathovars were identified. Finally, no statistically significant difference was observed in the occurrence of extended-spectrum beta lactamase (ESBL) production according to the adhesins expressed by the isolates (P = 0.09). Altogether, this study gives new insights into the relationship between adhesins, VF, and antimicrobial resistance in calf enteritis and supports the need for further standardization of methodologies for such approaches. PMID:25217019

  3. Methods for protein complex prediction and their contributions towards understanding the organisation, function and dynamics of complexes.

    PubMed

    Srihari, Sriganesh; Yong, Chern Han; Patil, Ashwini; Wong, Limsoon

    2015-09-14

    Complexes of physically interacting proteins constitute fundamental functional units responsible for driving biological processes within cells. A faithful reconstruction of the entire set of complexes is therefore essential to understand the functional organisation of cells. In this review, we discuss the key contributions of computational methods developed till date (approximately between 2003 and 2015) for identifying complexes from the network of interacting proteins (PPI network). We evaluate in depth the performance of these methods on PPI datasets from yeast, and highlight their limitations and challenges, in particular at detecting sparse and small or sub-complexes and discerning overlapping complexes. We describe methods for integrating diverse information including expression profiles and 3D structures of proteins with PPI networks to understand the dynamics of complex formation, for instance, of time-based assembly of complex subunits and formation of fuzzy complexes from intrinsically disordered proteins. Finally, we discuss methods for identifying dysfunctional complexes in human diseases, an application that is proving invaluable to understand disease mechanisms and to discover novel therapeutic targets. We hope this review aptly commemorates a decade of research on computational prediction of complexes and constitutes a valuable reference for further advancements in this exciting area. PMID:25913176

  4. Cable pili and the 22-kilodalton adhesin are required for Burkholderia cenocepacia binding to and transmigration across the squamous epithelium.

    PubMed

    Urban, Teresa A; Goldberg, Joanna B; Forstner, Janet F; Sajjan, Umadevi S

    2005-09-01

    Burkholderia cenocepacia strains expressing both cable (Cbl) pili and the 22-kDa adhesin bind to cytokeratin 13 (CK13) strongly and invade squamous epithelium efficiently. It has not been established, however, whether the gene encoding the adhesin is located in the cbl operon or what specific contribution the adhesin and Cbl pili lend to binding and transmigration or invasion capacity of B. cenocepacia. By immunoscreening an expression library of B. cenocepacia isolate BC7, we identified a large gene (adhA) that encodes the 22-kDa adhesin. Isogenic mutants lacking expression of either Cbl pili (cblA or cblS mutants) or the adhesin (adhA mutant) were constructed to assess the individual role of Cbl pili and the adhesin in mediating B. cenocepacia binding to and transmigration across squamous epithelium. Relative to the parent strain, mutants of Cbl pili showed reduced binding (50%) to isolated CK13, while the adhesin mutant showed almost no binding (0 to 8%). Mutants lacking either cable pili or the adhesin were compromised in their ability to bind to and transmigrate across the squamous epithelium compared to the wild-type strain, although this deficiency was most pronounced in the adhA mutant. These results indicate that both Cbl pili and the 22-kDa adhesin are necessary for the optimal binding to CK13 and transmigration properties of B. cenocepacia. PMID:16113259

  5. Pneumococcal Adhesins PavB and PspC Are Important for the Interplay with Human Thrombospondin-1.

    PubMed

    Binsker, Ulrike; Kohler, Thomas P; Krauel, Krystin; Kohler, Sylvia; Schwertz, Hansjörg; Hammerschmidt, Sven

    2015-06-01

    The human matricellular glycoprotein thrombospondin-1 (hTSP-1) is released by activated platelets and mediates adhesion of Gram-positive bacteria to various host cells. In staphylococci, the adhesins extracellular adherence protein (Eap) and autolysin (Atl), both surface-exposed proteins containing repeating structures, were shown to be involved in the acquisition of hTSP-1 to the bacterial surface. The interaction partner(s) on the pneumococcal surface was hitherto unknown. Here, we demonstrate for the first time that pneumococcal adherence and virulence factor B (PavB) and pneumococcal surface protein C (PspC) are key players for the interaction of Streptococcus pneumoniae with matricellular hTSP-1. PavB and PspC are pneumococcal surface-exposed adhesins and virulence factors exhibiting repetitive sequences in their core structure. Heterologously expressed fragments of PavB and PspC containing repetitive structures exhibit hTSP-1 binding activity as shown by ELISA and surface plasmon resonance studies. Binding of hTSP-1 is charge-dependent and inhibited by heparin. Importantly, the deficiency in PavB and PspC reduces the recruitment of soluble hTSP-1 by pneumococci and decreases hTSP-1-mediated pneumococcal adherence to human epithelial cells. Platelet activation assays suggested that PavB and PspC are not involved in the activation of purified human platelets by pneumococci. In conclusion, this study indicates a pivotal role of PavB and PspC for pneumococcal recruitment of soluble hTSP-1 to the bacterial surface and binding of pneumococci to host cell-bound hTSP-1 during adhesion. PMID:25897078

  6. Pneumococcal meningitis is promoted by single cocci expressing pilus adhesin RrgA.

    PubMed

    Iovino, Federico; Hammarlöf, Disa L; Garriss, Genevieve; Brovall, Sarah; Nannapaneni, Priyanka; Henriques-Normark, Birgitta

    2016-08-01

    Streptococcus pneumoniae (pneumococcus) is the primary cause of bacterial meningitis. Pneumococcal bacteria penetrates the blood-brain barrier (BBB), but the bacterial factors that enable this process are not known. Here, we determined that expression of pneumococcal pilus-1, which includes the pilus adhesin RrgA, promotes bacterial penetration through the BBB in a mouse model. S. pneumoniae that colonized the respiratory epithelium and grew in the bloodstream were chains of variable lengths; however, the pneumococci that entered the brain were division-competent, spherical, single cocci that expressed adhesive RrgA-containing pili. The cell division protein DivIVA, which is required for an ovoid shape, was localized at the poles and septum of pneumococcal chains of ovoid, nonseparated bacteria, but was absent in spherical, single cocci. In the bloodstream, a small percentage of pneumococci appeared as piliated, RrgA-expressing, DivIVA-negative single cocci, suggesting that only a minority of S. pneumoniae are poised to cross the BBB. Together, our data indicate that small bacterial cell size, which is signified by the absence of DivIVA, and the presence of an adhesive RrgA-containing pilus-1 mediate pneumococcal passage from the bloodstream through the BBB into the brain to cause lethal meningitis. PMID:27348589

  7. Catch-bond mechanism of the bacterial adhesin FimH

    PubMed Central

    Sauer, Maximilian M.; Jakob, Roman P.; Eras, Jonathan; Baday, Sefer; Eriş, Deniz; Navarra, Giulio; Bernèche, Simon; Ernst, Beat; Maier, Timm; Glockshuber, Rudi

    2016-01-01

    Ligand–receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell–cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force. PMID:26948702

  8. Catch-bond mechanism of the bacterial adhesin FimH.

    PubMed

    Sauer, Maximilian M; Jakob, Roman P; Eras, Jonathan; Baday, Sefer; Eriş, Deniz; Navarra, Giulio; Bernèche, Simon; Ernst, Beat; Maier, Timm; Glockshuber, Rudi

    2016-01-01

    Ligand-receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell-cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force. PMID:26948702

  9. Identifying protein complexes from interaction networks based on clique percolation and distance restriction

    PubMed Central

    2010-01-01

    Background Identification of protein complexes in large interaction networks is crucial to understand principles of cellular organization and predict protein functions, which is one of the most important issues in the post-genomic era. Each protein might be subordinate multiple protein complexes in the real protein-protein interaction networks. Identifying overlapping protein complexes from protein-protein interaction networks is a considerable research topic. Result As an effective algorithm in identifying overlapping module structures, clique percolation method (CPM) has a wide range of application in social networks and biological networks. However, the recognition accuracy of algorithm CPM is lowly. Furthermore, algorithm CPM is unfit to identifying protein complexes with meso-scale when it applied in protein-protein interaction networks. In this paper, we propose a new topological model by extending the definition of k-clique community of algorithm CPM and introduced distance restriction, and develop a novel algorithm called CP-DR based on the new topological model for identifying protein complexes. In this new algorithm, the protein complex size is restricted by distance constraint to conquer the shortcomings of algorithm CPM. The algorithm CP-DR is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Conclusion The proposed algorithm CP-DR based on clique percolation and distance restriction makes it possible to identify dense subgraphs in protein interaction networks, a large number of which correspond to known protein complexes. Compared to algorithm CPM, algorithm CP-DR has more outstanding performance. PMID:21047377

  10. The Network Organization of Cancer-associated Protein Complexes in Human Tissues

    NASA Astrophysics Data System (ADS)

    Zhao, Jing; Lee, Sang Hoon; Huss, Mikael; Holme, Petter

    2013-04-01

    Differential gene expression profiles for detecting disease genes have been studied intensively in systems biology. However, it is known that various biological functions achieved by proteins follow from the ability of the protein to form complexes by physically binding to each other. In other words, the functional units are often protein complexes rather than individual proteins. Thus, we seek to replace the perspective of disease-related genes by disease-related complexes, exemplifying with data on 39 human solid tissue cancers and their original normal tissues. To obtain the differential abundance levels of protein complexes, we apply an optimization algorithm to genome-wide differential expression data. From the differential abundance of complexes, we extract tissue- and cancer-selective complexes, and investigate their relevance to cancer. The method is supported by a clustering tendency of bipartite cancer-complex relationships, as well as a more concrete and realistic approach to disease-related proteomics.

  11. Stability and Immunogenicity of Hypoallergenic Peanut Protein-Polyphenol Complexes During In Vitro Pepsin Digestion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Allergenic peanut proteins are relatively resistant to digestion, and if digested, metabolized peptides tend to remain large and immunoreactive, triggering allergic reactions in sensitive individuals. In this study, the stability of hypoallergenic peanut protein-polyphenol complexes was evaluated d...

  12. Stability and Immunogenicity of Hypoallergenic Peanut Protein-Polyphenol Complexes during In Vitro Pepsin Diges

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Allergenic peanut proteins are relatively resistant to digestion, and if digested, metabolized peptides tend to remain large and immunoreactive, triggering allergic reactions in sensitive individuals. In this study, the stability of hypoallergenic peanut protein-polyphenol complexes was evaluated d...

  13. Folding analysis of the most complex Stevedore's protein knot.

    PubMed

    Wang, Iren; Chen, Szu-Yu; Hsu, Shang-Te Danny

    2016-01-01

    DehI is a homodimeric haloacid dehalogenase from Pseudomonas putida that contains the most complex 61 Stevedore's protein knot within its folding topology. To examine how DehI attains such an intricate knotted topology we combined far-UV circular dichroism (CD), intrinsic fluorescence spectroscopy and small angle X-ray scattering (SAXS) to investigate its folding mechanism. Equilibrium unfolding of DehI by chemical denaturation indicated the presence of two highly populated folding intermediates, I and I'. While the two intermediates vary in secondary structure contents and tertiary packing according to CD and intrinsic fluorescence, respectively, their overall dimension and compactness are similar according to SAXS. Three single-tryptophan variants (W34, W53, and W196) were generated to probe non-cooperative unfolding events localized around the three fluorophores. Kinetic fluorescence measurements indicated that the transition from the intermediate I' to the unfolded state is rate limiting. Our multiparametric folding analyses suggest that DehI unfolds through a linear folding pathway with two distinct folding intermediates by initial hydrophobic collapse followed by nucleation condensation, and that knotting precedes the formation of secondary structures. PMID:27527519

  14. Assembly of nuclear pore complexes mediated by major vault protein.

    PubMed

    Vollmar, Friederike; Hacker, Christian; Zahedi, René-Peiman; Sickmann, Albert; Ewald, Andrea; Scheer, Ulrich; Dabauvalle, Marie-Christine

    2009-03-15

    During interphase growth of eukaryotic cells, nuclear pore complexes (NPCs) are continuously incorporated into the intact nuclear envelope (NE) by mechanisms that are largely unknown. De novo formation of NPCs involves local fusion events between the inner and outer nuclear membrane, formation of a transcisternal membranous channel of defined diameter and the coordinated assembly of hundreds of nucleoporins into the characteristic NPC structure. Here we have used a cell-free system based on Xenopus egg extract, which allows the experimental separation of nuclear-membrane assembly and NPC formation. Nuclei surrounded by a closed double nuclear membrane, but devoid of NPCs, were first reconstituted from chromatin and a specific membrane fraction. Insertion of NPCs into the preformed pore-free nuclei required cytosol containing soluble nucleoporins or nucleoporin subcomplexes and, quite unexpectedly, major vault protein (MVP). MVP is the main component of vaults, which are ubiquitous barrel-shaped particles of enigmatic function. Our results implicate MVP, and thus also vaults, in NPC biogenesis and provide a functional explanation for the association of a fraction of vaults with the NE and specifically with NPCs in intact cells. PMID:19240118

  15. Evidence for a vasopressin receptor-GTP binding protein complex

    SciTech Connect

    Fitzgerald, T.J.; Uhing, R.J.; Exton, J.H.

    1986-05-01

    Plasma membranes from the livers of rats were able to hydrolyze the ..gamma..-phosphate from guanosine-5'-triphosphate (GTP). The rate of GTP hydrolysis could be decreased to 10% of its initial rate by the addition of adenosine-5'-triphosphate with a concomitant decrease in the K/sub m/ for GTP from approx. 10/sup -3/ M to 10/sup -6/ M. The low K/sub m/ GTPase activity was inhibited by the addition of nonhydrolyzable analogs of GTP. In addition, the GTPase activity was stimulated from 10 to 30% over basal by the addition of vasopressin. A dose dependency curve showed that the maximum stimulation was obtained with 10/sup -8/ M vasopressin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized in the presence of (Phenylalanyl-3,4,5-/sup 3/H(N))vasopressin were subjected to sucrose gradient centrifugation, the majority of bound (/sup 3/H)vasopressin migrated with an approximate molecular weight of 300,000. Moreover, there was a GTPase activity that migrated with the bound (/sup 3/H)vasopressin. This peak of bound (/sup 3/H)vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10/sup -5/ M guanosine-5'-O-(3-thiotriphosphate). These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.

  16. 193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology.

    PubMed

    Morrison, Lindsay J; Brodbelt, Jennifer S

    2016-08-31

    Protein-protein interfaces and architecture are critical to the function of multiprotein complexes. Mass spectrometry-based techniques have emerged as powerful strategies for characterization of protein complexes, particularly for heterogeneous mixtures of structures. In the present study, activation and dissociation of three tetrameric protein complexes (streptavidin, transthyretin, and hemoglobin) in the gas phase was undertaken by 193 nm ultraviolet photodissociation (UVPD) for the characterization of higher order structure. High pulse energy UVPD resulted in the production of dimers and low charged monomers exhibiting symmetrical charge partitioning among the subunits (the so-called symmetrical dissociation pathways), consistent with the subunit organization of the complexes. In addition, UVPD promoted backbone cleavages of the monomeric subunits, the abundances of which corresponded to the more flexible loop regions of the proteins. PMID:27480400

  17. The Staphylococcal Biofilm: Adhesins, Regulation, and Host Response.

    PubMed

    Paharik, Alexandra E; Horswill, Alexander R

    2016-04-01

    The staphylococci comprise a diverse genus of Gram-positive, nonmotile commensal organisms that inhabit the skin and mucous membranes of humans and other mammals. In general, staphylococci are benign members of the natural flora, but many species have the capacity to be opportunistic pathogens, mainly infecting individuals who have medical device implants or are otherwise immunocompromised. Staphylococcus aureus and Staphylococcus epidermidis are major sources of hospital-acquired infections and are the most common causes of surgical site infections and medical device-associated bloodstream infections. The ability of staphylococci to form biofilms in vivo makes them highly resistant to chemotherapeutics and leads to chronic diseases. These biofilm infections include osteomyelitis, endocarditis, medical device infections, and persistence in the cystic fibrosis lung. Here, we provide a comprehensive analysis of our current understanding of staphylococcal biofilm formation, with an emphasis on adhesins and regulation, while also addressing how staphylococcal biofilms interact with the immune system. On the whole, this review will provide a thorough picture of biofilm formation of the staphylococcus genus and how this mode of growth impacts the host. PMID:27227309

  18. Sticky Matrix: Adhesion Mechanism of the Staphylococcal Polysaccharide Intercellular Adhesin.

    PubMed

    Formosa-Dague, Cécile; Feuillie, Cécile; Beaussart, Audrey; Derclaye, Sylvie; Kucharíková, Soňa; Lasa, Iñigo; Van Dijck, Patrick; Dufrêne, Yves F

    2016-03-22

    The development of bacterial biofilms on surfaces leads to hospital-acquired infections that are difficult to fight. In Staphylococci, the cationic polysaccharide intercellular adhesin (PIA) forms an extracellular matrix that connects the cells together during biofilm formation, but the molecular forces involved are unknown. Here, we use advanced force nanoscopy techniques to unravel the mechanism of PIA-mediated adhesion in a clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strain. Nanoscale multiparametric imaging of the structure, adhesion, and elasticity of bacteria expressing PIA shows that the cells are surrounded by a soft and adhesive matrix of extracellular polymers. Cell surface softness and adhesion are dramatically reduced in mutant cells deficient for the synthesis of PIA or under unfavorable growth conditions. Single-cell force spectroscopy demonstrates that PIA promotes cell-cell adhesion via the multivalent electrostatic interaction with polyanionic teichoic acids on the S. aureus cell surface. This binding mechanism rationalizes, at the nanoscale, the well-known ability of PIA to strengthen intercellular adhesion in staphylococcal biofilms. Force nanoscopy offers promising prospects for understanding the fundamental forces in antibiotic-resistant biofilms and for designing anti-adhesion compounds targeting matrix polymers. PMID:26908275

  19. Qualitative and Quantitative Protein Complex Prediction Through Proteome-Wide Simulations.

    PubMed

    Rizzetto, Simone; Priami, Corrado; Csikász-Nagy, Attila

    2015-10-01

    Despite recent progress in proteomics most protein complexes are still unknown. Identification of these complexes will help us understand cellular regulatory mechanisms and support development of new drugs. Therefore it is really important to establish detailed information about the composition and the abundance of protein complexes but existing algorithms can only give qualitative predictions. Herein, we propose a new approach based on stochastic simulations of protein complex formation that integrates multi-source data--such as protein abundances, domain-domain interactions and functional annotations--to predict alternative forms of protein complexes together with their abundances. This method, called SiComPre (Simulation based Complex Prediction), achieves better qualitative prediction of yeast and human protein complexes than existing methods and is the first to predict protein complex abundances. Furthermore, we show that SiComPre can be used to predict complexome changes upon drug treatment with the example of bortezomib. SiComPre is the first method to produce quantitative predictions on the abundance of molecular complexes while performing the best qualitative predictions. With new data on tissue specific protein complexes becoming available SiComPre will be able to predict qualitative and quantitative differences in the complexome in various tissue types and under various conditions. PMID:26492574

  20. Affinity proteomics to study endogenous protein complexes: Pointers, pitfalls, preferences and perspectives

    PubMed Central

    LaCava, John; Molloy, Kelly R.; Taylor, Martin S.; Domanski, Michal; Chait, Brian T.; Rout, Michael P.

    2015-01-01

    Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls. PMID:25757543

  1. High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

    PubMed Central

    2012-01-01

    Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

  2. The identification of a heat-shock protein complex in chloroplasts of barley leaves.

    PubMed

    Clarke, A K; Critchley, C

    1992-12-01

    In vivo radiolabeling of chloroplast proteins in barley (Hordeum vulgare L. cv Corvette) leaves and their separation by one-dimensional electrophoresis revealed at least seven heat-shock proteins between 24 and 94 kD, of which most have not been previously identified in this C(3) species. Fractionation into stromal and thylakoid membrane components showed that all chloroplast heat-shock proteins were synthesized on cytoplasmic ribosomes, translocated into the chloroplast, and located in the stroma. Examination of stromal preparations by native (nondissociating) polyacrylamide gel electrophoresis revealed the presence of a high-molecular mass heat-shock protein complex in barley. This complex was estimated to be 250 to 265 kD in size. Dissociation by denaturing polyacrylamide gel electrophoresis revealed a single protein component, a 32-kD heat-shock protein. The synthesis of this protein and the formation of the heat-shock protein complex were dependent on functional cytoplasmic ribosomes. Immunological studies showed that the heat-shock protein complex did not contain any proteins homologous to the alpha-subunit of ribulose bisphosphate carboxylase oxygenase subunit-binding protein. Other features about the complex included the absence of nucleic acid (RNA or DNA) and its nondissociation in the presence of Mg(2+)/ATP. These results suggest that the heat-shock protein complex in barley chloroplasts is a homogeneous octamer of 32-kD subunits. PMID:16653243

  3. The Identification of a Heat-Shock Protein Complex in Chloroplasts of Barley Leaves 1

    PubMed Central

    Clarke, Adrian K.; Critchley, Christa

    1992-01-01

    In vivo radiolabeling of chloroplast proteins in barley (Hordeum vulgare L. cv Corvette) leaves and their separation by one-dimensional electrophoresis revealed at least seven heat-shock proteins between 24 and 94 kD, of which most have not been previously identified in this C3 species. Fractionation into stromal and thylakoid membrane components showed that all chloroplast heat-shock proteins were synthesized on cytoplasmic ribosomes, translocated into the chloroplast, and located in the stroma. Examination of stromal preparations by native (nondissociating) polyacrylamide gel electrophoresis revealed the presence of a high-molecular mass heat-shock protein complex in barley. This complex was estimated to be 250 to 265 kD in size. Dissociation by denaturing polyacrylamide gel electrophoresis revealed a single protein component, a 32-kD heat-shock protein. The synthesis of this protein and the formation of the heat-shock protein complex were dependent on functional cytoplasmic ribosomes. Immunological studies showed that the heat-shock protein complex did not contain any proteins homologous to the α-subunit of ribulose bisphosphate carboxylase oxygenase subunit-binding protein. Other features about the complex included the absence of nucleic acid (RNA or DNA) and its nondissociation in the presence of Mg2+/ATP. These results suggest that the heat-shock protein complex in barley chloroplasts is a homogeneous octamer of 32-kD subunits. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:16653243

  4. Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*

    PubMed Central

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-01-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  5. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    PubMed

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  6. PRISM: a web server and repository for prediction of protein-protein interactions and modeling their 3D complexes.

    PubMed

    Baspinar, Alper; Cukuroglu, Engin; Nussinov, Ruth; Keskin, Ozlem; Gursoy, Attila

    2014-07-01

    The PRISM web server enables fast and accurate prediction of protein-protein interactions (PPIs). The prediction algorithm is knowledge-based. It combines structural similarity and accounts for evolutionary conservation in the template interfaces. The predicted models are stored in its repository. Given two protein structures, PRISM will provide a structural model of their complex if a matching template interface is available. Users can download the complex structure, retrieve the interface residues and visualize the complex model. The PRISM web server is user friendly, free and open to all users at http://cosbi.ku.edu.tr/prism. PMID:24829450

  7. Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability

    PubMed Central

    Murchie, Ryan; Guo, Cong-Hui; Persaud, Avinash; Muise, Aleixo; Rotin, Daniela

    2014-01-01

    Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ−/− mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ−/− mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ−/− mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper–tyrosine-phosphorylated in the PTPσ−/− mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ−/− mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD. PMID:24385580

  8. Observation of two different fractal structures in nanoparticle, protein and surfactant complexes

    SciTech Connect

    Mehan, Sumit Kumar, Sugam Aswal, V. K.

    2014-04-24

    Small angle neutron scattering has been carried out from a complex of nanoparticle, protein and surfactant. Although all the components are similarly (anionic) charged, we have observed strong interactions in their complex formation. It is characterized by the coexistence of two different mass fractal structures. The first fractal structure is originated from the protein and surfactant interaction and second from the depletion effect of first fractal structure leading the nanoparticle aggregation. The fractal structure of protein-surfactant complex represents to bead necklace structure of micelle-like clusters of surfactant formed along the unfolded protein chain. Its fractal dimension depends on the surfactant to protein ratio (r) and decreases with the increase in r. However, fractal dimension of nanoparticle aggregates in nanoparticle-protein complex is found to be independent of protein concentration and governed by the diffusion limited aggregation like morphology.

  9. Observation of two different fractal structures in nanoparticle, protein and surfactant complexes

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Kumar, Sugam; Aswal, V. K.

    2014-04-01

    Small angle neutron scattering has been carried out from a complex of nanoparticle, protein and surfactant. Although all the components are similarly (anionic) charged, we have observed strong interactions in their complex formation. It is characterized by the coexistence of two different mass fractal structures. The first fractal structure is originated from the protein and surfactant interaction and second from the depletion effect of first fractal structure leading the nanoparticle aggregation. The fractal structure of protein-surfactant complex represents to bead necklace structure of micelle-like clusters of surfactant formed along the unfolded protein chain. Its fractal dimension depends on the surfactant to protein ratio (r) and decreases with the increase in r. However, fractal dimension of nanoparticle aggregates in nanoparticle-protein complex is found to be independent of protein concentration and governed by the diffusion limited aggregation like morphology.

  10. Identification and characterization of a surface protein-releasing activity in Streptococcus mutans and other pathogenic streptococci.

    PubMed Central

    Lee, S F

    1992-01-01

    Surface proteins of Streptococcus mutans have been reported to be released into the culture filtrate at concentrations that vary with the growth conditions. The reason for this is not clear. The present study attempts to investigate the mechanism of the protein release. The results showed that whole cells and raffinose-stabilized protoplasts of S. mutans NG8, when incubated in buffers, were capable of releasing their surface proteins in a pH-dependent manner with optimal release at pH 5 to 6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the released proteins were very complex. Two proteins, adhesin P1, which has been previously shown to interact with a human salivary agglutinin, and glucosyltransferase have been identified among the released proteins. The release of adhesin P1 and other proteins was found to be inhibited by heat, Cu2+,Zn2+, and thiol-blocking reagents. The inhibition by heat and Cu2+ was irreversible, whereas that by the thiol-blocking reagents was reversible. EDTA, phenylmethylsulfonyl fluoride, and N-p-tosyl-L-lysyl-chloromethyl ketone had no effect on the release of P1, indicating that the release was probably not due to proteolytic activity. Adhesin P1 from Cu(2+)-inactivated S. mutans NG8 protoplasts could be released by mixing with fresh whole cells and protoplasts, but not the culture filtrate, of a P1-negative mutant of NG8, suggesting that the enzyme is located on the cell surface. This P1-releasing activity was also detected in two other strains of S. mutans and one strain each of S. gordonii, S. agalactiae, S. pneumoniae, and S. pyogenes. The biological role(s) of this enzyme activity remains to be determined. However, owing to its ability to release virulent surface proteins from the cell, it may play an important role in cell surface modulation among the pathogenic streptococci. Images PMID:1398915

  11. Identification and characterization of a surface protein-releasing activity in Streptococcus mutans and other pathogenic streptococci.

    PubMed

    Lee, S F

    1992-10-01

    Surface proteins of Streptococcus mutans have been reported to be released into the culture filtrate at concentrations that vary with the growth conditions. The reason for this is not clear. The present study attempts to investigate the mechanism of the protein release. The results showed that whole cells and raffinose-stabilized protoplasts of S. mutans NG8, when incubated in buffers, were capable of releasing their surface proteins in a pH-dependent manner with optimal release at pH 5 to 6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the released proteins were very complex. Two proteins, adhesin P1, which has been previously shown to interact with a human salivary agglutinin, and glucosyltransferase have been identified among the released proteins. The release of adhesin P1 and other proteins was found to be inhibited by heat, Cu2+,Zn2+, and thiol-blocking reagents. The inhibition by heat and Cu2+ was irreversible, whereas that by the thiol-blocking reagents was reversible. EDTA, phenylmethylsulfonyl fluoride, and N-p-tosyl-L-lysyl-chloromethyl ketone had no effect on the release of P1, indicating that the release was probably not due to proteolytic activity. Adhesin P1 from Cu(2+)-inactivated S. mutans NG8 protoplasts could be released by mixing with fresh whole cells and protoplasts, but not the culture filtrate, of a P1-negative mutant of NG8, suggesting that the enzyme is located on the cell surface. This P1-releasing activity was also detected in two other strains of S. mutans and one strain each of S. gordonii, S. agalactiae, S. pneumoniae, and S. pyogenes. The biological role(s) of this enzyme activity remains to be determined. However, owing to its ability to release virulent surface proteins from the cell, it may play an important role in cell surface modulation among the pathogenic streptococci. PMID:1398915

  12. Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

    PubMed Central

    2015-01-01

    Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein

  13. Detection and identification of protein citrullination in complex biological systems.

    PubMed

    Clancy, Kathleen W; Weerapana, Eranthie; Thompson, Paul R

    2016-02-01

    Protein citrullination is a post-translational modification of arginine that is catalyzed by the Protein Arginine Deiminase (PAD) family of enzymes. Aberrantly increased citrullination is associated with a host of inflammatory diseases and cancer and PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis, lupus, atherosclerosis, and ulcerative colitis. In rheumatoid arthritis, citrullinated proteins serve as key antigens for rheumatoid arthritis-associated autoantibodies. These data suggest that citrullinated proteins may serve more generally as biomarkers of specific disease states, however, the identification of citrullinated residues remains challenging due to the small 1Da mass change that occurs upon citrullination. Herein, we highlight the available techniques to identify citrullinated proteins/residues focusing on advanced MS techniques as well as chemical derivatization strategies that are currently being employed to identify citrullinated proteins as well as the specific residues modified within those proteins. PMID:26517730

  14. Responses of two protein-protein complexes to solvent stress: does water play a role at the interface?

    PubMed Central

    Kornblatt, J A; Kornblatt, M J; Hoa, G H; Mauk, A G

    1993-01-01

    We have analyzed the stability of the cytochrome c-cytochrome b5 and cytochrome c-cytochrome c oxidase complexes as a function of solvent stress. High concentrations of glycerol were used to displace the two equilibria. Glycerol promotes complex formation between cytochrome c and cytochrome b5 but inhibits that between cytochrome c and cytochrome c oxidase. The results with cytochrome b5 and cytochrome c were expected; the association of this complex is largely entropy driven. Our interpretation is that the cytochrome c-cytochrome b5 complex excludes water. The results with the cytochrome c oxidase and cytochrome c couple were not expected. We interpret them to mean that either glycerol is binding to the oxidase, thereby displacing the cytochrome c, or that water is required at this protein-protein interface. A requirement for substantial quantities of water at the interface of some protein complexes is logical but has been reported only once. PMID:8241386

  15. Structural Features of the Pseudomonas fluorescens Biofilm Adhesin LapA Required for LapG-Dependent Cleavage, Biofilm Formation, and Cell Surface Localization

    PubMed Central

    Boyd, Chelsea D.; Smith, T. Jarrod; El-Kirat-Chatel, Sofiane; Newell, Peter D.; Dufrêne, Yves F.

    2014-01-01

    The localization of the LapA protein to the cell surface is a key step required by Pseudomonas fluorescens Pf0-1 to irreversibly attach to a surface and form a biofilm. LapA is a member of a diverse family of predicted bacterial adhesins, and although lacking a high degree of sequence similarity, family members do share common predicted domains. Here, using mutational analysis, we determine the significance of each domain feature of LapA in relation to its export and localization to the cell surface and function in biofilm formation. Our previous work showed that the N terminus of LapA is required for cleavage by the periplasmic cysteine protease LapG and release of the adhesin from the cell surface under conditions unfavorable for biofilm formation. We define an additional critical region of the N terminus of LapA required for LapG proteolysis. Furthermore, our results suggest that the domains within the C terminus of LapA are not absolutely required for biofilm formation, export, or localization to the cell surface, with the exception of the type I secretion signal, which is required for LapA export and cell surface localization. In contrast, deletion of the central repetitive region of LapA, consisting of 37 repeats of 100 amino acids, results in an inability to form a biofilm. We also used single-molecule atomic force microscopy to further characterize the role of these domains in biofilm formation on hydrophobic and hydrophilic surfaces. These studies represent the first detailed analysis of the domains of the LapA family of biofilm adhesin proteins. PMID:24837291

  16. Isolation of Human Mitotic Protein Phosphatase Complexes: Identification of a Complex between Protein Phosphatase 1 and the RNA Helicase Ddx21

    PubMed Central

    De Wever, Veerle; Lloyd, David C.; Nasa, Isha; Nimick, Mhairi; Trinkle-Mulcahy, Laura; Gourlay, Robert; Morrice, Nick; Moorhead, Greg B. G.

    2012-01-01

    Metazoan mitosis requires remodelling of sub-cellular structures to ensure proper division of cellular and genetic material. Faults often lead to genomic instability, cell cycle arrests and disease onset. These key structural changes are under tight spatial-temporal and post-translational control, with crucial roles for reversible protein phosphorylation. The phosphoprotein phosphatases PP1 and PP2A are paramount for the timely execution of mitotic entry and exit but their interaction partners and substrates are still largely unresolved. High throughput, mass-spectrometry based studies have limited sensitivity for the detection of low-abundance and transient complexes, a typical feature of many protein phosphatase complexes. Moreover, the limited timeframe during which mitosis takes place reduces the likelihood of identifying mitotic phosphatase complexes in asynchronous cells. Hence, numerous mitotic protein phosphatase complexes still await identification. Here we present a strategy to enrich and identify serine/threonine protein phosphatase complexes at the mitotic spindle. We thus identified a nucleolar RNA helicase, Ddx21/Gu, as a novel, direct PP1 interactor. Furthermore, our results place PP1 within the toposome, a Topoisomerase II alpha (TOPOIIα) containing complex with a key role in mitotic chromatin regulation and cell cycle progression, possibly via regulated protein phosphorylation. This study provides a strategy for the identification of further mitotic PP1 partners and the unravelling of PP1 functions during mitosis. PMID:22761809

  17. Gold Nanocluster and Quantum Dot Complex in Protein for Biofriendly White-Light-Emitting Material.

    PubMed

    Bhandari, Satyapriya; Pramanik, Sabyasachi; Khandelia, Rumi; Chattopadhyay, Arun

    2016-01-27

    We report the synthesis of a biofriendly highly luminescent white-light-emitting nanocomposite. The composite consisted of Au nanoclusters and ZnQ2 complex (on the surface of ZnS quantum dots) embedded in protein. The combination of red, green, and blue luminescence from clusters, complex, and protein, respectively, led to white light generation. PMID:26741861

  18. Tuning structure of oppositely charged nanoparticle and protein complexes

    SciTech Connect

    Kumar, Sugam Aswal, V. K.; Callow, P.

    2014-04-24

    Small-angle neutron scattering (SANS) has been used to probe the structures of anionic silica nanoparticles (LS30) and cationic lyszyme protein (M.W. 14.7kD, I.P. ∼ 11.4) by tuning their interaction through the pH variation. The protein adsorption on nanoparticles is found to be increasing with pH and determined by the electrostatic attraction between two components as well as repulsion between protein molecules. We show the strong electrostatic attraction between nanoparticles and protein molecules leads to protein-mediated aggregation of nanoparticles which are characterized by fractal structures. At pH 5, the protein adsorption gives rise to nanoparticle aggregation having surface fractal morphology with close packing of nanoparticles. The surface fractals transform to open structures of mass fractal morphology at higher pH (7 and 9) on approaching isoelectric point (I.P.)

  19. Another Role of Proline: Stabilization Interactions in Proteins and Protein Complexes Concerning Proline and Tryptophane

    SciTech Connect

    Biedermannova, Lada; Riley, Kevin E.; Berka, Karel; Hobza, Pavel; Vondrasek, Jiri

    2008-09-11

    Proline–tryptophan complexes derived from experimental structures are investigated by quantum chemical procedures known to properly describe the London dispersion energy. We study two geometrical arrangements: the “L-shaped”, stabilized by an H-bond, and the “stacked-like”, where the two residues are in parallel orientation without any H-bond. Interestingly, the interaction energies in both cases are comparable and very large (~7 kcal mol⁻¹). The strength of stabilization in the stacked arrangement is rather surprising considering the fact that only one partner has an aromatic character. The interaction energy decomposition using the SAPT method further demonstrates the very important role of dispersion energy in such arrangement. To elucidate the structural features responsible for this unexpectedly large stabilization we examined the role of the nitrogen heteroatom and the importance of the cyclicity of the proline residue. We show that the electrostatic interaction due to the presence of the dipole, caused by the nitrogen heteroatom, contributes largely to the strength of the interaction. Nevertheless, the cyclic arrangement of proline, which allows for the largest amount of dispersive contact with the aromatic partner, also has a notable-effect. Geometry optimizations carried out for the “stackedlike” complexes show that the arrangements derived from protein structure are close to their gas phase optimum geometry, suggesting that the environment has only a minor effect on the geometry of the interaction. We conclude that the strength of proline non-covalent interactions, combined with this residue’s rigidity, might be the explanation for its prominent role in protein stabilization and recognition processes.

  20. Theory of polyelectrolyte adsorption on heterogeneously charged surfaces applied to soluble protein-polyelectrolyte complexes

    NASA Astrophysics Data System (ADS)

    de Vries, R.; Weinbreck, F.; de Kruif, C. G.

    2003-03-01

    Existing theoretical approaches to polymer adsorption on heterogeneous surfaces are applied to the problems of polyelectrolyte and polyampholyte adsorption on randomly charged surfaces. Also, analytical estimates are developed for the critical pH at which weakly charged polyelectrolytes and globular proteins start forming soluble complexes. Below a critical salt concentration, soluble complexes form "on the wrong side" of the protein isoelectric point due to the heterogeneity of the protein surface charge distribution. The analytical estimates are consistent with experimental data on soluble complexes in mixtures of gum arabic and whey protein isolate.

  1. MICAL-Family Proteins: Complex Regulators of the Actin Cytoskeleton

    PubMed Central

    Giridharan, Sai Srinivas Panapakkam

    2014-01-01

    Abstract Significance: The molecules interacting with CasL (MICAL) family members participate in a multitude of activities, including axonal growth cone repulsion, membrane trafficking, apoptosis, and bristle development in flies. An interesting feature of MICAL proteins is the presence of an N-terminal flavo-mono-oxygenase domain. This mono-oxygenase domain generates redox potential with which MICALs can either oxidize proteins or produce reactive oxygen species (ROS). Actin is one such protein that is affected by MICAL function, leading to dramatic cytoskeletal rearrangements. This review describes the MICAL-family members, and discusses their mechanisms of actin-binding and regulation of actin cytoskeleton organization. Recent Advances: Recent studies show that MICALs directly induce oxidation of actin molecules, leading to actin depolymerization. ROS production by MICALs also causes oxidation of collapsin response mediator protein-2, a microtubule assembly promoter, which subsequently undergoes phosphorylation. Critical Issues: MICAL proteins oxidize proteins through two mechanisms: either directly by oxidizing methionine residues or indirectly via the production of ROS. It remains unclear whether MICAL proteins employ both mechanisms or whether the activity of MICAL-family proteins might vary with different substrates. Future Directions: The identification of additional substrates oxidized by MICAL will shed new light on MICAL protein function. Additional directions include expanding studies toward the MICAL-like homologs that lack flavin adenine dinucleotide domains and oxidation activity. Antioxid. Redox Signal. 20, 2059–2073. PMID:23834433

  2. Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex.

    PubMed

    Lee, In Hye; Lim, Hee Jung; Yoon, Suhyeon; Seong, Je Kyung; Bae, Duk Soo; Rhee, Sue Goo; Bae, Yun Soo

    2008-03-01

    We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase Cgamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A. PMID:18174170

  3. The electron conduction of photosynthetic protein complexes embedded in a membrane.

    PubMed

    Stamouli, A; Frenken, J W M; Oosterkamp, T H; Cogdell, R J; Aartsma, T J

    2004-02-27

    The conductivity of two photosynthetic protein-pigment complexes, a light harvesting 2 complex and a reaction center, was measured with an atomic force microscope capable of performing electrical measurements. Current-voltage measurements were performed on complexes embedded in their natural environment. Embedding the complexes in lipid bilayers made it possible to discuss the different conduction behaviors of the two complexes in light of their atomic structure. PMID:14988007

  4. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    NASA Astrophysics Data System (ADS)

    Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

    2014-07-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

  5. Determination of the structure and morphology of gold nanoparticle-HSA protein complexes

    NASA Astrophysics Data System (ADS)

    Capomaccio, Robin; Ojea Jimenez, Isaac; Colpo, Pascal; Gilliland, Douglas; Ceccone, Giacomo; Rossi, François; Calzolai, Luigi

    2015-10-01

    We propose a simple method to determine the structure and morphology of nanoparticle protein complexes. By combining a separation method with online size measurements, density measurements and circular dichroism, we could identify the number of proteins bound to each nanoparticle and their secondary structure changes in the complex. This method provides much-needed experimental information on the interaction of proteins with nanoparticles and on the behavior of nanoparticles in biological systems.We propose a simple method to determine the structure and morphology of nanoparticle protein complexes. By combining a separation method with online size measurements, density measurements and circular dichroism, we could identify the number of proteins bound to each nanoparticle and their secondary structure changes in the complex. This method provides much-needed experimental information on the interaction of proteins with nanoparticles and on the behavior of nanoparticles in biological systems. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05147a

  6. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2.

    PubMed

    Kovács, Krisztián A; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J; Cardinaux, Jean-René

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. PMID:26247811

  7. Immunogenicity of a prototype enterotoxigenic Escherichia coli adhesin vaccine in mice and nonhuman primates.

    PubMed

    Sincock, Stephanie A; Hall, Eric R; Woods, Colleen M; O'Dowd, Aisling; Poole, Steven T; McVeigh, Annette L; Nunez, Gladys; Espinoza, Nereyda; Miller, Milagros; Savarino, Stephen J

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) are the most common cause of bacterial diarrhea in young children in developing countries and in travelers. Efforts to develop an ETEC vaccine have intensified in the past decade, and intestinal colonization factors (CFs) are somatic components of most investigational vaccines. CFA/I and related Class 5 fimbrial CFs feature a major stalk-forming subunit and a minor, antigenically conserved tip adhesin. We hypothesized that the tip adhesin is critical for stimulating antibodies that specifically inhibit ETEC attachment to the small intestine. To address this, we compared the capacity of donor strand complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, and CFA/I fimbriae to elicit anti-adhesive antibodies in mice, using hemagglutination inhibition (HAI) as proxy for neutralization of intestinal adhesion. When given with genetically attenuated heat-labile enterotoxin LTR192G as adjuvant by intranasal (IN) or orogastric (OG) vaccination, dscCfaE exceeded CFA/I fimbriae in eliciting serum HAI titers and anti-CfaE antibody titers. Based on these findings, we vaccinated Aotus nancymaae nonhuman primates (NHP) with dscCfaE alone or admixed with one of two adjuvants, LTR192G and cholera toxin B-subunit, by IN and OG administration. Only IN vaccination with dscCfaE with either adjuvant elicited substantial serum HAI titers and IgA and IgG anti-adhesin responses, with the latter detectable a year after vaccination. In conclusion, we have shown that dscCfaE elicits robust HAI and anti-adhesin antibody responses in both mice and NHPs when given with adjuvant by IN vaccination, encouraging further evaluation of an ETEC adhesin-based vaccine approach. PMID:26597148

  8. The heat-resistant agglutinin family includes a novel adhesin from enteroaggregative Escherichia coli strain 60A.

    PubMed

    Mancini, Justin; Weckselblatt, Brooke; Chung, Yoonjie K; Durante, Julia C; Andelman, Steven; Glaubman, Jessica; Dorff, Justin D; Bhargava, Samhita; Lijek, Rebeccah S; Unger, Katherine P; Okeke, Iruka N

    2011-09-01

    Heat-resistant agglutinin 1 (Hra1) is an accessory colonization factor of enteroaggregative Escherichia coli (EAEC) strain 042. Tia, a close homolog of Hra1, is an invasin and adhesin that has been described in enterotoxigenic E. coli. We devised a PCR-restriction fragment length polymorphism screen for the associated genes and found that they occur among 55 (36.7%) of the enteroaggregative E. coli isolates screened, as well as lower proportions of enterotoxigenic, enteropathogenic, enterohemorrhagic, and commensal E. coli isolates. Overall, 25%, 8%, and 3% of 150 EAEC strains harbored hra1 alone, tia alone, or both genes, respectively. One EAEC isolate, 60A, produced an amplicon with a unique restriction profile, distinct from those of hra1 and tia. We cloned and sequenced the full-length agglutinin gene from strain 60A and have designated it hra2. The hra2 gene was not detected in any of 257 diarrheagenic E. coli isolates in our collection but is present in the genome of Salmonella enterica serovar Heidelberg strain SL476. The cloned hra2 gene from strain 60A, which encodes a predicted amino acid sequence that is 64% identical to that of Hra1 and 68% identical to that of Tia, was sufficient to confer adherence on E. coli K-12. We constructed an hra2 deletion mutant of EAEC strain 60A. The mutant was deficient in adherence but not autoaggregation or invasion, pointing to a functional distinction from the autoagglutinin Hra1 and the Tia invasin. Hra1, Tia, and the novel accessory adhesin Hra2 are members of a family of integral outer membrane proteins that confer different colonization-associated phenotypes. PMID:21764925

  9. Production of unstable proteins through the formation of stable core complexes

    PubMed Central

    Levy, Nicolas; Eiler, Sylvia; Pradeau-Aubreton, Karine; Maillot, Benoit; Stricher, François; Ruff, Marc

    2016-01-01

    Purification of proteins that participate in large transient complexes is impeded by low amounts, heterogeneity, instability and poor solubility. To circumvent these difficulties we set up a methodology that enables the production of stable complexes for structural and functional studies. This procedure is benchmarked and applied to two challenging protein families: the human steroid nuclear receptors (SNR) and the HIV-1 pre-integration complex. In the context of transcriptional regulation studies, we produce and characterize the ligand-binding domains of the glucocorticoid nuclear receptor and the oestrogen receptor beta in complex with a TIF2 (transcriptional intermediary factor 2) domain containing the three SNR-binding motifs. In the context of retroviral integration, we demonstrate the stabilization of the HIV-1 integrase by formation of complexes with partner proteins and DNA. This procedure provides a powerful research tool for structural and functional studies of proteins participating in non-covalent macromolecular complexes. PMID:26983699

  10. Production of unstable proteins through the formation of stable core complexes.

    PubMed

    Levy, Nicolas; Eiler, Sylvia; Pradeau-Aubreton, Karine; Maillot, Benoit; Stricher, François; Ruff, Marc

    2016-01-01

    Purification of proteins that participate in large transient complexes is impeded by low amounts, heterogeneity, instability and poor solubility. To circumvent these difficulties we set up a methodology that enables the production of stable complexes for structural and functional studies. This procedure is benchmarked and applied to two challenging protein families: the human steroid nuclear receptors (SNR) and the HIV-1 pre-integration complex. In the context of transcriptional regulation studies, we produce and characterize the ligand-binding domains of the glucocorticoid nuclear receptor and the oestrogen receptor beta in complex with a TIF2 (transcriptional intermediary factor 2) domain containing the three SNR-binding motifs. In the context of retroviral integration, we demonstrate the stabilization of the HIV-1 integrase by formation of complexes with partner proteins and DNA. This procedure provides a powerful research tool for structural and functional studies of proteins participating in non-covalent macromolecular complexes. PMID:26983699

  11. Towards a Hierarchical Strategy to Explore Multi-Scale IP/MS Data for Protein Complexes

    PubMed Central

    Kutzera, Joachim; Smilde, Age K.; Wilderjans, Tom F.; Hoefsloot, Huub C. J.

    2015-01-01

    Protein interaction in cells can be described at different levels. At a low interaction level, proteins function together in small, stable complexes and at a higher level, in sets of interacting complexes. All interaction levels are crucial for the living organism, and one of the challenges in proteomics is to measure the proteins at their different interaction levels. One common method for such measurements is immunoprecipitation followed by mass spectrometry (IP/MS), which has the potential to probe the different protein interaction forms. However, IP/MS data are complex because proteins, in their diverse interaction forms, manifest themselves in different ways in the data. Numerous bioinformatic tools for finding protein complexes in IP/MS data are currently available, but most tools do not provide information about the interaction level of the discovered complexes, and no tool is geared specifically to unraveling and visualizing these different levels. We present a new bioinformatic tool to explore IP/MS datasets for protein complexes at different interaction levels and show its performance on several real–life datasets. Our tool creates clusters that represent protein complexes, but unlike previous methods, it arranges them in a tree–shaped structure, reporting why specific proteins are predicted to build a complex and where it can be divided into smaller complexes. In every data analysis method, parameters have to be chosen. Our method can suggest values for its parameters and comes with adapted visualization tools that display the effect of the parameters on the result. The tools provide fast graphical feedback and allow the user to interact with the data by changing the parameters and examining the result. The tools also allow for exploring the different organizational levels of the protein complexes in a given dataset. Our method is available as GNU-R source code and includes examples at www.bdagroup.nl. PMID:26448546

  12. Structural Basis for the Coevolution of a Viral RNA-Protein Complex

    SciTech Connect

    Chao,J.; Patskovsky, Y.; Almo, S.; Singer, R.

    2008-01-01

    The cocrystal structure of the PP7 bacteriophage coat protein in complex with its translational operator identifies a distinct mode of sequence-specific RNA recognition when compared to the well-characterized MS2 coat protein-RNA complex. The structure reveals the molecular basis of the PP7 coat protein's ability to selectively bind its cognate RNA, and it demonstrates that the conserved beta-sheet surface is a flexible architecture that can evolve to recognize diverse RNA hairpins.

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

    PubMed

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

    2016-04-01

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

  14. A complex of three related membrane proteins is conserved on malarial merozoites

    PubMed Central

    Rayavara, Kempaiah; Rajapandi, Thavamani; Wollenberg, Kurt; Kabat, Juraj; Fischer, Elizabeth R.; Desai, Sanjay A.

    2009-01-01

    Invasion of human red blood cells by the malaria parasite P. falciparum is a coordinated, multi-step process. Here, we describe three novel integral membrane proteins that colocalize on the inner membrane complex immediately beneath the merozoite plasma membrane. Each has 6 predicted transmembrane domains and is conserved in diverse apicomplexan parasites. Immunoprecipitation studies using specific antibodies reveal that these proteins assemble into a heteromeric complex. Each protein was also expressed on insect cells using the baculovirus vector system with a truncated SUMO tag that facilitates maximal expression and protein purification while permitting cleavage with SUMO protease to release unmodified parasite protein. The expressed proteins were successfully reconstituted into artificial liposomes, but were not recognized by human immune sera. Because all three genes are highly conserved in apicomplexan parasites, the complex formed by their encoded proteins likely serves an essential role for invasive merozoites. PMID:19465059

  15. Fractionation of Thylakoid Membranes with the Nonionic Detergent Octyl-beta-d-glucopyranoside: RESOLUTION OF CHLOROPHYLL-PROTEIN COMPLEX II INTO TWO CHLOROPHYLL-PROTEIN COMPLEXES.

    PubMed

    Camm, E L; Green, B R

    1980-09-01

    The detergent octyl-beta-d-glucopyranoside (30 millimolar in 2 millimolar Tris-maleate, pH 7.0) preferentially extracts complexes containing protein and chlorophylls a plus b (CP) from spinach, leaving a residue highly enriched in CP I (P700-chlorophyll a protein). Use of the detergent results in a relatively gentle extraction since little free chlorophyll is formed and since sodium dodecyl sulfate-gel electrophoresis (on 10% acrylamide) of the extract also reveals the presence of two minor chlorophyll a complexes (apparent molecular weight, 47,000 and 43,000) instead of the usual single complex. The major complex preserved is CP 64, a chlorophyll a/b complex (apparent molecular weight, 64,000) which is an oligomer of another chlorophyll a/b complex, CP 27, the light-harvesting complex (apparent molecular weight, 27,000). Dissociation of each complex reveals two polypeptides (molecular weight, 32,000 and 28,000) and limited proteolysis confirms that those of CP 64 have the same structure as those of CP 27. An additional chlorophyll a/b complex (apparent molecular weight, 29,000) is clearly separable from CP 27, and differs from it and CP 64 in having a higher chlorophyll a/b ratio and a single polypeptide (molecular weight, 29,000) which differs structurally from those of the other complexes. PMID:16661449

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

    PubMed

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

    2016-06-01

    Nanoparticles (NP) have capability to adsorb proteins from biological fluids and form protein layer, which is called protein corona. As the cell sees corona coated NPs, the protein corona can dictate biological response to NPs. The composition of protein corona is varied by physicochemical properties of NPs including size, shape, surface chemistry. Processing of protein adsorption is dynamic phenomena; to that end, a protein may desorb or leave a surface vacancy that is rapidly filled by another protein and cause changes in the corona composition mainly by the Vroman effect. In this review, we discuss the interaction between NP and proteins and the available techniques for identification of NP-bound proteins. Also we review current developed computational methods for understanding the NP-protein complex interactions. PMID:26873405

  17. Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by nonflammable solvents.

    PubMed

    Lin, Miao-Fang; Williams, Christie; Murray, Michael V; Ropp, Philip A

    2005-02-25

    During the recovery of recombinant proteins from gram negative bacteria, many of the methods used to extract proteins from cells release lipopolysaccharides (LPS, endotoxin) along with the protein of interest. In many instances, LPS will co-purify with the target protein due to specific or non-specific protein-LPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on ion exchange chromatographic resins. Proteins were complexed with fluorescently labeled LPS and bound to ion exchange resin. Alkanediol washes of the resins were preformed and the proteins eluted. Column eluates were monitored for LPS and protein by fluorescence and UV spectroscopy, respectively. Alkanediols were effective agents for dissociating LPS from protein-LPS complexes. The efficiency of LPS removal increased with increasing alkanediol chain length. The 1,2-alkanediol isomers were more effective than terminal alkanediol isomers in the separation of LPS from protein-LPS complexes, while the separation of LPS from protein-LPS complexes was more efficient on cation exchangers than on anion exchangers. In addition, it was noted during these investigations that the 1,2-alkanediols increased the retention time of the proteins on the ion exchange resins. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile for the separation of LPS from protein due to their lower toxicity and decreased inflammability. In addition, they are less costly than many of the detergents that have been used for similar purposes. PMID:15664347

  18. In vivo protein complex topologies: Sights through a cross-linking lens

    PubMed Central

    Bruce, James E.

    2013-01-01

    Proteins are a remarkable class of molecules that exhibit wide diversity of shapes or topological features that underpin protein interactions and give rise to biological function. In addition to quantitation of abundance levels of proteins in biological systems under a variety of conditions, the field of proteome research has as a primary mission the assignment of function for proteins and if possible, illumination of factors that enable function. For many years, chemical cross-linking methods have been used to provide structural data on single purified proteins and purified protein complexes. However, these methods also offer the alluring possibility to extend capabilities to complex biological samples such as cell lysates or intact living cells where proteins may exhibit native topological features that do not exist in purified form. Recent efforts are beginning to provide glimpses of protein complexes and topologies in cells that suggest continued development will yield novel capabilities to view functional topological features of many protein and complexes as they exist in cells, tissues or other complex samples. This review will describe rationale, challenges and a few success stories along the path of development of cross-linking technologies for measurement of in vivo protein interaction topologies. PMID:22610688

  19. An updated version of NPIDB includes new classifications of DNA-protein complexes and their families.

    PubMed

    Zanegina, Olga; Kirsanov, Dmitriy; Baulin, Eugene; Karyagina, Anna; Alexeevski, Andrei; Spirin, Sergey

    2016-01-01

    The recent upgrade of nucleic acid-protein interaction database (NPIDB, http://npidb.belozersky.msu.ru/) includes a newly elaborated classification of complexes of protein domains with double-stranded DNA and a classification of families of related complexes. Our classifications are based on contacting structural elements of both DNA: the major groove, the minor groove and the backbone; and protein: helices, beta-strands and unstructured segments. We took into account both hydrogen bonds and hydrophobic interaction. The analyzed material contains 1942 structures of protein domains from 748 PDB entries. We have identified 97 interaction modes of individual protein domain-DNA complexes and 17 DNA-protein interaction classes of protein domain families. We analyzed the sources of diversity of DNA-protein interaction modes in different complexes of one protein domain family. The observed interaction mode is sometimes influenced by artifacts of crystallization or diversity in secondary structure assignment. The interaction classes of domain families are more stable and thus possess more biological sense than a classification of single complexes. Integration of the classification into NPIDB allows the user to browse the database according to the interacting structural elements of DNA and protein molecules. For each family, we present average DNA shape parameters in contact zones with domains of the family. PMID:26656949

  20. Evidence for a structural role for chlorophyll in chlorophyll-protein complexes.

    PubMed

    Jennings, R C; Garlaschi, F M; Forti, G; Gerola, P D

    1979-11-23

    1. Chymotrypsin treatment of spinach chloroplast membranes does not change the electrophoretic mobility of either chlorophyll-protein complex 1 or 2. 2. The extraction of lipids with 80% acetone after treatment of the membranes with chymotrypsin reveals that the polypeptide components of both chlorophyll-protein complexes had been extensively digested. The extraction of carotenes with petroleum ether under the same conditions does not change the electrophoretic mobility of the chlorophyll-protein complexes. 3. Fluorescence polarisation studies of chlorophyll-protein complex 2 reveal that the chymotrypsin digestion of this complex does not result in changes of mutual orientation or distance apart of chlorophyll a, chlorophyll b or carotenoid. 4. Two polypeptide components have been detected after lipid extraction of electrophoretically purified chlorophyll-protein complexes 1 and 2. The SDS molecular weights are 24 000 and 27 000 for complex 2, and 68 000 and 64 000 for complex 1. 5. We conclude that chlorophyll performs an important structural function in both chlorophyll-protein complexes. PMID:508798

  1. Native gel analysis of macromolecular protein complexes in cultured mammalian cells.

    PubMed

    Munawar, Nayla; Olivero, Giorgio; Jerman, Emilia; Doyle, Benjamin; Streubel, Gundula; Wynne, Kieran; Bracken, Adrian; Cagney, Gerard

    2015-11-01

    Native gel electrophoresis enables separation of cellular proteins in their non-denatured state. In experiments aimed at analysing proteins in higher order or multimeric assemblies (i.e. protein complexes) it offers some advantages over rival approaches, particularly as an interface technology with mass spectrometry. Here we separated fractions from HEK293 cells by native electrophoresis in order to survey protein complexes in the cytoplasmic, nuclear and chromatin environments, finding 689 proteins distributed among 217 previously described complexes. As expected, different fractions contained distinct combinations of macromolecular complexes, with subunits of the same complex tending to co-migrate. Exceptions to this observation could often be explained by the presence of subunits shared among different complexes. We investigated one identified complex, the Polycomb Repressor Complex 2 (PRC2), in more detail following affinity purification of the EZH2 subunit. This approach resulted in the identification of all previously reported members of PRC2. Overall, this work demonstrates that the use of native gel electrophoresis as an upstream separating step is an effective approach for analysis of the components and cellular distribution of protein complexes. PMID:26223664

  2. A Three-Hybrid System to Probe In Vivo Protein-Protein Interactions: Application to the Essential Proteins of the RD1 Complex of M. tuberculosis

    PubMed Central

    Bhalla, Kuhulika; Ghosh, Anamika; Kumar, Krishan; Kumar, Sushil; Ranganathan, Anand

    2011-01-01

    Background Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes. Methodology/Principal Findings The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3. Conclusions/Significance The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes. PMID:22087330

  3. A novel approach to analyze membrane proteins by laser mass spectrometry: from protein subunits to the integral complex.

    PubMed

    Morgner, Nina; Kleinschroth, Thomas; Barth, Hans-Dieter; Ludwig, Bernd; Brutschy, Bernhard

    2007-08-01

    A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices. PMID:17544294

  4. Domain stealing by receptors in a protein transport complex.

    PubMed

    Hulett, Joanne M; Walsh, Peter; Lithgow, Trevor

    2007-09-01

    The mitochondrion is an essential cellular compartment in eukaryotes. The mitochondrial proteins Tom20 and Tom22 are receptors that ensure recognition and binding of proteins imported for mitochondrial biogenesis. Comparison of the sequence for the Tom20 and Tom22 subunits in the yeasts Saccharomyces cerevisiae and Saccharomyces castellii, show a rare case of domain stealing, where in Saccharomyces castellii Tom22 has lost an acidic domain, and Tom20 has gained one. This example of domain stealing is a snapshot of evolution in action and provides excellent evidence that Tom20 and Tom22 are subunits of a single, composite receptor that binds precursor proteins for import into mitochondria. PMID:17586602

  5. Protein-protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids.

    PubMed

    Lau, Julia B; Stork, Simone; Moog, Daniel; Schulz, Julian; Maier, Uwe G

    2016-04-01

    Most secondary plastids of red algal origin are surrounded by four membranes and nucleus-encoded plastid proteins have to traverse these barriers. Translocation across the second outermost plastid membrane, the periplastidal membrane (PPM), is facilitated by a ERAD-(ER-associated degradation) derived machinery termed SELMA (symbiont-specific ERAD-like machinery). In the last years, important subunits of this translocator have been identified, which clearly imply compositional similarities between SELMA and ERAD. Here we investigated, via protein-protein interaction studies, if the composition of SELMA is comparable to the known ERAD complex. As a result, our data suggest that the membrane proteins of SELMA, the derlin proteins, are linked to the soluble sCdc48 complex via the UBX protein sUBX. This is similar to the ERAD machinery whereas the additional SELMA components, sPUB und a second Cdc48 copy might indicate the influence of functional constraints in developing a translocation machinery from ERAD-related factors. In addition, we show for the first time that a rhomboid protease is a central interaction partner of the membrane proteins of the SELMA system in complex plastids. PMID:26712034

  6. Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

    NASA Technical Reports Server (NTRS)

    Cannone, Jaime J.; Barnes, Cindy L.; Achari, Aniruddha; Kundrot, Craig E.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

  7. Single-molecule study of protein-DNA target search mechanisms for dimer-active protein complexes

    NASA Astrophysics Data System (ADS)

    Landry, Markita; Huang, Wai Mun; Chemla, Yann

    2012-02-01

    Protein-DNA interactions are essential to cellular processes, many of which require proteins to recognize a specific DNA target-site. This search process is well-documented for monomeric proteins, but not as well understood for systems that require dimerization at the target site for activity. We present a single-molecule study of the target-search mechanism of Protelomerase TelK, a recombinase-like protein that is only active as a dimer. We observe that TelK undergoes 1D diffusion on non-target DNA as a monomer, as expected, but becomes immobile on DNA as a dimer or oligomer despite the absence of its target site. We further show that TelK condenses non-target DNA upon dimerization, forming a tightly bound nucleo-protein complex. Together with simulations, our results suggest a search model whereby monomers diffuse along DNA, and subsequently dimerize to form an active complex on target DNA. These results show that target-finding occurs faster than nonspecific dimerization at biologically relevant protein concentrations. This model may provide insights into the search mechanisms of proteins that are active as multimeric complexes for a more accurate and comprehensive model for the target-search process by sequence specific proteins.

  8. Tyrosine-selective protein alkylation using pi-allylpalladium complexes.

    PubMed

    Tilley, S David; Francis, Matthew B

    2006-02-01

    A new protein modification reaction has been developed based on a palladium-catalyzed allylic alkylation of tyrosine residues. This technique employs electrophilic pi-allyl intermediates derived from allylic acetate and carbamate precursors and can be used to modify proteins in aqueous solution at room temperature. To facilitate the detection of modified proteins using SDS-PAGE analysis, a fluorescent allyl acetate was synthesized and coupled to chymotrypsinogen A and bacteriophage MS2. The tyrosine selectivity of the reaction was confirmed through trypsin digest analysis. The utility of the reaction was demonstrated by using taurine-derived carbamates as water solubilizing groups that are cleaved upon protein functionalization. This solubility switching technique was used to install hydrophobic farnesyl and C(17) chains on chymotrypsinogen A in water using little or no cosolvent. Following this, the C(17) alkylated proteins were found to associate with lipid vesicles. In addition to providing a new protein modification strategy targeting an under-utilized amino acid side chain, this method provides convenient access to synthetic lipoproteins. PMID:16433516

  9. Kojak: Efficient analysis of chemically cross-linked protein complexes

    PubMed Central

    Hoopmann, Michael R.; Zelter, Alex; Johnson, Richard S.; Riffle, Michael; MacCoss, Michael J.; Davis, Trisha N.; Moritz, Robert L.

    2015-01-01

    Protein chemical cross-linking and mass spectrometry enable the analysis of protein-protein interactions and protein topologies, however complicated cross-linked peptide spectra require specialized algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein-protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms, and can identify cross-linked peptides using many different chemical cross-linkers, with or without heavy isotope labels. Kojak was used to analyze both novel and existing datasets, and was compared with existing cross-linking algorithms. The algorithm provided increased cross-link identifications over existing algorithms, and equally importantly, the results in a fraction of computational time. The Kojak algorithm is open-source, cross-platform, and freely available. This software provides both existing and new cross-linking researchers alike an effective way to derive additional cross-link identifications from new or existing datasets. For new users, it provides a simple analytical resource resulting in more cross-link identifications than other methods. PMID:25812159

  10. Monte Carlo simulations of flexible polyanions complexing with whey proteins at their isoelectric point

    NASA Astrophysics Data System (ADS)

    de Vries, R.

    2004-02-01

    Electrostatic complexation of flexible polyanions with the whey proteins α-lactalbumin and β-lactoglobulin is studied using Monte Carlo simulations. The proteins are considered at their respective isoelectric points. Discrete charges on the model polyelectrolytes and proteins interact through Debye-Hückel potentials. Protein excluded volume is taken into account through a coarse-grained model of the protein shape. Consistent with experimental results, it is found that α-lactalbumin complexes much more strongly than β-lactoglobulin. For α-lactalbumin, strong complexation is due to localized binding to a single large positive "charge patch," whereas for β-lactoglobulin, weak complexation is due to diffuse binding to multiple smaller charge patches.

  11. Identification of a Predicted Trimeric Autotransporter Adhesin Required for Biofilm Formation of Burkholderia pseudomallei

    PubMed Central

    Lazar Adler, Natalie R.; Dean, Rachel E.; Saint, Richard J.; Stevens, Mark P.; Prior, Joann L.; Atkins, Timothy P.; Galyov, Edouard E.

    2013-01-01

    The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A. PMID:24223950

  12. Glycosaminoglycan binding by Borrelia burgdorferi adhesin BBK32 specifically and uniquely promotes joint colonization

    PubMed Central

    Lin, Yi-Pin; Chen, Qiang; Ritchie, Jennifer A.; Dufour, Nicholas P.; Fischer, Joshua R.; Coburn, Jenifer; Leong, John M.

    2014-01-01

    SUMMARY Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins that mediate attachment to cells and/or extracellular matrix in target organs. Many of these ‘adhesins’ bind to multiple ligands, complicating efforts to understand the role of each ligand-binding activity. Borrelia burgdorferi, the causative agent of Lyme disease, produces BBK32, first identified as a fibronectin-binding adhesin that promotes skin and joint colonization. BBK32 also binds to glycosaminoglycan (GAG), which, like fibronectin is ubiquitously present on cell surfaces. To determine which binding activity is relevant for BBK32-promoted infectivity, we generated a panel of BBK32 truncation and internal deletion mutants, and identified variants specifically defective for binding to either fibronectin or GAG. These variants promoted bacterial attachment to different mammalian cell types in vitro, suggesting that fibronectin and GAG binding may play distinct roles during infection. Intravenous inoculation of mice with a high-passage non-infectious B. burgdorferi strain that produced wild type BBK32 or BBK32 mutants defective for GAG or fibronectin binding, revealed that only GAG-binding activity was required for significant localization to joints at 60 minutes post-infection. An otherwise infectious B. burgdorferi strain producing BBK32 specifically deficient in fibronectin binding was fully capable of both skin and joint colonization in the murine model, whereas a strain producing BBK32 selectively attenuated for GAG binding colonized the inoculation site but not knee or tibiotarsus joints. Thus, the BBK32 fibronectin- and GAG-binding activities are separable in vivo, and BBK32-mediated GAG binding, but not fibronectin binding, contributes to joint colonization. PMID:25486989

  13. Identification of a Novel Trimeric Autotransporter Adhesin in the Cryptic Genospecies of Haemophilus▿

    PubMed Central

    Sheets, Amanda J.; Grass, Susan A.; Miller, Sara E.; St. Geme, Joseph W.

    2008-01-01

    Haemophilus biotype IV strains belonging to the recently recognized Haemophilus cryptic genospecies are an important cause of maternal genital tract and neonatal systemic infections and initiate infection by colonizing the genital or respiratory epithelium. To gain insight into the mechanism of Haemophilus cryptic genospecies colonization, we began by examining prototype strain 1595 and three other strains for adherence to genital and respiratory epithelial cell lines. Strain 1595 and two of the three other strains demonstrated efficient adherence to all of the cell lines tested. With a stably adherent variant of strain 1595, we generated a Mariner transposon library and identified 16 nonadherent mutants. All of these mutants lacked surface fibers and contained an insertion in the same open reading frame, which encodes a 157-kDa protein designated Cha for cryptic haemophilus adhesin. Analysis of the predicted amino acid sequence of Cha revealed the presence of an N-terminal signal peptide and a C-terminal domain bearing homology to YadA-like and Hia-like trimeric autotransporters. Examination of the C-terminal 120 amino acids of Cha demonstrated mobility as a trimer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the capacity to present the passenger domain of the Hia trimeric autotransporter on the bacterial surface. Southern analysis revealed that the gene that encodes Cha is conserved among clinical isolates of the Haemophilus cryptic genospecies and is absent from the closely related species Haemophilus influenzae. We speculate that Cha is important in the pathogenesis of disease due to the Haemophilus cryptic genospecies and is in part responsible for the apparent tissue tropism of this organism. PMID:18424521

  14. Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1:1 syntaxin/SNAP-25 complex

    PubMed Central

    Weninger, Keith; Bowen, Mark E.; Choi, Ucheor B.; Chu, Steven; Brunger, Axel T.

    2010-01-01

    Summary Formation of a binary complex between syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) at the active zone is believed to precede assembly of the ternary SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex that is essential for neurotransmitter release. Despite its importance in models of synaptic neurotransmitter release, this binary complex has been difficult to characterize by bulk methods due to the prevalence of a 2:1 dead-end species. Here we used single molecule fluorescence resonance energy transfer (smFRET) to study the structure and dynamics of the 1:1 syntaxin/SNAP-25 binary complex. The binary complex is conformationally variable with FRET efficiency states often changing on the second timescale. One state corresponds to a parallel three-helix bundle configuration, while other states correspond to configurations with one of the SNAP-25 SNARE domains dissociated. All configurations of the binary complex are rapidly locked into the single three-helix bundle configuration by the addition of synaptobrevin. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex serves as a dynamic acceptor for synaptobrevin binding, and interactions with accessory proteins stabilize this acceptor. In a high protein density cellular environment the syntaxin/SNAP-25 complex is therefore expected to be in the configuration where it can rapidly interact with synaptobrevin so its formation is unlikely a limiting step for SNARE-mediated neurotransmitter release. PMID:18275821

  15. SVP-like MADS-box protein from Carya cathayensis forms higher-order complexes.

    PubMed

    Wang, Jingjing; Hou, Chuanming; Huang, Jianqin; Wang, Zhengjia; Xu, Yingwu

    2015-03-01

    To properly regulate plant flowering time and construct floral pattern, MADS-domain containing transcription factors must form multimers including homo- and hetero-dimers. They are also active in forming hetero-higher-order complexes with three to five different molecules. However, it is not well known if a MADS-box protein can also form homo-higher-order complex. In this study a biochemical approach is utilized to provide insight into the complex formation for an SVP-like MADS-box protein cloned from hickory. The results indicated that the protein is a heterogeneous higher-order complex with the peak population containing over 20 monomers. Y2H verified the protein to form homo-complex in yeast cells. Western blot of the hickory floral bud sample revealed that the protein exists in higher-order polymers in native. Deletion assays indicated that the flexible C-terminal residues are mainly responsible for the higher-order polymer formation and the heterogeneity. Current results provide direct biochemical evidences for an active MADS-box protein to be a high order complex, much higher than a quartermeric polymer. Analysis suggests that a MADS-box subset may be able to self-assemble into large complexes, and thereby differentiate one subfamily from the other in a higher-order structural manner. Present result is a valuable supplement to the action of mechanism for MADS-box proteins in plant development. PMID:25602439

  16. MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae

    PubMed Central

    Robinson, Mark W.; Buchtmann, Kyle A.; Jenkins, Cheryl; Tacchi, Jessica L.; Raymond, Benjamin B. A.; To, Joyce; Roy Chowdhury, Piklu; Woolley, Lauren K.; Labbate, Maurizio; Turnbull, Lynne; Whitchurch, Cynthia B.; Padula, Matthew P.; Djordjevic, Steven P.

    2013-01-01

    Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis. PMID:23594879

  17. Analysis of the Mycoplasma genitalium MgpB Adhesin to Predict Membrane Topology, Investigate Antibody Accessibility, Characterize Amino Acid Diversity, and Identify Functional and Immunogenic Epitopes

    PubMed Central

    Iverson-Cabral, Stefanie L.; Wood, Gwendolyn E.; Totten, Patricia A.

    2015-01-01

    Mycoplasma genitalium is a sexually transmitted pathogen and is associated with reproductive tract disease that can be chronic in nature despite the induction of a strong antibody response. Persistent infection exacerbates the likelihood of transmission, increases the risk of ascension to the upper tract, and suggests that M. genitalium may possess immune evasion mechanism(s). Antibodies from infected patients predominantly target the MgpB adhesin, which is encoded by a gene that recombines with homologous donor sequences, thereby generating sequence variation within and among strains. We have previously characterized mgpB heterogeneity over the course of persistent infection and have correlated the induction of variant-specific antibodies with the loss of that particular variant from the infected host. In the current study, we examined the membrane topology, antibody accessibility, distribution of amino acid diversity, and the location of functional and antigenic epitopes within the MgpB adhesin. Our results indicate that MgpB contains a single transmembrane domain, that the majority of the protein is surface exposed and antibody accessible, and that the attachment domain is located within the extracellular C-terminus. Not unexpectedly, amino acid diversity was concentrated within and around the three previously defined variable regions (B, EF, and G) of MgpB; while nonsynonymous mutations were twice as frequent as synonymous mutations in regions B and G, region EF had equal numbers of nonsynonymous and synonymous mutations. Interestingly, antibodies produced during persistent infection reacted predominantly with the conserved C-terminus and variable region B. In contrast, infection-induced antibodies reacted poorly with the N-terminus, variable regions EF and G, and intervening conserved regions despite the presence of predicted B cell epitopes. Overall, this study provides an important foundation to define how different segments of the MgpB adhesin contribute to

  18. Single-molecule observation of protein folding in symmetric GroEL-(GroES)2 complexes.

    PubMed

    Takei, Yodai; Iizuka, Ryo; Ueno, Taro; Funatsu, Takashi

    2012-11-30

    The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding. PMID:23048033

  19. Single-molecule Observation of Protein Folding in Symmetric GroEL-(GroES)2 Complexes*

    PubMed Central

    Takei, Yodai; Iizuka, Ryo; Ueno, Taro; Funatsu, Takashi

    2012-01-01

    The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)2 complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding. PMID:23048033

  20. Mass spectrometry-based shotgun proteomic analysis of C. elegans protein complexes.

    PubMed

    Fonslow, Bryan R; Moresco, James J; Tu, Patricia G; Aalto, Antti P; Pasquinelli, Amy E; Dillin, Andrew G; Yates, John R

    2014-01-01

    Mass spectrometry (MS)-based shotgun proteomics is an enabling technology for the study of C. elegans proteins. When coupled with co-immunoprecipitation (CoIP), new interactions and functions among proteins can be discovered. We provide a general background on protein complexes and methods for their analysis, along with the lifecycle and interaction types of proteins that ultimately define the identifiable components of protein complexes. We highlight traditional biochemical methods to evaluate whether the complexes are sufficiently pure and abundant for analysis with shotgun proteomics. We present two CoIP-MS case studies of protein complexes from C. elegans, using both endogenous and fusion protein antibodies to illustrate the important aspects of their analyses. We discuss results from mass spectrometers with differences in mass accuracy and resolution, along with the relevant information that can be extracted from the data generated, such as protein relative abundance, post-translational modifications, and identification confidence. Finally, we illustrate how comparative analysis can reveal candidate binding partners for biological follow-up and validation. This chapter should act as a complement and extension to the WormBook chapter Biochemistry and molecular biology, which describes tandem affinity purification (TAP) of protein complexes for analysis by mass spectrometry. PMID:24967700

  1. Production of Protein Complexes in Non-methylotrophic and Methylotrophic Yeasts : Nonmethylotrophic and Methylotrophic Yeasts.

    PubMed

    Fernández, Francisco J; López-Estepa, Miguel; Querol-García, Javier; Vega, M Cristina

    2016-01-01

    Protein complexes can be produced in multimilligram quantities using nonmethylotrophic and methylotrophic yeasts such as Saccharomyces cerevisiae and Komagataella (Pichia) pastoris. Yeasts have distinct advantages as hosts for recombinant protein production owing to their cost efficiency, ease of cultivation and genetic manipulation, fast growth rates, capacity to introduce post-translational modifications, and high protein productivity (yield) of correctly folded protein products. Despite those advantages, yeasts have surprisingly lagged behind other eukaryotic hosts in their use for the production of multisubunit complexes. As our knowledge of the metabolic and genomic bottlenecks that yeast microorganisms face when overexpressing foreign proteins expands, new possibilities emerge for successfully engineering yeasts as superb expression hosts. In this chapter, we describe the current state of the art and discuss future possibilities for the development of yeast-based systems for the production of protein complexes. PMID:27165323

  2. Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS.

    PubMed

    Gubbens, Jacob; Slijper, Monique; de Kruijff, Ben; de Kroon, Anton I P M

    2008-12-01

    Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins. PMID:18817900

  3. Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

    PubMed Central

    Rehman, Asma; Archbold, Julia K.; Hu, Shu-Hong; Norwood, Suzanne J.; Collins, Brett M.; Martin, Jennifer L.

    2014-01-01

    Membrane fusion is essential for human health, playing a vital role in processes as diverse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for example through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins. PMID:25485130

  4. Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation

    PubMed Central

    Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd

    2016-01-01

    Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380

  5. Sulfated glycoconjugate receptors for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and mapping of the heparin-binding domain on FHA.

    PubMed Central

    Hannah, J H; Menozzi, F D; Renauld, G; Locht, C; Brennan, M J

    1994-01-01

    Filamentous hemagglutinin (FHA) is a major adhesin present on the surface of the gram-negative respiratory pathogen Bordetella pertussis. A number of binding mechanisms have been described for the interaction of FHA with eukaryotic cells. We have focused on its function as a sulfated polysaccharide-binding protein and on identifying potential receptors for FHA on the epithelial cell surface. Using a thin-layer overlay technique, we found that FHA binds specifically to sulfated glycolipids but not to gangliosides or other neutral glycolipids. These results suggest that epithelial cell surface sulfated glycolipids function as receptors for FHA. Further studies demonstrated that a Chinese hamster ovary (CHO) cell strain deficient in glycosaminoglycan expression exhibits greatly diminished attachment to FHA. By FHA-Affi-Gel chromatography, a putative receptor for FHA that has characteristics consistent with a heparan sulfate proteoglycan was isolated from epithelial cell extracts. In addition, by using recombinant FHA fusion proteins, a specific glycosaminoglycan-binding domain located near the N terminus of the FHA molecule was identified. Our results indicate that the B. pertussis adhesin FHA may utilize sulfated glycolipids and proteoglycans commonly found on the surface of human cells and tissues to initiate infection. Images PMID:7927782

  6. Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

    PubMed Central

    Hillenbrand, Matthias; Schori, Christian; Schöppe, Jendrik; Plückthun, Andreas

    2015-01-01

    Agonist binding to G-protein–coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1 or αsL and all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1 and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes. PMID:25733868

  7. The alternative oxidases: simple oxidoreductase proteins with complex functions.

    PubMed

    Young, Luke; Shiba, Tomoo; Harada, Shigeharu; Kita, Kiyoshi; Albury, Mary S; Moore, Anthony L

    2013-10-01

    The alternative oxidases are membrane-bound monotopic terminal electron transport proteins found in all plants and in some agrochemically important fungi and parasites including Trypansoma brucei, which is the causative agent of trypanosomiasis. They are integral membrane proteins and reduce oxygen to water in a four electron process. The recent elucidation of the crystal structure of the trypanosomal alternative oxidase at 2.85 Å (1 Å=0.1 nm) has revealed salient structural features necessary for its function. In the present review we compare the primary and secondary ligation spheres of the alternative oxidases with other di-iron carboxylate proteins and propose a mechanism for the reduction of oxygen to water. PMID:24059524

  8. Modifications in structure and interaction of nanoparticle-protein-surfactant complexes in electrolyte solution

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Kumar, S.; Aswal, V. K.; Schweins, R.

    2016-05-01

    SANS experiments of three-component system of anionic silica nanoparticles, anionic BSA protein and anionic SDS surfactants have been carried out without and with electrolyte in aqueous solution. In both the cases, the interaction of surfactant with protein results in formation of bead-necklace structure of protein-surfactant complexes in solution. These protein-surfactant complexes interact very differently with nanoparticles in absence and presence of electrolyte. In absence of electrolyte, nanoparticles remain in dispersed phase in solution, whereas with the addition of electrolyte the nanoparticles fractal aggregates are formed. SANS describes the phase behavior to be governed by competition of electrostatic and depletion interactions among the components solution.

  9. Probing protein complexes inside living cells using a silicon nanowire-based pull-down assay.

    PubMed

    Choi, Sojoong; Kim, Hyunju; Kim, So Yeon; Yang, Eun Gyeong

    2016-06-01

    Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells. PMID:27198202

  10. NMR techniques in drug delivery: application to zein protein complexes.

    PubMed

    Sousa, F F O; Luzardo-Álvarez, Asteria; Blanco-Méndez, José; Martín-Pastor, Manuel

    2012-12-15

    Zein is a protein containing a large amount of nonpolar amino acids, which has shown the ability to form aggregates and entrap solutes, such as drugs and amino acids. NMR techniques were used to detect binding interactions and measure affinity between zein and three different drugs: tetracycline, amoxicillin and indomethacin. The release study of zein microparticle formulations containing any of these drugs was confronted with the affinity results, showing a remarkable correlation. The feasible methodology employed, focused in the functionality of the protein-drug interaction, can be very promising for the rational design of appropriate drug vehicles for drug delivery. PMID:23041651

  11. Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex.

    PubMed

    Tunc-Ozdemir, Meral; Urano, Daisuke; Jaiswal, Dinesh Kumar; Clouse, Steven D; Jones, Alan M

    2016-07-01

    Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1. PMID:27235398

  12. Isolation and properties of the complex between the enhancer binding protein NIFA and the sensor NIFL.

    PubMed

    Money, T; Jones, T; Dixon, R; Austin, S

    1999-08-01

    In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation. PMID:10419940

  13. Exposing the subunit diversity within protein complexes: a mass spectrometry approach.

    PubMed

    Rozen, Shelly; Tieri, Alessandra; Ridner, Gabriela; Stark, Ann-Kathrin; Schmaler, Tilo; Ben-Nissan, Gili; Dubiel, Wolfgang; Sharon, Michal

    2013-03-01

    Identifying the list of subunits that make up protein complexes constitutes an important step towards understanding their biological functions. However, such knowledge alone does not reveal the full complexity of protein assemblies, as each subunit can take on multiple forms. Proteins can be post-translationally modified or cleaved, multiple products of alternative splicing can exist, and a single subunit may be encoded by more than one gene. Thus, for a complete description of a protein complex, it is necessary to expose the diversity of its subunits. Adding this layer of information is an important step towards understanding the mechanisms that regulate the activity of protein assemblies. Here, we describe a mass spectrometry-based approach that exposes the array of protein variants that comprise protein complexes. Our method relies on denaturing the protein complex, and separating its constituent subunits on a monolithic column prepared in-house. Following the subunit elution from the column, the flow is split into two fractions, using a Triversa NanoMate robot. One fraction is directed straight into an on-line ESI-QToF mass spectrometer for intact protein mass measurements, while the rest of the flow is fractionated into a 96-well plate for subsequent proteomic analysis. The heterogeneity of subunit composition is then exposed by correlating the subunit sequence identity with the accurate mass. Below, we describe in detail the methodological setting of this approach, its application on the endogenous human COP9 signalosome complex, and the significance of the method for structural mass spectrometry analysis of intact protein complexes. PMID:23296018

  14. A cryptobiosis-specific 19S protein complex of Artemia salina gastrulae.

    PubMed

    De Herdt, E; De Voeght, F; Clauwaert, J; Kondo, M; Slegers, H

    1981-01-15

    The postribosomal supernatant of Artemia salina cryptobiotic embryos contains a large quantity of a 19S protein complex. An amount of 3.6 mg/g of cysts is measured by immunoprecipitation with anti-(19S protein complex) antibody. The quantity of this complex decreases during further development to nauplius larvae to only 15% of the quantity present in cryptobiotic embryos. The complex was no longer detectable after 7 days of growth. The 27000-Mr protein subunit of the 19S complex is not synthesized by mRNA isolated from cryptobiotic embryos. The cryptobiosis-specific complex has Mr 573000 and 610000 as calculated from light-scattering and sedimentation-diffusion measurements respectively. The 19S homocomplex contains 20-23 27000-Mr proteins and has no function in the translation of homologous mRNA. From hydrodynamic data a hydration of 1.25 g of water/g of protein is calculated. The abundant presence of the 19S protein complex in cryptobiotic embryos and the absence of synthesis during development to nauplius larvae indicate a functional role during the cryptobiotic process in early embryogenesis. A role in maintaining the water content of the cytoplasm above a critical threshold during desiccation is suggested. PMID:7305995

  15. Structural Analysis of Protein-RNA Complexes in Solution Using NMR Paramagnetic Relaxation Enhancements.

    PubMed

    Hennig, Janosch; Warner, Lisa R; Simon, Bernd; Geerlof, Arie; Mackereth, Cameron D; Sattler, Michael

    2015-01-01

    Biological activity in the cell is predominantly mediated by large multiprotein and protein-nucleic acid complexes that act together to ensure functional fidelity. Nuclear magnetic resonance (NMR) spectroscopy is the only method that can provide information for high-resolution three-dimensional structures and the conformational dynamics of these complexes in solution. Mapping of binding interfaces and molecular interactions along with the characterization of conformational dynamics is possible for very large protein complexes. In contrast, de novo structure determination by NMR becomes very time consuming and difficult for protein complexes larger than 30 kDa as data are noisy and sparse. Fortunately, high-resolution structures are often available for individual domains or subunits of a protein complex and thus sparse data can be used to define their arrangement and dynamics within the assembled complex. In these cases, NMR can therefore be efficiently combined with complementary solution techniques, such as small-angle X-ray or neutron scattering, to provide a comprehensive description of the structure and dynamics of protein complexes in solution. Particularly useful are NMR-derived paramagnetic relaxation enhancements (PREs), which provide long-range distance restraints (ca. 20Å) for structural analysis of large complexes and also report on conformational dynamics in solution. Here, we describe the use of PREs from sample production to structure calculation, focusing on protein-RNA complexes. On the basis of recent examples from our own research, we demonstrate the utility, present protocols, and discuss potential pitfalls when using PREs for studying the structure and dynamic features of protein-RNA complexes. PMID:26068746

  16. Novel Cul3 binding proteins function to remodel E3 ligase complexes

    PubMed Central

    2014-01-01

    Background Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein. Results Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of Cul3-bound proteins that contain either the LRR or WD40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor. To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex. Conclusion Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC

  17. Restricted dynamics of water around a protein-carbohydrate complex: Computer simulation studies

    NASA Astrophysics Data System (ADS)

    Jana, Madhurima; Bandyopadhyay, Sanjoy

    2012-08-01

    Water-mediated protein-carbohydrate interaction is a complex phenomenon responsible for different biological processes in cellular environment. One of the unexplored but important issues in this area is the role played by water during the recognition process and also in controlling the microscopic properties of the complex. In this study, we have carried out atomistic molecular dynamics simulations of a protein-carbohydrate complex formed between the hyaluronan binding domain of the murine Cd44 protein and the octasaccharide hyaluronan in explicit water. Efforts have been made to explore the heterogeneous influence of the complex on the dynamic properties of water present in different regions around it. It is revealed from our analyses that the heterogeneous dynamics of water around the complex are coupled with differential time scales of formation and breaking of hydrogen bonds at the interface. Presence of a highly rigid thin layer of motionally restricted water molecules bridging the protein and the carbohydrate in the common region of the complex has been identified. Such water molecules are expected to play a crucial role in controlling properties of the complex. Importantly, it is demonstrated that the formation of the protein-carbohydrate complex affects the transverse and longitudinal degrees of freedom of the interfacial water molecules in a heterogeneous manner.

  18. Signal peptide peptidase (SPP) assembles with substrates and misfolded membrane proteins into distinct oligomeric complexes

    PubMed Central

    Schrul, Bianca; Kapp, Katja; Sinning, Irmgard; Dobberstein, Bernhard

    2010-01-01

    SPP (signal peptide peptidase) is an aspartyl intramembrane cleaving protease, which processes a subset of signal peptides, and is linked to the quality control of ER (endoplasmic reticulum) membrane proteins. We analysed SPP interactions with signal peptides and other membrane proteins by co-immunoprecipitation assays. We found that SPP interacts specifically and tightly with a large range of newly synthesized membrane proteins, including signal peptides, preproteins and misfolded membrane proteins, but not with all co-expressed type II membrane proteins. Signal peptides are trapped by the catalytically inactive SPP mutant SPPD/A. Preproteins and misfolded membrane proteins interact with both SPP and the SPPD/A mutant, and are not substrates for SPP-mediated intramembrane proteolysis. Proteins interacting with SPP are found in distinct complexes of different sizes. A signal peptide is mainly trapped in a 200 kDa SPP complex, whereas a preprotein is predominantly found in a 600 kDa SPP complex. A misfolded membrane protein is detected in 200, 400 and 600 kDa SPP complexes. We conclude that SPP not only processes signal peptides, but also collects preproteins and misfolded membrane proteins that are destined for disposal. PMID:20196774

  19. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

    SciTech Connect

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion

    2010-08-18

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{sub 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

  20. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of α- and PPII-helices

    PubMed Central

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion

    2010-01-01

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 Å) crystal structure of the A3VP1 fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 Å) through the interaction of two noncontiguous regions in the primary sequence. The A3 repeat of the alanine-rich domain adopts an extended α-helix that intertwines with the P1 repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A3 and P1 helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length. PMID:20231452

  1. Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

  2. Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

    PubMed Central

    Stefan, E.; Aquin, S.; Berger, N.; Landry, C. R.; Nyfeler, B.; Bouvier, M.; Michnick, S. W.

    2007-01-01

    The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Gαs protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive β-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades. PMID:17942691

  3. Conservation and Variability of Synaptonemal Complex Proteins in Phylogenesis of Eukaryotes

    PubMed Central

    Grishaeva, Tatiana M.; Bogdanov, Yuri F.

    2014-01-01

    The problems of the origin and evolution of meiosis include the enigmatic variability of the synaptonemal complexes (SCs) which, being morphology similar, consist of different proteins in different eukaryotic phyla. Using bioinformatics methods, we monitored all available eukaryotic proteomes to find proteins similar to known SC proteins of model organisms. We found proteins similar to SC lateral element (LE) proteins and possessing the HORMA domain in the majority of the eukaryotic taxa and assume them the most ancient among all SC proteins. Vertebrate LE proteins SYCP2, SYCP3, and SC65 proved to have related proteins in many invertebrate taxa. Proteins of SC central space are most evolutionarily variable. It means that different protein-protein interactions can exist to connect LEs. Proteins similar to the known SC proteins were not found in Euglenophyta, Chrysophyta, Charophyta, Xanthophyta, Dinoflagellata, and primitive Coelomata. We conclude that different proteins whose common feature is the presence of domains with a certain conformation are involved in the formation of the SC in different eukaryotic phyla. This permits a targeted search for orthologs of the SC proteins using phylogenetic trees. Here we consider example of phylogenetic trees for protozoans, fungi, algae, mosses, and flowering plants. PMID:25147749

  4. Conservation and variability of synaptonemal complex proteins in phylogenesis of eukaryotes.

    PubMed

    Grishaeva, Tatiana M; Bogdanov, Yuri F

    2014-01-01

    The problems of the origin and evolution of meiosis include the enigmatic variability of the synaptonemal complexes (SCs) which, being morphology similar, consist of different proteins in different eukaryotic phyla. Using bioinformatics methods, we monitored all available eukaryotic proteomes to find proteins similar to known SC proteins of model organisms. We found proteins similar to SC lateral element (LE) proteins and possessing the HORMA domain in the majority of the eukaryotic taxa and assume them the most ancient among all SC proteins. Vertebrate LE proteins SYCP2, SYCP3, and SC65 proved to have related proteins in many invertebrate taxa. Proteins of SC central space are most evolutionarily variable. It means that different protein-protein interactions can exist to connect LEs. Proteins similar to the known SC proteins were not found in Euglenophyta, Chrysophyta, Charophyta, Xanthophyta, Dinoflagellata, and primitive Coelomata. We conclude that different proteins whose common feature is the presence of domains with a certain conformation are involved in the formation of the SC in different eukaryotic phyla. This permits a targeted search for orthologs of the SC proteins using phylogenetic trees. Here we consider example of phylogenetic trees for protozoans, fungi, algae, mosses, and flowering plants. PMID:25147749

  5. The beginning of a beautiful friendship: Cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes

    PubMed Central

    Rappsilber, Juri

    2011-01-01

    After more than a decade of method development, cross-linking in combination with mass spectrometry and bioinformatics is finally coming of age. This technology now provides improved opportunities for modelling by mapping structural details of functional complexes in solution. The structure of proteins or protein complexes is ascertained by identifying amino acid pairs that are positioned in close proximity to each other. The validity of this technique has recently been benchmarked for large multi-protein complexes, by comparing cross-link data with that from a crystal structure of RNA polymerase II. Here, the specific nature of this cross-linking data will be discussed to assess the technical challenges and opportunities for model building. We believe that once remaining technological challenges of cross-linking/mass spectrometry have been addressed and cross-linking/mass spectrometry data has been incorporated into modelling algorithms it will quickly become an indispensable companion of protein and protein complex modelling and a corner-stone of integrated structural biology. PMID:21029779

  6. Fractal and complex network analyses of protein molecular dynamics

    NASA Astrophysics Data System (ADS)

    Zhou, Yuan-Wu; Liu, Jin-Long; Yu, Zu-Guo; Zhao, Zhi-Qin; Anh, Vo

    2014-12-01

    Based on protein molecular dynamics, we investigate the fractal properties of energy, pressure and volume time series using the multifractal detrended fluctuation analysis (MF-DFA) and the topological and fractal properties of their converted horizontal visibility graphs (HVGs). The energy parameters of protein dynamics we considered are bonded potential, angle potential, dihedral potential, improper potential, kinetic energy, Van der Waals potential, electrostatic potential, total energy and potential energy. The shape of the h(q) curves from MF-DFA indicates that these time series are multifractal. The numerical values of the exponent h(2) of MF-DFA show that the series of total energy and potential energy are non-stationary and anti-persistent; the other time series are stationary and persistent apart from series of pressure (with H≈0.5 indicating the absence of long-range correlation). The degree distributions of their converted HVGs show that these networks are exponential. The results of fractal analysis show that fractality exists in these converted HVGs. For each energy, pressure or volume parameter, it is found that the values of h(2) of MF-DFA on the time series, exponent λ of the exponential degree distribution and fractal dimension dB of their converted HVGs do not change much for different proteins (indicating some universality). We also found that after taking average over all proteins, there is a linear relationship between (from MF-DFA on time series) and of the converted HVGs for different energy, pressure and volume.

  7. Capillary electrophoresis methods for the determination of covalent polyphenol-protein complexes.

    PubMed

    Trombley, John D; Loegel, Thomas N; Danielson, Neil D; Hagerman, Ann E

    2011-09-01

    The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine. PMID:21400190

  8. An integrated approach to identify protein complex based on best neighbour and modularity increment.

    PubMed

    Shen, Xianjun; Zhao, Yanli; Li, Yanan; Yi, Yang; He, Tingting; Yang, Jincai

    2015-01-01

    In order to overcome the limitations of global modularity and the deficiency of local modularity, we propose a hybrid modularity measure Local-Global Quantification (LGQ) which considers global modularity and local modularity together. LGQ adopts a suitable module feature adjustable parameter to control the balance of global detecting capability and local search capability in Protein-Protein Interactions (PPI) Network. Furthermore, we develop a new protein complex mining algorithm called Best Neighbour and Local-Global Quantification (BN-LGQ) which integrates the best neighbour node and modularity increment. BN-LGQ expands the protein complex by fast searching the best neighbour node of the current cluster and by calculating the modularity increment as a metric to determine whether the best neighbour node can join the current cluster. The experimental results show BN-LGQ performs a better accuracy on predicting protein complexes and has a higher match with the reference protein complexes than MCL and MCODE algorithms. Moreover, BN-LGQ can effectively discover protein complexes with better biological significance in the PPI network. PMID:26336669

  9. Influence of pea protein aggregates on the structure and stability of pea protein/soybean polysaccharide complex emulsions.

    PubMed

    Yin, Baoru; Zhang, Rujing; Yao, Ping

    2015-01-01

    The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI) with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS), and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry. PMID:25803397

  10. Application of model bread baking in the examination of arabinoxylan-protein complexes in rye bread.

    PubMed

    Buksa, Krzysztof

    2016-09-01

    The changes in molecular mass of arabinoxylan (AX) and protein caused by bread baking process were examined using a model rye bread. Instead of the normal flour, the dough contained starch, water-extractable AX and protein which were isolated from rye wholemeal. From the crumb of selected model breads, starch was removed releasing AX-protein complexes, which were further examined by size exclusion chromatography. On the basis of the research, it was concluded that optimum model mix can be composed of 3-6% AX and 3-6% rye protein isolate at 94-88% of rye starch meaning with the most similar properties to low extraction rye flour. Application of model rye bread allowed to examine the interactions between AX and proteins. Bread baked with a share of AX, rye protein and starch, from which the complexes of the highest molar mass were isolated, was characterized by the strongest structure of the bread crumb. PMID:27185141

  11. Wrap-and-Strip Technology of Protein-Polyelectrolyte Complex for Biomedical Application.

    PubMed

    Shiraki, Kentaro; Kurinomaru, Takaaki; Tomita, Shunsuke

    2016-01-01

    A polyelectrolyte is a polymer composed of repeating units of an electrolyte group that enables reversible complex formation with proteins in aqueous solutions. This review introduces "wrap-and-strip" technology of protein-polyelectrolyte complex (PPC) by noncovalent interaction. Storage: protein is stabilized against physical and chemical stresses. Enrichment: precipitation through PPC can be used as an enrichment method without irreversible unfolding. Catalytic activity switch: a complementary charged pair of polyelectrolytes functions as a reversible enzyme activity switch. Hyperactivation: a specific combination of a polyelectrolyte and substrate enhances enzyme activity by one order of magnitude compared with an enzyme alone. Stabilization: PPC increases protein stability against chemical and physical stresses, such as covalently modified polyethylene glycosylated protein. Simple PPC-based technology can expand the applicable fields of soluble proteins in aqueous solutions. PMID:26630921

  12. Probing protein complexes inside living cells using a silicon nanowire-based pull-down assay

    NASA Astrophysics Data System (ADS)

    Choi, Sojoong; Kim, Hyunju; Kim, So Yeon; Yang, Eun Gyeong

    2016-06-01

    Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells.Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells. Electronic supplementary information (ESI) available: Materials, experimental methods and Fig. S1-S8. See DOI: 10.1039/c6nr00171h

  13. Binding Linkage in a Telomere DNA–Protein Complex at the Ends of Oxytricha nova Chromosomes

    PubMed Central

    Buczek, Pawel; Orr, Rochelle S.; Pyper, Sean R.; Shum, Mili; Ota, Emily Kimmel Irene; Gerum, Shawn E.; Horvath, Martin P.

    2005-01-01

    Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein–protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (KD-DNA=1.4 nM). Another fusion protein, constructed without the C-terminal protein–protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (KD-DNA=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA–protein stability to protein–protein contacts at a remote site may provide a trigger point for DNA–protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase. PMID:15967465

  14. Robust assessment of protein complex formation in vivo via single-molecule intensity distributions of autofluorescent proteins

    NASA Astrophysics Data System (ADS)

    Meckel, Tobias; Semrau, Stefan; Schaaf, Marcel J. M.; Schmidt, Thomas

    2011-07-01

    The formation of protein complexes or clusters in the plasma membrane is essential for many biological processes, such as signaling. We develop a tool, based on single-molecule microscopy, for following cluster formation in vivo. Detection and tracing of single autofluorescent proteins have become standard biophysical techniques. The determination of the number of proteins in a cluster, however, remains challenging. The reasons are (i) the poor photophysical stability and complex photophysics of fluorescent proteins and (ii) noise and autofluorescent background in live cell recordings. We show that, despite those obstacles, the accurate fraction of signals in which a certain (or set) number of labeled proteins reside, can be determined in an accurate an robust way in vivo. We define experimental conditions under which fluorescent proteins exhibit predictable distributions of intensity and quantify the influence of noise. Finally, we confirm our theoretical predictions by measurements of the intensities of individual enhanced yellow fluorescent protein (EYFP) molecules in living cells. Quantification of the average number of EYFP-C10HRAS chimeras in diffraction-limited spots finally confirm that the membrane anchor of human Harvey rat sarcoma (HRAS) heterogeneously distributes in the plasma membrane of living Chinese hamster ovary cells.

  15. An informatic framework for decoding protein complexes by top-down mass spectrometry

    PubMed Central

    Skinner, Owen S.; Havugimana, Pierre C.; Haverland, Nicole A.; Fornelli, Luca; Early, Bryan P.; Greer, Joseph B.; Fellers, Ryan T.; Durbin, Kenneth R.; Do Vale, Luis H. F.; Melani, Rafael D.; Seckler, Henrique S.; Nelp, Micah T.; Belov, Mikhail E.; Horning, Stevan R.; Makarov, Alexander A.; LeDuc, Richard D.; Bandarian, Vahe; Compton, Philip D.; Kelleher, Neil L.

    2015-01-01

    Efforts to map the human protein interactome have resulted in information about hundreds to thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we combined the CORUM and UniProt databases to create candidates for an error-tolerant search engine designed for hierarchical top-down analyses, identification, and scoring of multi-proteoform complexes by native mass spectrometry. PMID:26780093

  16. An informatic framework for decoding protein complexes by top-down mass spectrometry.