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Sample records for protein folding secretion

  1. Secretion of slow-folding proteins by a Type 1 secretion system.

    PubMed

    Schwarz, Christian K W; Lenders, Michael H H; Smits, Sander H J; Schmitt, Lutz

    2012-01-01

    Protein production through dedicated secretion systems might offer an potential alternative to the conventional cytoplasmical expression. The application of Type 1 secretion systems of Gram-negative bacteria, however, where often not successful in the past for a wide range of proteins. Recently, two studies using the E. coli maltose binding protein (MalE) and the rat intestinal fatty acid binding protein (IFABP) revealed a rational to circumvent these limitations. Here, wild-type passenger proteins were not secreted, while folding mutants with decreased folding kinetics were efficiently exported to the extracellular space. Subsequently, an one-step purification protocol yielded homogeneous and active protein. Taken together, theses two studies suggest that the introduction of slow-folding mutations into a protein sequence might be the key to use Type 1 secretion systems for the biotechnological production of proteins.

  2. Yersinia enterocolitica type III secretion: evidence for the ability to transport proteins that are folded prior to secretion.

    PubMed

    Wilharm, Gottfried; Lehmann, Verena; Neumayer, Wibke; Trcek, Janja; Heesemann, Jürgen

    2004-07-12

    Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a type three secretion system (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2-3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of specific Yop chaperones (called Sycs). In this study we have fused YopE secretion signals of various length to the mouse dihydrofolate reductase (DHFR) in order to analyse the DHFR folding state prior to secretion. We could demonstrate that secretion-deficient as well as secretion-competent YopE-DHFR fusions complexed to SycE can be efficiently purified from Yersinia cytosol by affinity chromatography using methotrexate-agarose. This implies the folding of the DHFR fusion moiety despite SycE binding and contradicts the previously presented model of folding inhibition by chaperone binding. Secretion-deficient YopE-DHFR fusions caused severe jamming of the TTSS. This observation contradicts the co-translational secretion model. We present evidence that the Yersinia TTSS is familiar with the processing of transport substrates which are folded prior to secretion. We therefore predict that an unfoldase is involved in type III secretion.

  3. The Salmonella Type III Secretion System Inner Rod Protein PrgJ Is Partially Folded*

    PubMed Central

    Zhong, Dalian; Lefebre, Matthew; Kaur, Kawaljit; McDowell, Melanie A.; Gdowski, Courtney; Jo, Sunhwan; Wang, Yu; Benedict, Stephen H.; Lea, Susan M.; Galan, Jorge E.; De Guzman, Roberto N.

    2012-01-01

    The type III secretion system (T3SS) is essential in the pathogenesis of many bacteria. The inner rod is important in the assembly of the T3SS needle complex. However, the atomic structure of the inner rod protein is currently unknown. Based on computational methods, others have suggested that the Salmonella inner rod protein PrgJ is highly helical, forming a folded 3 helix structure. Here we show by CD and NMR spectroscopy that the monomeric form of PrgJ lacks a tertiary structure, and the only well-structured part of PrgJ is a short α-helix at the C-terminal region from residues 65–82. Disruption of this helix by glycine or proline mutation resulted in defective assembly of the needle complex, rendering bacteria incapable of secreting effector proteins. Likewise, CD and NMR data for the Shigella inner rod protein MxiI indicate this protein lacks a tertiary structure as well. Our results reveal that the monomeric forms of the T3SS inner rod proteins are partially folded. PMID:22654099

  4. Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

    SciTech Connect

    Davis, Jamaine; Wang, Jiawei; Tropea, Joseph E.; Zhang, Di; Dauter, Zbigniew; Waugh, David S.; Wlodawer, Alexander

    2009-01-28

    VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave {alpha}-tubulin, thus facilitating intracellular spreading. We have now determined the crystal structure of VirA at 3.0 {angstrom} resolution. The shape of the molecule resembles the letter 'V,' with the residues in the N-terminal third of the 45-kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its Escherichia coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.

  5. Secretion of the Intimin Passenger Domain Is Driven by Protein Folding*

    PubMed Central

    Leo, Jack C.; Oberhettinger, Philipp; Yoshimoto, Shogo; Udatha, D. B. R. K. Gupta; Morth, J. Preben; Schütz, Monika; Hori, Katsutoshi

    2016-01-01

    Intimin is an essential adhesin of attaching and effacing organisms such as entropathogenic Escherichia coli. It is also the prototype of type Ve secretion or inverse autotransport, where the extracellular C-terminal region or passenger is exported with the help of an N-terminal transmembrane β-barrel domain. We recently reported a stalled secretion intermediate of intimin, where the passenger is located in the periplasm but the β-barrel is already inserted into the membrane. Stalling of this mutant is due to the insertion of an epitope tag at the very N terminus of the passenger. Here, we examined how this insertion disrupts autotransport and found that it causes misfolding of the N-terminal immunoglobulin (Ig)-like domain D00. We could also stall the secretion by making an internal deletion in D00, and introducing the epitope tag into the second Ig-like domain, D0, also resulted in reduced passenger secretion. In contrast to many classical autotransporters, where a proximal folding core in the passenger is required for secretion, the D00 domain is dispensable, as the passenger of an intimin mutant lacking D00 entirely is efficiently exported. Furthermore, the D00 domain is slightly less stable than the D0 and D1 domains, unfolding at ∼200 piconewtons (pN) compared with ∼250 pN for D0 and D1 domains as measured by atomic force microscopy. Our results support a model where the secretion of the passenger is driven by sequential folding of the extracellular Ig-like domains, leading to vectorial transport of the passenger domain across the outer membrane in an N to C direction. PMID:27466361

  6. THE DELICATE BALANCE BETWEEN SECRETED PROTEIN FOLDING AND ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION IN HUMAN PHYSIOLOGY

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.

    2014-01-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates. PMID:22535891

  7. The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology.

    PubMed

    Guerriero, Christopher J; Brodsky, Jeffrey L

    2012-04-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding "problem," as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates.

  8. Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter.

    PubMed

    Skillman, Kristen M; Barnard, Travis J; Peterson, Janine H; Ghirlando, Rodolfo; Bernstein, Harris D

    2005-11-01

    Bacterial autotransporters are proteins that contain a small C-terminal 'beta domain' that facilitates translocation of a large N-terminal 'passenger domain' across the outer membrane (OM) by an unknown mechanism. Here we used EspP, an autotransporter produced by Escherichia coli 0157:H7, as a model protein to gain insight into the transport reaction. Initially we found that the passenger domain of a truncated version of EspP (EspPDelta1-851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspPDelta1-851 behaves as a compact monomer and strongly suggest that the channel formed by the beta domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N-terminus of EspPDelta1-851 was efficiently translocated across the OM. Further analysis revealed that the passenger domain of wild-type EspP also folds at least partially in the periplasm. To reconcile these data, we propose that the EspP beta domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.

  9. Calcium-Driven Folding of RTX Domain β-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts.

    PubMed

    Bumba, Ladislav; Masin, Jiri; Macek, Pavel; Wald, Tomas; Motlova, Lucia; Bibova, Ilona; Klimova, Nela; Bednarova, Lucie; Veverka, Vaclav; Kachala, Michael; Svergun, Dmitri I; Barinka, Cyril; Sebo, Peter

    2016-04-07

    Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. The LcrG Tip Chaperone Protein of the Yersinia pestis Type III Secretion System Is Partially Folded.

    PubMed

    Chaudhury, Sukanya; de Azevedo Souza, Clarice; Plano, Gregory V; De Guzman, Roberto N

    2015-09-25

    The type III secretion system (T3SS) is essential in the pathogenesis of Yersinia pestis, the causative agent of plague. A small protein, LcrG, functions as a chaperone to the tip protein LcrV, and the LcrG-LcrV interaction is important in regulating protein secretion through the T3SS. The atomic structure of the LcrG family is currently unknown. However, because of its predicted helical propensity, many have suggested that the LcrG family forms a coiled-coil structure. Here, we show by NMR and CD spectroscopy that LcrG lacks a tertiary structure and it consists of three partially folded α-helices spanning residues 7-38, 41-46, and 58-73. NMR titrations of LcrG with LcrV show that the entire length of a truncated LcrG (residues 7-73) is involved in binding to LcrV. However, there is regional variation in how LcrG binds to LcrV. The C-terminal region of a truncated LcrG (residues 52-73) shows tight binding interaction with LcrV while the N-terminal region (residues 7-51) shows weaker interaction with LcrV. This suggests that there are at least two binding events when LcrG binds to LcrV. Biological assays and mutagenesis indicate that the C-terminal region of LcrG (residues 52-73) is important in blocking protein secretion through the T3SS. Our results reveal structural and mechanistic insights into the atomic conformation of LcrG and how it binds to LcrV. Copyright © 2015. Published by Elsevier Ltd.

  11. Genetic ablation of N-linked glycosylation reveals two key folding pathways for R345W fibulin-3, a secreted protein associated with retinal degeneration

    PubMed Central

    Hulleman, John D.; Kelly, Jeffery W.

    2015-01-01

    An R345W mutation in the N-glycoprotein, fibulin-3 (F3), results in inefficient F3 folding/secretion and higher intracellular F3 levels. Inheritance of this mutation causes the retinal dystrophy malattia leventinese. N-Linked glycosylation is a common cotranslational protein modification that can regulate protein folding efficiency and energetics. Therefore, we explored how N-glycosylation alters the protein homeostasis or proteostasis of wild-type (WT) and R345W F3 in ARPE-19 cells. Enzymatic and lectin binding assays confirmed that WT and R345W F3 are both primarily N-glycosylated at Asn249. Tunicamycin treatment selectively reduced R345W F3 secretion by 87% (vs. WT F3). Genetic elimination of F3 N-glycosylation (via an N249Q mutation) caused R345W F3 to aggregate intracellularly and adopt an altered secreted conformation. The endoplasmic reticulum (ER) chaperones GRP78 (glucose-regulated protein 78) and GRP94 (glucose-regulated protein 94), and the ER lectins calnexin and calreticulin were identified as F3 binding partners by immunoprecipitation. Significantly more N249Q and N249Q/R345W F3 interacted with GRP94, while substantially less N249Q and N249Q/R345W interacted with the ER lectins than their N-glycosylated counterparts. Inhibition of GRP94 ATPase activity reduced only N249Q/R345W F3 secretion (by 62%), demonstrating this variant’s unique reliance on GRP94 for secretion. These observations suggest that R345W F3, but not WT F3, requires N-glycosylation to acquire a stable, native-like structure.—Hulleman, J. D., Kelly, J. W. Genetic ablation of N-linked glycosylation reveals two key folding pathways for R345W fibulin-3, a secreted protein associated with retinal degeneration. PMID:25389134

  12. The Protein Folding Problem

    PubMed Central

    Dill, Ken A.; Ozkan, S. Banu; Shell, M. Scott; Weikl, Thomas R.

    2008-01-01

    The “protein folding problem” consists of three closely related puzzles: (a) What is the folding code? (b) What is the folding mechanism? (c) Can we predict the native structure of a protein from its amino acid sequence? Once regarded as a grand challenge, protein folding has seen great progress in recent years. Now, foldable proteins and nonbiological polymers are being designed routinely and moving toward successful applications. The structures of small proteins are now often well predicted by computer methods. And, there is now a testable explanation for how a protein can fold so quickly: A protein solves its large global optimization problem as a series of smaller local optimization problems, growing and assembling the native structure from peptide fragments, local structures first. PMID:18573083

  13. Fast protein folding kinetics.

    PubMed

    Gelman, Hannah; Gruebele, Martin

    2014-05-01

    Fast-folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast-folding proteins has provided insight into the mechanisms, which allow some proteins to find their native conformation well <1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even 'slow' folding processes: fast folders are small; relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast-folding proteins and provides an overview of the major findings of fast-folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general, as well as some work that is left to do.

  14. Fast protein folding kinetics

    PubMed Central

    Gelman, Hannah; Gruebele, Martin

    2014-01-01

    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  15. Protein Folding: Detailed Models

    NASA Astrophysics Data System (ADS)

    Pande, Vijay

    Proteins play a fundamental role in biology. With their ability to perform numerous biological roles, including acting as catalysts, antibodies, and molecular signals, proteins today realize many of the goals that modern nanotechnology aspires to. However, before proteins can carry out these remarkable molecular functions, they must perform another amazing feat — they must assemble themselves. This process of protein self-assembly into a particular shape, or "fold" is called protein folding. Due to the importance of the folded state in the biological activity of proteins, recent interest from misfolding related diseases [1], as well as a fascination of just how this process occurs [2-4], there has been much work performed in order to unravel the mechanism of protein folding [5].

  16. Structural mapping of the ClpB ATPases of Plasmodium falciparum: Targeting protein folding and secretion for antimalarial drug design.

    PubMed

    AhYoung, Andrew P; Koehl, Antoine; Cascio, Duilio; Egea, Pascal F

    2015-09-01

    Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains-a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs. © 2015 The Protein Society.

  17. Structural mapping of the ClpB ATPases of Plasmodium falciparum: Targeting protein folding and secretion for antimalarial drug design

    PubMed Central

    AhYoung, Andrew P; Koehl, Antoine; Cascio, Duilio; Egea, Pascal F

    2015-01-01

    Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains—a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs. PMID:26130467

  18. Genetic ablation of N-linked glycosylation reveals two key folding pathways for R345W fibulin-3, a secreted protein associated with retinal degeneration.

    PubMed

    Hulleman, John D; Kelly, Jeffery W

    2015-02-01

    An R345W mutation in the N-glycoprotein, fibulin-3 (F3), results in inefficient F3 folding/secretion and higher intracellular F3 levels. Inheritance of this mutation causes the retinal dystrophy malattia leventinese. N-Linked glycosylation is a common cotranslational protein modification that can regulate protein folding efficiency and energetics. Therefore, we explored how N-glycosylation alters the protein homeostasis or proteostasis of wild-type (WT) and R345W F3 in ARPE-19 cells. Enzymatic and lectin binding assays confirmed that WT and R345W F3 are both primarily N-glycosylated at Asn249. Tunicamycin treatment selectively reduced R345W F3 secretion by 87% (vs. WT F3). Genetic elimination of F3 N-glycosylation (via an N249Q mutation) caused R345W F3 to aggregate intracellularly and adopt an altered secreted conformation. The endoplasmic reticulum (ER) chaperones GRP78 (glucose-regulated protein 78) and GRP94 (glucose-regulated protein 94), and the ER lectins calnexin and calreticulin were identified as F3 binding partners by immunoprecipitation. Significantly more N249Q and N249Q/R345W F3 interacted with GRP94, while substantially less N249Q and N249Q/R345W interacted with the ER lectins than their N-glycosylated counterparts. Inhibition of GRP94 ATPase activity reduced only N249Q/R345W F3 secretion (by 62%), demonstrating this variant's unique reliance on GRP94 for secretion. These observations suggest that R345W F3, but not WT F3, requires N-glycosylation to acquire a stable, native-like structure.

  19. Folding kinetics and thermodynamics of Pseudomonas syringae effector protein AvrPto provide insight into translocation via the type III secretion system.

    PubMed

    Dawson, Jennifer E; Nicholson, Linda K

    2008-07-01

    In order to infect their hosts, many Gram-negative bacteria translocate agents of infection, called effector proteins, through the type III secretion system (TTSS) into the host cytoplasm. This process is thought to require at least partial unfolding of these agents, raising the question of how an effector protein might unfold to enable its translocation and then refold once it reaches the host cytoplasm. AvrPto is a well-studied effector protein of Pseudomonas syringae pv tomato. The presence of a readily observed unfolded population of AvrPto in aqueous solution and the lack of a known secretion chaperone make it ideal for studying the kinetic and thermodynamic characteristics that facilitate translocation. Application of Nzz exchange spectroscopy revealed a global, two-state folding equilibrium with 16% unfolded population, a folding rate of 1.8 s(-1), and an unfolding rate of 0.33 s(-1) at pH 6.1. TrAvrPto stability increases with increasing pH, with only 2% unfolded population observed at pH 7.0. The R(1) relaxation of TrAvrPto, which is sensitive to both the global anisotropy of folded TrAvrPto and slow exchange between folded and unfolded conformations, provided independent verification of the global kinetic rate constants. Given the acidic apoplast in which the pathogen resides and the more basic host cytoplasm, these results offer an intriguing mechanism by which the pH dependence of stability and slow folding kinetics of AvrPto would allow efficient translocation of the unfolded form through the TTSS and refolding into its functional folded form once inside the host.

  20. Protein folding and misfolding

    NASA Astrophysics Data System (ADS)

    Dobson, Christopher M.

    2003-12-01

    The manner in which a newly synthesized chain of amino acids transforms itself into a perfectly folded protein depends both on the intrinsic properties of the amino-acid sequence and on multiple contributing influences from the crowded cellular milieu. Folding and unfolding are crucial ways of regulating biological activity and targeting proteins to different cellular locations. Aggregation of misfolded proteins that escape the cellular quality-control mechanisms is a common feature of a wide range of highly debilitating and increasingly prevalent diseases.

  1. Autotransporter protein secretion.

    PubMed

    Tame, Jeremy R H

    2011-12-01

    Autotransporter proteins are a large family of virulence factors secreted from Gram-negative bacteria by a unique mechanism. First described in the 1980s, these proteins have a C-terminal region that folds into a β-barrel in the bacterial outer membrane. The so-called passenger domain attached to this barrel projects away from the cell surface and may be liberated from the cell by self-cleavage or surface proteases. Although the majority of passenger domains have a similar β-helical structure, they carry a variety of sub-domains, allowing them to carry out widely differing functions related to pathogenesis. Considerable biochemical and structural characterisation of the barrel domain has shown that 'autotransporters' in fact require a conserved and essential protein complex in the outer membrane for correct folding. Although the globular domains of this complex projecting into the periplasmic space have also been structurally characterised, the overall secretion pathway of the autotransporters remains highly puzzling. It was presumed for many years that the passenger domain passed through the centre of the barrel domain to reach the cell surface, driven at least in part by folding. This picture is complicated by conflicting data, and there is currently little hard information on the true nature of the secretion intermediates. As well as their medical importance therefore, autotransporters are proving to be an excellent system to study the folding and membrane insertion of outer membrane proteins in general. This review focuses on structural aspects of autotransporters; their many functions in pathogenesis are beyond its scope.

  2. Protein Flexibilty and Folding

    NASA Astrophysics Data System (ADS)

    Thorpe, Michael

    2003-10-01

    In this talk we apply a novel approach to the exploration of energy landscapes of macromolecules and proteins that uses constraint theory. Constraints fix the bond lengths and bond angles and allow the use of theorems from graph theory to perform a rigid region decomposition of the network of atoms, which identifies the rigid regions, the flexible joints between them and also the stressed regions. We will show movies of the diffusive motion of various proteins. The protein unfolding transition is an example of a rigid to floppy transition and is shown to be more first order than second order because of the self-organized nature of the cross-linked polypeptide chain in the native protein. This approach emphasizes the universality in protein unfolding and allows the folding core and the transition state to be identified. Useful reference are: M.F. Thorpe, Ming Lei, A.J. Rader, Donald J. Jacobs and Leslie A. Kuhn Protein Flexibility Predictions using Graph Theory, Proteins 44, 150 - 165, (2001). A. J. Rader, Brandon M. Hespenheide, Leslie A. Kuhn and M. F. Thorpe Protein Unfolding: Rigidity Lost Proceedings of the National Academy of Sciences 99, 3540-3545 (2002). More details of this work can be found via http://physics.asu.edu/mfthorpe

  3. Protein Folding: Then and Now

    PubMed Central

    Chen, Yiwen; Ding, Feng; Nie, Huifen; Serohijos, Adrian W.; Sharma, Shantanu; Wilcox, Kyle C.; Yin, Shuangye; Dokholyan, Nikolay V.

    2007-01-01

    Over the past three decades the protein folding field has undergone monumental changes. Originally a purely academic question, how a protein folds has now become vital in understanding diseases and our abilities to rationally manipulate cellular life by engineering protein folding pathways. We review and contrast past and recent developments in the protein folding field. Specifically, we discuss the progress in our understanding of protein folding thermodynamics and kinetics, the properties of evasive intermediates, and unfolded states. We also discuss how some abnormalities in protein folding lead to protein aggregation and human diseases. PMID:17585870

  4. Regulation of protein secretion by ... protein secretion?

    PubMed

    Atmakuri, Krishnamohan; Fortune, Sarah M

    2008-09-11

    Mycobacterium tuberculosis (Mtb) requires an alternative protein secretion system, ESX1, for virulence. Recently, Raghavan et al. (2008) reported a new regulatory circuit that may explain how ESX1 activity is controlled during infection. Mtb appears to regulate ESX1 by modulating transcription of associated genes rather than structural components of the secretion system itself.

  5. How do chaperonins fold protein?

    PubMed Central

    Motojima, Fumihiro

    2015-01-01

    Protein folding is a biological process that is essential for the proper functioning of proteins in all living organisms. In cells, many proteins require the assistance of molecular chaperones for their folding. Chaperonins belong to a class of molecular chaperones that have been extensively studied. However, the mechanism by which a chaperonin mediates the folding of proteins is still controversial. Denatured proteins are folded in the closed chaperonin cage, leading to the assumption that denatured proteins are completely encapsulated inside the chaperonin cage. In contrast to the assumption, we recently found that denatured protein interacts with hydrophobic residues at the subunit interfaces of the chaperonin, and partially protrude out of the cage. In this review, we will explain our recent results and introduce our model for the mechanism by which chaperonins accelerate protein folding, in view of recent findings. PMID:27493521

  6. Evolutionary Optimization of Protein Folding

    PubMed Central

    Debès, Cédric; Wang, Minglei; Caetano-Anollés, Gustavo; Gräter, Frauke

    2013-01-01

    Nature has shaped the make up of proteins since their appearance, 3.8 billion years ago. However, the fundamental drivers of structural change responsible for the extraordinary diversity of proteins have yet to be elucidated. Here we explore if protein evolution affects folding speed. We estimated folding times for the present-day catalog of protein domains directly from their size-modified contact order. These values were mapped onto an evolutionary timeline of domain appearance derived from a phylogenomic analysis of protein domains in 989 fully-sequenced genomes. Our results show a clear overall increase of folding speed during evolution, with known ultra-fast downhill folders appearing rather late in the timeline. Remarkably, folding optimization depends on secondary structure. While alpha-folds showed a tendency to fold faster throughout evolution, beta-folds exhibited a trend of folding time increase during the last 1.5 billion years that began during the “big bang” of domain combinations. As a consequence, these domain structures are on average slow folders today. Our results suggest that fast and efficient folding of domains shaped the universe of protein structure. This finding supports the hypothesis that optimization of the kinetic and thermodynamic accessibility of the native fold reduces protein aggregation propensities that hamper cellular functions. PMID:23341762

  7. Changes of protein stiffness during folding detect protein folding intermediates.

    PubMed

    Małek, Katarzyna E; Szoszkiewicz, Robert

    2014-01-01

    Single-molecule force-quench atomic force microscopy (FQ-AFM) is used to detect folding intermediates of a simple protein by detecting changes of molecular stiffness of the protein during its folding process. Those stiffness changes are obtained from shape and peaks of an autocorrelation of fluctuations in end-to-end length of the folding molecule. The results are supported by predictions of the equipartition theorem and agree with existing Langevin dynamics simulations of a simplified model of a protein folding. In the light of the Langevin simulations the experimental data probe an ensemble of random-coiled collapsed states of the protein, which are present both in the force-quench and thermal-quench folding pathways.

  8. The protein folding network

    NASA Astrophysics Data System (ADS)

    Rao, Francesco; Caflisch, Amedeo

    2004-03-01

    Networks are everywhere. The conformation space of a 20-residue antiparallel beta-sheet peptide [1], sampled by molecular dynamics simulations, is mapped to a network. Conformations are nodes of the network, and the transitions between them are links. As previously found for the World-Wide Web as well as for social and biological networks , the conformation space contains highly connected hubs like the native state which is the most populated free energy basin. Furthermore, the network shows a hierarchical modularity [2] which is consistent with the funnel mechanism of folding [3] and is not observed for a random heteropolymer lacking a native state. Here we show that the conformation space network describes the free energy landscape without requiring projections into arbitrarily chosen reaction coordinates. The network analysis provides a basis for understanding the heterogeneity of the folding transition state and the existence of multiple pathways. [1] P. Ferrara and A. Caflisch, Folding simulations of a three-stranded antiparallel beta-sheet peptide, PNAS 97, 10780-10785 (2000). [2] Ravasz, E. and Barabási, A. L. Hierarchical organization in complex networks. Phys. Rev. E 67, 026112 (2003). [3] Dill, K. and Chan, H From Levinthal to pathways to funnels. Nature Struct. Biol. 4, 10-19 (1997)

  9. Protein folding in the cell

    NASA Astrophysics Data System (ADS)

    Gething, Mary-Jane; Sambrook, Joseph

    1992-01-01

    In the cell, as in vitro, the final conformation of a protein is determined by its amino-acid sequence. But whereas some isolated proteins can be denatured and refolded in vitro in the absence of other macromolecular cellular components, folding and assembly of polypeptides in vivo involves other proteins, many of which belong to families that have been highly conserved during evolution.

  10. Protein folding by motion planning

    NASA Astrophysics Data System (ADS)

    Thomas, Shawna; Song, Guang; Amato, Nancy M.

    2005-12-01

    We investigate a novel approach for studying protein folding that has evolved from robotics motion planning techniques called probabilistic roadmap methods (PRMs). Our focus is to study issues related to the folding process, such as the formation of secondary and tertiary structures, assuming we know the native fold. A feature of our PRM-based framework is that the large sets of folding pathways in the roadmaps it produces, in just a few hours on a desktop PC, provide global information about the protein's energy landscape. This is an advantage over other simulation methods such as molecular dynamics or Monte Carlo methods which require more computation and produce only a single trajectory in each run. In our initial studies, we obtained encouraging results for several small proteins. In this paper, we investigate more sophisticated techniques for analyzing the folding pathways in our roadmaps. In addition to more formally revalidating our previous results, we present a case study showing that our technique captures known folding differences between the structurally similar proteins G and L. This research was supported in part by NSF CAREER Award CCR-9624315, NSF Grants ACI-9872126, EIA-9975018, EIA-0103742, EIA-9805823, ACR-0113971, CCR-0113974, EIA-9810937, EIA-0079874 and the Texas Higher Education Coordinating Board grant ATP-000512-0261-2001. ST was supported in part by an NSF Graduate Research Fellowship. GS was supported in part by an IBM PhD Fellowship.

  11. Protein folding by motion planning.

    PubMed

    Thomas, Shawna; Song, Guang; Amato, Nancy M

    2005-11-09

    We investigate a novel approach for studying protein folding that has evolved from robotics motion planning techniques called probabilistic roadmap methods (PRMs). Our focus is to study issues related to the folding process, such as the formation of secondary and tertiary structures, assuming we know the native fold. A feature of our PRM-based framework is that the large sets of folding pathways in the roadmaps it produces, in just a few hours on a desktop PC, provide global information about the protein's energy landscape. This is an advantage over other simulation methods such as molecular dynamics or Monte Carlo methods which require more computation and produce only a single trajectory in each run. In our initial studies, we obtained encouraging results for several small proteins. In this paper, we investigate more sophisticated techniques for analyzing the folding pathways in our roadmaps. In addition to more formally revalidating our previous results, we present a case study showing that our technique captures known folding differences between the structurally similar proteins G and L.

  12. Use of Protein Folding Reagents.

    PubMed

    2016-04-01

    The reagents and methods for purification and use of the most commonly used denaturants, guanidine hydrochloride (guanidine-HCl) and urea, are described. Other protein denaturants and reagents used to fold proteins are briefly mentioned. Sulfhydryl reagents (reducing agents) and "oxido-shuffling" (or oxidative regeneration) systems are also described.

  13. Turbulent phenomena in protein folding.

    PubMed

    Kalgin, Igor V; Chekmarev, Sergei F

    2011-01-01

    Protein folding and hydrodynamic turbulence are two long-standing challenges, in molecular biophysics and fluid dynamics, respectively. The theories of these phenomena have been developed independently and used different formalisms. Here we show that the protein folding flows can be surprisingly similar to turbulent fluid flows. Studying a benchmark model protein (an SH3 domain), we have found that the flows for the slow folding trajectories of the protein, in which a partly formed N- and C-terminal β sheet hinders the RT loop from attaching to the protein core, have many properties of turbulent flows of a fluid. The flows are analyzed in a three-dimensional (3D) space of collective variables, which are the numbers of native contacts between the terminal β strands, between the RT loop and the protein core, and the rest of the native contacts. We have found that the flows have fractal nature and are filled with 3D eddies; the latter contain strange attractors, at which the tracer flow paths behave as saddle trajectories. Two regions of the space increment have been observed, in which the flux variations are self-similar with the scaling exponent h=1/3, in surprising agreement with the Kolmogorov inertial range theory of turbulence. In one region, the cascade of protein rearrangements is directed from larger to smaller scales (net folding), and in the other, it is oppositely directed (net unfolding). Folding flows for the fast trajectories are essentially "laminar" and do not have the property of self-similarity. Based on the results of our study, we infer, and support this inference by simulations, that the origin of the similarity between the protein folding and turbulent motion of a fluid is in a cascade mechanism of structural transformations in the systems that underlies these phenomena.

  14. Assembly-induced folding regulates interleukin 12 biogenesis and secretion.

    PubMed

    Reitberger, Susanne; Haimerl, Pascal; Aschenbrenner, Isabel; Esser-von Bieren, Julia; Feige, Matthias J

    2017-05-12

    Members of the IL-12 family perform essential functions in immunoregulation by connecting innate and adaptive immunity and are emerging therapeutic targets. They are unique among other interleukins in forming heterodimers that arise from extensive subunit sharing within the family, leading to the production of at least four functionally distinct heterodimers from only five subunits. This raises important questions about how the assembly of IL-12 family members is regulated and controlled in the cell. Here, using cell-biological approaches, we have dissected basic principles that underlie the biogenesis of the founding member of the family, IL-12. Within the native IL-12 heterodimer, composed of IL-12α and IL-12β, IL-12α possesses three intramolecular and one intermolecular disulfide bridges. We show that, in isolation, IL-12α fails to form its native structure but, instead, misfolds, forming incorrect disulfide bonds. Co-expression of its β subunit inhibits misfolding and thus allows secretion of biologically active heterodimeric IL-12. On the basis of these findings, we identified the disulfide bonds in IL-12α that are critical for assembly-induced secretion and biological activity of IL-12 versus misfolding and degradation of IL-12α. Surprisingly, two of the three disulfide bridges in IL-12α are dispensable for IL-12 secretion, stability, and biological activity. Extending our findings, we show that misfolding also occurs for IL-23α, another IL-12 family protein. Our results indicate that assembly-induced folding is key in IL-12 family biogenesis and secretion. The identification of essential disulfide bonds that underlie this process lays the basis for a simplified yet functional IL-12 cytokine. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Protein photo-folding and quantum folding theory.

    PubMed

    Luo, Liaofu

    2012-06-01

    The rates of protein folding with photon absorption or emission and the cross section of photon -protein inelastic scattering are calculated from quantum folding theory by use of a field-theoretical method. All protein photo-folding processes are compared with common protein folding without the interaction of photons (non-radiative folding). It is demonstrated that there exists a common factor (thermo-averaged overlap integral of the vibration wave function, TAOI) for protein folding and protein photo-folding. Based on this finding it is predicted that (i) the stimulated photo-folding rates and the photon-protein resonance Raman scattering sections show the same temperature dependence as protein folding; (ii) the spectral line of the electronic transition is broadened to a band that includes an abundant vibration spectrum without and with conformational transitions, and the width of each vibration spectral line is largely reduced. The particular form of the folding rate-temperature relation and the abundant spectral structure imply the existence of quantum tunneling between protein conformations in folding and photo-folding that demonstrates the quantum nature of the motion of the conformational-electronic system.

  16. Analysis of secreted proteins.

    PubMed

    Severino, Valeria; Farina, Annarita; Chambery, Angela

    2013-01-01

    Most biological processes including growth, proliferation, differentiation, and apoptosis are coordinated by tightly regulated signaling pathways, which also involve secreted proteins acting in an autocrine and/or paracrine manner. In addition, extracellular signaling molecules affect local niche biology and influence the cross-talking with the surrounding tissues. The understanding of this molecular language may provide an integrated and broader view of cellular regulatory networks under physiological and pathological conditions. In this context, the profiling at a global level of cell secretomes (i.e., the subpopulations of a proteome secreted from the cell) has become an active area of research. The current interest in secretome research also deals with its high potential for the biomarker discovery and the identification of new targets for therapeutic strategies. Several proteomic and mass spectrometry platforms and methodologies have been applied to secretome profiling of conditioned media of cultured cell lines and primary cells. Nevertheless, the analysis of secreted proteins is still a very challenging task, because of the technical difficulties that may hamper the subsequent mass spectrometry analysis. This chapter describes a typical workflow for the analysis of proteins secreted by cultured cells. Crucial issues related to cell culture conditions for the collection of conditioned media, secretome preparation, and mass spectrometry analysis are discussed. Furthermore, an overview of quantitative LC-MS-based approaches, computational tools for data analysis, and strategies for validation of potential secretome biomarkers is also presented.

  17. Folding superfunnel to describe cooperative folding of interacting proteins.

    PubMed

    Smeller, László

    2016-07-01

    This paper proposes a generalization of the well-known folding funnel concept of proteins. In the funnel model the polypeptide chain is treated as an individual object not interacting with other proteins. Since biological systems are considerably crowded, protein-protein interaction is a fundamental feature during the life cycle of proteins. The folding superfunnel proposed here describes the folding process of interacting proteins in various situations. The first example discussed is the folding of the freshly synthesized protein with the aid of chaperones. Another important aspect of protein-protein interactions is the folding of the recently characterized intrinsically disordered proteins, where binding to target proteins plays a crucial role in the completion of the folding process. The third scenario where the folding superfunnel is used is the formation of aggregates from destabilized proteins, which is an important factor in case of several conformational diseases. The folding superfunnel constructed here with the minimal assumption about the interaction potential explains all three cases mentioned above. Proteins 2016; 84:1009-1016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Understanding Protein Non-Folding

    PubMed Central

    Uversky, Vladimir N.; Dunker, A. Keith

    2010-01-01

    This review describes the family of intrinsically disordered proteins, members of which fail to form rigid 3-D structures under physiological conditions, either along their entire lengths or only in localized regions. Instead, these intriguing proteins/regions exist as dynamic ensembles within which atom positions and backbone Ramachandran angles exhibit extreme temporal fluctuations without specific equilibrium values. Many of these intrinsically disordered proteins are known to carry out important biological functions which, in fact, depend on the absence of specific 3-D structure. The existence of such proteins does not fit the prevailing structure-function paradigm, which states that unique 3-D structure is a prerequisite to function. Thus, the protein structure-function paradigm has to be expanded to include intrinsically disordered proteins and alternative relationships among protein sequence, structure, and function. This shift in the paradigm represents a major breakthrough for biochemistry, biophysics and molecular biology, as it opens new levels of understanding with regard to the complex life of proteins. This review will try to answer the following questions: How were intrinsically disordered proteins discovered? Why don't these proteins fold? What is so special about intrinsic disorder? What are the functional advantages of disordered proteins/regions? What is the functional repertoire of these proteins? What are the relationships between intrinsically disordered proteins and human diseases? PMID:20117254

  19. Hydrodynamic interactions in protein folding

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek; Niewieczerzał, Szymon

    2009-03-01

    We incorporate hydrodynamic interactions (HIs) in a coarse-grained and structure-based model of proteins by employing the Rotne-Prager hydrodynamic tensor. We study several small proteins and demonstrate that HIs facilitate folding. We also study HIV-1 protease and show that HIs make the flap closing dynamics faster. The HIs are found to affect time correlation functions in the vicinity of the native state even though they have no impact on same time characteristics of the structure fluctuations around the native state.

  20. Predicting protein folds with fold-specific PSSM libraries.

    PubMed

    Hong, Yoojin; Chintapalli, Sree Vamsee; Ko, Kyung Dae; Bhardwaj, Gaurav; Zhang, Zhenhai; van Rossum, Damian; Patterson, Randen L

    2011-01-01

    Accurately assigning folds for divergent protein sequences is a major obstacle to structural studies. Herein, we outline an effective method for fold recognition using sets of PSSMs, each of which is constructed for different protein folds. Our analyses demonstrate that FSL (Fold-specific Position Specific Scoring Matrix Libraries) can predict/relate structures given only their amino acid sequences of highly divergent proteins. This ability to detect distant relationships is dependent on low-identity sequence alignments obtained from FSL. Results from our experiments demonstrate that FSL perform well in recognizing folds from the "twilight-zone" SABmark dataset. Further, this method is capable of accurate fold prediction in newly determined structures. We suggest that by building complete PSSM libraries for all unique folds within the Protein Database (PDB), FSL can be used to rapidly and reliably annotate a large subset of protein folds at proteomic level. The related programs and fold-specific PSSMs for our FSL are publicly available at: http://ccp.psu.edu/download/FSLv1.0/.

  1. Chaperonin-mediated Protein Folding

    PubMed Central

    Horwich, Arthur L.

    2013-01-01

    We have been studying chaperonins these past twenty years through an initial discovery of an action in protein folding, analysis of structure, and elucidation of mechanism. Some of the highlights of these studies were presented recently upon sharing the honor of the 2013 Herbert Tabor Award with my early collaborator, Ulrich Hartl, at the annual meeting of the American Society for Biochemistry and Molecular Biology in Boston. Here, some of the major findings are recounted, particularly recognizing my collaborators, describing how I met them and how our great times together propelled our thinking and experiments. PMID:23803606

  2. Protein secretion in Bacillus species.

    PubMed Central

    Simonen, M; Palva, I

    1993-01-01

    Bacilli secrete numerous proteins into the environment. Many of the secretory proteins, their export signals, and their processing steps during secretion have been characterized in detail. In contrast, the molecular mechanisms of protein secretion have been relatively poorly characterized. However, several components of the protein secretion machinery have been identified and cloned recently, which is likely to lead to rapid expansion of the knowledge of the protein secretion mechanism in Bacillus species. Comparison of the presently known export components of Bacillus species with those of Escherichia coli suggests that the mechanism of protein translocation across the cytoplasmic membrane is conserved among gram-negative and gram-positive bacteria differences are found in steps preceding and following the translocation process. Many of the secretory proteins of bacilli are produced industrially, but several problems have been encountered in the production of Bacillus heterologous secretory proteins. In the final section we discuss these problems and point out some possibilities to overcome them. PMID:8464403

  3. How Does Your Protein Fold? Elucidating the Apomyoglobin Folding Pathway.

    PubMed

    Dyson, H Jane; Wright, Peter E

    2017-01-17

    Although each type of protein fold and in some cases individual proteins within a fold classification can have very different mechanisms of folding, the underlying biophysical and biochemical principles that operate to cause a linear polypeptide chain to fold into a globular structure must be the same. In an aqueous solution, the protein takes up the thermodynamically most stable structure, but the pathway along which the polypeptide proceeds in order to reach that structure is a function of the amino acid sequence, which must be the final determining factor, not only in shaping the final folded structure, but in dictating the folding pathway. A number of groups have focused on a single protein or group of proteins, to determine in detail the factors that influence the rate and mechanism of folding in a defined system, with the hope that hypothesis-driven experiments can elucidate the underlying principles governing the folding process. Our research group has focused on the folding of the globin family of proteins, and in particular on the monomeric protein apomyoglobin. Apomyoglobin (apoMb) folds relatively slowly (∼2 s) via an ensemble of obligatory intermediates that form rapidly after the initiation of folding. The folding pathway can be dissected using rapid-mixing techniques, which can probe processes in the millisecond time range. Stopped-flow measurements detected by circular dichroism (CD) or fluorescence spectroscopy give information on the rates of folding events. Quench-flow experiments utilize the differential rates of hydrogen-deuterium exchange of amide protons protected in parts of the structure that are folded early; protection of amides can be detected by mass spectrometry or proton nuclear magnetic resonance spectroscopy (NMR). In addition, apoMb forms an intermediate at equilibrium at pH ∼ 4, which is sufficiently stable for it to be structurally characterized by solution methods such as CD, fluorescence and NMR spectroscopies, and the

  4. Extracellular secretion of recombinant proteins

    SciTech Connect

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  5. Improving protein fold recognition by random forest

    PubMed Central

    2014-01-01

    Background Recognizing the correct structural fold among known template protein structures for a target protein (i.e. fold recognition) is essential for template-based protein structure modeling. Since the fold recognition problem can be defined as a binary classification problem of predicting whether or not the unknown fold of a target protein is similar to an already known template protein structure in a library, machine learning methods have been effectively applied to tackle this problem. In our work, we developed RF-Fold that uses random forest - one of the most powerful and scalable machine learning classification methods - to recognize protein folds. Results RF-Fold consists of hundreds of decision trees that can be trained efficiently on very large datasets to make accurate predictions on a highly imbalanced dataset. We evaluated RF-Fold on the standard Lindahl's benchmark dataset comprised of 976 × 975 target-template protein pairs through cross-validation. Compared with 17 different fold recognition methods, the performance of RF-Fold is generally comparable to the best performance in fold recognition of different difficulty ranging from the easiest family level, the medium-hard superfamily level, and to the hardest fold level. Based on the top-one template protein ranked by RF-Fold, the correct recognition rate is 84.5%, 63.4%, and 40.8% at family, superfamily, and fold levels, respectively. Based on the top-five template protein folds ranked by RF-Fold, the correct recognition rate increases to 91.5%, 79.3% and 58.3% at family, superfamily, and fold levels. Conclusions The good performance achieved by the RF-Fold demonstrates the random forest's effectiveness for protein fold recognition. PMID:25350499

  6. Simple Model of Protein Folding Kinetics

    NASA Astrophysics Data System (ADS)

    Zwanzig, Robert

    1995-10-01

    A simple model of the kinetics of protein folding is presented. The reaction coordinate is the "correctness" of a configuration compared with the native state. The model has a gap in the energy spectrum, a large configurational entropy, a free energy barrier between folded and partially folded states, and a good thermodynamic folding transition. Folding kinetics is described by a master equation. The folding time is estimated by means of a local thermodynamic equilibrium assumption and then is calculated both numerically and analytically by solving the master equation. The folding time has a maximum near the folding transition temperature and can have a minimum at a lower temperature.

  7. Protein folding at single-molecule resolution

    PubMed Central

    Ferreon, Allan Chris M.; Deniz, Ashok A.

    2011-01-01

    The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms. PMID:21303706

  8. Macromolecule-Assisted de novo Protein Folding

    PubMed Central

    Choi, Seong Il; Son, Ahyun; Lim, Keo-Heun; Jeong, Hotcherl; Seong, Baik L.

    2012-01-01

    In the processes of protein synthesis and folding, newly synthesized polypeptides are tightly connected to the macromolecules, such as ribosomes, lipid bilayers, or cotranslationally folded domains in multidomain proteins, representing a hallmark of de novo protein folding environments in vivo. Such linkage effects on the aggregation of endogenous polypeptides have been largely neglected, although all these macromolecules have been known to effectively and robustly solubilize their linked heterologous proteins in fusion or display technology. Thus, their roles in the aggregation of linked endogenous polypeptides need to be elucidated and incorporated into the mechanisms of de novo protein folding in vivo. In the classic hydrophobic interaction-based stabilizing mechanism underlying the molecular chaperone-assisted protein folding, it has been assumed that the macromolecules connected through a simple linkage without hydrophobic interactions and conformational changes would make no effect on the aggregation of their linked polypeptide chains. However, an increasing line of evidence indicates that the intrinsic properties of soluble macromolecules, especially their surface charges and excluded volume, could be important and universal factors for stabilizing their linked polypeptides against aggregation. Taken together, these macromolecules could act as folding helpers by keeping their linked nascent chains in a folding-competent state. The folding assistance provided by these macromolecules in the linkage context would give new insights into de novo protein folding inside the cell. PMID:22949867

  9. Structural features of protein folding nuclei.

    PubMed

    Garbuzynskiy, S O; Kondratova, M S

    2008-03-05

    A crucial event of protein folding is the formation of a folding nucleus. We demonstrate the presence of a considerable coincidence between the location of folding nuclei and the location of so-called "root structural motifs", which have unique overall folds and handedness. In the case of proteins with a single root structural motif, the involvement in the formation of a folding nucleus is in average significantly higher for amino acids residues that are in root structural motifs, compared to residues in other parts of the protein. The tests carried out revealed that the observed difference is statistically reliable. Thus, a structural feature that corresponds to the protein folding nucleus is now found.

  10. Protein Folding and Self-Organized Criticality

    NASA Astrophysics Data System (ADS)

    Bajracharya, Arun; Murray, Joelle

    Proteins are known to fold into tertiary structures that determine their functionality in living organisms. However, the complex dynamics of protein folding and the way they consistently fold into the same structures is not fully understood. Self-organized criticality (SOC) has provided a framework for understanding complex systems in various systems (earthquakes, forest fires, financial markets, and epidemics) through scale invariance and the associated power law behavior. In this research, we use a simple hydrophobic-polar lattice-bound computational model to investigate self-organized criticality as a possible mechanism for generating complexity in protein folding.

  11. Accelerated molecular dynamics simulations of protein folding.

    PubMed

    Miao, Yinglong; Feixas, Ferran; Eun, Changsun; McCammon, J Andrew

    2015-07-30

    Folding of four fast-folding proteins, including chignolin, Trp-cage, villin headpiece and WW domain, was simulated via accelerated molecular dynamics (aMD). In comparison with hundred-of-microsecond timescale conventional molecular dynamics (cMD) simulations performed on the Anton supercomputer, aMD captured complete folding of the four proteins in significantly shorter simulation time. The folded protein conformations were found within 0.2-2.1 Å of the native NMR or X-ray crystal structures. Free energy profiles calculated through improved reweighting of the aMD simulations using cumulant expansion to the second-order are in good agreement with those obtained from cMD simulations. This allows us to identify distinct conformational states (e.g., unfolded and intermediate) other than the native structure and the protein folding energy barriers. Detailed analysis of protein secondary structures and local key residue interactions provided important insights into the protein folding pathways. Furthermore, the selections of force fields and aMD simulation parameters are discussed in detail. Our work shows usefulness and accuracy of aMD in studying protein folding, providing basic references in using aMD in future protein-folding studies. © 2015 Wiley Periodicals, Inc.

  12. Under-folded proteins: Conformational ensembles and their roles in protein folding, function, and pathogenesis.

    PubMed

    Uversky, Vladimir N

    2013-11-01

    For decades, protein function was intimately linked to the presence of a unique, aperiodic crystal-like structure in a functional protein. The two only places for conformational ensembles of under-folded (or partially folded) protein forms in this picture were either the end points of the protein denaturation processes or transiently populated folding intermediates. Recent years witnessed dramatic change in this perception and conformational ensembles, which the under-folded proteins are, have moved from the shadow. Accumulated to date data suggest that a protein can exist in at least three global forms-functional and folded, functional and intrinsically disordered (nonfolded), and nonfunctional and misfolded/aggregated. Under-folded protein states are crucial for each of these forms, serving as important folding intermediates of ordered proteins, or as functional states of intrinsically disordered proteins (IDPs) and IDP regions (IDPRs), or as pathology triggers of misfolded proteins. Based on these observations, conformational ensembles of under-folded proteins can be classified as transient (folding and misfolding intermediates) and permanent (IDPs and stable misfolded proteins). Permanently under-folded proteins can further be split into intentionally designed (IDPs and IDPRs) and unintentionally designed (misfolded proteins). Although intrinsic flexibility, dynamics, and pliability are crucial for all under-folded proteins, the different categories of under-foldedness are differently encoded in protein amino acid sequences. Copyright © 2013 Wiley Periodicals, Inc.

  13. Frustration in Condensed Matter and Protein Folding

    NASA Astrophysics Data System (ADS)

    Li, Z.; Tanner, S.; Conroy, B.; Owens, F.; Tran, M. M.; Boekema, C.

    2014-03-01

    By means of computer modeling, we are studying frustration in condensed matter and protein folding, including the influence of temperature and Thomson-figure formation. Frustration is due to competing interactions in a disordered state. The key issue is how the particles interact to reach the lowest frustration. The relaxation for frustration is mostly a power function (randomly assigned pattern) or an exponential function (regular patterns like Thomson figures). For the atomic Thomson model, frustration is predicted to decrease with the formation of Thomson figures at zero kelvin. We attempt to apply our frustration modeling to protein folding and dynamics. We investigate the homogeneous protein frustration that would cause the speed of the protein folding to increase. Increase of protein frustration (where frustration and hydrophobicity interplay with protein folding) may lead to a protein mutation. Research is supported by WiSE@SJSU and AFC San Jose.

  14. Protein Folding and Mechanisms of Proteostasis

    PubMed Central

    Díaz-Villanueva, José Fernando; Díaz-Molina, Raúl; García-González, Victor

    2015-01-01

    Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis. PMID:26225966

  15. Ultrafast protein folding in cages and zippers

    NASA Astrophysics Data System (ADS)

    Qiu, Linlin; Hagen, Stephen J.

    2003-03-01

    The smallest, fastest-folding proteins fold on the ˜μ s time scale, where state-of-the-art molecular dynamics (MD) simulation can finally overlap with the fastest experimental probes such as laser temperature-jump spectroscopy. For such proteins, one can now ask whether molecular dynamics correctly predicts the native structure and/or the folding speed. We will present experimental measurements of folding speed in two small proteins that acquire a stable tertiary fold rapidly enough to have been simulated in MD: (a) The 20-residue tryptophan (Trp) cage, which constitutes both the smallest truly protein-like molecule and also the fastest-folding [Neidigh et al., Nat. Struct. Biol. 9 425 (2002); Qiu et al., JACS 124 12952 (2002)], and (b) the 12-residue Trp zippers (e.g. TrpZip1), monomeric β-hairpins engineered by Cochran et al. [PNAS 98 5578 (2001)]. Both proteins fold in a cooperative, two-state transition at rates exceeding 10^5 s-1 (τ < 10 μs). We will compare the folding kinetics of these proteins with the predictions of MD simulations.

  16. Simultaneous Alignment and Folding of Protein Sequences

    PubMed Central

    Waldispühl, Jérôme; O'Donnell, Charles W.; Will, Sebastian; Devadas, Srinivas; Backofen, Rolf

    2014-01-01

    Abstract Accurate comparative analysis tools for low-homology proteins remains a difficult challenge in computational biology, especially sequence alignment and consensus folding problems. We present partiFold-Align, the first algorithm for simultaneous alignment and consensus folding of unaligned protein sequences; the algorithm's complexity is polynomial in time and space. Algorithmically, partiFold-Align exploits sparsity in the set of super-secondary structure pairings and alignment candidates to achieve an effectively cubic running time for simultaneous pairwise alignment and folding. We demonstrate the efficacy of these techniques on transmembrane β-barrel proteins, an important yet difficult class of proteins with few known three-dimensional structures. Testing against structurally derived sequence alignments, partiFold-Align significantly outperforms state-of-the-art pairwise and multiple sequence alignment tools in the most difficult low-sequence homology case. It also improves secondary structure prediction where current approaches fail. Importantly, partiFold-Align requires no prior training. These general techniques are widely applicable to many more protein families (partiFold-Align is available at http://partifold.csail.mit.edu/). PMID:24766258

  17. Protein vivisection reveals elusive intermediates in folding

    PubMed Central

    Zheng, Zhongzhou; Sosnick, Tobin R.

    2010-01-01

    Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu→Glu−) to destabilize and unfold a specific region of the protein. We apply this strategy to Ubiquitin, reversibly trapping a folding intermediate in which the β5 strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high energy states. PMID:20144618

  18. Hydrophobic folding units at protein-protein interfaces: implications to protein folding and to protein-protein association.

    PubMed Central

    Tsai, C. J.; Nussinov, R.

    1997-01-01

    A hydrophobic folding unit cutting algorithm, originally developed for dissecting single-chain proteins, has been applied to a dataset of dissimilar two-chain protein-protein interfaces. Rather than consider each individual chain separately, the two-chain complex has been treated as a single chain. The two-chain parsing results presented in this work show hydrophobicity to be a critical attribute of two-state versus three-state protein-protein complexes. The hydrophobic folding units at the interfaces of two-state complexes suggest that the cooperative nature of the two-chain protein folding is the outcome of the hydrophobic effect, similar to its being the driving force in a single-chain folding. In analogy to the protein-folding process, the two-chain, two-state model complex may correspond to the formation of compact, hydrophobic nuclei. On the other hand, the three-state model complex involves binding of already folded monomers, similar to the association of the hydrophobic folding units within a single chain. The similarity between folding entities in protein cores and in two-state protein-protein interfaces, despite the absence of some chain connectivities in the latter, indicates that chain linkage does not necessarily affect the native conformation. This further substantiates the notion that tertiary, non-local interactions play a critical role in protein folding. These compact, hydrophobic, two-chain folding units, derived from structurally dissimilar protein-protein interfaces, provide a rich set of data useful in investigations of the role played by chain connectivity and by tertiary interactions in studies of binding and of folding. Since they are composed of non-contiguous pieces of protein backbones, they may also aid in defining folding nuclei. PMID:9232644

  19. Network measures for protein folding state discrimination

    PubMed Central

    Menichetti, Giulia; Fariselli, Piero; Remondini, Daniel

    2016-01-01

    Proteins fold using a two-state or multi-state kinetic mechanisms, but up to now there is not a first-principle model to explain this different behavior. We exploit the network properties of protein structures by introducing novel observables to address the problem of classifying the different types of folding kinetics. These observables display a plain physical meaning, in terms of vibrational modes, possible configurations compatible with the native protein structure, and folding cooperativity. The relevance of these observables is supported by a classification performance up to 90%, even with simple classifiers such as discriminant analysis. PMID:27464796

  20. Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

    PubMed Central

    Metcalf, Kevin J.; Finnerty, Casey; Azam, Anum; Valdivia, Elias

    2014-01-01

    The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28 ± 9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant. PMID:25038096

  1. The robustness and innovability of protein folds.

    PubMed

    Tóth-Petróczy, Agnes; Tawfik, Dan S

    2014-06-01

    Assignment of protein folds to functions indicates that >60% of folds carry out one or two enzymatic functions, while few folds, for example, the TIM-barrel and Rossmann folds, exhibit hundreds. Are there structural features that make a fold amenable to functional innovation (innovability)? Do these features relate to robustness--the ability to readily accumulate sequence changes? We discuss several hypotheses regarding the relationship between the architecture of a protein and its evolutionary potential. We describe how, in a seemingly paradoxical manner, opposite properties, such as high stability and rigidity versus conformational plasticity and structural order versus disorder, promote robustness and/or innovability. We hypothesize that polarity--differentiation and low connectivity between a protein's scaffold and its active-site--is a key prerequisite for innovability. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Stochastic Resonance in Protein Folding Dynamics.

    PubMed

    Davtyan, Aram; Platkov, Max; Gruebele, Martin; Papoian, Garegin A

    2016-05-04

    Although protein folding reactions are usually studied under static external conditions, it is likely that proteins fold in a locally fluctuating cellular environment in vivo. To mimic such behavior in in vitro experiments, the local temperature of the solvent can be modulated either harmonically or using correlated noise. In this study, coarse-grained molecular simulations are used to investigate these possibilities, and it is found that both periodic and correlated random fluctuations of the environment can indeed accelerate folding kinetics if the characteristic frequencies of the applied fluctuations are commensurate with the internal timescale of the folding reaction; this is consistent with the phenomenon of stochastic resonance observed in many other condensed-matter processes. To test this theoretical prediction, the folding dynamics of phosphoglycerate kinase under harmonic temperature fluctuations are experimentally probed using Förster resonance energy transfer fluorescence measurements. To analyze these experiments, a combination of theoretical approaches is developed, including stochastic simulations of folding kinetics and an analytical mean-field kinetic theory. The experimental observations are consistent with the theoretical predictions of stochastic resonance in phosphoglycerate kinase folding. When combined with an alternative experiment on the protein VlsE using a power spectrum analysis, elaborated in Dave et al., ChemPhysChem 2016, 10.1002/cphc.201501041, the overall data overwhelmingly point to the experimental confirmation of stochastic resonance in protein folding dynamics.

  3. Protein folding: When ribosomes pick the structure

    NASA Astrophysics Data System (ADS)

    Sivertsson, Elin M.; Itzhaki, Laura S.

    2014-05-01

    Anfinsen's principle tells us that the folded structure of a protein is determined solely by its sequence. Now, it has been shown that the rate at which a polypeptide chain is synthesized in the cell can affect which of two alternative folded structures it adopts.

  4. Local vs global motions in protein folding

    PubMed Central

    Maisuradze, Gia G.; Liwo, Adam; Senet, Patrick; Scheraga, Harold A.

    2013-01-01

    It is of interest to know whether local fluctuations in a polypeptide chain play any role in the mechanism by which the chain folds to the native structure of a protein. This question is addressed by analyzing folding and non-folding trajectories of a protein; as an example, the analysis is applied to the 37-residue triple β-strand WW domain from the Formin binding protein 28 (FBP28) (PDB ID: 1E0L). Molecular dynamics (MD) trajectories were generated with the coarse-grained united-residue force field, and one- and two-dimensional free-energy landscapes (FELs) along the backbone virtual-bond angle θ and backbone virtual-bond-dihedral angle γ of each residue, and principal components, respectively, were analyzed. The key residues involved in the folding of the FBP28 WW domain are elucidated by this analysis. The correlations between local and global motions are found. It is shown that most of the residues in the folding trajectories of the system studied here move in a concerted fashion, following the dynamics of the whole system. This demonstrates how the choice of a pathway has to involve concerted movements in order for this protein to fold. This finding also sheds light on the effectiveness of principal component analysis (PCA) for the description of the folding dynamics of the system studied. It is demonstrated that the FEL along the PCs, computed by considering only several critically-placed residues, can correctly describe the folding dynamics. PMID:23914144

  5. Protein folding and misfolding: mechanism and principles.

    PubMed

    Englander, S Walter; Mayne, Leland; Krishna, Mallela M G

    2007-11-01

    Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors

  6. Cooperativity and modularity in protein folding

    PubMed Central

    Sasai, Masaki; Chikenji, George; Terada, Tomoki P.

    2016-01-01

    A simple statistical mechanical model proposed by Wako and Saitô has explained the aspects of protein folding surprisingly well. This model was systematically applied to multiple proteins by Muñoz and Eaton and has since been referred to as the Wako-Saitô-Muñoz-Eaton (WSME) model. The success of the WSME model in explaining the folding of many proteins has verified the hypothesis that the folding is dominated by native interactions, which makes the energy landscape globally biased toward native conformation. Using the WSME and other related models, Saitô emphasized the importance of the hierarchical pathway in protein folding; folding starts with the creation of contiguous segments having a native-like configuration and proceeds as growth and coalescence of these segments. The Φ-values calculated for barnase with the WSME model suggested that segments contributing to the folding nucleus are similar to the structural modules defined by the pattern of native atomic contacts. The WSME model was extended to explain folding of multi-domain proteins having a complex topology, which opened the way to comprehensively understanding the folding process of multi-domain proteins. The WSME model was also extended to describe allosteric transitions, indicating that the allosteric structural movement does not occur as a deterministic sequential change between two conformations but as a stochastic diffusive motion over the dynamically changing energy landscape. Statistical mechanical viewpoint on folding, as highlighted by the WSME model, has been renovated in the context of modern methods and ideas, and will continue to provide insights on equilibrium and dynamical features of proteins. PMID:28409080

  7. Folding funnels, binding funnels, and protein function.

    PubMed Central

    Tsai, C. J.; Kumar, S.; Ma, B.; Nussinov, R.

    1999-01-01

    Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly

  8. Cotranslational folding of deeply knotted proteins

    NASA Astrophysics Data System (ADS)

    Chwastyk, Mateusz; Cieplak, Marek

    2015-09-01

    Proper folding of deeply knotted proteins has a very low success rate even in structure-based models which favor formation of the native contacts but have no topological bias. By employing a structure-based model, we demonstrate that cotranslational folding on a model ribosome may enhance the odds to form trefoil knots for protein YibK without any need to introduce any non-native contacts. The ribosome is represented by a repulsive wall that keeps elongating the protein. On-ribosome folding proceeds through a a slipknot conformation. We elucidate the mechanics and energetics of its formation. We show that the knotting probability in on-ribosome folding is a function of temperature and that there is an optimal temperature for the process. Our model often leads to the establishment of the native contacts without formation of the knot.

  9. Cotranslational folding of deeply knotted proteins.

    PubMed

    Chwastyk, Mateusz; Cieplak, Marek

    2015-09-09

    Proper folding of deeply knotted proteins has a very low success rate even in structure-based models which favor formation of the native contacts but have no topological bias. By employing a structure-based model, we demonstrate that cotranslational folding on a model ribosome may enhance the odds to form trefoil knots for protein YibK without any need to introduce any non-native contacts. The ribosome is represented by a repulsive wall that keeps elongating the protein. On-ribosome folding proceeds through a a slipknot conformation. We elucidate the mechanics and energetics of its formation. We show that the knotting probability in on-ribosome folding is a function of temperature and that there is an optimal temperature for the process. Our model often leads to the establishment of the native contacts without formation of the knot.

  10. The nature of protein folding pathways

    PubMed Central

    Englander, S. Walter; Mayne, Leland

    2014-01-01

    How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the “new view” model for protein folding. Emergent macroscopic foldon–foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the “how” and the “why” questions. The protein folding pathway depends on the same foldon units and foldon–foldon interactions that construct the native structure. PMID:25326421

  11. The hydrogen exchange core and protein folding.

    PubMed Central

    Li, R.; Woodward, C.

    1999-01-01

    A database of hydrogen-deuterium exchange results has been compiled for proteins for which there are published rates of out-exchange in the native state, protection against exchange during folding, and out-exchange in partially folded forms. The question of whether the slow exchange core is the folding core (Woodward C, 1993, Trends Biochem Sci 18:359-360) is reexamined in a detailed comparison of the specific amide protons (NHs) and the elements of secondary structure on which they are located. For each pulsed exchange or competition experiment, probe NHs are shown explicitly; the large number and broad distribution of probe NHs support the validity of comparing out-exchange with pulsed-exchange/competition experiments. There is a strong tendency for the same elements of secondary structure to carry NHs most protected in the native state, NHs first protected during folding, and NHs most protected in partially folded species. There is not a one-to-one correspondence of individual NHs. Proteins for which there are published data for native state out-exchange and theta values are also reviewed. The elements of secondary structure containing the slowest exchanging NHs in native proteins tend to contain side chains with high theta values or be connected to a turn/loop with high theta values. A definition for a protein core is proposed, and the implications for protein folding are discussed. Apparently, during folding and in the native state, nonlocal interactions between core sequences are favored more than other possible nonlocal interactions. Other studies of partially folded bovine pancreatic trypsin inhibitor (Barbar E, Barany G, Woodward C, 1995, Biochemistry 34:11423-11434; Barber E, Hare M, Daragan V, Barany G, Woodward C, 1998, Biochemistry 37:7822-7833), suggest that developing cores have site-specific energy barriers between microstates, one disordered, and the other(s) more ordered. PMID:10452602

  12. A Brevibacillus choshinensis system that secretes cytoplasmic proteins.

    PubMed

    Horne, Irene; Williams, Michelle; Sutherland, Tara D; Russell, Robyn J; Oakeshott, John G

    2004-01-01

    Brevibacillus choshinensis has previously been shown to be a useful strain for the secretion of heterologous proteins via the Sec secretory pathway. This pathway involves the secretion of proteins prior to folding, whereas the alternative TAT (twin-arginine translocation) pathway enables pre-folded proteins to be secreted. We have modified the signal peptide of the Brevibacillus expression vector pNCMO2 to accommodate a Sec avoidance signal as well as the twin arginines required for secretion via the TAT system. Use of this modified signal peptide with the phosphotriesterase OpdA enabled B. choshinensis transformants to express and secrete the enzyme in an active and substantially pure form. The system was also used successfully to secrete two cytoplasmic proteins, the phosphotriesterase HocA from Pseudomonas monteilii and the phenylcarbamate-degrading enzyme, PCD, from Arthrobacter oxydans. The inhibitors carbonyl cyanide m-chlorophenyl hydrazine and sodium azide were used to confirm that secretion was occurring via the TAT secretion pathway. The modified B. choshinensis system we have developed may have general utility in secreting a wide range of heterologous proteins in active and conveniently processed form.

  13. Protein folding and de novo protein design for biotechnological applications

    PubMed Central

    Khoury, George A.; Smadbeck, James; Kieslich, Chris A.; Floudas, Christodoulos A.

    2014-01-01

    In the post-genomic era, the medical/biological fields are advancing faster than ever. However, before the power of full-genome sequencing can be fully realized, the connection between amino acid sequence and protein structure, known as the protein folding problem, needs to be elucidated. The protein folding problem remains elusive, with significant difficulties still arising when modeling amino acid sequences lacking an identifiable template. Understanding protein folding will allow for unforeseen advances in protein design, often referred as the inverse protein folding problem. Despite challenges in protein folding, de novo protein design has recently demonstrated significant success via computational techniques. We review advances and challenges in protein structure prediction and de novo protein design, and highlight their interplay in successful biotechnological applications. PMID:24268901

  14. The Ribosome Modulates Nascent Protein Folding

    PubMed Central

    Kaiser, Christian M.; Goldman, Daniel H.; Chodera, John D.; Tinoco, Ignacio; Bustamante, Carlos

    2014-01-01

    Proteins are synthesized by the ribosome and generally must fold to become functionally active. Although it is commonly assumed that the ribosome affects the folding process, this idea has been extremely difficult to demonstrate. We have developed an experimental system to investigate the folding of single ribosome-bound stalled nascent polypeptides with optical tweezers. In T4 lysozyme, synthesized in a reconstituted in vitro translation system, the ribosome slows the formation of stable tertiary interactions and the attainment of the native state relative to the free protein. Incomplete T4 lysozyme polypeptides misfold and aggregate when free in solution, but they remain folding-competent near the ribosomal surface. Altogether, our results suggest that the ribosome not only decodes the genetic information and synthesizes polypeptides, but also promotes efficient de novo attainment of the native state. PMID:22194581

  15. Visualizing chaperone-assisted protein folding

    PubMed Central

    Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; Ahlstrom, Logan S.; Martin, Raoul; Quan, Shu; Afonine, Pavel V.; van den Bedem, Henry; Wang, Lili; Xu, Qingping; Trievel, Raymond C.; Brooks, Charles L.; Bardwell, James CA

    2016-01-01

    Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding, where obtaining structural ensembles of chaperone:substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a novel structural biology approach based on X-ray crystallography, termed Residual Electron and Anomalous Density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the E. coli chaperone Spy. This study resulted in a series of snapshots depicting the various folding states of Im7 while bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded and native-like states, and reveals how a substrate can explore its folding landscape while bound to a chaperone. PMID:27239796

  16. Visualizing chaperone-assisted protein folding

    SciTech Connect

    Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; Ahlstrom, Logan S.; Martin, Raoul; Quan, Shu; Afonine, Pavel V.; van den Bedem, Henry; Wang, Lili; Xu, Qingping; Trievel, Raymond C.; Brooks, Charles L.; Bardwell, James C. A.

    2016-05-30

    We present that challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperone Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.

  17. Visualizing chaperone-assisted protein folding

    DOE PAGES

    Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; ...

    2016-05-30

    We present that challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperonemore » Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.« less

  18. Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment

    PubMed Central

    Peterson, Janine H.; Tian, Pu; Ieva, Raffaele; Dautin, Nathalie; Bernstein, Harris D.

    2010-01-01

    Autotransporters are bacterial virulence factors consisting of an N-terminal “passenger domain” that is secreted in a C- to-N-terminal direction and a C-terminal “β domain” that resides in the outer membrane (OM). Although passenger domain secretion does not appear to use ATP, the energy source for this reaction is unknown. Here, we show that efficient secretion of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP requires the stable folding of a C-terminal ≈17-kDa passenger domain segment. We found that mutations that perturb the folding of this segment do not affect its translocation across the OM but impair the secretion of the remainder of the passenger domain. Interestingly, an examination of kinetic folding mutants strongly suggested that the ≈17-kDa segment folds in the extracellular space. By mutagenizing the ≈17-kDa segment, we also fortuitously isolated a unique translocation intermediate. Analysis of this intermediate suggests that a heterooligomer that facilitates the membrane integration of OM proteins (the Bam complex) also promotes the surface exposure of the ≈17-kDa segment. Our results provide direct evidence that protein folding can drive translocation and help to clarify the mechanism of autotransporter secretion. PMID:20876094

  19. GroEL-mediated protein folding.

    PubMed Central

    Fenton, W. A.; Horwich, A. L.

    1997-01-01

    I. Architecture of GroEL and GroES and the reaction pathway A. Architecture of the chaperonins B. Reaction pathway of GroEL-GroES-mediated folding II. Polypeptide binding A. A parallel network of chaperones binding polypeptides in vivo B. Polypeptide binding in vitro 1. Role of hydrophobicity in recognition 2. Homologous proteins with differing recognition-differences in primary structure versus effects on folding pathway 3. Conformations recognized by GroEL a. Refolding studies b. Binding of metastable intermediates c. Conformations while stably bound at GroEL 4. Binding constants and rates of association 5. Conformational changes in the substrate protein associated with binding by GroEL a. Observations b. Kinetic versus thermodynamic action of GroEL in mediating unfolding c. Crossing the energy landscape in the presence of GroEL III. ATP binding and hydrolysis-driving the reaction cycle IV. GroEL-GroES-polypeptide ternary complexes-the folding-active cis complex A. Cis and trans ternary complexes B. Symmetric complexes C. The folding-active intermediate of a chaperonin reaction-cis ternary complex D. The role of the cis space in the folding reaction E. Folding governed by a "timer" mechanism F. Release of nonnative polypeptides during the GroEL-GroES reaction G. Release of both native and nonnative forms under physiologic conditions H. A role for ATP binding, as well as hydrolysis, in the folding cycle V. Concluding remarks. PMID:9098884

  20. Fast-Folding Proteins under Stress

    PubMed Central

    Dave, Kapil; Gruebele, Martin

    2015-01-01

    Proteins are subject to a variety of stresses in biological organisms, including pressure and temperature, which are the easiest stresses to simulate by molecular dynamics. We discuss the effect of pressure and thermal stress on very fast folding model proteins, whose in vitro folding can be fully simulated on computers and compared with experiments. We then discuss experiments that can be used to subject proteins to low and high temperature unfolding, as well as low and high pressure unfolding. Pressure and temperature are prototypical perturbations that illustrate how close many proteins are to instability, a property that cells can exploit to control protein function. We conclude by reviewing some recent in-cell experiments, and progress being made in simulating and measuring protein stability and function inside live cells. PMID:26231095

  1. Optimum folding pathways for growing protein chains.

    PubMed

    Senturk, Serife; Baday, Sefer; Arkun, Yaman; Erman, Burak

    2007-11-26

    The folding of a protein is studied as it grows residue by residue from the N-terminus and enters an environment that stabilizes the folded state. This mode of folding of a growing chain is different from refolding where the full chain folds from a disordered initial configuration to the native state. We propose a sequential dynamic optimization method that computes the evolution of optimum folding pathways as amino acid residues are added to the peptide chain one by one. The dynamic optimization formulation is deterministic and uses Newton's equations of motion and a Go-type potential that establishes the native contacts and excluded volume effects. The method predicts the optimal energy-minimizing path among all the alternative feasible pathways. As two examples, the folding of the chicken villin headpiece, a 36-residue protein, and chymotrypsin inhibitor 2 (CI2), a 64-residue protein, are studied. Results on the villin headpiece show significant differences from the refolding of the same chain studied previously. Results on CI2 mostly agree with the results of refolding experiments and computational work.

  2. Folding mechanism of proteins and protein-like polymers

    NASA Astrophysics Data System (ADS)

    Pande, Vijay

    2000-03-01

    Proteins are amazing biomaterials: they both perform biological activity as well as assemble themselves. In order to understand how proteins fold and to design synthetic polymers with protein-like properties, we need to understand how these molecules assemble themselves. I will discuss results from recent simulations of proteins and protein-like polymers in order to examine which is common and potentially ``universal'' about the folding (self-assembly) mechanism. These results may shed light on protein and protein-like polymer design, experiments on folding, as well as areas in which misfolding may be important such as many neurodegenerative diseases.

  3. Protein Folding:. Physics on Products of Evolution

    NASA Astrophysics Data System (ADS)

    Go, Nobuhiro

    2001-09-01

    Proteins are self-assembling molecular systems. A polypeptide chain of a protein molecule folds into a globular three-dimensional structure, which is specific to the amino acid sequence of the chain. A protein molecule is in the "native state" when folded into its specific three-dimensional structure. Only in the native state, a protein molecule carries out its biological function. This extraordinary self-assembly ability of proteins can be explained based on the three generally accepted empirical observations in proteins: (1) Two-state character; Folding and unfolding transitions in small globular proteins are generally of the two-state character. (2) Consistency principle; Various components of intra-molecular interactions responsible for stabilizing the native state of globular proteins are consistent to each other in their native state. (3) Principle of marginal stability; The native folded states of globular proteins are generally only marginally stable against their unfolded states. Deduction of the self-assembly ability from the three observations is a problem of physical nature. Very sophisticated theories have been developed recently as to this point. I shall give a very simple and intuitive discussion on this point. Asking why protein molecules show the three observations is another problem. Observation (1) can be derived from the globularity of native states. Observations (2) and (3) can be understood only by considering the evolutionary history of protein molecules, i.e., only polypeptide chains with very specific amino acid sequences selected during the history of evolution show properties of observations (2) and (3). Here we see a case where the mechanism of an extraordinary ability of biopolymers is elucidated in terms of physics, and physics expects that only a very small fraction of amino acid sequences have such an ability. Nature has left the job of finding able sequences to the history of evolution.

  4. Microfluidic Mixers for Studying Protein Folding

    PubMed Central

    Waldauer, Steven A.; Wu, Ling; Yao, Shuhuai; Bakajin, Olgica; Lapidus, Lisa J.

    2012-01-01

    The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 μs2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4. Laminar flow is achieved for Reynolds numbers Re ≤100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 μm wide and 10 μm deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 μm in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 μL/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 μM (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM. The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 μm in 1 ms

  5. Ubiquitylation Directly Induces Fold Destabilization of Proteins

    PubMed Central

    Morimoto, Daichi; Walinda, Erik; Fukada, Harumi; Sugase, Kenji; Shirakawa, Masahiro

    2016-01-01

    Ubiquitin is a common post-translational modifier and its conjugation is a key signal for proteolysis by the proteasome. Because the molecular mass of ubiquitin is larger than that of other modifiers such as phosphate, acetyl, or methyl groups, ubiquitylation not only influences biochemical signaling, but also may exert physical effects on its substrate proteins by increasing molecular volume and altering shape anisotropy. Here we show that ubiquitylation destabilizes the fold of two proteins, FKBP12 and FABP4, and that elongation of the conjugated ubiquitin chains further enhances this destabilization effect. Moreover, NMR relaxation analysis shows that ubiquitylation induces characteristic structural fluctuations in the backbone of both proteins. These results suggest that the ubiquitylation-driven structural fluctuations lead to fold destabilization of its substrate proteins. Thus, physical destabilization by ubiquitylation may facilitate protein degradation by the proteasome. PMID:27991582

  6. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  7. Computational and theoretical methods for protein folding.

    PubMed

    Compiani, Mario; Capriotti, Emidio

    2013-12-03

    A computational approach is essential whenever the complexity of the process under study is such that direct theoretical or experimental approaches are not viable. This is the case for protein folding, for which a significant amount of data are being collected. This paper reports on the essential role of in silico methods and the unprecedented interplay of computational and theoretical approaches, which is a defining point of the interdisciplinary investigations of the protein folding process. Besides giving an overview of the available computational methods and tools, we argue that computation plays not merely an ancillary role but has a more constructive function in that computational work may precede theory and experiments. More precisely, computation can provide the primary conceptual clues to inspire subsequent theoretical and experimental work even in a case where no preexisting evidence or theoretical frameworks are available. This is cogently manifested in the application of machine learning methods to come to grips with the folding dynamics. These close relationships suggested complementing the review of computational methods within the appropriate theoretical context to provide a self-contained outlook of the basic concepts that have converged into a unified description of folding and have grown in a synergic relationship with their computational counterpart. Finally, the advantages and limitations of current computational methodologies are discussed to show how the smart analysis of large amounts of data and the development of more effective algorithms can improve our understanding of protein folding.

  8. Unconventional protein secretion: an evolving mechanism

    PubMed Central

    Malhotra, Vivek

    2013-01-01

    The process by which proteins are secreted without entering the classical endoplasmic reticulum (ER)–Golgi complex pathway, in eukaryotic cells, is conveniently called unconventional protein secretion. Recent studies on one such protein called Acb1 have revealed a number of components involved in its secretion. Interestingly, conditions that promote the secretion of Acb1 trigger the biogenesis of a new compartment called CUPS (Compartment for Unconventional Protein Secretion). CUPS form near the ER exit site but lack ER-specific proteins. Other proteins that share some of the features common with the secretion of Acb1 are interleukin-1β and tissue transglutaminase. Here I will review recent advances made in the field and propose a new model for unconventional protein secretion. PMID:23665917

  9. Folding of the Tau Protein on Microtubules.

    PubMed

    Kadavath, Harindranath; Jaremko, Mariusz; Jaremko, Łukasz; Biernat, Jacek; Mandelkow, Eckhard; Zweckstetter, Markus

    2015-08-24

    Microtubules are regulated by microtubule-associated proteins. However, little is known about the structure of microtubule-associated proteins in complex with microtubules. Herein we show that the microtubule-associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a β-sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Energy landscape in protein folding and unfolding

    PubMed Central

    Mallamace, Francesco; Corsaro, Carmelo; Mallamace, Domenico; Vasi, Sebastiano; Vasi, Cirino; Baglioni, Piero; Buldyrev, Sergey V.; Chen, Sow-Hsin; Stanley, H. Eugene

    2016-01-01

    We use 1H NMR to probe the energy landscape in the protein folding and unfolding process. Using the scheme ⇄ reversible unfolded (intermediate) → irreversible unfolded (denatured) state, we study the thermal denaturation of hydrated lysozyme that occurs when the temperature is increased. Using thermal cycles in the range 295protein energy surface, we observe that the hydrophilic (the amide NH) and hydrophobic (methyl CH3 and methine CH) peptide groups evolve and exhibit different behaviors. We also discuss the role of water and hydrogen bonding in the protein configurational stability. PMID:26957601

  11. Energy landscape in protein folding and unfolding.

    PubMed

    Mallamace, Francesco; Corsaro, Carmelo; Mallamace, Domenico; Vasi, Sebastiano; Vasi, Cirino; Baglioni, Piero; Buldyrev, Sergey V; Chen, Sow-Hsin; Stanley, H Eugene

    2016-03-22

    We use (1)H NMR to probe the energy landscape in the protein folding and unfolding process. Using the scheme ⇄ reversible unfolded (intermediate) → irreversible unfolded (denatured) state, we study the thermal denaturation of hydrated lysozyme that occurs when the temperature is increased. Using thermal cycles in the range 295 < T < 365 K and following different trajectories along the protein energy surface, we observe that the hydrophilic (the amide NH) and hydrophobic (methyl CH3 and methine CH) peptide groups evolve and exhibit different behaviors. We also discuss the role of water and hydrogen bonding in the protein configurational stability.

  12. Communication: Folding of glycosylated proteins under confinement

    NASA Astrophysics Data System (ADS)

    Shental-Bechor, Dalit; Levy, Yaakov

    2011-10-01

    Conjugating flexible polymers (such as oligosaccharides) to proteins or confining a protein in a restricted volume often increases protein thermal stability. In this communication, we investigate the interplay between conjugation and confinement which is not trivial as the magnitude and the mechanism of stabilization are different in each instance. Using coarse-grained computational approach the folding biophysics is studied when the protein is placed in a sphere of variable radius and is conjugated to 0-6 mono- or penta-saccharides. We observe a synergistic effect on thermal stability when short oligosaccharides are attached and the modified protein is confined in a small cage. However, when large oligosaccharides are added, a conflict between confinement and glycosylation arises as the stabilizing effect of the cage is dramatically reduced and it is almost impossible to further stabilize the protein beyond the mild stabilization induced by the sugars.

  13. PREFACE Protein folding: lessons learned and new frontiers Protein folding: lessons learned and new frontiers

    NASA Astrophysics Data System (ADS)

    Pappu, Rohit V.; Nussinov, Ruth

    2009-03-01

    In appropriate physiological milieux proteins spontaneously fold into their functional three-dimensional structures. The amino acid sequences of functional proteins contain all the information necessary to specify the folds. This remarkable observation has spawned research aimed at answering two major questions. (1) Of all the conceivable structures that a protein can adopt, why is the ensemble of native-like structures the most favorable? (2) What are the paths by which proteins manage to robustly and reproducibly fold into their native structures? Anfinsen's thermodynamic hypothesis has guided the pursuit of answers to the first question whereas Levinthal's paradox has influenced the development of models for protein folding dynamics. Decades of work have led to significant advances in the folding problem. Mean-field models have been developed to capture our current, coarse grain understanding of the driving forces for protein folding. These models are being used to predict three-dimensional protein structures from sequence and stability profiles as a function of thermodynamic and chemical perturbations. Impressive strides have also been made in the field of protein design, also known as the inverse folding problem, thereby testing our understanding of the determinants of the fold specificities of different sequences. Early work on protein folding pathways focused on the specific sequence of events that could lead to a simplification of the search process. However, unifying principles proved to be elusive. Proteins that show reversible two-state folding-unfolding transitions turned out to be a gift of natural selection. Focusing on these simple systems helped researchers to uncover general principles regarding the origins of cooperativity in protein folding thermodynamics and kinetics. On the theoretical front, concepts borrowed from polymer physics and the physics of spin glasses led to the development of a framework based on energy landscape theories. These

  14. Using the folding landscapes of proteins to understand protein function.

    PubMed

    Giri Rao, V V Hemanth; Gosavi, Shachi

    2016-02-01

    Proteins fold on a biologically-relevant timescale because of a funnel-shaped energy landscape. This landscape is sculpted through evolution by selecting amino-acid sequences that stabilize native interactions while suppressing stable non-native interactions that occur during folding. However, there is strong evolutionary selection for functional residues and these cannot be chosen to optimize folding. Their presence impacts the folding energy landscape in a variety of ways. Here, we survey the effects of functional residues on folding by providing several examples. We then review how such effects can be detected computationally and be used as assays for protein function. Overall, an understanding of how functional residues modulate folding should provide insights into the design of natural proteins and their homeostasis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Arsenic(III) species inhibit oxidative protein folding in vitro.

    PubMed

    Ramadan, Danny; Rancy, Pumtiwitt C; Nagarkar, Radhika P; Schneider, Joel P; Thorpe, Colin

    2009-01-20

    The success of arsenic trioxide in the treatment of acute promyelocytic leukemia has renewed interest in the cellular targets of As(III) species. The effects of arsenicals are usually attributed to their ability to bind vicinal thiols or thiol selenols in prefolded proteins thereby compromising cellular function. The present studies suggest an additional, more pleiotropic, contribution to the biological effects of arsenicals. As(III) species, by avid coordination to the cysteine residues of unfolded reduced proteins, can compromise protein folding pathways. Three representative As(III) compounds (arsenite, monomethylarsenous acid (MMA), and an aryl arsenical (PSAO)) have been tested with three reduced secreted proteins (lysozyme, ribonuclease A, and riboflavin binding protein (RfBP)). Using absorbance, fluorescence, and pre-steady-state methods, we show that arsenicals bind tightly to low micromolar concentrations of these unfolded proteins with stoichiometries of 1 As(III) per 2 thiols for MMA and PSAO and 1 As(III) for every 3 thiols with arsenite. Arsenicals, at 10 microM, strongly disrupt the oxidative folding of RfBP even in the presence of 5 mM reduced glutathione, a competing ligand for As(III) species. MMA catalyzes the formation of amyloid-like monodisperse fibrils using reduced RNase. These in vitro data show that As(III) species can slow, or even derail, protein folding pathways. In vivo, the propensity of As(III) species to bind to unfolded cysteine-containing proteins may contribute to oxidative and protein folding stresses that are prominent features of the cellular response to arsenic exposure.

  16. Is Protein Folding Sub-Diffusive?

    PubMed Central

    Krivov, Sergei V.

    2010-01-01

    Protein folding dynamics is often described as diffusion on a free energy surface considered as a function of one or few reaction coordinates. However, a growing number of experiments and models show that, when projected onto a reaction coordinate, protein dynamics is sub-diffusive. This raises the question as to whether the conventionally used diffusive description of the dynamics is adequate. Here, we numerically construct the optimum reaction coordinate for a long equilibrium folding trajectory of a Go model of a -repressor protein. The trajectory projected onto this coordinate exhibits diffusive dynamics, while the dynamics of the same trajectory projected onto a sub-optimal reaction coordinate is sub-diffusive. We show that the higher the (cut-based) free energy profile for the putative reaction coordinate, the more diffusive the dynamics become when projected on this coordinate. The results suggest that whether the projected dynamics is diffusive or sub-diffusive depends on the chosen reaction coordinate. Protein folding can be described as diffusion on the free energy surface as function of the optimum reaction coordinate. And conversely, the conventional reaction coordinates, even though they might be based on physical intuition, are often sub-optimal and, hence, show sub-diffusive dynamics. PMID:20862361

  17. A Simple Model for Protein Folding

    NASA Astrophysics Data System (ADS)

    Henry, Eric R.; Eaton, William A.

    We describe a simple Ising-like statistical mechanical model for folding proteins based on the α-carbon contact map of the native structure. In this model residues can adopt two microscopic states corresponding to the native and non-native conformations. In order to exactly enumerate the large number of possible configurations, structure is considered to grow as continuous sequences of native residues, with no more than two sequences in each molecule. Inter-residue contacts can only form within each sequence and between residues of the two native sequences. As structure grows there is a tradeoff between the stabilizing effect of inter-residue contacts and the entropy losses from ordering residues in their native conformation and from forming a disordered loop to connect two continuous sequences. Folding kinetics are calculated from the dynamics on the free energy profile, as in Kramers' reaction rate theory. Although non-native interactions responsible for roughness in the energy landscape are not explicitly considered in the model, they are implicitly included by determining the absolute rates for motion on the free energy profile. With the exception of α-helical proteins, the kinetic progress curves exhibit single exponential time courses, consistent with two state behavior, as observed experimentally. The calculated folding rates are in remarkably good agreement with the measured values for the 25 two-state proteins investigated, with a correlation coefficient of 0.8. With its coarse-grained description of both the energy and entropy, and only three independently adjustable parameters, the model may be regarded as the simplest possible analytical model of protein folding capable of predicting experimental properties of specific proteins.

  18. Folding mechanism of a multiple independently-folding domain protein: double B domain of protein A.

    PubMed

    Arora, Pooja; Hammes, Gordon G; Oas, Terrence G

    2006-10-10

    The antibody binding properties of staphylococcal protein A (SpA) can be attributed to the presence of five highly homologous domains (E, D, A, B, and C). Although the folding of the B domain of protein A (BdpA) is well-characterized, the folding behavior of this domain in the context of full-length SpA in the cell remains unexplored. The sequence of the B domain is 89 and 91% identical to those of domains A and C, respectively. We have fused B domain sequences (BBdpA) as a close approximation of the A-B or B-C portion of SpA. Circular dichroism and fluorescence-detected denaturation curves of BBdpA are experimentally indistinguishable from those of BdpA. The rate constants for folding and unfolding from NMR line shape analysis for the single- and double-domain proteins are the same within experimental uncertainties (+/-20%). These results support the designation of SpA as a multiple independently-folding domain (MIFD) protein. We develop a mathematical model that describes the folding thermodynamics and kinetics of MIFD proteins. The model depicts MIFD protein folding and unfolding as a parallel network and explicitly calculates the flux through all parallel pathways. These fluxes are combined to give a complete description of the global thermodynamics and kinetics of the folding and unfolding of MIFD proteins. The global rates for complete folding and unfolding of a MIFD protein and those of the individual domains depend on the stability of the protein. We show that the global unfolding rate of a MIFD protein may be many orders of magnitude slower than that of the constituent domains.

  19. The role of ascorbate in protein folding.

    PubMed

    Szarka, András; Lőrincz, Tamás

    2014-05-01

    Ascorbate was linked to protein folding a long time ago. At the first level of this connection, it had been shown that ascorbate functions as an essential cofactor in the hydroxylation enzymes involved in collagen synthesis. Although the hydroxylation reactions catalyzed by the members of the prolyl 4-hydroxylase family are considered to be ascorbate dependent, the hydroxylation of proline alone does not need ascorbate. Prolyl 4-hydroxylases participate in two catalytic reactions: one in which proline residues are hydroxylated, while 2-oxoglutarate is decarboxylated and molecular oxygen is consumed. This reaction is ascorbate independent. However, in another reaction, prolyl 4-hydroxylases catalyze the decarboxylation of 2-oxoglutarate uncoupled from proline hydroxylation but still needing molecular oxygen. At this time, ferrous iron is oxidized and the protein is rendered catalytically inactive until reduced by ascorbate. At the second level of the connection, the oxidation and the oxidized form of ascorbate, dehydroascorbate, is involved in the formation of disulfide bonds of secretory proteins. The significance of the dehydroascorbate reductase activity of protein disulfide isomerase was debated because protein disulfide isomerase as a dehydroascorbate reductase was found to be too slow to be the major route for the reduction of dehydroascorbate (and formation of disulfides) in the endoplasmic reticulum lumen. However, very recently, low tissue ascorbate levels and a noncanonical scurvy were observed in endoplasmic reticulum thiol oxidase- and peroxiredoxin 4-compromised mice. This novel observation implies that ascorbate may be involved in oxidative protein folding and creates a link between the disulfide bond formation (oxidative protein folding) and hydroxylation.

  20. Secretion by numbers: protein traffic in prokaryotes

    PubMed Central

    Economou, Anastasias; Christie, Peter J.; Fernandez, Rachel C.; Palmer, Tracy; Plano, Greg V.; Pugsley, Anthony P.

    2013-01-01

    Summary Almost all aspects of protein traffic in bacteria were covered at the ASM-FEMS meeting on the topic in Iraklio, Crete in May 2006. The studies presented ranged from mechanistic analysis of specific events leading proteins to their final destinations to the physiological roles of the targeted proteins. Among the highlights from the meeting that are reviewed here are the molecular dynamics of SecA protein, membrane protein insertion, type III secretion needles and chaperones, type IV secretion, the two partner and autosecretion systems, the ‘secretion competent state’, and the recently discovered type VI secretion system. PMID:17020575

  1. Exploring the Levinthal limit in protein folding.

    PubMed

    Cruzeiro, Leonor; Degrève, Léo

    2017-03-01

    According to the thermodynamic hypothesis, the native state of proteins is uniquely defined by their amino acid sequence. On the other hand, according to Levinthal, the native state is just a local minimum of the free energy and a given amino acid sequence, in the same thermodynamic conditions, can assume many, very different structures that are as thermodynamically stable as the native state. This is the Levinthal limit explored in this work. Using computer simulations, we compare the interactions that stabilize the native state of four different proteins with those that stabilize three non-native states of each protein and find that the nature of the interactions is very similar for all such 16 conformers. Furthermore, an enhancement of the degree of fluctuation of the non-native conformers can be explained by an insufficient relaxation to their local free energy minimum. These results favor Levinthal's hypothesis that protein folding is a kinetic non-equilibrium process.

  2. Microcanonical versus Canonical Analysis of Protein Folding

    NASA Astrophysics Data System (ADS)

    Hernández-Rojas, J.; Gomez Llorente, J. M.

    2008-06-01

    The microcanonical analysis is shown to be a powerful tool to characterize the protein folding transition and to neatly distinguish between good and bad folders. An off-lattice model with parameter chosen to represent polymers of these two types is used to illustrate this approach. Both canonical and microcanonical ensembles are employed. The required calculations were performed using parallel tempering Monte Carlo simulations. The most revealing features of the folding transition are related to its first-order-like character, namely, the S-bend pattern in the caloric curve, which gives rise to negative microcanonical specific heats, and the bimodality of the energy distribution function at the transition temperatures. Models for a good folder are shown to be quite robust against perturbations in the interaction potential parameters.

  3. Mutations in HlyD, Part of the Type 1 Translocator for Hemolysin Secretion, Affect the Folding of the Secreted Toxin

    PubMed Central

    Pimenta, A. L.; Racher, K.; Jamieson, L.; Blight, M. A.; Holland, I. B.

    2005-01-01

    HlyD, a member of the membrane fusion protein family, is essential for the secretion of the RTX hemolytic toxin HlyA from Escherichia coli. Random point mutations affecting HlyA secretion were obtained, distributed in most periplasmic regions of the HlyD molecule. Analysis of the secretion phenotypes of different mutants allowed the identification of regions in HlyD involved in different steps of HlyA translocation. Four mutants, V349-I, T85-I, V334-I and L165-Q, were conditionally defective, a phenotype shown to be linked to the presence of inhibitory concentrations of Ca2+ in extracellular medium. Hly mutant T85-I was defective at an early stage in secretion, while mutants V334-I and L165-Q appeared to accumulate HlyA in the cell envelope, indicating a block at an intermediate step. Mutants V349-I, V334-I, and L165-Q were only partially defective in secretion, allowing significant levels of HlyA to be transported, but in the case of V349-I and L165-Q the HlyA molecules secreted showed greatly reduced hemolytic activity. Hemolysin molecules secreted from V349-I and V334-I are defective in normal folding and can be reactivated in vitro to the same levels as HlyA secreted from the wild-type translocator. Both V349-I and V334-I mutations mapped to the C-terminal lipoyl repeat motif, involved in the switching from the helical hairpin to the extended form of HlyD during assembly of the functional transport channel. These results suggest that HlyD is an integral component of the transport pathway, whose integrity is essential for the final folding of secreted HlyA into its active form. PMID:16237030

  4. Deletional Protein Engineering Based on Stable Fold

    PubMed Central

    Sokalingam, Sriram; Yun, Hyungdon; Lee, Sun-Gu

    2012-01-01

    Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191–196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability. PMID:23240034

  5. Computational Solutions to the Protein Folding Problem,

    DTIC Science & Technology

    1994-05-19

    A TRIDENT SCHOLAR oN PROJECT REPORT 0 NO. 223 "Computational Solutions to the Protein Folding Problem" L T -’ ’r i SEP 2 7 1994 ýV UNITED STATES...potential energy function (Chapter II), 25 1 2 2 U = X• k( l 1 -lo) 2+ X.ko (8,-8o) 2+X.-[1l + cos (Pip + )] Equation 4.1 xei (C ¶±~12.4 a where ri, is...iterative process, a set of k >_ 2"t+ l distinct local minima are computed. This can be done with rela- tive ease by using an efficient unconstrained

  6. Distinguishing between sequential and nonsequentially folded proteins: implications for folding and misfolding.

    PubMed Central

    Tsai, C. J.; Maizel, J. V.; Nussinov, R.

    1999-01-01

    We describe here an algorithm for distinguishing sequential from nonsequentially folding proteins. Several experiments have recently suggested that most of the proteins that are synthesized in the eukaryotic cell may fold sequentially. This proposed folding mechanism in vivo is particularly advantageous to the organism. In the absence of chaperones, the probability that a sequentially folding protein will misfold is reduced significantly. The problem we address here is devising a procedure that would differentiate between the two types of folding patterns. Footprints of sequential folding may be found in structures where consecutive fragments of the chain interact with each other. In such cases, the folding complexity may be viewed as being lower. On the other hand, higher folding complexity suggests that at least a portion of the polypeptide backbone folds back upon itself to form three-dimensional (3D) interactions with noncontiguous portion(s) of the chain. Hence, we look at the mechanism of folding of the molecule via analysis of its complexity, that is, through the 3D interactions formed by contiguous segments on the polypeptide chain. To computationally splice the structure into consecutively interacting fragments, we either cut it into compact hydrophobic folding units or into a set of hypothetical, transient, highly populated, contiguous fragments ("building blocks" of the structure). In sequential folding, successive building blocks interact with each other from the amino to the carboxy terminus of the polypeptide chain. Consequently, the results of the parsing differentiate between sequentially vs. nonsequentially folded chains. The automated assessment of the folding complexity provides insight into both the likelihood of misfolding and the kinetic folding rate of the given protein. In terms of the funnel free energy landscape theory, a protein that truly follows the mechanism of sequential folding, in principle, encounters smoother free energy barriers

  7. Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion.

    PubMed

    Ling Lin Fu; Zi Rong Xu; Wei Fen Li; Jiang Bing Shuai; Ping Lu; Chun Xia Hu

    2007-01-01

    The absence of an outer membrane in Bacillus subtilis can simplify the protein secretion pathways and allow the organism to secrete high levels of extracellular proteins. Of the three known secretory routes, Sec-SRP pathway can direct the majority of secretory proteins into the growth medium. Alternatively, a small number of exoproteins with specific functions are secreted via Tat pathway or ABC transporters in B. subtilis. The discriminating function of precursor proteins among these pathways is largely attributed to the distinct structure of their cleavable signal peptides. Individual secretion machinery components with their special functions are involved in the total flow of proteins from the cytoplasm to the medium. Notably, multiple regulators with signal transduction functions can affect expression of secretion machinery as well as their post-transcriptional actions for protein secretion, resulting in the complicated networks in B. subtilis. Ultimately, according to the available knowledge of secretion machinery, several approaches aimed at optimizing protein secretion are discussed.

  8. Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae.

    PubMed

    Hou, Jin; Tyo, Keith E J; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

    2012-08-01

    The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, and it is also provides a platform for the production of many heterologous proteins of medical or industrial interest. Therefore, many studies have focused on metabolic engineering S. cerevisiae to improve the recombinant protein production, and with the development of systems biology, it is interesting to see how this approach can be applied both to gain further insight into protein production and secretion and to further engineer the cell for improved production of valuable proteins. In this review, the protein post-translational modification such as folding, trafficking, and secretion, steps that are traditionally studied in isolation will here be described in the context of the whole system of protein secretion. Furthermore, examples of engineering secretion pathways, high-throughput screening and systems biology applications of studying protein production and secretion are also given to show how the protein production can be improved by different approaches. The objective of the review is to describe individual biological processes in the context of the larger, complex protein synthesis network.

  9. Analyzing the Role of Periplasmic Folding Factors in the Biogenesis of OMPs and Members of the Type V Secretion System.

    PubMed

    Bodelón, Gustavo; Marín, Elvira; Fernández, Luis Ángel

    2015-01-01

    The outer membrane (OM) of gram-negative bacteria is highly packed with OM proteins (OMPs) and the trafficking and assembly of OMPs in gram-negative bacteria is a subject of intense research. Structurally, OMPs vary in the number of β-strands and in the size and complexity of extra-membrane domains, with extreme examples being the members of the type V protein secretion system (T5SS), such as the autotransporter (AT) and intimin/invasin families of secreted proteins, in which a large extracellular "passenger" domain is linked to a β-barrel that inserts in the OM. Despite their structural and functional diversity, OMPs interact in the periplasm with a relatively small set of protein chaperones that facilitate their transport from the inner membrane (IM) to the β-barrel assembly machinery (BAM complex), preventing aggregation and assisting their folding in various aspects including disulfide bond formation. This chapter is focused on the periplasmic folding factors involved in the biogenesis of integral OMPs and members of T5SS in E. coli, which are used as a model system in this field. Background information on these periplasmic folding factors is provided along with genetic methods to generate conditional mutants that deplete these factors from E. coli and biochemical methods to analyze the folding, surface display, disulfide formation and oligomerization state of OMPs/T5SS in these mutants.

  10. Hydrophobic-hydrophilic forces in protein folding.

    PubMed

    Durell, Stewart R; Ben-Naim, Arieh

    2017-08-01

    The process of protein folding is obviously driven by forces exerted on the atoms of the amino-acid chain. These forces arise from interactions with other parts of the protein itself (direct forces), as well as from interactions with the solvent (solvent-induced forces). We present a statistical-mechanical formalism that describes both these direct and indirect, solvent-induced thermodynamic forces on groups of the protein. We focus on 2 kinds of protein groups, commonly referred to as hydrophobic and hydrophilic. Analysis of this result leads to the conclusion that the forces on hydrophilic groups are in general stronger than on hydrophobic groups. This is then tested and verified by a series of molecular dynamics simulations, examining both hydrophobic alkanes of different sizes and hydrophilic moieties represented by polar-neutral hydroxyl groups. The magnitude of the force on assemblies of hydrophilic groups is dependent on their relative orientation: with 2 to 4 times larger forces on groups that are able to form one or more direct hydrogen bonds. © 2017 Wiley Periodicals, Inc.

  11. Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand.

    PubMed

    Chang, Yu-Chu; Oas, Terrence G

    2010-06-29

    Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.

  12. Some physical approaches to protein folding

    NASA Astrophysics Data System (ADS)

    Bascle, J.; Garel, T.; Orland, H.

    1993-02-01

    To understand how a protein folds is a problem which has important biological implications. In this article, we would like to present a physics-oriented point of view, which is twofold. First of all, we introduce simple statistical mechanics models which display, in the thermodynamic limit, folding and related transitions. These models can be divided into (i) crude spin glass-like models (with their Mattis analogs), where one may look for possible correlations between the chain self-interactions and the folded structure, (ii) glass-like models, where one emphasizes the geometrical competition between one- or two-dimensional local order (mimicking α helix or β sheet structures), and the requirement of global compactness. Both models are too simple to predict the spatial organization of a realistic protein, but are useful for the physicist and should have some feedback in other glassy systems (glasses, collapsed polymers .... ). These remarks lead us to the second physical approach, namely a new Monte-Carlo method, where one grows the protein atom-by-atom (or residue-by-residue), using a standard form (CHARMM .... ) for the total energy. A detailed comparison with other Monte-Carlo schemes, or Molecular Dynamics calculations, is then possible; we will sketch such a comparison for poly-alanines. Our twofold approach illustrates some of the difficulties one encounters in the protein folding problem, in particular those associated with the existence of a large number of metastable states. Le repliement des protéines est un problème qui a de nombreuses implications biologiques. Dans cet article, nous présentons, de deux façons différentes, un point de vue de physicien. Nous introduisons tout d'abord des modèles simples de mécanique statistique qui exhibent, à la limite thermodynamique, des transitions de repliement. Ces modèles peuvent être divisés en (i) verres de spin (éventuellement à la Mattis), où l'on peut chercher des corrélations entre les

  13. Progress towards mapping the universe of protein folds

    PubMed Central

    Grant, Alastair; Lee, David; Orengo, Christine

    2004-01-01

    Although the precise aims differ between the various international structural genomics initiatives currently aiming to illuminate the universe of protein folds, many selectively target protein families for which the fold is unknown. How well can the current set of known protein families and folds be used to estimate the total number of folds in nature, and will structural genomics initiatives yield representatives for all the major protein families within a reasonable time scale? PMID:15128436

  14. How the hydrophobic factor drives protein folding

    PubMed Central

    Baldwin, Robert L.; Rose, George D.

    2016-01-01

    How hydrophobicity (HY) drives protein folding is studied. The 1971 Nozaki–Tanford method of measuring HY is modified to use gases as solutes, not crystals, and this makes the method easy to use. Alkanes are found to be much more hydrophobic than rare gases, and the two different kinds of HY are termed intrinsic (rare gases) and extrinsic (alkanes). The HY values of rare gases are proportional to solvent-accessible surface area (ASA), whereas the HY values of alkanes depend on special hydration shells. Earlier work showed that hydration shells produce the hydration energetics of alkanes. Evidence is given here that the transfer energetics of alkanes to cyclohexane [Wolfenden R, Lewis CA, Jr, Yuan Y, Carter CW, Jr (2015) Proc Natl Acad Sci USA 112(24):7484–7488] measure the release of these shells. Alkane shells are stabilized importantly by van der Waals interactions between alkane carbon and water oxygen atoms. Thus, rare gases cannot form this type of shell. The very short (approximately picoseconds) lifetime of the van der Waals interaction probably explains why NMR efforts to detect alkane hydration shells have failed. The close similarity between the sizes of the opposing energetics for forming or releasing alkane shells confirms the presence of these shells on alkanes and supports Kauzmann's 1959 mechanism of protein folding. A space-filling model is given for the hydration shells on linear alkanes. The model reproduces the n values of Jorgensen et al. [Jorgensen WL, Gao J, Ravimohan C (1985) J Phys Chem 89:3470–3473] for the number of waters in alkane hydration shells. PMID:27791131

  15. How the hydrophobic factor drives protein folding.

    PubMed

    Baldwin, Robert L; Rose, George D

    2016-11-01

    How hydrophobicity (HY) drives protein folding is studied. The 1971 Nozaki-Tanford method of measuring HY is modified to use gases as solutes, not crystals, and this makes the method easy to use. Alkanes are found to be much more hydrophobic than rare gases, and the two different kinds of HY are termed intrinsic (rare gases) and extrinsic (alkanes). The HY values of rare gases are proportional to solvent-accessible surface area (ASA), whereas the HY values of alkanes depend on special hydration shells. Earlier work showed that hydration shells produce the hydration energetics of alkanes. Evidence is given here that the transfer energetics of alkanes to cyclohexane [Wolfenden R, Lewis CA, Jr, Yuan Y, Carter CW, Jr (2015) Proc Natl Acad Sci USA 112(24):7484-7488] measure the release of these shells. Alkane shells are stabilized importantly by van der Waals interactions between alkane carbon and water oxygen atoms. Thus, rare gases cannot form this type of shell. The very short (approximately picoseconds) lifetime of the van der Waals interaction probably explains why NMR efforts to detect alkane hydration shells have failed. The close similarity between the sizes of the opposing energetics for forming or releasing alkane shells confirms the presence of these shells on alkanes and supports Kauzmann's 1959 mechanism of protein folding. A space-filling model is given for the hydration shells on linear alkanes. The model reproduces the n values of Jorgensen et al. [Jorgensen WL, Gao J, Ravimohan C (1985) J Phys Chem 89:3470-3473] for the number of waters in alkane hydration shells.

  16. Crystal structure of a defective folding protein

    PubMed Central

    Saul, Frederick A.; Mourez, Michaël; Vulliez-le Normand, Brigitte; Sassoon, Nathalie; Bentley, Graham A.; Betton, Jean-Michel

    2003-01-01

    Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32→Asp and Ile 33→Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 Å resolution. The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first α-helix to the first β-strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system. PMID:12592028

  17. Crystal structure of a defective folding protein.

    PubMed

    Saul, Frederick A; Mourez, Michaël; Vulliez-Le Normand, Brigitte; Sassoon, Nathalie; Bentley, Graham A; Betton, Jean-Michel

    2003-03-01

    Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32-->Asp and Ile 33-->Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 A resolution. The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first alpha-helix to the first beta-strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system.

  18. Understanding the folding-function tradeoff in proteins.

    PubMed

    Gosavi, Shachi

    2013-01-01

    When an amino-acid sequence cannot be optimized for both folding and function, folding can get compromised in favor of function. To understand this tradeoff better, we devise a novel method for extracting the "function-less" folding-motif of a protein fold from a set of structurally similar but functionally diverse proteins. We then obtain the β-trefoil folding-motif, and study its folding using structure-based models and molecular dynamics simulations. CompariA protein sequence serves two purpson with the folding of wild-type β-trefoil proteins shows that function affects folding in two ways: In the slower folding interleukin-1β, binding sites make the fold more complex, increase contact order and slow folding. In the faster folding hisactophilin, residues which could have been part of the folding-motif are used for function. This reduces the density of native contacts in functional regions and increases folding rate. The folding-motif helps identify subtle structural deviations which perturb folding. These may then be used for functional annotation. Further, the folding-motif could potentially be used as a first step in the sequence design of function-less scaffold proteins. Desired function can then be engineered into these scaffolds.

  19. Secreted Proteins Defy the Expression Level-Evolutionary Rate Anticorrelation.

    PubMed

    Feyertag, Felix; Berninsone, Patricia M; Alvarez-Ponce, David

    2017-03-01

    The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E-R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein-protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E-R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E-R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E-R anticorrelation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Purification of secreted recombinant proteins from Escherichia coli.

    PubMed

    Le, H V; Trotta, P P

    1991-01-01

    Secretion systems engineered for the expression of heterologous protein in E. coli provide several advantages for subsequent isolation of purified product. Proteins released from the periplasmic space, which represent a small fraction (i.e., 4-10%) of total cell protein, can readily be separated from other cellular proteins by centrifugation of the remaining cellular debris or cross-flow ultrafiltration. The starting material derived from secretion systems is generally of higher purity than comparable material produced from strains expressing cytoplasmically for systems exhibiting similar expression levels. The available evidence suggests that recombinant proteins derived from the periplasm are generally, but not always (44-46), soluble in a nonaggregated form. Consequently, simple purification protocols can be effectively employed for producing homogeneous product with a high yield. The majority of the secreted recombinant proteins reviewed in this chapter were purified by simple one- or two-step chromatography procedures. High-resolution techniques such as reversed phase HPLC were found necessary only in cases where the secreted polypeptides were contaminated with proteolytic degradation variants, e.g., hirudin (51) and beta-endorphin (22). The fact that a high level of biological activity has been shown to be characteristic of purified recombinant proteins secreted into the periplasmic space suggests the presence of a native conformation stabilized by the expected disulfide linkages. Intramolecular disulfide bonds most probably form either as the polypeptide is translocated through the cytoplasmic membrane into the periplasm or within the periplasmic compartment, which has a higher oxidation potential than that found in the cytoplasm (57). Studies performed with hGH (31) and muIL-2 (35) provide excellent examples of differences observed in protein folding and disulfide bond formation between heterologous proteins expressed in the cytoplasmic and periplasmic

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

    PubMed Central

    Schneewind, Olaf; Missiakas, Dominique M.

    2012-01-01

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

  2. Fold assessment for comparative protein structure modeling.

    PubMed

    Melo, Francisco; Sali, Andrej

    2007-11-01

    Accurate and automated assessment of both geometrical errors and incompleteness of comparative protein structure models is necessary for an adequate use of the models. Here, we describe a composite score for discriminating between models with the correct and incorrect fold. To find an accurate composite score, we designed and applied a genetic algorithm method that searched for a most informative subset of 21 input model features as well as their optimized nonlinear transformation into the composite score. The 21 input features included various statistical potential scores, stereochemistry quality descriptors, sequence alignment scores, geometrical descriptors, and measures of protein packing. The optimized composite score was found to depend on (1) a statistical potential z-score for residue accessibilities and distances, (2) model compactness, and (3) percentage sequence identity of the alignment used to build the model. The accuracy of the composite score was compared with the accuracy of assessment by single and combined features as well as by other commonly used assessment methods. The testing set was representative of models produced by automated comparative modeling on a genomic scale. The composite score performed better than any other tested score in terms of the maximum correct classification rate (i.e., 3.3% false positives and 2.5% false negatives) as well as the sensitivity and specificity across the whole range of thresholds. The composite score was implemented in our program MODELLER-8 and was used to assess models in the MODBASE database that contains comparative models for domains in approximately 1.3 million protein sequences.

  3. Fold assessment for comparative protein structure modeling

    PubMed Central

    Melo, Francisco; Sali, Andrej

    2007-01-01

    Accurate and automated assessment of both geometrical errors and incompleteness of comparative protein structure models is necessary for an adequate use of the models. Here, we describe a composite score for discriminating between models with the correct and incorrect fold. To find an accurate composite score, we designed and applied a genetic algorithm method that searched for a most informative subset of 21 input model features as well as their optimized nonlinear transformation into the composite score. The 21 input features included various statistical potential scores, stereochemistry quality descriptors, sequence alignment scores, geometrical descriptors, and measures of protein packing. The optimized composite score was found to depend on (1) a statistical potential z-score for residue accessibilities and distances, (2) model compactness, and (3) percentage sequence identity of the alignment used to build the model. The accuracy of the composite score was compared with the accuracy of assessment by single and combined features as well as by other commonly used assessment methods. The testing set was representative of models produced by automated comparative modeling on a genomic scale. The composite score performed better than any other tested score in terms of the maximum correct classification rate (i.e., 3.3% false positives and 2.5% false negatives) as well as the sensitivity and specificity across the whole range of thresholds. The composite score was implemented in our program MODELLER-8 and was used to assess models in the MODBASE database that contains comparative models for domains in approximately 1.3 million protein sequences. PMID:17905832

  4. The folding of an ``average'' beta trefoil protein.

    NASA Astrophysics Data System (ADS)

    Gosavi, Shachi; Jennings, Pat; Onuchic, Jose

    2007-03-01

    The beta-trefoil fold is characterized by twelve beta strands folded into three similar beta-beta-beta-loop-beta (trefoil) units. The overall fold has pseudo-threefold symmetry and consists of a six stranded-barrel, capped by a triangular hairpin triplet. The loops connecting the beta-strands vary in length and structure. It is these loops that give the fold its varied binding capability and the binding sites lie in different parts of the fold. The beta-trefoil proteins have little sequence similarity (sometimes less than 17%) and bind a range of molecules, including other proteins, DNA, membranes and carbohydrates. Protein folding experiments have been performed on four of the beta trefoils, namely, interleukin-1 (IL1B), acidic and basic fibroblast growth factors (FGF-1 and FGF-2) and hisactophilin (HIS). These experiments indicate that the proteins fold by different routes. Folding simulations of the proteins identify the possible folding routes and also show that the shapes of the barriers are different for the different proteins. In this work, we design a model protein which contains only the core fold elements of the beta-trefoil fold. We compare the folding of this ``average'' protein to the folding of His, FGF and IL1B and make some connections with function.

  5. Spin glasses and the statistical mechanics of protein folding.

    PubMed Central

    Bryngelson, J D; Wolynes, P G

    1987-01-01

    The theory of spin glasses was used to study a simple model of protein folding. The phase diagram of the model was calculated, and the results of dynamics calculations are briefly reported. The relation of these results to folding experiments, the relation of these hypotheses to previous protein folding theories, and the implication of these hypotheses for protein folding prediction schemes are discussed. PMID:3478708

  6. A light-triggered protein secretion system.

    PubMed

    Chen, Daniel; Gibson, Emily S; Kennedy, Matthew J

    2013-05-13

    Optical control of protein interactions has emerged as a powerful experimental paradigm for manipulating and studying various cellular processes. Tools are now available for controlling a number of cellular functions, but some fundamental processes, such as protein secretion, have been difficult to engineer using current optical tools. Here we use UVR8, a plant photoreceptor protein that forms photolabile homodimers, to engineer the first light-triggered protein secretion system. UVR8 fusion proteins were conditionally sequestered in the endoplasmic reticulum, and a brief pulse of light triggered robust forward trafficking through the secretory pathway to the plasma membrane. UVR8 was not responsive to excitation light used to image cyan, green, or red fluorescent protein variants, allowing multicolor visualization of cellular markers and secreted protein cargo as it traverses the cellular secretory pathway. We implemented this novel tool in neurons to demonstrate restricted, local trafficking of secretory cargo near dendritic branch points.

  7. Improving Protein Fold Recognition by Deep Learning Networks

    NASA Astrophysics Data System (ADS)

    Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

    2015-12-01

    For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl’s benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.

  8. Proteins with Highly Similar Native Folds Can Show Vastly Dissimilar Folding Behavior When Desolvated**

    PubMed Central

    Schennach, Moritz; Breuker, Kathrin

    2014-01-01

    Proteins can be exposed to vastly different environments such as the cytosol or membranes, but the delicate balance between external factors and intrinsic determinants of protein structure, stability, and folding is only poorly understood. Here we used electron capture dissociation to study horse and tuna heart Cytochromes c in the complete absence of solvent. The significantly different stability of their highly similar native folds after transfer into the gas phase, and their strikingly different folding behavior in the gas phase, can be rationalized on the basis of electrostatic interactions such as salt bridges. In the absence of hydrophobic bonding, protein folding is far slower and more complex than in solution. PMID:24259450

  9. Dual folding pathways of an α /β protein from all-atom ab initio folding simulations

    NASA Astrophysics Data System (ADS)

    Lei, Hongxing; Wang, Zhi-Xiang; Wu, Chun; Duan, Yong

    2009-10-01

    Successful ab initio folding of proteins with both α-helix and β-sheet requires a delicate balance among a variety of forces in the simulation model, which may explain that the successful folding of any α /β proteins to within experimental error has yet to be reported. Here we demonstrate that it is an achievable goal to fold α /β proteins with a force field emphasizing the balance between the two major secondary structures. Using our newly developed force field, we conducted extensive ab initio folding simulations on an α /β protein full sequence design (FSD) employing both conventional molecular dynamics and replica exchange molecular dynamics in combination with a generalized-Born solvation model. In these simulations, the folding of FSD to the native state with high population (>64.2%) and high fidelity (Cα-Root Mean Square Deviation of 1.29 Å for the most sampled conformation when compared to the experimental structure) was achieved. The folding of FSD was found to follow two pathways. In the major pathway, the folding started from the formation of the helix. In the minor pathway, however, folding of the β-hairpin started first. Further examination revealed that the helix initiated from the C-terminus and propagated toward the N-terminus. The formation of the hydrophobic contacts coincided with the global folding. Therefore the hydrophobic force does not appear to be the driving force of the folding of this protein.

  10. Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins.

    PubMed

    De Jaco, Antonella; Dubi, Noga; Camp, Shelley; Taylor, Palmer

    2012-12-01

    The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination.

  11. Understanding the role of the topology in protein folding by computational inverse folding experiments.

    PubMed

    Mucherino, Antonio; Costantini, Susan; di Serafino, Daniela; D'Apuzzo, Marco; Facchiano, Angelo; Colonna, Giovanni

    2008-08-01

    Recent studies suggest that protein folding should be revisited as the emergent property of a complex system and that the nature allows only a very limited number of folds that seem to be strongly influenced by geometrical properties. In this work we explore the principles underlying this new view and show how helical protein conformations can be obtained starting from simple geometric considerations. We generated a large data set of C-alpha traces made of 65 points, by computationally solving a backbone model that takes into account only topological features of the all-alpha proteins; then, we built corresponding tertiary structures, by using the sequences associated to the crystallographic structures of four small globular all-alpha proteins from PDB, and analysed them in terms of structural and energetic properties. In this way we obtained four poorly populated sets of structures that are reasonably similar to the conformational states typical of the experimental PDB structures. These results show that our computational approach can capture the native topology of all-alpha proteins; furthermore, it generates backbone folds without the influence of the side chains and uses the protein sequence to select a specific fold among the generated folds. This agrees with the recent view that the backbone plays an important role in the protein folding process and that the amino acid sequence chooses its own fold within a limited total number of folds.

  12. Multiple folding pathways of proteins with shallow knots and co-translational folding

    NASA Astrophysics Data System (ADS)

    Chwastyk, Mateusz; Cieplak, Marek

    2015-07-01

    We study the folding process in the shallowly knotted protein MJ0366 within two variants of a structure-based model. We observe that the resulting topological pathways are much richer than identified in previous studies. In addition to the single knot-loop events, we find novel, and dominant, two-loop mechanisms. We demonstrate that folding takes place in a range of temperatures and the conditions of most successful folding are at temperatures which are higher than those required for the fastest folding. We also demonstrate that nascent conditions are more favorable to knotting than off-ribosome folding.

  13. Protein-Folding Landscapes in Multi-Chain Systems

    SciTech Connect

    Cellmer, Troy; Bratko, Dusan; Prausnitz, John M.; Blanch, Harvey

    2005-06-20

    Computational studies of proteins have significantly improved our understanding of protein folding. These studies are normally carried out using chains in isolation. However, in many systems of practical interest, proteins fold in the presence of other molecules. To obtain insight into folding in such situations, we compare the thermodynamics of folding for a Miyazawa-Jernigan model 64-mer in isolation to results obtained in the presence of additional chains. The melting temperature falls as the chain concentration increases. In multi-chain systems, free-energy landscapes for folding show an increased preference for misfolded states. Misfolding is accompanied by an increase in inter-protein interactions; however, near the folding temperature, the transition from folded chains to misfolded and associated chains isentropically driven. A majority of the most probable inter-protein contacts are also native contacts, suggesting that native topology plays a role in early stages of aggregation.

  14. Protein fold classification with genetic algorithms and feature selection.

    PubMed

    Chen, Peng; Liu, Chunmei; Burge, Legand; Mahmood, Mohammad; Southerland, William; Gloster, Clay

    2009-10-01

    Protein fold classification is a key step to predicting protein tertiary structures. This paper proposes a novel approach based on genetic algorithms and feature selection to classifying protein folds. Our dataset is divided into a training dataset and a test dataset. Each individual for the genetic algorithms represents a selection function of the feature vectors of the training dataset. A support vector machine is applied to each individual to evaluate the fitness value (fold classification rate) of each individual. The aim of the genetic algorithms is to search for the best individual that produces the highest fold classification rate. The best individual is then applied to the feature vectors of the test dataset and a support vector machine is built to classify protein folds based on selected features. Our experimental results on Ding and Dubchak's benchmark dataset of 27-class folds show that our approach achieves an accuracy of 71.28%, which outperforms current state-of-the-art protein fold predictors.

  15. Transition paths, diffusive processes, and preequilibria of protein folding.

    PubMed

    Zhang, Zhuqing; Chan, Hue Sun

    2012-12-18

    Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.

  16. CoinFold: a web server for protein contact prediction and contact-assisted protein folding.

    PubMed

    Wang, Sheng; Li, Wei; Zhang, Renyu; Liu, Shiwang; Xu, Jinbo

    2016-07-08

    CoinFold (http://raptorx2.uchicago.edu/ContactMap/) is a web server for protein contact prediction and contact-assisted de novo structure prediction. CoinFold predicts contacts by integrating joint multi-family evolutionary coupling (EC) analysis and supervised machine learning. This joint EC analysis is unique in that it not only uses residue coevolution information in the target protein family, but also that in the related families which may have divergent sequences but similar folds. The supervised learning further improves contact prediction accuracy by making use of sequence profile, contact (distance) potential and other information. Finally, this server predicts tertiary structure of a sequence by feeding its predicted contacts and secondary structure to the CNS suite. Tested on the CASP and CAMEO targets, this server shows significant advantages over existing ones of similar category in both contact and tertiary structure prediction. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Mechanism of Action of Secreted Newt Anterior Gradient Protein

    PubMed Central

    Grassme, Kathrin S.; Garza-Garcia, Acely; Delgado, Jean-Paul; Godwin, James W.; Kumar, Anoop; Gates, Phillip B.; Brockes, Jeremy P.

    2016-01-01

    Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family. PMID:27100463

  18. Novel Protein Folding Pathways for Protein Salvage and Recycling

    DTIC Science & Technology

    2013-08-26

    Life. The Archaea have many molecular properties that are found universally in modern lineages of both Bacteria and Archaea, and many species are...eukarya or bacteria . In hyperthermophiles, the chaperonin (Cpn60) is the only ATP dependent protein folding complex. It is a 1 mDa molecular machine... fermentation physiology for formate and carbon monoxide. In Fig. 3 below, actual gene replacement and knockouts of the chaperonin HSP60 loci in

  19. Start2Fold: a database of hydrogen/deuterium exchange data on protein folding and stability

    PubMed Central

    Pancsa, Rita; Varadi, Mihaly; Tompa, Peter; Vranken, Wim F.

    2016-01-01

    Proteins fulfil a wide range of tasks in cells; understanding how they fold into complex three-dimensional (3D) structures and how these structures remain stable while retaining sufficient dynamics for functionality is essential for the interpretation of overall protein behaviour. Since the 1950's, solvent exchange-based methods have been the most powerful experimental means to obtain information on the folding and stability of proteins. Considerable expertise and care were required to obtain the resulting datasets, which, despite their importance and intrinsic value, have never been collected, curated and classified. Start2Fold is an openly accessible database (http://start2fold.eu) of carefully curated hydrogen/deuterium exchange (HDX) data extracted from the literature that is open for new submissions from the community. The database entries contain (i) information on the proteins investigated and the underlying experimental procedures and (ii) the classification of the residues based on their exchange protection levels, also allowing for the instant visualization of the relevant residue groups on the 3D structures of the corresponding proteins. By providing a clear hierarchical framework for the easy sharing, comparison and (re-)interpretation of HDX data, Start2Fold intends to promote a better understanding of how the protein sequence encodes folding and structure as well as the development of new computational methods predicting protein folding and stability. PMID:26582925

  20. Modeling Protein Folding and Applying It to a Relevant Activity

    ERIC Educational Resources Information Center

    Nelson, Allan; Goetze, Jim

    2004-01-01

    The different levels of protein structure that can be easily understood by creating a model that simulates protein folding, which can then be evaluated by applying it to a relevant activity, is presented. The materials required and the procedure for constructing a protein folding model are mentioned.

  1. Ligand-Promoted Protein Folding by Biased Kinetic Partitioning

    PubMed Central

    Hingorani, Karan S.; Metcalf, Matthew C.; Deming, Derrick T.; Garman, Scott C.; Powers, Evan T.; Gierasch, Lila M.

    2017-01-01

    Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems. PMID:28218913

  2. Atom-by-atom analysis of global downhill protein folding

    NASA Astrophysics Data System (ADS)

    Sadqi, Mourad; Fushman, David; Muñoz, Victor

    2006-07-01

    Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.

  3. Cotranslational Protein Folding inside the Ribosome Exit Tunnel.

    PubMed

    Nilsson, Ola B; Hedman, Rickard; Marino, Jacopo; Wickles, Stephan; Bischoff, Lukas; Johansson, Magnus; Müller-Lucks, Annika; Trovato, Fabio; Puglisi, Joseph D; O'Brien, Edward P; Beckmann, Roland; von Heijne, Gunnar

    2015-09-08

    At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Cotranslational Protein Folding inside the Ribosome Exit Tunnel

    PubMed Central

    Nilsson, Ola B.; Hedman, Rickard; Marino, Jacopo; Wickles, Stephan; Bischoff, Lukas; Johansson, Magnus; Müller-Lucks, Annika; Trovato, Fabio; Puglisi, Joseph D.; O’Brien, Edward P.; Beckmann, Roland; von Heijne, Gunnar

    2015-01-01

    Summary At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. PMID:26321634

  5. Milk secretion: The role of SNARE proteins.

    PubMed

    Truchet, Sandrine; Chat, Sophie; Ollivier-Bousquet, Michèle

    2014-03-01

    During lactation, polarized mammary epithelial secretory cells (MESCs) secrete huge quantities of the nutrient molecules that make up milk, i.e. proteins, fat globules and soluble components such as lactose and minerals. Some of these nutrients are only produced by the MESCs themselves, while others are to a great extent transferred from the blood. MESCs can thus be seen as a crossroads for both the uptake and the secretion with cross-talks between intracellular compartments that enable spatial and temporal coordination of the secretion of the milk constituents. Although the physiology of lactation is well understood, the molecular mechanisms underlying the secretion of milk components remain incompletely characterized. Major milk proteins, namely caseins, are secreted by exocytosis, while the milk fat globules are released by budding, being enwrapped by the apical plasma membrane. Prolactin, which stimulates the transcription of casein genes, also induces the production of arachidonic acid, leading to accelerated casein transport and/or secretion. Because of their ability to form complexes that bridge two membranes and promote their fusion, SNARE (Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins are involved in almost all intracellular trafficking steps and exocytosis. As SNAREs can bind arachidonic acid, they could be the effectors of the secretagogue effect of prolactin in MESCs. Indeed, some SNAREs have been observed between secretory vesicles and lipid droplets suggesting that these proteins could not only orchestrate the intracellular trafficking of milk components but also act as key regulators for both the coupling and coordination of milk product secretion in response to hormones.

  6. Thermodynamics and kinetics of protein folding: an evolutionary perspective.

    PubMed

    Demetrius, Lloyd

    2002-08-07

    This article appeals to an evolutionary model which postulates that primordial proteins were described by small polypeptide chains which (i) lack disulfide bridges, and (ii) display slow folding rates with multi-state kinetics, to determine relations between structural properties of proteins and their folding kinetics. We parameterize the energy landscape of proteins in terms of thermodynamic activation variables. The model studies evolutionary changes in these thermodynamic parameters, and we invoke relations between these activation variables and structural properties of the protein to predict the following correspondence between protein structure and folding kinetics. 1. Proteins with inter- and intra-chain disulfide bridges: large variability in both folding rates and stability of intermediates, multi-state kinetics. 2. Proteins which lack inter and intra-chain disulfide bridges. 2.1 Single-domain chains: fast folding rates; unstable intermediates; two-state kinetics. 2.2 Multi-domain monomers: intermediate rates; metastable intermediates; multi-state kinetics. 2.3 Multi-domain oligomers: slow rates; metastable intermediates; multi-state kinetics. The evolutionary model thus provides a kinetic characterization of one important subfamily of proteins which we describe by the following properties: Folding dynamics of single-domain proteins which lack disulfide bridges are described by two-state kinetics. Folding rate of this class of proteins is positively correlated with the thermodynamic stability of the folded state.

  7. Designing pH induced fold switch in proteins

    NASA Astrophysics Data System (ADS)

    Baruah, Anupaul; Biswas, Parbati

    2015-05-01

    This work investigates the computational design of a pH induced protein fold switch based on a self-consistent mean-field approach by identifying the ensemble averaged characteristics of sequences that encode a fold switch. The primary challenge to balance the alternative sets of interactions present in both target structures is overcome by simultaneously optimizing two foldability criteria corresponding to two target structures. The change in pH is modeled by altering the residual charge on the amino acids. The energy landscape of the fold switch protein is found to be double funneled. The fold switch sequences stabilize the interactions of the sites with similar relative surface accessibility in both target structures. Fold switch sequences have low sequence complexity and hence lower sequence entropy. The pH induced fold switch is mediated by attractive electrostatic interactions rather than hydrophobic-hydrophobic contacts. This study may provide valuable insights to the design of fold switch proteins.

  8. Molecular Recognition by Templated Folding of an Intrinsically Disordered Protein

    NASA Astrophysics Data System (ADS)

    Toto, Angelo; Camilloni, Carlo; Giri, Rajanish; Brunori, Maurizio; Vendruscolo, Michele; Gianni, Stefano

    2016-02-01

    Intrinsically disordered proteins often become structured upon interacting with their partners. The mechanism of this ‘folding upon binding’ process, however, has not been fully characterised yet. Here we present a study of the folding of the intrinsically disordered transactivation domain of c-Myb (c-Myb) upon binding its partner KIX. By determining the structure of the folding transition state for the binding of wild-type and three mutational variants of KIX, we found a remarkable plasticity of the folding pathway of c-Myb. To explain this phenomenon, we show that the folding of c-Myb is templated by the structure of KIX. This adaptive folding behaviour, which occurs by heterogeneous nucleation, differs from the robust homogeneous nucleation typically observed for globular proteins. We suggest that this templated folding mechanism may enable intrinsically disordered proteins to achieve specific and reliable binding with multiple partners while avoiding aberrant interactions.

  9. Designing pH induced fold switch in proteins.

    PubMed

    Baruah, Anupaul; Biswas, Parbati

    2015-05-14

    This work investigates the computational design of a pH induced protein fold switch based on a self-consistent mean-field approach by identifying the ensemble averaged characteristics of sequences that encode a fold switch. The primary challenge to balance the alternative sets of interactions present in both target structures is overcome by simultaneously optimizing two foldability criteria corresponding to two target structures. The change in pH is modeled by altering the residual charge on the amino acids. The energy landscape of the fold switch protein is found to be double funneled. The fold switch sequences stabilize the interactions of the sites with similar relative surface accessibility in both target structures. Fold switch sequences have low sequence complexity and hence lower sequence entropy. The pH induced fold switch is mediated by attractive electrostatic interactions rather than hydrophobic-hydrophobic contacts. This study may provide valuable insights to the design of fold switch proteins.

  10. Molecular Recognition by Templated Folding of an Intrinsically Disordered Protein

    PubMed Central

    Toto, Angelo; Camilloni, Carlo; Giri, Rajanish; Brunori, Maurizio; Vendruscolo, Michele; Gianni, Stefano

    2016-01-01

    Intrinsically disordered proteins often become structured upon interacting with their partners. The mechanism of this ‘folding upon binding’ process, however, has not been fully characterised yet. Here we present a study of the folding of the intrinsically disordered transactivation domain of c-Myb (c-Myb) upon binding its partner KIX. By determining the structure of the folding transition state for the binding of wild-type and three mutational variants of KIX, we found a remarkable plasticity of the folding pathway of c-Myb. To explain this phenomenon, we show that the folding of c-Myb is templated by the structure of KIX. This adaptive folding behaviour, which occurs by heterogeneous nucleation, differs from the robust homogeneous nucleation typically observed for globular proteins. We suggest that this templated folding mechanism may enable intrinsically disordered proteins to achieve specific and reliable binding with multiple partners while avoiding aberrant interactions. PMID:26912067

  11. Sampling Kinetic Protein Folding Pathways using All-Atom Models

    NASA Astrophysics Data System (ADS)

    Bolhuis, P. G.

    This chapter summarizes several computational strategies to study the kinetics of two-state protein folding using all atom models. After explaining the background of two state folding using energy landscapes I introduce common protein models and computational tools to study folding thermodynamics and kinetics. Free energy landscapes are able to capture the thermodynamics of two-state protein folding, and several methods for efficient sampling of these landscapes are presented. An accurate estimate of folding kinetics, the main topic of this chapter, is more difficult to achieve. I argue that path sampling methods are well suited to overcome the problems connected to the sampling of folding kinetics. Some of the major issues are illustrated in the case study on the folding of the GB1 hairpin.

  12. Evolution, Energy Landscapes and the Paradoxes of Protein Folding

    PubMed Central

    Wolynes, Peter G.

    2014-01-01

    Protein folding has been viewed as a difficult problem of molecular self-organization. The search problem involved in folding however has been simplified through the evolution of folding energy landscapes that are funneled. The funnel hypothesis can be quantified using energy landscape theory based on the minimal frustration principle. Strong quantitative predictions that follow from energy landscape theory have been widely confirmed both through laboratory folding experiments and from detailed simulations. Energy landscape ideas also have allowed successful protein structure prediction algorithms to be developed. The selection constraint of having funneled folding landscapes has left its imprint on the sequences of existing protein structural families. Quantitative analysis of co-evolution patterns allows us to infer the statistical characteristics of the folding landscape. These turn out to be consistent with what has been obtained from laboratory physicochemical folding experiments signalling a beautiful confluence of genomics and chemical physics. PMID:25530262

  13. Toward understanding driving forces in membrane protein folding.

    PubMed

    Hong, Heedeok

    2014-12-15

    α-Helical membrane proteins are largely composed of nonpolar residues that are embedded in the lipid bilayer. An enigma in the folding of membrane proteins is how a polypeptide chain can be condensed into the compact folded state in the environment where the hydrophobic effect cannot strongly drive molecular interactions. Probably other forces such as van der Waals packing, hydrogen bonding, and weakly polar interactions, which are regarded less important in the folding of water-soluble proteins, should emerge. However, it is not clearly understood how those individual forces operate and how they are balanced for stabilizing membrane proteins. Studying this problem is not a trivial task mainly because of the methodological challenges in controlling the reversible folding of membrane proteins in the lipid bilayer. Overcoming the hurdles, meaningful progress has been made in the field in the last few decades. This review will focus on recent studies tackling the problem of driving forces in membrane protein folding.

  14. Spectroscopic studies of protein folding: Linear and nonlinear methods

    PubMed Central

    Serrano, Arnaldo L; Waegele, Matthias M; Gai, Feng

    2012-01-01

    Although protein folding is a simple outcome of the underlying thermodynamics, arriving at a quantitative and predictive understanding of how proteins fold nevertheless poses huge challenges. Therefore, both advanced experimental and computational methods are continuously being developed and refined to probe and reveal the atomistic details of protein folding dynamics and mechanisms. Herein, we provide a concise review of recent developments in spectroscopic studies of protein folding, with a focus on new triggering and probing methods. In particular, we describe several laser-based techniques for triggering protein folding/unfolding on the picosecond and/or nanosecond timescales and various linear and nonlinear spectroscopic techniques for interrogating protein conformations, conformational transitions, and dynamics. PMID:22109973

  15. Untangling the Influence of a Protein Knot on Folding.

    PubMed

    Capraro, Dominique T; Jennings, Patricia A

    2016-03-08

    Entanglement and knots occur across all aspects of the physical world. Despite the common belief that knots are too complicated for incorporation into proteins, knots have been identified in the native fold of a growing number of proteins. The discovery of proteins with this unique backbone characteristic has challenged the preconceptions about the complexity of biological structures, as well as current folding theories. Given the intricacies of the knotted geometry, the interplay between a protein's fold, structure, and function is of particular interest. Interestingly, for most of these proteins, the knotted region appears critical both in folding and function, although full understanding of these contributions is still incomplete. Here, we experimentally reveal the impact of the knot on the landscape, the origin of the bistable nature of the knotted protein, and broaden the view of knot formation as uniquely decoupled from folding.

  16. Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins.

    PubMed

    Das, Indrajit; Png, Chin Wen; Oancea, Iulia; Hasnain, Sumaira Z; Lourie, Rohan; Proctor, Martina; Eri, Rajaraman D; Sheng, Yong; Crane, Denis I; Florin, Timothy H; McGuckin, Michael A

    2013-06-03

    Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with colitis in mice and inflammatory bowel disease (IBD). Endogenous intestinal glucocorticoids are important for homeostasis and glucocorticoid drugs are efficacious in IBD. In Winnie mice with intestinal ER stress caused by misfolding of the Muc2 mucin, the glucocorticoid dexamethasone (DEX) suppressed ER stress and activation of the unfolded protein response (UPR), substantially restoring goblet cell Muc2 production. In mice lacking inflammation, a glucocorticoid receptor antagonist increased ER stress, and DEX suppressed ER stress induced by the N-glycosylation inhibitor, tunicamycin (Tm). In cultured human intestinal secretory cells, in a glucocorticoid receptor-dependent manner, DEX suppressed ER stress and UPR activation induced by blocking N-glycosylation, reducing ER Ca(2+) or depleting glucose. DEX up-regulated genes encoding chaperones and elements of ER-associated degradation (ERAD), including EDEM1. Silencing EDEM1 partially inhibited DEX's suppression of misfolding-induced ER stress, showing that DEX enhances ERAD. DEX inhibited Tm-induced MUC2 precursor accumulation, promoted production of mature mucin, and restored ER exit and secretion of Winnie mutant recombinant Muc2 domains, consistent with enhanced protein folding. In IBD, glucocorticoids are likely to ameliorate ER stress by promoting correct folding of secreted proteins and enhancing removal of misfolded proteins from the ER.

  17. Folding of multidomain proteins: biophysical consequences of tethering even in apparently independent folding.

    PubMed

    Arviv, Oshrit; Levy, Yaakov

    2012-12-01

    Most eukaryotic and a substantial fraction of prokaryotic proteins are composed of more than one domain. The tethering of these evolutionary, structural, and functional units raises, among others, questions regarding the folding process of conjugated domains. Studying the folding of multidomain proteins in silico enables one to identify and isolate the tethering-induced biophysical determinants that govern crosstalks generated between neighboring domains. For this purpose, we carried out coarse-grained and atomistic molecular dynamics simulations of two two-domain constructs from the immunoglobulin-like β-sandwich fold. Each of these was experimentally shown to behave as the "sum of its parts," that is, the thermodynamic and kinetic folding behavior of the constituent domains of these constructs seems to occur independently, with the folding of each domain uncoupled from the folding of its partner in the two-domain construct. We show that the properties of the individual domains can be significantly affected by conjugation to another domain. The tethering may be accompanied by stabilizing as well as destabilizing factors whose magnitude depends on the size of the interface, the length, and the flexibility of the linker, and the relative stability of the domains. Accordingly, the folding of a multidomain protein should not be viewed as the sum of the folding patterns of each of its parts, but rather, it involves abrogating several effects that lead to this outcome. An imbalance between these effects may result in either stabilization or destabilization owing to the tethering. Copyright © 2012 Wiley Periodicals, Inc.

  18. Lattice model for rapidly folding protein-like heteropolymers.

    PubMed Central

    Shrivastava, I; Vishveshwara, S; Cieplak, M; Maritan, A; Banavar, J R

    1995-01-01

    Protein folding is a relatively fast process considering the astronomical number of conformations in which a protein could find itself. Within the framework of a lattice model, we show that one can design rapidly folding sequences by assigning the strongest attractive couplings to the contacts present in a target native state. Our protein design can be extended to situations with both attractive and repulsive contacts. Frustration is minimized by ensuring that all the native contacts are again strongly attractive. Strikingly, this ensures the inevitability of folding and accelerates the folding process by an order of magnitude. The evolutionary implications of our findings are discussed. PMID:7568102

  19. Effects of confinement on protein folding and protein stability

    NASA Astrophysics Data System (ADS)

    Ping, G.; Yuan, J. M.; Vallieres, M.; Dong, H.; Sun, Z.; Wei, Y.; Li, F. Y.; Lin, S. H.

    2003-05-01

    In a cell, proteins exist in crowded environments; these environments influence their stability and dynamics. Similarly, for an enzyme molecule encapsulated in an inorganic cavity as in biosensors or biocatalysts, confinement and even surface effects play important roles in its stability and dynamics. Using a minimalist model (two-dimensional HP lattice model), we have carried out Monte Carlo simulations to study confinement effects on protein stability. We have calculated heat capacity as a function of temperature using the histogram method and results obtained show that confinement tends to stabilize the folded conformations, consistent with experimental results (some reported here) and previous theoretical analyses. Furthermore, for a protein molecule tethered to a solid surface the stabilization effect can be even greater. We have also investigated the effects of confinement on the kinetics of the refolding and unfolding processes as functions of temperature and box size. As expected, unfolding time increases as box size decreases, however, confinement affects folding times in a more complicated way. Our theoretical results agree with our experimentally observed trends that thermal stability of horseradish peroxidase and acid phosphatase, encapsulated in mesoporous silica, increases as the pore size of the silica matrix decreases.

  20. ProFold: Protein Fold Classification with Additional Structural Features and a Novel Ensemble Classifier

    PubMed Central

    2016-01-01

    Protein fold classification plays an important role in both protein functional analysis and drug design. The number of proteins in PDB is very large, but only a very small part is categorized and stored in the SCOPe database. Therefore, it is necessary to develop an efficient method for protein fold classification. In recent years, a variety of classification methods have been used in many protein fold classification studies. In this study, we propose a novel classification method called proFold. We import protein tertiary structure in the period of feature extraction and employ a novel ensemble strategy in the period of classifier training. Compared with existing similar ensemble classifiers using the same widely used dataset (DD-dataset), proFold achieves 76.2% overall accuracy. Another two commonly used datasets, EDD-dataset and TG-dataset, are also tested, of which the accuracies are 93.2% and 94.3%, higher than the existing methods. ProFold is available to the public as a web-server. PMID:27660761

  1. Proteins with highly similar native folds can show vastly dissimilar folding behavior when desolvated.

    PubMed

    Schennach, Moritz; Breuker, Kathrin

    2014-01-03

    Proteins can be exposed to vastly different environments such as the cytosol or membranes, but the delicate balance between external factors and intrinsic determinants of protein structure, stability, and folding is only poorly understood. Here we used electron capture dissociation to study horse and tuna heart Cytochromes c in the complete absence of solvent. The significantly different stability of their highly similar native folds after transfer into the gas phase, and their strikingly different folding behavior in the gas phase, can be rationalized on the basis of electrostatic interactions such as salt bridges. In the absence of hydrophobic bonding, protein folding is far slower and more complex than in solution. © 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  2. Effects of confinement and crowding on folding of model proteins.

    PubMed

    Wojciechowski, M; Cieplak, Marek

    2008-12-01

    We perform molecular dynamics simulations for a simple coarse-grained model of crambin placed inside of a softly repulsive sphere of radius R. The confinement makes folding at the optimal temperature slower and affects the folding scenarios, but both effects are not dramatic. The influence of crowding on folding are studied by placing several identical proteins within the sphere, denaturing them, and then by monitoring refolding. If the interactions between the proteins are dominated by the excluded volume effects, the net folding times are essentially like for a single protein. An introduction of inter-proteinic attractive contacts hinders folding when the strength of the attraction exceeds about a half of the value of the strength of the single protein contacts. The bigger the strength of the attraction, the more likely is the occurrence of aggregation and misfolding.

  3. Dodging the crisis of folding proteins with knots.

    PubMed

    Sułkowska, Joanna I; Sułkowski, Piotr; Onuchic, José

    2009-03-03

    Proteins with nontrivial topology, containing knots and slipknots, have the ability to fold to their native states without any additional external forces invoked. A mechanism is suggested for folding of these proteins, such as YibK and YbeA, that involves an intermediate configuration with a slipknot. It elucidates the role of topological barriers and backtracking during the folding event. It also illustrates that native contacts are sufficient to guarantee folding in approximately 1-2% of the simulations, and how slipknot intermediates are needed to reduce the topological bottlenecks. As expected, simulations of proteins with similar structure but with knot removed fold much more efficiently, clearly demonstrating the origin of these topological barriers. Although these studies are based on a simple coarse-grained model, they are already able to extract some of the underlying principles governing folding in such complex topologies.

  4. Dodging the crisis of folding proteins with knots

    NASA Astrophysics Data System (ADS)

    Sulkowska, Joanna

    2009-03-01

    Proteins with nontrivial topology, containing knots and slipknots, have the ability to fold to their native states without any additional external forces invoked. A mechanism is suggested for folding of these proteins, such as YibK and YbeA, which involves an intermediate configuration with a slipknot. It elucidates the role of topological barriers and backtracking during the folding event. It also illustrates that native contacts are sufficient to guarantee folding in around 1-2% of the simulations, and how slipknot intermediates are needed to reduce the topological bottlenecks. As expected, simulations of proteins with similar structure but with knot removed fold much more efficiently, clearly demonstrating the origin of these topological barriers. Although these studies are based on a simple coarse-grained model, they are already able to extract some of the underlying principles governing folding in such complex topologies.

  5. Dodging the crisis of folding proteins with knots

    PubMed Central

    Sułkowska, Joanna I.; Sułkowski, Piotr; Onuchic, José

    2009-01-01

    Proteins with nontrivial topology, containing knots and slipknots, have the ability to fold to their native states without any additional external forces invoked. A mechanism is suggested for folding of these proteins, such as YibK and YbeA, that involves an intermediate configuration with a slipknot. It elucidates the role of topological barriers and backtracking during the folding event. It also illustrates that native contacts are sufficient to guarantee folding in ≈1–2% of the simulations, and how slipknot intermediates are needed to reduce the topological bottlenecks. As expected, simulations of proteins with similar structure but with knot removed fold much more efficiently, clearly demonstrating the origin of these topological barriers. Although these studies are based on a simple coarse-grained model, they are already able to extract some of the underlying principles governing folding in such complex topologies. PMID:19211785

  6. Self-organized critical model for protein folding

    NASA Astrophysics Data System (ADS)

    Moret, M. A.

    2011-09-01

    The major factor that drives a protein toward collapse and folding is the hydrophobic effect. At the folding process a hydrophobic core is shielded by the solvent-accessible surface area of the protein. We study the fractal behavior of 5526 protein structures present in the Brookhaven Protein Data Bank. Power laws of protein mass, volume and solvent-accessible surface area are measured independently. The present findings indicate that self-organized criticality is an alternative explanation for the protein folding. Also we note that the protein packing is an independent and constant value because the self-similar behavior of the volumes and protein masses have the same fractal dimension. This power law guarantees that a protein is a complex system. From the analyzed data, q-Gaussian distributions seem to fit well this class of systems.

  7. Translation and folding of single proteins in real time.

    PubMed

    Wruck, Florian; Katranidis, Alexandros; Nierhaus, Knud H; Büldt, Georg; Hegner, Martin

    2017-05-30

    Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide's vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein's amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein's amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.

  8. Inferring the rate-length law of protein folding.

    PubMed

    Lane, Thomas J; Pande, Vijay S

    2013-01-01

    We investigate the rate-length scaling law of protein folding, a key undetermined scaling law in the analytical theory of protein folding. Available data yield statistically significant evidence for the existence of a rate-length law capable of predicting folding times to within about two orders of magnitude (over 9 decades of variation). Unambiguous determination of the functional form of such a law could provide key mechanistic insight into folding. Four proposed laws from literature (power law, exponential, and two stretched exponentials) are tested against one another, and it is found that the power law best explains the data by a modest margin. We conclude that more data is necessary to unequivocally infer the rate-length law. Such data could be obtained through a small number of protein folding experiments on large protein domains.

  9. High-resolution protein folding with a transferable potential.

    PubMed

    Hubner, Isaac A; Deeds, Eric J; Shakhnovich, Eugene I

    2005-12-27

    A generalized computational method for folding proteins with a fully transferable potential and geometrically realistic all-atom model is presented and tested on seven helix bundle proteins. The protocol, which includes graph-theoretical analysis of the ensemble of resulting folded conformations, was systematically applied and consistently produced structure predictions of approximately 3 A without any knowledge of the native state. To measure and understand the significance of the results, extensive control simulations were conducted. Graph theoretic analysis provides a means for systematically identifying the native fold and provides physical insight, conceptually linking the results to modern theoretical views of protein folding. In addition to presenting a method for prediction of structure and folding mechanism, our model suggests that an accurate all-atom amino acid representation coupled with a physically reasonable atomic interaction potential and hydrogen bonding are essential features for a realistic protein model.

  10. NMR monitoring of accumulation and folding of 15N-labeled protein overexpressed in Pichia pastoris.

    PubMed

    de Lamotte, F; Boze, H; Blanchard, C; Klein, C; Moulin, G; Gautier, M F; Delsuc, M A

    2001-07-01

    Postgenomic studies have led to an increasing demand for isotope-labeled proteins. We present a method for producing large quantities of truly native (15)N-labeled protein. Based on the secretion capabilities of the yeast Pichia pastoris, the recombinant protein is easily purified in a single step as it is secreted. Control of all nitrogen sources permits very high labeling yields. As a result, accumulation and folding of the recombinant protein can be monitored by heteronuclear NMR without purification. Comparison of sample spectra with the spectrum of the purified recombinant protein allows detection of the secreted protein in the culture and monitoring of its folding, from the start of the induction phase. The detection limit for a (15)N-labeled protein is estimated as 20 microM and corresponds, for a 10-kDa protein, to a load of 40 mg/liter in the fermentor. This concentration is reached by most reported preparations in P. pastoris. Further concentration by ultrafiltration would compensate for lower production. This procedure may be useful in many structural genomics and combinatorial chemistry screening projects where most protein productions meet the requirements for this method. Copyright 2001 Academic Press.

  11. Fluorescence of Alexa Fluor Dye Tracks Protein Folding

    PubMed Central

    Lindhoud, Simon; Westphal, Adrie H.; Visser, Antonie J. W. G.; Borst, Jan Willem; van Mierlo, Carlo P. M.

    2012-01-01

    Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding. PMID:23056480

  12. Effects of tethering a multistate folding protein to a surface

    NASA Astrophysics Data System (ADS)

    Wei, Shuai; Knotts, Thomas A.

    2011-05-01

    Protein/surface interactions are important in a variety of fields and devices, yet fundamental understanding of the relevant phenomena remains fragmented due to resolution limitations of experimental techniques. Molecular simulation has provided useful answers, but such studies have focused on proteins that fold through a two-state process. This study uses simulation to show how surfaces can affect proteins which fold through a multistate process by investigating the folding mechanism of lysozyme (PDB ID: 7LZM). The results demonstrate that in the bulk 7LZM folds through a process with four stable states: the folded state, the unfolded state, and two stable intermediates. The folding mechanism remains the same when the protein is tethered to a surface at most residues; however, in one case the folding mechanism changes in such a way as to eliminate one of the intermediates. An analysis of the molecular configurations shows that tethering at this site is advantageous for protein arrays because the active site is both presented to the bulk phase and stabilized. Taken as a whole, the results offer hope that rational design of protein arrays is possible once the behavior of the protein on the surface is ascertained.

  13. Effects of tethering a multistate folding protein to a surface.

    PubMed

    Wei, Shuai; Knotts, Thomas A

    2011-05-14

    Protein/surface interactions are important in a variety of fields and devices, yet fundamental understanding of the relevant phenomena remains fragmented due to resolution limitations of experimental techniques. Molecular simulation has provided useful answers, but such studies have focused on proteins that fold through a two-state process. This study uses simulation to show how surfaces can affect proteins which fold through a multistate process by investigating the folding mechanism of lysozyme (PDB ID: 7LZM). The results demonstrate that in the bulk 7LZM folds through a process with four stable states: the folded state, the unfolded state, and two stable intermediates. The folding mechanism remains the same when the protein is tethered to a surface at most residues; however, in one case the folding mechanism changes in such a way as to eliminate one of the intermediates. An analysis of the molecular configurations shows that tethering at this site is advantageous for protein arrays because the active site is both presented to the bulk phase and stabilized. Taken as a whole, the results offer hope that rational design of protein arrays is possible once the behavior of the protein on the surface is ascertained.

  14. Fluorescence of Alexa fluor dye tracks protein folding.

    PubMed

    Lindhoud, Simon; Westphal, Adrie H; Visser, Antonie J W G; Borst, Jan Willem; van Mierlo, Carlo P M

    2012-01-01

    Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.

  15. Protein Solubility and Folding Enhancement by Interaction with RNA

    PubMed Central

    Choi, Seong Il; Han, Kyoung Sim; Kim, Chul Woo; Ryu, Ki-Sun; Kim, Byung Hee; Kim, Kyun-Hwan; Kim, Seo-Il; Kang, Tae Hyun; Shin, Hang-Cheol; Lim, Keo-Heun; Kim, Hyo Kyung; Hyun, Jeong-Min; Seong, Baik L.

    2008-01-01

    While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo. PMID:18628952

  16. Protein folding, protein structure and the origin of life: Theoretical methods and solutions of dynamical problems

    NASA Technical Reports Server (NTRS)

    Weaver, D. L.

    1982-01-01

    Theoretical methods and solutions of the dynamics of protein folding, protein aggregation, protein structure, and the origin of life are discussed. The elements of a dynamic model representing the initial stages of protein folding are presented. The calculation and experimental determination of the model parameters are discussed. The use of computer simulation for modeling protein folding is considered.

  17. Unfolded protein ensembles, folding trajectories, and refolding rate prediction

    NASA Astrophysics Data System (ADS)

    Das, A.; Sin, B. K.; Mohazab, A. R.; Plotkin, S. S.

    2013-09-01

    Computer simulations can provide critical information on the unfolded ensemble of proteins under physiological conditions, by explicitly characterizing the geometrical properties of the diverse conformations that are sampled in the unfolded state. A general computational analysis across many proteins has not been implemented however. Here, we develop a method for generating a diverse conformational ensemble, to characterize properties of the unfolded states of intrinsically disordered or intrinsically folded proteins. The method allows unfolded proteins to retain disulfide bonds. We examined physical properties of the unfolded ensembles of several proteins, including chemical shifts, clustering properties, and scaling exponents for the radius of gyration with polymer length. A problem relating simulated and experimental residual dipolar couplings is discussed. We apply our generated ensembles to the problem of folding kinetics, by examining whether the ensembles of some proteins are closer geometrically to their folded structures than others. We find that for a randomly selected dataset of 15 non-homologous 2- and 3-state proteins, quantities such as the average root mean squared deviation between the folded structure and unfolded ensemble correlate with folding rates as strongly as absolute contact order. We introduce a new order parameter that measures the distance travelled per residue, which naturally partitions into a smooth "laminar" and subsequent "turbulent" part of the trajectory. This latter conceptually simple measure with no fitting parameters predicts folding rates in 0 M denaturant with remarkable accuracy (r = -0.95, p = 1 × 10-7). The high correlation between folding times and sterically modulated, reconfigurational motion supports the rapid collapse of proteins prior to the transition state as a generic feature in the folding of both two-state and multi-state proteins. This method for generating unfolded ensembles provides a powerful approach to

  18. Protein folding pathology in domestic animals*

    PubMed Central

    Gruys, Erik

    2004-01-01

    Fibrillar proteins form structural elements of cells and the extracellular matrix. Pathological lesions of fibrillar microanatomical structures, or secondary fibrillar changes in globular proteins are well known. A special group concerns histologically amorphous deposits, amyloid. The major characteristics of amyloid are: apple green birefringence after Congo red staining of histological sections, and non-branching 7–10 nm thick fibrils on electron microscopy revealing a high content of cross beta pleated sheets. About 25 different types of amyloid have been characterised. In animals, AA-amyloid is the most frequent type. Other types of amyloid in animals represent: AIAPP (in cats), AApoAI, AApoAII, localised AL-amyloid, amyloid in odontogenic or mammary tumors and amyloid in the brain. In old dogs Aβ and in sheep APrPsc-amyloid can be encountered. AA-amyloidosis is a systemic disorder with a precursor in blood, acute phase serum amyloid A (SAA). In chronic inflammatory processes AA-amyloid can be deposited. A rapid crystallization of SAA to amyloid fibrils on small beta-sheeted fragments, the ‘amyloid enhancing factor’ (AEF), is known and the AEF has been shown to penetrate the enteric barrier. Amyloid fibrils can aggregate from various precursor proteins in vitro in particular at acidic pH and when proteolytic fragments are formed. Molecular chaperones influence this process. Tissue data point to amyloid fibrillogenesis in lysosomes and near cell surfaces. A comparison can be made of the fibrillogenesis in prion diseases and in enhanced AA-amyloidosis. In the reactive form, acute phase SAA is the supply of the precursor protein, whereas in the prion diseases, cell membrane proteins form a structural source. Aβ-amyloid in brain tissue of aged dogs showing signs of dementia forms a canine counterpart of senile dementia of the Alzheimer type (ccSDAT) in man. Misfolded proteins remain potential food hazards. Developments concerning prevention of

  19. Polymer Uncrossing and Knotting in Protein Folding, and Their Role in Minimal Folding Pathways

    PubMed Central

    Mohazab, Ali R.; Plotkin, Steven S.

    2013-01-01

    We introduce a method for calculating the extent to which chain non-crossing is important in the most efficient, optimal trajectories or pathways for a protein to fold. This involves recording all unphysical crossing events of a ghost chain, and calculating the minimal uncrossing cost that would have been required to avoid such events. A depth-first tree search algorithm is applied to find minimal transformations to fold , , , and knotted proteins. In all cases, the extra uncrossing/non-crossing distance is a small fraction of the total distance travelled by a ghost chain. Different structural classes may be distinguished by the amount of extra uncrossing distance, and the effectiveness of such discrimination is compared with other order parameters. It was seen that non-crossing distance over chain length provided the best discrimination between structural and kinetic classes. The scaling of non-crossing distance with chain length implies an inevitable crossover to entanglement-dominated folding mechanisms for sufficiently long chains. We further quantify the minimal folding pathways by collecting the sequence of uncrossing moves, which generally involve leg, loop, and elbow-like uncrossing moves, and rendering the collection of these moves over the unfolded ensemble as a multiple-transformation “alignment”. The consensus minimal pathway is constructed and shown schematically for representative cases of an , , and knotted protein. An overlap parameter is defined between pathways; we find that proteins have minimal overlap indicating diverse folding pathways, knotted proteins are highly constrained to follow a dominant pathway, and proteins are somewhere in between. Thus we have shown how topological chain constraints can induce dominant pathway mechanisms in protein folding. PMID:23365638

  20. Milk protein concentrations in galactorrhoeic mammary secretions.

    PubMed

    Yap, P L; Pryde, E A; McClelland, D B

    1980-02-01

    Milk protein concentrations were determined either by double antibody radioimmunoassay (IgA) or single radial immunodiffusion (IgG, lactoferrin, lysozyme and albumin) in the mammayr secretions of one nulliparous and three parous female patients with galactorrhoea due to hyperprolactinaemia. Concentrations of all the proteins studied were found to be similar to the concentrations observed in post-partum colostrum. In particular, secretory IgA was the only form of IgA detected in galactorrhoeic secretions. It is suggested that hyperprolactinaemia alone can result in increased mammary synthesis of the milk proteins since the steroid changes associated with a full-term pregnancy and delivery of the placenta did not immediately precede the galactorrhoea in three of the four patients studied.

  1. In situ protein folding and activation in bacterial inclusion bodies.

    PubMed

    Gonzalez-Montalban, Nuria; Natalello, Antonino; García-Fruitós, Elena; Villaverde, Antonio; Doglia, Silvia Maria

    2008-07-01

    Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK.

  2. THEORY OF PROTEIN FOLDING: The Energy Landscape Perspective

    NASA Astrophysics Data System (ADS)

    Onuchic, Jose Nelson; Luthey-Schulten, Zaida; Wolynes, Peter G.

    1997-10-01

    The energy landscape theory of protein folding is a statistical description of a protein's potential surface. It assumes that folding occurs through organizing an ensemble of structures rather than through only a few uniquely defined structural intermediates. It suggests that the most realistic model of a protein is a minimally frustrated heteropolymer with a rugged funnel-like landscape biased toward the native structure. This statistical description has been developed using tools from the statistical mechanics of disordered systems, polymers, and phase transitions of finite systems. We review here its analytical background and contrast the phenomena in homopolymers, random heteropolymers, and protein-like heteropolymers that are kinetically and thermodynamically capable of folding. The connection between these statistical concepts and the results of minimalist models used in computer simulations is discussed. The review concludes with a brief discussion of how the theory helps in the interpretation of results from fast folding experiments and in the practical task of protein structure prediction.

  3. Protein folding by distributed computing and the denatured state ensemble.

    PubMed

    Marianayagam, Neelan J; Fawzi, Nicolas L; Head-Gordon, Teresa

    2005-11-15

    The distributed computing (DC) paradigm in conjunction with the folding@home (FH) client server has been used to study the folding kinetics of small peptides and proteins, giving excellent agreement with experimentally measured folding rates, although pathways sampled in these simulations are not always consistent with the folding mechanism. In this study, we use a coarse-grain model of protein L, whose two-state kinetics have been characterized in detail by using long-time equilibrium simulations, to rigorously test a FH protocol using approximately 10,000 short-time, uncoupled folding simulations starting from an extended state of the protein. We show that the FH results give non-Poisson distributions and early folding events that are unphysical, whereas longer folding events experience a correct barrier to folding but are not representative of the equilibrium folding ensemble. Using short-time, uncoupled folding simulations started from an equilibrated denatured state ensemble (DSE), we also do not get agreement with the equilibrium two-state kinetics because of overrepresented folding events arising from higher energy subpopulations in the DSE. The DC approach using uncoupled short trajectories can make contact with traditionally measured experimental rates and folding mechanism when starting from an equilibrated DSE, when the simulation time is long enough to sample the lowest energy states of the unfolded basin and the simulated free-energy surface is correct. However, the DC paradigm, together with faster time-resolved and single-molecule experiments, can also reveal the breakdown in the two-state approximation due to observation of folding events from higher energy subpopulations in the DSE.

  4. Simulating protein folding and aggregation on the 10 second timescale

    NASA Astrophysics Data System (ADS)

    Pande, Vijay

    2007-03-01

    Understanding how proteins self-assemble or ``fold'' is a fundamental problem in biophysics. Moreover, the ability to understand and quantitatively predict folding kinetics would have many implications, especially in the area of diseases related to protein misfolding, such as Alzheimer's Disease. However, there are many challenges to simulating folding, most notably the great computational challenges of simulating protein folding with models with sufficient accuracy to make quantitative predictions of experiments. In my talk, I will discuss our recent work to combine distributed computing with a new theoretical technique (Markov State Models) in order to simulate folding on long timescales as well as the direct and quantitative experimental tests of these methods. I will conclude with the application of these methods to the study of the Abeta peptide, whose aggregation has been directly implicated as the toxic element in Alzheimer's Disease.

  5. Viral capsid proteins are segregated in structural fold space.

    PubMed

    Cheng, Shanshan; Brooks, Charles L

    2013-01-01

    Viral capsid proteins assemble into large, symmetrical architectures that are not found in complexes formed by their cellular counterparts. Given the prevalence of the signature jelly-roll topology in viral capsid proteins, we are interested in whether these functionally unique capsid proteins are also structurally unique in terms of folds. To explore this question, we applied a structure-alignment based clustering of all protein chains in VIPERdb filtered at 40% sequence identity to identify distinct capsid folds, and compared the cluster medoids with a non-redundant subset of protein domains in the SCOP database, not including the viral capsid entries. This comparison, using Template Modeling (TM)-score, identified 2078 structural "relatives" of capsid proteins from the non-capsid set, covering altogether 210 folds following the definition in SCOP. The statistical significance of the 210 folds shared by two sets of the same sizes, estimated from 10,000 permutation tests, is less than 0.0001, which is an upper bound on the p-value. We thus conclude that viral capsid proteins are segregated in structural fold space. Our result provides novel insight on how structural folds of capsid proteins, as opposed to their surface chemistry, might be constrained during evolution by requirement of the assembled cage-like architecture. Also importantly, our work highlights a guiding principle for virus-based nanoplatform design in a wide range of biomedical applications and materials science.

  6. Viral Capsid Proteins Are Segregated in Structural Fold Space

    PubMed Central

    Cheng, Shanshan; Brooks, Charles L.

    2013-01-01

    Viral capsid proteins assemble into large, symmetrical architectures that are not found in complexes formed by their cellular counterparts. Given the prevalence of the signature jelly-roll topology in viral capsid proteins, we are interested in whether these functionally unique capsid proteins are also structurally unique in terms of folds. To explore this question, we applied a structure-alignment based clustering of all protein chains in VIPERdb filtered at 40% sequence identity to identify distinct capsid folds, and compared the cluster medoids with a non-redundant subset of protein domains in the SCOP database, not including the viral capsid entries. This comparison, using Template Modeling (TM)-score, identified 2078 structural “relatives” of capsid proteins from the non-capsid set, covering altogether 210 folds following the definition in SCOP. The statistical significance of the 210 folds shared by two sets of the same sizes, estimated from 10,000 permutation tests, is less than 0.0001, which is an upper bound on the p-value. We thus conclude that viral capsid proteins are segregated in structural fold space. Our result provides novel insight on how structural folds of capsid proteins, as opposed to their surface chemistry, might be constrained during evolution by requirement of the assembled cage-like architecture. Also importantly, our work highlights a guiding principle for virus-based nanoplatform design in a wide range of biomedical applications and materials science. PMID:23408879

  7. Pachytene spermatocytes regulate the secretion of Sertoli cell protein(s) which stimulate Leydig cell steroidogenesis.

    PubMed

    Onoda, M; Djakiew, D; Papadopoulos, V

    1991-05-01

    The influence of germ cells (pachytene spermatocytes and round spermatids) on the secretion by Sertoli cells of the proteinaceous factor(s) which stimulates Leydig cell steroid biosynthesis was investigated. Sertoli cells from immature rats were cultured on plastic dishes or on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Immature rat Sertoli cell secreted proteins (rSCSP; MW greater than 10,000), from conventional cultures, stimulated 4- to 5-fold steroid biosynthesis in normal rat and MA-10 mouse tumor Leydig cells, respectively. MA-10 cells were then used as a bioassay system for most studies, although purified rat Leydig cells were used in some cases to further confirm results obtained with MA-10 cells. rSCSP collected from both the apical and basal compartment of the chambers were examined for their ability to stimulate Leydig cell steroidogenesis. The Leydig cell stimulatory activity from Sertoli cells was found to be secreted in a polarized manner, with 80% of the total bioactivity found in the basal rSCSP. Addition of pachytene spermatocyte proteins (PSP) in the apical compartment of the chambers inhibited, in a time- and concentration-dependent manner, the basally directed Sertoli cell secretion of the Leydig cell stimulatory protein(s) by 85%. Similar results were obtained when freshly isolated pachytene spermatocytes were directly added on top of Sertoli cell epithelial sheets in the apical compartment of the chambers. In contrast, round spermatid proteins (RSP) did not exhibit a comparable effect to that of PSP in regulating the Sertoli cell secretion of the Leydig cell stimulatory activity. These results demonstrate that the Sertoli cell secreted protein(s) which stimulates Leydig cell steroid biosynthesis is secreted in a basally polarized direction, and its secretion is specifically modulated by pachytene spermatocytes.

  8. Transient misfolding dominates multidomain protein folding

    PubMed Central

    Borgia, Alessandro; Kemplen, Katherine R.; Borgia, Madeleine B.; Soranno, Andrea; Shammas, Sarah; Wunderlich, Bengt; Nettels, Daniel; Best, Robert B.; Clarke, Jane; Schuler, Benjamin

    2015-01-01

    Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated. PMID:26572969

  9. Transient misfolding dominates multidomain protein folding

    NASA Astrophysics Data System (ADS)

    Borgia, Alessandro; Kemplen, Katherine R.; Borgia, Madeleine B.; Soranno, Andrea; Shammas, Sarah; Wunderlich, Bengt; Nettels, Daniel; Best, Robert B.; Clarke, Jane; Schuler, Benjamin

    2015-11-01

    Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.

  10. Learning generative models for protein fold families.

    PubMed

    Balakrishnan, Sivaraman; Kamisetty, Hetunandan; Carbonell, Jaime G; Lee, Su-In; Langmead, Christopher James

    2011-04-01

    We introduce a new approach to learning statistical models from multiple sequence alignments (MSA) of proteins. Our method, called GREMLIN (Generative REgularized ModeLs of proteINs), learns an undirected probabilistic graphical model of the amino acid composition within the MSA. The resulting model encodes both the position-specific conservation statistics and the correlated mutation statistics between sequential and long-range pairs of residues. Existing techniques for learning graphical models from MSA either make strong, and often inappropriate assumptions about the conditional independencies within the MSA (e.g., Hidden Markov Models), or else use suboptimal algorithms to learn the parameters of the model. In contrast, GREMLIN makes no a priori assumptions about the conditional independencies within the MSA. We formulate and solve a convex optimization problem, thus guaranteeing that we find a globally optimal model at convergence. The resulting model is also generative, allowing for the design of new protein sequences that have the same statistical properties as those in the MSA. We perform a detailed analysis of covariation statistics on the extensively studied WW and PDZ domains and show that our method out-performs an existing algorithm for learning undirected probabilistic graphical models from MSA. We then apply our approach to 71 additional families from the PFAM database and demonstrate that the resulting models significantly out-perform Hidden Markov Models in terms of predictive accuracy.

  11. Protein Secretion in Gram-Positive Bacteria: From Multiple Pathways to Biotechnology.

    PubMed

    Anné, Jozef; Economou, Anastassios; Bernaerts, Kristel

    2016-11-25

    A number of Gram-positive bacteria are important players in industry as producers of a diverse array of economically interesting metabolites and proteins. As discussed in this overview, several Gram-positive bacteria are valuable hosts for the production of heterologous proteins. In contrast to Gram-negative bacteria, proteins secreted by Gram-positive bacteria are released into the culture medium where conditions for correct folding are more appropriate, thus facilitating the isolation and purification of active proteins. Although seven different protein secretion pathways have been identified in Gram-positive bacteria, the majority of heterologous proteins are produced via the general secretion or Sec pathway. Not all proteins are equally well secreted, because heterologous protein production often faces bottlenecks including hampered secretion, susceptibility to proteases, secretion stress, and metabolic burden. These bottlenecks are associated with reduced yields leading to non-marketable products. In this chapter, besides a general overview of the different protein secretion pathways, possible hurdles that may hinder efficient protein secretion are described and attempts to improve yield are discussed including modification of components of the Sec pathway. Attention is also paid to omics-based approaches that may offer a more rational approach to optimize production of heterologous proteins.

  12. SVM-Fold: a tool for discriminative multi-class protein fold and superfamily recognition

    PubMed Central

    Melvin, Iain; Ie, Eugene; Kuang, Rui; Weston, Jason; Stafford, William Noble; Leslie, Christina

    2007-01-01

    Background Predicting a protein's structural class from its amino acid sequence is a fundamental problem in computational biology. Much recent work has focused on developing new representations for protein sequences, called string kernels, for use with support vector machine (SVM) classifiers. However, while some of these approaches exhibit state-of-the-art performance at the binary protein classification problem, i.e. discriminating between a particular protein class and all other classes, few of these studies have addressed the real problem of multi-class superfamily or fold recognition. Moreover, there are only limited software tools and systems for SVM-based protein classification available to the bioinformatics community. Results We present a new multi-class SVM-based protein fold and superfamily recognition system and web server called SVM-Fold, which can be found at . Our system uses an efficient implementation of a state-of-the-art string kernel for sequence profiles, called the profile kernel, where the underlying feature representation is a histogram of inexact matching k-mer frequencies. We also employ a novel machine learning approach to solve the difficult multi-class problem of classifying a sequence of amino acids into one of many known protein structural classes. Binary one-vs-the-rest SVM classifiers that are trained to recognize individual structural classes yield prediction scores that are not comparable, so that standard "one-vs-all" classification fails to perform well. Moreover, SVMs for classes at different levels of the protein structural hierarchy may make useful predictions, but one-vs-all does not try to combine these multiple predictions. To deal with these problems, our method learns relative weights between one-vs-the-rest classifiers and encodes information about the protein structural hierarchy for multi-class prediction. In large-scale benchmark results based on the SCOP database, our code weighting approach significantly improves

  13. Identifying folding nucleus based on residue contact networks of proteins.

    PubMed

    Li, Jie; Wang, Jun; Wang, Wei

    2008-06-01

    In the native structure of a protein, all the residues are tightly parked together in a specific order following its folding and every residue contacts with some spatially neighbor residues. A residue contact network can be constructed by defining the residues as nodes and the native contacts as edges. During the folding of small single-domain proteins, there is a set of contacts (or bonds), defined as the folding nucleus (FN), which is formed around the transition state, i.e., a rate-limiting barrier located at about the middle between the unfolded states and the native state on the free energy landscape. Such a FN plays an essential role in the folding dynamics and the residues, which form the related contacts called as folding nucleus residues (FNRs). In this work, the FNRs in proteins are identified by using quantities which characterize the topology of residue contact networks of proteins. By comparing the specificities of residues with the network quantities K(R), L(R), and D(R), up to 90% FNRs of six typical proteins found experimentally are identified. It is found that the FNRs behave the full-closeness centrals rather than degree or closeness centers in the residue contact network, implying that they are important to the folding cooperativity of proteins. Our study shows that the FNRs can be identified solely from the native structures of proteins based on the analysis of residue contact network without any knowledge of the transition state ensemble. (c) 2008 Wiley-Liss, Inc.

  14. How cooperative are protein folding and unfolding transitions?

    PubMed

    Malhotra, Pooja; Udgaonkar, Jayant B

    2016-11-01

    A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two-state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non-cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier-less "downhill" folding, as well as for continuous "uphill" unfolding transitions, indicate that gradual non-cooperative processes may be ubiquitous features on the free energy landscape of protein folding.

  15. Solitons and protein folding: An In Silico experiment

    NASA Astrophysics Data System (ADS)

    Ilieva, N.; Dai, J.; Sieradzan, A.; Niemi, A.

    2015-10-01

    Protein folding [1] is the process of formation of a functional 3D structure from a random coil — the shape in which amino-acid chains leave the ribosome. Anfinsen's dogma states that the native 3D shape of a protein is completely determined by protein's amino acid sequence. Despite the progress in understanding the process rate and the success in folding prediction for some small proteins, with presently available physics-based methods it is not yet possible to reliably deduce the shape of a biologically active protein from its amino acid sequence. The protein-folding problem endures as one of the most important unresolved problems in science; it addresses the origin of life itself. Furthermore, a wrong fold is a common cause for a protein to lose its function or even endanger the living organism. Soliton solutions of a generalized discrete non-linear Schrödinger equation (GDNLSE) obtained from the energy function in terms of bond and torsion angles κ and τ provide a constructive theoretical framework for describing protein folds and folding patterns [2]. Here we study the dynamics of this process by means of molecular-dynamics simulations. The soliton manifestation is the pattern helix-loop-helix in the secondary structure of the protein, which explains the importance of understanding loop formation in helical proteins. We performed in silico experiments for unfolding one subunit of the core structure of gp41 from the HIV envelope glycoprotein (PDB ID: 1AIK [3]) by molecular-dynamics simulations with the MD package GROMACS. We analyzed 80 ns trajectories, obtained with one united-atom and two different all-atom force fields, to justify the side-chain orientation quantification scheme adopted in the studies and to eliminate force-field based artifacts. Our results are compatible with the soliton model of protein folding and provide first insight into soliton-formation dynamics.

  16. Hierarchical classification of protein folds using a novel ensemble classifier.

    PubMed

    Lin, Chen; Zou, Ying; Qin, Ji; Liu, Xiangrong; Jiang, Yi; Ke, Caihuan; Zou, Quan

    2013-01-01

    The analysis of biological information from protein sequences is important for the study of cellular functions and interactions, and protein fold recognition plays a key role in the prediction of protein structures. Unfortunately, the prediction of protein fold patterns is challenging due to the existence of compound protein structures. Here, we processed the latest release of the Structural Classification of Proteins (SCOP, version 1.75) database and exploited novel techniques to impressively increase the accuracy of protein fold classification. The techniques proposed in this paper include ensemble classifying and a hierarchical framework, in the first layer of which similar or redundant sequences were deleted in two manners; a set of base classifiers, fused by various selection strategies, divides the input into seven classes; in the second layer of which, an analogous ensemble method is adopted to predict all protein folds. To our knowledge, it is the first time all protein folds can be intelligently detected hierarchically. Compared with prior studies, our experimental results demonstrated the efficiency and effectiveness of our proposed method, which achieved a success rate of 74.21%, which is much higher than results obtained with previous methods (ranging from 45.6% to 70.5%). When applied to the second layer of classification, the prediction accuracy was in the range between 23.13% and 46.05%. This value, which may not be remarkably high, is scientifically admirable and encouraging as compared to the relatively low counts of proteins from most fold recognition programs. The web server Hierarchical Protein Fold Prediction (HPFP) is available at http://datamining.xmu.edu.cn/software/hpfp.

  17. Hierarchical Classification of Protein Folds Using a Novel Ensemble Classifier

    PubMed Central

    Qin, Ji; Liu, Xiangrong; Jiang, Yi; Ke, Caihuan; Zou, Quan

    2013-01-01

    The analysis of biological information from protein sequences is important for the study of cellular functions and interactions, and protein fold recognition plays a key role in the prediction of protein structures. Unfortunately, the prediction of protein fold patterns is challenging due to the existence of compound protein structures. Here, we processed the latest release of the Structural Classification of Proteins (SCOP, version 1.75) database and exploited novel techniques to impressively increase the accuracy of protein fold classification. The techniques proposed in this paper include ensemble classifying and a hierarchical framework, in the first layer of which similar or redundant sequences were deleted in two manners; a set of base classifiers, fused by various selection strategies, divides the input into seven classes; in the second layer of which, an analogous ensemble method is adopted to predict all protein folds. To our knowledge, it is the first time all protein folds can be intelligently detected hierarchically. Compared with prior studies, our experimental results demonstrated the efficiency and effectiveness of our proposed method, which achieved a success rate of 74.21%, which is much higher than results obtained with previous methods (ranging from 45.6% to 70.5%). When applied to the second layer of classification, the prediction accuracy was in the range between 23.13% and 46.05%. This value, which may not be remarkably high, is scientifically admirable and encouraging as compared to the relatively low counts of proteins from most fold recognition programs. The web server Hierarchical Protein Fold Prediction (HPFP) is available at http://datamining.xmu.edu.cn/software/hpfp. PMID:23437146

  18. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants

    PubMed Central

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: http://dx.doi.org/10.7554/eLife.03421.001 PMID:25073928

  19. Protein folding simulations and structure predictions

    NASA Astrophysics Data System (ADS)

    Okamoto, Yuko

    2001-12-01

    In complex systems such as spin glasses and proteins, conventional simulations in the canonical ensemble will get trapped in states of energy local minima. We employ the simulated annealing method and generalized-ensemble algorithms in order to overcome this multiple-minima problem. Besides simulated annealing, three well-known generalized-ensemble algorithms, namely, multicanonical algorithm, simulated tempering, and replica-exchange method, are described. We then present three new generalized-ensemble algorithms based on the combinations of the three methods.

  20. Desolvation effects and topology-dependent protein folding

    NASA Astrophysics Data System (ADS)

    Ferguson, Allison; Liu, Zhirong; Chan, Hue Sun

    2007-03-01

    As a protein folds, water molecules must be excluded from the hydrophobic core, and thus desolvation barriers between the protein's constituents must be crossed in order to reach the final folded state. Previous research on continuum Go-like protein models has demonstrated that pairwise-additive desolvation potentials lead to more thermodynamically and kinetically cooperative folding/unfolding transitions (Z. Liu and H. S. Chan, Phys. Biol. 2, S75-S85, 2005). The present work focuses on the role of this elementary desolvation potential in improving predictions of the well-known topology-folding rate relationship (K. W. Plaxco et al, J. Mol. Biol. 277, 985-994, 1998) of small single-domain proteins. Recent computational studies without desolvation barriers have shown (S. Wallin and H. S. Chan, J. Phys.: Condens. Matt. 18, S307-S328, 2006) that the observed correlation between topological parameters and folding rates is because these parameters may be proxies for rate-determining properties of the transition state, such as the activation free energy δG^ and activation conformational entropy δS^. Including the desolvation barrier in the model results in stronger correlations between measures of topology and simulated folding rates / transition state properties, reinforcing the theory that even simple representations of the desolvation effect are important for understanding crucial features of protein folding.

  1. Molecular Origins of Internal Friction Effects on Protein Folding Rates

    PubMed Central

    Sirur, Anshul

    2014-01-01

    Recent experiments on protein folding dynamics have revealed strong evidence for internal friction effects. That is, observed relaxation times are not simply proportional to the solvent viscosity as might be expected if the solvent were the only source of friction. However, a molecular interpretation of this remarkable phenomenon is currently lacking. Here, we use all-atom simulations of peptide and protein folding in explicit solvent, to probe the origin of the unusual viscosity dependence. We find that an important contribution to this effect, explaining the viscosity dependence of helix formation and the folding of a helix-containing protein, is the insensitivity of torsion angle isomerization to solvent friction. The influence of this landscape roughness can, in turn, be quantitatively explained by a rate theory including memory friction. This insensitivity of local barrier crossing to solvent friction is expected to contribute to the viscosity dependence of folding rates in larger proteins. PMID:24986114

  2. Folding and self-assembly of a small protein complex

    PubMed Central

    Sieradzan, Adam K.; Liwo, Adam; Hansmann, Ulrich H.E.

    2012-01-01

    The synthetic homotetrameric ββα (BBAT1) protein possesses a stable quaternary structure with a ββα fold. Because of its small size (a total of 84 residues), the homotetramer is an excellent model system with which to study the self-assembly and protein-protein interactions. We find from replica exchange molecular dynamics simulations with the coarse-grain UNRES force field that the folding and association pathway consists of three well-separated steps, where that association to a tetramer precedes and facilitates folding of the four chains. At room temperature the tetramer exists in an ensemble of diverse structures. The crystal structure becomes energetically favored only when the molecule is put in a dense and crystal-like environment. The observed picture of folding promoted by association may mirror the mechanism according to which intrinsically unfolded proteins assume their functional structure. PMID:24039552

  3. Nonsymmetric Two-Body Score Function for Protein Fold Recognition:

    NASA Astrophysics Data System (ADS)

    Heo, Muyoung; Cheon, Mookyung; Chang, Iksoo

    The usual two-body score (energy) function to recognize native folds of proteins is Miyazawa-Jernigan (MJ) pairwise-contact function. The pairwise-contact parameters between two amino acids in MJ function are symmetric in a sense that a directional order of amino acids sequence along the backbone of a protein is ignored in constructing score parameters. Here we report that we succeeded in constructing a nonsymmetric two-body score function, capturing a directional order of amino acids sequence, by a perceptron learning and a protein threading. We considered pairs of two adjacent amino acids that are separated by two consecutive peptide bonds with the backbone directionality from the N-terminus to the C-terminus of a protein. We also considered the local environmental character, such as the secondary structures and the hydrophobicity (solvation), of amino acids in protein structures. The score is a corresponding propensity for a directional alignment of these two adjacent amino acids with their local environments. The resulting score function simultaneously recognized native folds of 1006 proteins covering all representative proteins with a homology less than 30% among them. The quality of this score function was validated by a threading test of new distinct 382 proteins with a homology less than 90% among them, and it entailed a high success ratio for recognizing native folds of 364 (95.3%) proteins. It showed a good feasibility of designing protein score functions for protein fold recognition by a perceptron learning and a protein threading.

  4. Mechanical Modeling and Computer Simulation of Protein Folding

    ERIC Educational Resources Information Center

    Prigozhin, Maxim B.; Scott, Gregory E.; Denos, Sharlene

    2014-01-01

    In this activity, science education and modern technology are bridged to teach students at the high school and undergraduate levels about protein folding and to strengthen their model building skills. Students are guided from a textbook picture of a protein as a rigid crystal structure to a more realistic view: proteins are highly dynamic…

  5. Mechanical Modeling and Computer Simulation of Protein Folding

    ERIC Educational Resources Information Center

    Prigozhin, Maxim B.; Scott, Gregory E.; Denos, Sharlene

    2014-01-01

    In this activity, science education and modern technology are bridged to teach students at the high school and undergraduate levels about protein folding and to strengthen their model building skills. Students are guided from a textbook picture of a protein as a rigid crystal structure to a more realistic view: proteins are highly dynamic…

  6. Basic units of protein structure, folding, and function.

    PubMed

    Berezovsky, Igor N; Guarnera, Enrico; Zheng, Zejun

    2017-09-01

    Study of the hierarchy of domain structure with alternative sets of domains and analysis of discontinuous domains, consisting of remote segments of the polypeptide chain, raised a question about the minimal structural unit of the protein domain. The hypothesis on the decisive role of the polypeptide backbone in determining the elementary units of globular proteins have led to the discovery of closed loops. It is reviewed here how closed loops form the loop-n-lock structure of proteins, providing the foundation for stability and designability of protein folds/domain and underlying their co-translational folding. Simplified protein sequences are considered here with the aim to explore the basic principles that presumably dominated the folding and stability of proteins in the early stages of structural evolution. Elementary functional loops (EFLs), closed loops with one or few catalytic residues, are, in turn, units of the protein function. They are apparent descendants of the prebiotic ring-like peptides, which gave rise to the first functional folds/domains being fused in the beginning of the evolution of protein structure. It is also shown how evolutionary relations between protein functional superfamilies and folds delineated with the help of EFLs can contribute to establishing the rules for design of desired enzymatic functions. Generalized descriptors of the elementary functions are proposed to be used as basic units in the future computational design. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Engineering the protein folding landscape in gram-negative bacteria.

    PubMed

    Mansell, Thomas J; Fisher, Adam C; DeLisa, Matthew P

    2008-04-01

    Gram-negative bacteria, especially Escherichia coli, are often the preferred hosts for recombinant protein production because of their fast doubling times, ability to grow to high cell density, propensity for high recombinant protein titers and straightforward protein purification techniques. The utility of simple bacteria in such studies continues to improve as a result of an ever-increasing body of knowledge regarding their native protein biogenesis machinery. From translation on the ribosome to interaction with cytosolic accessory factors to transport across the inner membrane into the periplasmic space, cellular proteins interact with many different types of cellular machinery and each interaction can have a profound effect on the protein folding process. This review addresses key aspects of cellular protein folding, solubility and expression in E. coli with particular focus on the elegant biological machinery that orchestrates the transition from nascent polypeptide to folded, functional protein. Specifically highlighted are a variety of different techniques to intentionally alter the folding environment of the cell as a means to understand and engineer intracellular protein folding and stability.

  8. Thermodynamics of protein folding: a random matrix formulation.

    PubMed

    Shukla, Pragya

    2010-10-20

    The process of protein folding from an unfolded state to a biologically active, folded conformation is governed by many parameters, e.g. the sequence of amino acids, intermolecular interactions, the solvent, temperature and chaperon molecules. Our study, based on random matrix modeling of the interactions, shows, however, that the evolution of the statistical measures, e.g. Gibbs free energy, heat capacity, and entropy, is single parametric. The information can explain the selection of specific folding pathways from an infinite number of possible ways as well as other folding characteristics observed in computer simulation studies.

  9. Solitons and protein folding: An In Silico experiment

    SciTech Connect

    Ilieva, N.; Dai, J.; Sieradzan, A.; Niemi, A.

    2015-10-28

    Protein folding [1] is the process of formation of a functional 3D structure from a random coil — the shape in which amino-acid chains leave the ribosome. Anfinsen’s dogma states that the native 3D shape of a protein is completely determined by protein’s amino acid sequence. Despite the progress in understanding the process rate and the success in folding prediction for some small proteins, with presently available physics-based methods it is not yet possible to reliably deduce the shape of a biologically active protein from its amino acid sequence. The protein-folding problem endures as one of the most important unresolved problems in science; it addresses the origin of life itself. Furthermore, a wrong fold is a common cause for a protein to lose its function or even endanger the living organism. Soliton solutions of a generalized discrete non-linear Schrödinger equation (GDNLSE) obtained from the energy function in terms of bond and torsion angles κ and τ provide a constructive theoretical framework for describing protein folds and folding patterns [2]. Here we study the dynamics of this process by means of molecular-dynamics simulations. The soliton manifestation is the pattern helix–loop–helix in the secondary structure of the protein, which explains the importance of understanding loop formation in helical proteins. We performed in silico experiments for unfolding one subunit of the core structure of gp41 from the HIV envelope glycoprotein (PDB ID: 1AIK [3]) by molecular-dynamics simulations with the MD package GROMACS. We analyzed 80 ns trajectories, obtained with one united-atom and two different all-atom force fields, to justify the side-chain orientation quantification scheme adopted in the studies and to eliminate force-field based artifacts. Our results are compatible with the soliton model of protein folding and provide first insight into soliton-formation dynamics.

  10. Origination of the Protein Fold Repertoire from Oily Pluripotent Peptides

    PubMed Central

    Mannige, Ranjan V.

    2014-01-01

    While the repertoire of protein folds that exists today underlies most of life’s capabilities, our mechanistic picture of protein fold origination is incomplete. This paper discusses a hypothetical mechanism for the emergence of the protein fold repertoire from highly dynamic and collapsed peptides, exemplified by peptides with high oil content or hydrophobicity. These peptides are called pluripotent to emphasize their capacity to evolve into numerous folds transiently available to them. As evidence, the paper will discuss previous simulation work on the superior fold evolvability of oily peptides, trace (“fossil”) evidence within proteomes seen today, and a general relationship between protein dynamism and evolvability. Aside from implications on the origination of protein folds, the hypothesis implies that the vanishing utility of a random peptide in protein origination may be relatively exaggerated, as some random peptides with a certain composition (e.g., oily) may fare better than others. In later sections, the hypothesis is discussed in the context of existing discussions regarding the spontaneous origination of biomolecules. PMID:28250375

  11. Unconventional protein secretion (UPS) pathways in plants.

    PubMed

    Ding, Yu; Robinson, David G; Jiang, Liwen

    2014-08-01

    As in yeast and mammalian cells, novel unconventional protein secretion (UPS) or unconventional membrane trafficking pathways are now known to operate in plants. UPS in plants is generally associated with stress conditions such as pathogen attack, but little is known about its underlying mechanism and function. Here, we present an update on the current knowledge of UPS in the plants in terms of its transport pathways, possible functions and its relationship to autophagy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Folding propensity of intrinsically disordered proteins by osmotic stress

    SciTech Connect

    Mansouri, Amanda L.; Grese, Laura N.; Rowe, Erica L.; Pino, James C.; Chennubhotla, S. Chakra; Ramanathan, Arvind; O'Neill, Hugh Michael; Berthelier, Valerie; Stanley, Christopher B.

    2016-10-11

    Proteins imparted with intrinsic disorder conduct a range of essential cellular functions. To better understand the folding and hydration properties of intrinsically disordered proteins (IDPs), we used osmotic stress to induce conformational changes in nuclear co-activator binding domain (NCBD) and activator for thyroid hormone and retinoid receptor (ACTR). Osmotic stress was applied by the addition of small and polymeric osmolytes, where we discovered that water contributions to NCBD folding always exceeded those for ACTR. Both NCBD and ACTR were found to gain a-helical structure with increasing osmotic stress, consistent with their folding upon NCBD/ACTR complex formation. Using small-angle neutron scattering (SANS), we further characterized NCBD structural changes with the osmolyte ethylene glycol. Here a large reduction in overall size initially occurred before substantial secondary structural change. In conclusion, by focusing on folding propensity, and linked hydration changes, we uncover new insights that may be important for how IDP folding contributes to binding.

  13. Enhanced protein folding by removal of kinetic traps

    NASA Astrophysics Data System (ADS)

    Liu, Yanxin; Chapagain, Prem; Parra, Jose; Gerstman, Bernard

    2007-03-01

    The presence of non-native kinetic traps along the free energy landscape of a protein may significantly lengthen the overall folding time so that the folding process becomes unreliable. We used a computational 3-D lattice model to investigate the free energy landscape of a model alpha helical hairpin peptide. We used two slightly different sequences and show that strategic substitutions of only a few amino acid residues greatly enhance the folding process. These strategic substitutions prevent the formation of long-lived misfolded configurations which not only lengthen the folding time but also may cause unwanted aggregation. Detailed kinetic and thermodynamic analysis was carried out for the folding of these two sequences and the results are consistent with the experimental and molecular dynamics simulations of small helical bundle proteins.

  14. Predicting folding-unfolding transitions in proteins without a priori knowledge of the folded state

    NASA Astrophysics Data System (ADS)

    Okan, Osman; Turgut, Deniz; Garcia, Angel; Ozisik, Rahmi

    2013-03-01

    The common computational method of studying folding transitions in proteins is to compare simulated conformations against the folded structure, but this method obviously requires the folded structure to be known beforehand. In the current study, we show that the use of bond orientational order parameter (BOOP) Ql [Steinhardt PJ, Nelson DR, Ronchetti M, Phys. Rev. B 1983, 28, 784] is a viable alternative to the commonly adopted root mean squared distance (RMSD) measure in probing conformational transitions. Replica exchange molecular dynamics simulations of the trp-cage protein (with 20 residues) in TIP-3P water were used to compare BOOP against RMSD. The results indicate that the correspondence between BOOP and RMSD time series become stronger with increasing l. We finally show that robust linear models that incorporate different Ql can be parameterized from a given replica run and can be used to study other replica trajectories. This work is partially supported by NSF DUE-1003574.

  15. Orchestration of secretory protein folding by ER chaperones

    PubMed Central

    Gidalevitz, Tali; Stevens, Fred; Argon, Yair

    2013-01-01

    The endoplasmic reticulum is a major compartment of protein biogenesis in the cell, dedicated to production of secretory, membrane and organelle proteins. The secretome has distinct structural and post-translational characteristics, since folding in the ER occurs in an environment that is distinct in terms of its ionic composition, dynamics and requirements for quality contol. The folding machinery in the ER therefore includes chaperones and folding enzymes that introduce, monitor and react to disulfide bonds, glycans, and fluctuations of luminal calcium. We describe the major chaperone networks in the lumen and discuss how they have distinct modes of operation that enable cells to accomplish highly efficient production of the secretome. PMID:23507200

  16. Thermally triggered self-assembly of folded proteins into vesicles.

    PubMed

    Park, Won Min; Champion, Julie A

    2014-12-31

    We report thermally triggered self-assembly of folded proteins into vesicles that incorporates globular proteins as building blocks. Leucine zipper coiled coils were combined with either globular proteins or elastin-like polypeptides as recombinant fusion proteins, which form "rod-coil" and "globule-rod-coil" protein complex amphiphiles. In aqueous solution, they self-assembled into hollow vesicles via temperature-responsive inverse phase transition. The characteristic of the protein vesicle membranes enables preferential encapsulation of simultaneously formed protein coacervate. Furthermore, the type of encapsulated cargo extends to small molecules and nanoparticles. Our approach offers a versatile strategy to create protein vesicles as vehicles with biological functionality.

  17. Getting Folded: Chaperone proteins in muscle development, maintenance and disease

    PubMed Central

    Smith, Daniel A.; Carland, Carmen R.; Guo, Yiming; Bernstein, Sanford I.

    2014-01-01

    Chaperone proteins are critical for protein folding and stability, and hence are necessary for normal cellular organization and function. Recent studies have begun to interrogate the role of this specialized class of proteins in muscle biology. During development, chaperone-mediated folding of client proteins enables their integration into nascent sarcomeres. In addition to assisting with muscle differentiation, chaperones play a key role in maintenance of muscle tissues. Further, disruption of the chaperone network can result in neuromuscular disease. In this review, we discuss how chaperones are involved in myofibrillogenesis, sarcomere maintenance and muscle disorders. We also consider the possibilities of therapeutically targeting chaperones to treat muscle disease. PMID:25125177

  18. Folding and Stabilization of Native-Sequence-Reversed Proteins

    PubMed Central

    Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

    2016-01-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844

  19. Folding and Stabilization of Native-Sequence-Reversed Proteins

    NASA Astrophysics Data System (ADS)

    Zhang, Yuanzhao; Weber, Jeffrey K.; Zhou, Ruhong

    2016-04-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols.

  20. Self-consistent calculation of protein folding pathways

    NASA Astrophysics Data System (ADS)

    Orioli, S.; a Beccara, S.; Faccioli, P.

    2017-08-01

    We introduce an iterative algorithm to efficiently simulate protein folding and other conformational transitions, using state-of-the-art all-atom force fields. Starting from the Langevin equation, we obtain a self-consistent stochastic equation of motion, which directly yields the reaction pathways. From the solution of this set of equations we derive a stochastic estimate of the reaction coordinate. We validate this approach against the results of plain MD simulations of the folding of a small protein, which were performed on the Anton supercomputer. In order to explore the computational efficiency of this algorithm, we apply it to generate a folding pathway of a protein that consists of 130 amino acids and has a folding rate of the order of s-1.

  1. Modern Analysis of Protein Folding by Differential Scanning Calorimetry.

    PubMed

    Ibarra-Molero, Beatriz; Naganathan, Athi N; Sanchez-Ruiz, Jose M; Muñoz, Victor

    2016-01-01

    Differential scanning calorimetry (DSC) is a very powerful tool for investigating protein folding and stability because its experimental output reflects the energetics of all conformations that become minimally populated during thermal unfolding. Accordingly, analysis of DSC experiments with simple thermodynamic models has been key for developing our understanding of protein stability during the past five decades. The discovery of ultrafast folding proteins, which have naturally broad conformational ensembles and minimally cooperative unfolding, opens the possibility of probing the complete folding free energy landscape, including those conformations at the top of the barrier to folding, via DSC. Exploiting this opportunity requires high-quality experiments and the implementation of novel analytical methods based on statistical mechanics. Here, we cover the recent exciting developments in this front, describing the new analytical procedures in detail as well as providing experimental guidelines for performing such analysis. © 2016 Elsevier Inc. All rights reserved.

  2. The Energy Computation Paradox and ab initio Protein Folding

    PubMed Central

    Faver, John C.; Benson, Mark L.; He, Xiao; Roberts, Benjamin P.; Wang, Bing; Marshall, Michael S.; Sherrill, C. David; Merz, Kenneth M.

    2011-01-01

    The routine prediction of three-dimensional protein structure from sequence remains a challenge in computational biochemistry. It has been intuited that calculated energies from physics-based scoring functions are able to distinguish native from nonnative folds based on previous performance with small proteins and that conformational sampling is the fundamental bottleneck to successful folding. We demonstrate that as protein size increases, errors in the computed energies become a significant problem. We show, by using error probability density functions, that physics-based scores contain significant systematic and random errors relative to accurate reference energies. These errors propagate throughout an entire protein and distort its energy landscape to such an extent that modern scoring functions should have little chance of success in finding the free energy minima of large proteins. Nonetheless, by understanding errors in physics-based score functions, they can be reduced in a post-hoc manner, improving accuracy in energy computation and fold discrimination. PMID:21541343

  3. Protein folding in HP model on hexagonal lattices with diagonals

    PubMed Central

    2014-01-01

    Three dimensional structure prediction of a protein from its amino acid sequence, known as protein folding, is one of the most studied computational problem in bioinformatics and computational biology. Since, this is a hard problem, a number of simplified models have been proposed in literature to capture the essential properties of this problem. In this paper we introduce the hexagonal lattices with diagonals to handle the protein folding problem considering the well researched HP model. We give two approximation algorithms for protein folding on this lattice. Our first algorithm is a 53-approximation algorithm, which is based on the strategy of partitioning the entire protein sequence into two pieces. Our next algorithm is also based on partitioning approaches and improves upon the first algorithm. PMID:24564789

  4. Topology and structural self-organization in folded proteins

    NASA Astrophysics Data System (ADS)

    Lundgren, M.; Krokhotin, Andrey; Niemi, Antti J.

    2013-10-01

    Topological methods are indispensable in theoretical studies of particle physics, condensed matter physics, and gravity. These powerful techniques have also been applied to biological physics. For example, knowledge of DNA topology is pivotal to the understanding as to how living cells function. Here, the biophysical repertoire of topological methods is extended, with the aim to understand and characterize the global structure of a folded protein. For this, the elementary concept of winding number of a vector field on a plane is utilized to introduce a topological quantity called the folding index of a crystallographic protein. It is observed that in the case of high resolution protein crystals, the folding index, when evaluated over the entire length of the crystallized protein backbone, has a very clear and strong propensity towards integer values. The observation proposes that the way how a protein folds into its biologically active conformation is a structural self-organization process with a topological facet that relates to the concept of solitons. It is proposed that the folding index has a potential to become a useful tool for the global, topological characterization of the folding pathways.

  5. Intermediates and the folding of proteins L and G

    SciTech Connect

    Brown, Scott; Head-Gordon, Teresa

    2003-07-01

    We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G that are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation-condensation mechanism, each of which is described as helix-assisted {beta}-1 and {beta}-2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate-limiting barrier of folding and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contacts involving the third {beta}-strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two-step first order reversible reaction, proving that protein G folding involves an on-pathway early intermediate, and should be populated and therefore observable by experiment.

  6. From Helix–Coil Transitions to Protein Folding

    PubMed Central

    Scheraga, Harold A.

    2009-01-01

    An evolution of procedures to simulate protein structure and folding pathways is described. From an initial focus on the helix–coil transition and on hydrogen-bonding and hydrophobic interactions, our original attempts to determine protein structure and folding pathways were based on an experimental approach. Experiments on the oxidative folding of reduced bovine pancreatic ribonuclease A (RNase A) led to a mechanism by which the molecule folded to the native structure by a minimum of four different pathways. The experiments with RNase A were followed by development of a molecular mechanics approach, first, making use of global optimization procedures and then with molecular dynamics (MD), evolving from an all-atom to a united-residue model. This hierarchical MD approach facilitated probing of the folding trajectory to longer time scales than with all-atom MD, and hence led to the determination of complete folding trajectories, thus far for a protein containing as many as 75 amino acid residues. With increasing refinement of the computational procedures, the computed results are coming closer to experimental observations, providing an understanding as to how physics directs the folding process. PMID:18008324

  7. GroEL stimulates protein folding through forced unfolding

    PubMed Central

    Lin, Zong; Madan, Damian; Rye, Hays S

    2013-01-01

    Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroEL’s physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding. PMID:18311152

  8. Folding of Small Proteins Using Constrained Molecular Dynamics

    PubMed Central

    Balaraman, Gouthaman S.; Park, In-Hee; Jain, Abhinandan; Vaidehi, Nagarajan

    2011-01-01

    The focus of this paper is to examine whether conformational search using constrained molecular dynamics (MD) method is more enhanced and enriched towards “native-like” structures compared to all-atom MD for the protein folding as a model problem. Constrained MD methods provide an alternate MD tool for protein structure prediction and structure refinement. It is computationally expensive to perform all-atom simulations of protein folding because the processes occur on a timescale of microseconds. Compared to the all-atom MD simulation, constrained MD methods have the advantage that stable dynamics can be achieved for larger time steps and the number of degrees of freedom is an order of magnitude smaller, leading to a decrease in computational cost. We have developed a generalized constrained MD method that allows the user to “freeze and thaw” torsional degrees of freedom as fit for the problem studied. We have used this method to perform all-torsion constrained MD in implicit solvent coupled with the replica exchange method to study folding of small proteins with various secondary structural motifs such as, α-helix (polyalanine, WALP16), β-turn (1E0Q), and a mixed motif protein (Trp-cage). We demonstrate that constrained MD replica exchange method exhibits a wider conformational search than all-atom MD with increased enrichment of near native structures. “Hierarchical” constrained MD simulations, where the partially formed helical regions in the initial stretch of the all-torsion folding simulation trajectory of Trp-cage were frozen, showed a better sampling of near native structures than all-torsion constrained MD simulations. This is in agreement with the zipping-and-assembly folding model put forth by Dill and coworkers for folding proteins. The use of hierarchical “freeze and thaw” clustering schemes in constrained MD simulation can be used to sample conformations that contribute significantly to folding of proteins. PMID:21591767

  9. Effects of osmolytes on protein folding and aggregation in cells.

    PubMed

    Ignatova, Zoya; Gierasch, Lila M

    2007-01-01

    Nature has developed many strategies to ensure that the complex and challenging protein folding reaction occurs in vivo with adequate efficiency and fidelity for the success of the organism. Among the strategies widely employed in a huge range of species and cell types is the elaboration of small organic molecules called osmolytes that offset the potentially damaging effects of osmotic stress. While considerable knowledge has been gained in vitro regarding the influence of osmolytes on protein structure and folding, it is of great interest to probe the effects of osmolytes in cells. We have developed an in-cell fluorescent-labeling method that enables the study of protein stability and also protein aggregation in vivo. We utilize a genetically encoded tag called a tetra-Cys motif that binds specifically to a bis-arsenical fluorescein-based dye "FlAsH"; we inserted the tetra-Cys motif into a protein of interest in such a way that the FlAsH signal reported on the state of folding or aggregation of the protein. Then, we designed protocols to assess how various osmolytes influence the stability and propensity to aggregate of our protein of interest. These are described here. Not only are there potential biotechnological applications of osmolytes in the quest to produce greater quantities of well-folded proteins, but also osmolytes may serve as tools and points of departure for therapeutic intervention in protein folding and aggregation diseases. Having in vivo methods to analyze how osmolytes affect folding and aggregation enhances our ability to further these goals greatly.

  10. Learning To Fold Proteins Using Energy Landscape Theory

    PubMed Central

    Schafer, N.P.; Kim, B.L.; Zheng, W.; Wolynes, P.G.

    2014-01-01

    This review is a tutorial for scientists interested in the problem of protein structure prediction, particularly those interested in using coarse-grained molecular dynamics models that are optimized using lessons learned from the energy landscape theory of protein folding. We also present a review of the results of the AMH/AMC/AMW/AWSEM family of coarse-grained molecular dynamics protein folding models to illustrate the points covered in the first part of the article. Accurate coarse-grained structure prediction models can be used to investigate a wide range of conceptual and mechanistic issues outside of protein structure prediction; specifically, the paper concludes by reviewing how AWSEM has in recent years been able to elucidate questions related to the unusual kinetic behavior of artificially designed proteins, multidomain protein misfolding, and the initial stages of protein aggregation. PMID:25308991

  11. Chaperone networks: Tipping the balance in protein folding diseases

    PubMed Central

    Voisine, Cindy; Pedersen, Jesper Søndergaard; Morimoto, Richard I.

    2012-01-01

    Adult-onset neurodegeneration and other protein conformational diseases are associated with the appearance, persistence, and accumulation of misfolded and aggregation prone proteins. To protect the proteome from long-term damage, the cell expresses a highly integrated protein homeostasis (proteostasis) machinery to ensure that proteins are properly expressed, folded, and cleared, and to recognize damaged proteins. Molecular chaperones have a central role in proteostasis as they have been shown to be essential to prevent the accumulation of alternate folded proteotoxic states as occurs in protein conformation diseases exemplified by neurodegeneration. Studies using invertebrate models expressing proteins associated with Huntington's disease, Alzheimer's disease, ALS, and Parkinson's disease have provided insights into the genetic networks and stress signaling pathways that regulate the proteostasis machinery to prevent cellular dysfunction, tissue pathology, and organismal failure. These events appear to be further amplified by aging and provide evidence that age-related failures in proteostasis may be a common element in many diseases. PMID:20472062

  12. Protein folding: Vexing debates on a fundamental problem.

    PubMed

    Gianni, Stefano; Jemth, Per

    2016-05-01

    The folding of proteins has been at the heart of protein chemistry and biophysics ever since the pioneering experiments by the labs of Fred Richards and Christian Anfinsen. But, despite nearly 60 years of intense research, there are unresolved issues and a lively debate regarding some aspects of this fundamental problem. In this review we give a personal account on some key topics in the field: (i) the nature of the denatured state of a protein, (ii) nucleation sites in the folding reaction, and (iii) the time it takes for individual molecules to traverse the transition state.

  13. Engineering chimaeric proteins from fold fragments: 'hopeful monsters' in protein design.

    PubMed

    Höcker, Birte

    2013-10-01

    Modern highly complex proteins evolved from much simpler and less specialized subunits. The same concept can be applied in protein engineering to construct new well-folded proteins. Hybrid proteins or chimaeras can be built from contemporary protein fragments through illegitimate recombination. Even parts from different globular folds can be fitted together using rational design methodologies. Furthermore, intrinsic functional properties encoded in the fold fragments allow rapid adaptation of the new proteins and thus provide interesting starting scaffolds for further redesign.

  14. Riboflavin deficiency impairs oxidative folding and secretion of apolipoprotein B-100 in HepG2 cells, triggering stress response systems.

    PubMed

    Manthey, Karoline C; Chew, Yap Ching; Zempleni, Janos

    2005-05-01

    Secretory proteins such as apolipoprotein B-100 (apoB) undergo oxidative folding (formation of disulfide bonds) in the endoplasmic reticulum (ER) before secretion. Oxidative folding depends on flavoproteins in eukaryotes. Here, human liver (HepG2) cells were used to model effects of riboflavin concentrations in culture media on folding and secretion of apoB. Cells were cultured in media containing 3.1, 12.6, and 300 nmol/L of riboflavin, representing moderately deficient, physiological, and pharmacological plasma concentrations in humans, respectively. When cells were cultured in riboflavin-deficient medium, secretion of apoB decreased by >80% compared with controls cultured in physiological medium. The nuclear translocation of the transcription factor ATF-6 increased by >180% in riboflavin-deficient cells compared with physiological controls; this is consistent with ER stress. Nuclear translocation of ATF-6 was associated with activation of the unfolded protein response. Expression of stress-response genes coding for ubiquitin-activating enzyme 1, growth arrest and DNA damage inducible gene, and glucose regulated protein of 78 kDa was greater in riboflavin-deficient cells compared with other treatment groups. Finally, phosphorylation of the eukaryotic initiation factor (eukaryotic initiation factor 2alpha) increased in riboflavin-deficient cells, consistent with decreased translational activity. We conclude 1) that riboflavin deficiency causes ER stress and activation of unfolded protein response in HepG2 cells, and 2) that riboflavin deficiency decreases protein secretion in HepG2 cells. Decreased secretion of apoB in riboflavin-deficient cells might interfere with lipid homeostasis in vivo.

  15. Prediction of the protein folding core: application to the immunoglobulin fold.

    PubMed

    Prudhomme, Nicolas; Chomilier, Jacques

    2009-01-01

    We propose an algorithm that allows predicting residues important for the formation of the structure of globular proteins. It relies on a simulation that detects the amino acids presenting a maximum number of neighbours during the early steps of the folding process. They have been called MIR (Most Interacting Residues). Independently, description of the protein structures in fragments with closed ends shows the correlation between these extremities and the core of the globules. These fragments are of rather constant length, typically between 20 and 25 amino acids, and we have previously shown that their extremities are preferentially occupied by MIR. Introduction of rules derived from this fragment analysis of tertiary structures allows to smooth the distribution of MIR, for a better match between TEF ends and MIR. In order to assess this prediction of the folding core, a large family of structures has been used, with sequences as different as possible. A dataset of 56 immunoglobulin structures of various functions but common fold has been used in this study. This fold was chosen because it is one of the most populated with a large amount of data available on its nucleus. In the immunoglobulin domain, "functional and structural load is clearly separated: loops are responsible for binding and recognition while interactions between several residues of the buried core provide stability and fast folding"[1]. We then determined the positions susceptible of high importance for the folding process to occur and compared them to published data, either to High Throw Out Order (HTOO), Conservatism of Conservatism (CoC) or Phi value experiments. It results a reasonable agreement between the positions that we predict and experimental data. Besides, our prediction goes beyond the simple use of a null solvent accessibility of amino acids as a criterion to predict the core. We find the same quality of our prediction on the flavodoxin like superfamily.

  16. Accurate prediction of cellular co-translational folding indicates proteins can switch from post- to co-translational folding

    NASA Astrophysics Data System (ADS)

    Nissley, Daniel A.; Sharma, Ajeet K.; Ahmed, Nabeel; Friedrich, Ulrike A.; Kramer, Günter; Bukau, Bernd; O'Brien, Edward P.

    2016-02-01

    The rates at which domains fold and codons are translated are important factors in determining whether a nascent protein will co-translationally fold and function or misfold and malfunction. Here we develop a chemical kinetic model that calculates a protein domain's co-translational folding curve during synthesis using only the domain's bulk folding and unfolding rates and codon translation rates. We show that this model accurately predicts the course of co-translational folding measured in vivo for four different protein molecules. We then make predictions for a number of different proteins in yeast and find that synonymous codon substitutions, which change translation-elongation rates, can switch some protein domains from folding post-translationally to folding co-translationally--a result consistent with previous experimental studies. Our approach explains essential features of co-translational folding curves and predicts how varying the translation rate at different codon positions along a transcript's coding sequence affects this self-assembly process.

  17. On the polymer physics origins of protein folding thermodynamics.

    PubMed

    Taylor, Mark P; Paul, Wolfgang; Binder, Kurt

    2016-11-07

    A remarkable feature of the spontaneous folding of many small proteins is the striking similarity in the thermodynamics of the folding process. This process is characterized by simple two-state thermodynamics with large and compensating changes in entropy and enthalpy and a funnel-like free energy landscape with a free-energy barrier that varies linearly with temperature. One might attribute the commonality of this two-state folding behavior to features particular to these proteins (e.g., chain length, hydrophobic/hydrophilic balance, attributes of the native state) or one might suspect that this similarity in behavior has a more general polymer-physics origin. Here we show that this behavior is also typical for flexible homopolymer chains with sufficiently short range interactions. Two-state behavior arises from the presence of a low entropy ground (folded) state separated from a set of high entropy disordered (unfolded) states by a free energy barrier. This homopolymer model exhibits a funneled free energy landscape that reveals a complex underlying dynamics involving competition between folding and non-folding pathways. Despite the presence of multiple pathways, this simple physics model gives the robust result of two-state thermodynamics for both the cases of folding from a basin of expanded coil states and from a basin of compact globule states.

  18. On the polymer physics origins of protein folding thermodynamics

    NASA Astrophysics Data System (ADS)

    Taylor, Mark P.; Paul, Wolfgang; Binder, Kurt

    2016-11-01

    A remarkable feature of the spontaneous folding of many small proteins is the striking similarity in the thermodynamics of the folding process. This process is characterized by simple two-state thermodynamics with large and compensating changes in entropy and enthalpy and a funnel-like free energy landscape with a free-energy barrier that varies linearly with temperature. One might attribute the commonality of this two-state folding behavior to features particular to these proteins (e.g., chain length, hydrophobic/hydrophilic balance, attributes of the native state) or one might suspect that this similarity in behavior has a more general polymer-physics origin. Here we show that this behavior is also typical for flexible homopolymer chains with sufficiently short range interactions. Two-state behavior arises from the presence of a low entropy ground (folded) state separated from a set of high entropy disordered (unfolded) states by a free energy barrier. This homopolymer model exhibits a funneled free energy landscape that reveals a complex underlying dynamics involving competition between folding and non-folding pathways. Despite the presence of multiple pathways, this simple physics model gives the robust result of two-state thermodynamics for both the cases of folding from a basin of expanded coil states and from a basin of compact globule states.

  19. Stable folding core in the folding transition state of an alpha-helical integral membrane protein.

    PubMed

    Curnow, Paul; Di Bartolo, Natalie D; Moreton, Kathleen M; Ajoje, Oluseye O; Saggese, Nicholas P; Booth, Paula J

    2011-08-23

    Defining the structural features of a transition state is important in understanding a folding reaction. Here, we use Φ-value and double mutant analyses to probe the folding transition state of the membrane protein bacteriorhodopsin. We focus on the final C-terminal helix, helix G, of this seven transmembrane helical protein. Φ-values could be derived for 12 amino acid residues in helix G, most of which have low or intermediate values, suggesting that native structure is disrupted at these amino acid positions in the transition state. Notably, a cluster of residues between E204 and M209 all have Φ-values close to zero. Disruption of helix G is further confirmed by a low Φ-value of 0.2 between residues T170 on helix F and S226 on helix G, suggesting the absence of a native hydrogen bond between helices F and G. Φ-values for paired mutations involved in four interhelical hydrogen bonds revealed that all but one of these bonds is absent in the transition state. The unstructured helix G contrasts with Φ-values along helix B that are generally high, implying native structure in helix B in the transition state. Thus helix B seems to constitute part of a stable folding nucleus while the consolidation of helix G is a relatively late folding event. Polarization of secondary structure correlates with sequence position, with a structured helix B near the N terminus contrasting with an unstructured C-terminal helix G.

  20. New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens

    PubMed Central

    English, Grant; Trunk, Katharina; Rao, Vincenzo A; Srikannathasan, Velupillai; Hunter, William N; Coulthurst, Sarah J

    2012-01-01

    Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS-secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (‘Rap’ proteins) included two specifically conferring self-resistance (‘immunity’) against T6SS-dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate Ssp–Rap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS-secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS-secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches. PMID:22957938

  1. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties

    PubMed Central

    Fessart, Delphine; Domblides, Charlotte; Avril, Tony; Eriksson, Leif A; Begueret, Hugues; Pineau, Raphael; Malrieux, Camille; Dugot-Senant, Nathalie; Lucchesi, Carlo; Chevet, Eric; Delom, Frederic

    2016-01-01

    The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.13887.001 PMID:27240165

  2. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties.

    PubMed

    Fessart, Delphine; Domblides, Charlotte; Avril, Tony; Eriksson, Leif A; Begueret, Hugues; Pineau, Raphael; Malrieux, Camille; Dugot-Senant, Nathalie; Lucchesi, Carlo; Chevet, Eric; Delom, Frederic

    2016-05-30

    The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis.

  3. Probing the physical determinants of thermal expansion of folded proteins.

    PubMed

    Dellarole, Mariano; Kobayashi, Kei; Rouget, Jean-Baptiste; Caro, José Alfredo; Roche, Julien; Islam, Mohammad M; Garcia-Moreno E, Bertrand; Kuroda, Yutaka; Royer, Catherine A

    2013-10-24

    The magnitude and sign of the volume change upon protein unfolding are strongly dependent on temperature. This temperature dependence reflects differences in the thermal expansivity of the folded and unfolded states. The factors that determine protein molar expansivities and the large differences in thermal expansivity for proteins of similar molar volume are not well understood. Model compound studies have suggested that a major contribution is made by differences in the molar volume of water molecules as they transfer from the protein surface to the bulk upon heating. The expansion of internal solvent-excluded voids upon heating is another possible contributing factor. Here, the contribution from hydration density to the molar thermal expansivity of a protein was examined by comparing bovine pancreatic trypsin inhibitor and variants with alanine substitutions at or near the protein-water interface. Variants of two of these proteins with an additional mutation that unfolded them under native conditions were also examined. A modest decrease in thermal expansivity was observed in both the folded and unfolded states for the alanine variants compared with the parent protein, revealing that large changes can be made to the external polarity of a protein without causing large ensuing changes in thermal expansivity. This modest effect is not surprising, given the small molar volume of the alanine residue. Contributions of the expansion of the internal void volume were probed by measuring the thermal expansion for cavity-containing variants of a highly stable form of staphylococcal nuclease. Significantly larger (2-3-fold) molar expansivities were found for these cavity-containing proteins relative to the reference protein. Taken together, these results suggest that a key determinant of the thermal expansivities of folded proteins lies in the expansion of internal solvent-excluded voids.

  4. A deterministic algorithm for constrained enumeration of transmembrane protein folds.

    SciTech Connect

    Brown, William Michael; Young, Malin M.; Sale, Kenneth L.; Faulon, Jean-Loup Michel; Schoeniger, Joseph S.

    2004-07-01

    A deterministic algorithm for enumeration of transmembrane protein folds is presented. Using a set of sparse pairwise atomic distance constraints (such as those obtained from chemical cross-linking, FRET, or dipolar EPR experiments), the algorithm performs an exhaustive search of secondary structure element packing conformations distributed throughout the entire conformational space. The end result is a set of distinct protein conformations, which can be scored and refined as part of a process designed for computational elucidation of transmembrane protein structures.

  5. Thermodynamic stability and folding of proteins from hyperthermophilic organisms.

    PubMed

    Luke, Kathryn A; Higgins, Catherine L; Wittung-Stafshede, Pernilla

    2007-08-01

    Life grows almost everywhere on earth, including in extreme environments and under harsh conditions. Organisms adapted to high temperatures are called thermophiles (growth temperature 45-75 degrees C) and hyperthermophiles (growth temperature >or= 80 degrees C). Proteins from such organisms usually show extreme thermal stability, despite having folded structures very similar to their mesostable counterparts. Here, we summarize the current data on thermodynamic and kinetic folding/unfolding behaviors of proteins from hyperthermophilic microorganisms. In contrast to thermostable proteins, rather few (i.e. less than 20) hyperthermostable proteins have been thoroughly characterized in terms of their in vitro folding processes and their thermodynamic stability profiles. Examples that will be discussed include co-chaperonin proteins, iron-sulfur-cluster proteins, and DNA-binding proteins from hyperthermophilic bacteria (i.e. Aquifex and Theromotoga) and archea (e.g. Pyrococcus, Thermococcus, Methanothermus and Sulfolobus). Despite the small set of studied systems, it is clear that super-slow protein unfolding is a dominant strategy to allow these proteins to function at extreme temperatures.

  6. Improved method for predicting protein fold patterns with ensemble classifiers.

    PubMed

    Chen, W; Liu, X; Huang, Y; Jiang, Y; Zou, Q; Lin, C

    2012-01-27

    Protein folding is recognized as a critical problem in the field of biophysics in the 21st century. Predicting protein-folding patterns is challenging due to the complex structure of proteins. In an attempt to solve this problem, we employed ensemble classifiers to improve prediction accuracy. In our experiments, 188-dimensional features were extracted based on the composition and physical-chemical property of proteins and 20-dimensional features were selected using a coupled position-specific scoring matrix. Compared with traditional prediction methods, these methods were superior in terms of prediction accuracy. The 188-dimensional feature-based method achieved 71.2% accuracy in five cross-validations. The accuracy rose to 77% when we used a 20-dimensional feature vector. These methods were used on recent data, with 54.2% accuracy. Source codes and dataset, together with web server and software tools for prediction, are available at: http://datamining.xmu.edu.cn/main/~cwc/ProteinPredict.html.

  7. An Introduction to Research in Protein Folding for Undergraduates

    NASA Astrophysics Data System (ADS)

    Jones, Colleen M.

    1997-11-01

    The objective of this article is to introduce students to current research activity on protein folding via experimentation and a literature survey. Major effort in the field of biophysical chemistry today is focused on elucidating those factors controlling the transformation of a protein from a nascent polypeptide chain to a unique, functionally active three-dimensional structure. The possible involvement of misfolded or aggregated proteins in diseases such as Altzheimer's, cystic fibrosis, and cataracts as well as various neurodegenerative diseases has increased the incentive to solve the "protein folding problem". In this experiment the guanidine-hydrochloride induced protein unfolding of horse heart metmyoglobin is monitored spectrophotometrically via the protein fluorescence emission. The data are analyzed using a simple thermodynamic model which assumes a two-state system and fitted using nonlinear curve fitting. Background information on protein structure, protein fluorescence, simple models for folding, and the use of chaotropic agents is also presented. The experiment is suitable for students in advanced undergraduate chemistry courses such as physical or biophysical chemistry.

  8. Electrostatically Accelerated Coupled Binding and Folding of Intrinsically Disordered Proteins

    PubMed Central

    Ganguly, Debabani; Otieno, Steve; Waddell, Brett; Iconaru, Luigi; Kriwacki, Richard W.; Chen, Jianhan

    2012-01-01

    Intrinsically disordered proteins (IDPs) are now recognized to be prevalent in biology, and many potential functional benefits have been discussed. However, the frequent requirement of peptide folding in specific interactions of IDPs could impose a kinetic bottleneck, which could be overcome only by efficient folding upon encounter. Intriguingly, existing kinetic data suggest that specific binding of IDPs is generally no slower than that of globular proteins. Here, we exploited the cell cycle regulator p27Kip1 (p27) as a model system to understand how IDPs might achieve efficient folding upon encounter for facile recognition. Combining experiments and coarse-grained modeling, we demonstrate that long-range electrostatic interactions between enriched charges on p27 and near its binding site on cyclin A not only enhance the encounter rate (i.e., electrostatic steering), but also promote folding-competent topologies in the encounter complexes, allowing rapid subsequent formation of short-range native interactions en route to the specific complex. In contrast, nonspecific hydrophobic interactions, while hardly affecting the encounter rate, can significantly reduce the efficiency of folding upon encounter and lead to slower binding kinetics. Further analysis of charge distributions in a set of known IDP complexes reveals that, although IDP binding sites tend to be more hydrophobic compared to the rest of the target surface, their vicinities are frequently enriched with charges to complement those on IDPs. This observation suggests that electrostatically accelerated encounter and induced folding might represent a prevalent mechanism for promoting facile IDP recognition. PMID:22721951

  9. SecretP: a new method for predicting mammalian secreted proteins.

    PubMed

    Yu, Lezheng; Guo, Yanzhi; Zhang, Zheng; Li, Yizhou; Li, Menglong; Li, Gongbing; Xiong, Wenjia; Zeng, Yuhong

    2010-04-01

    In contrast to a large number of classically secreted proteins (CSPs) and non-secreted proteins (NSPs), only a few proteins have been experimentally proved to enter non-classical secretory pathways. So it is difficult to identify non-classically secreted proteins (NCSPs), and no methods are available for distinguishing the three types of proteins simultaneously. In order to solve this problem, a data mining has been taken firstly, and mammalian proteins exported via ER-Golgi-independent pathways are collected through extensive literature searches. In this paper, a support vector machine (SVM)-based ternary classifier named SecretP is proposed to predict mammalian secreted proteins by using pseudo-amino acid composition (PseAA) and five additional features. When distinguishing the three types of proteins, SecretP yielded an accuracy of 88.79%. Evaluating the performance of our method by an independent test set of 92 human proteins, 76 of them are correctly predicted as NCSPs. When performed on another public independent data set, the prediction result of SecretP is comparable to those of other existing computational methods. Therefore, SecretP can be a useful supplementary tool for future secretome studies. The web server SecretP and all supplementary tables listed in this paper are freely available at http://cic.scu.edu.cn/bioinformatics/secretp/index.htm. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  10. Periodic and stochastic thermal modulation of protein folding kinetics

    SciTech Connect

    Platkov, Max; Gruebele, Martin

    2014-07-21

    Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

  11. Periodic and stochastic thermal modulation of protein folding kinetics.

    PubMed

    Platkov, Max; Gruebele, Martin

    2014-07-21

    Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

  12. Dali/FSSP classification of three-dimensional protein folds.

    PubMed

    Holm, L; Sander, C

    1997-01-01

    The FSSP database presents a continuously updated structural classification of three-dimensional protein folds. It is derived using an automatic structure comparison program (Dali) for the all-against-all comparison of over 6000 three-dimensional coordinate sets in the Protein Data Bank (PDB). Sequence-related protein families are covered by a representative set of 813 protein chains. Hierachical clustering based on structural similarities yields a fold tree that defines 253 fold classes. For each representative protein chain, there is a database entry containing structure-structure alignments with its structural neighbours in the PDB. The database is accessible online through World Wide Web browsers and by anonymous ftp (file transfer protocol). The overview of fold space and the individual data sets provide a rich source of information for the study of both divergent and convergent aspects of molecular evolution, and define useful test sets and a standard of truth for assessing the correctness of sequence-sequence or sequence-structure alignments.

  13. Dynamics of protein folding: probing the kinetic network of folding-unfolding transitions with experiment and theory.

    PubMed

    Buchner, Ginka S; Murphy, Ronan D; Buchete, Nicolae-Viorel; Kubelka, Jan

    2011-08-01

    The problem of spontaneous folding of amino acid chains into highly organized, biologically functional three-dimensional protein structures continues to challenge the modern science. Understanding how proteins fold requires characterization of the underlying energy landscapes as well as the dynamics of the polypeptide chains in all stages of the folding process. In recent years, important advances toward these goals have been achieved owing to the rapidly growing interdisciplinary interest and significant progress in both experimental techniques and theoretical methods. Improvements in the experimental time resolution led to determination of the timescales of the important elementary events in folding, such as formation of secondary structure and tertiary contacts. Sensitive single molecule methods made possible probing the distributions of the unfolded and folded states and following the folding reaction of individual protein molecules. Discovery of proteins that fold in microseconds opened the possibility of atomic-level theoretical simulations of folding and their direct comparisons with experimental data, as well as of direct experimental observation of the barrier-less folding transition. The ultra-fast folding also brought new questions, concerning the intrinsic limits of the folding rates and experimental signatures of barrier-less "downhill" folding. These problems will require novel approaches for even more detailed experimental investigations of the folding dynamics as well as for the analysis of the folding kinetic data. For theoretical simulations of folding, a main challenge is how to extract the relevant information from overwhelmingly detailed atomistic trajectories. New theoretical methods have been devised to allow a systematic approach towards a quantitative analysis of the kinetic network of folding-unfolding transitions between various configuration states of a protein, revealing the transition states and the associated folding pathways at

  14. Persistent homology analysis of protein structure, flexibility and folding

    PubMed Central

    Xia, Kelin; Wei, Guo-Wei

    2014-01-01

    Proteins are the most important biomolecules for living organisms. The understanding of protein structure, function, dynamics and transport is one of most challenging tasks in biological science. In the present work, persistent homology is, for the first time, introduced for extracting molecular topological fingerprints (MTFs) based on the persistence of molecular topological invariants. MTFs are utilized for protein characterization, identification and classification. The method of slicing is proposed to track the geometric origin of protein topological invariants. Both all-atom and coarse-grained representations of MTFs are constructed. A new cutoff-like filtration is proposed to shed light on the optimal cutoff distance in elastic network models. Based on the correlation between protein compactness, rigidity and connectivity, we propose an accumulated bar length generated from persistent topological invariants for the quantitative modeling of protein flexibility. To this end, a correlation matrix based filtration is developed. This approach gives rise to an accurate prediction of the optimal characteristic distance used in protein B-factor analysis. Finally, MTFs are employed to characterize protein topological evolution during protein folding and quantitatively predict the protein folding stability. An excellent consistence between our persistent homology prediction and molecular dynamics simulation is found. This work reveals the topology-function relationship of proteins. PMID:24902720

  15. Genomic determinants of protein folding thermodynamics in prokaryotic organisms.

    PubMed

    Bastolla, Ugo; Moya, Andrés; Viguera, Enrique; van Ham, Roeland C H J

    2004-11-05

    Here we investigate how thermodynamic properties of orthologous proteins are influenced by the genomic environment in which they evolve. We performed a comparative computational study of 21 protein families in 73 prokaryotic species and obtained the following main results. (i) Protein stability with respect to the unfolded state and with respect to misfolding are anticorrelated. There appears to be a trade-off between these two properties, which cannot be optimized simultaneously. (ii) Folding thermodynamic parameters are strongly correlated with two genomic features, genome size and G+C composition. In particular, the normalized energy gap, an indicator of folding efficiency in statistical mechanical models of protein folding, is smaller in proteins of organisms with a small genome size and a compositional bias towards A+T. Such genomic features are characteristic for bacteria with an intracellular lifestyle. We interpret these correlations in light of mutation pressure and natural selection. A mutational bias toward A+T at the DNA level translates into a mutational bias toward more hydrophobic (and in general more interactive) proteins, a consequence of the structure of the genetic code. Increased hydrophobicity renders proteins more stable against unfolding but less stable against misfolding. Proteins with high hydrophobicity and low stability against misfolding occur in organisms with reduced genomes, like obligate intracellular bacteria. We argue that they are fixed because these organisms experience weaker purifying selection due to their small effective population sizes. This interpretation is supported by the observation of a high expression level of chaperones in these bacteria. Our results indicate that the mutational spectrum of a genome and the strength of selection significantly influence protein folding thermodynamics.

  16. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

    PubMed Central

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.; Song, Albert S.; Boomsma, Wouter; Bandyopadhyay, Pradip K.; Gruber, Christian W.; Purcell, Anthony W.; Yandell, Mark; Olivera, Baldomero M.

    2016-01-01

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea. PMID:26957604

  17. Collective aspects of protein folding illustrated by a toy model

    SciTech Connect

    Stillinger, F.H.; Head-Gordon, T.

    1995-09-01

    A simple toy model for polypeptides serves as a testbed to illuminate some nonlocal, or collective, aspects of protein folding phenomena. The model is two dimensional and has only two amino acids, but involves a continuous range of backbone bend angles. Global potential energy minima and their folding structures have been determined for leading members of two special and contrasting polypeptide sequences, center doped and Fibonacci, named descriptively for their primary structures. The results display the presence of spontaneous symmetry breaking, elastic strain, and substantial conformational variation for specific embedded amino acid strings. We conclude that collective variables generated by the primary amino acid structure may be required for fully effective protein folding predictors, including those based on neural networks.

  18. Effect of surfaces in modulating protein folding mechanisms

    NASA Astrophysics Data System (ADS)

    Shea, Joan

    2014-03-01

    Protein-surface interactions are ubiquitous in the crowded cytosol, where proteins encounter a variety of surfaces, ranging from membranes surfaces, to the surfaces presented by chaperone molecules. Protein-surface interactions are also at the heart of a number of emerging technologies, including protein micro-arrays, biosensors and biomaterials. The effect of surfaces on protein structure and stability can vary substantially depending on the chemical composition of the surface. In this talk, I will present detailed atomistic simulations of the folding of a small beta-sheet protein in the presence of graphite and titanium oxide surfaces. The role of water-mediated and direct protein-surface interactions in governing protein conformations will be discussed.

  19. Protein folding and misfolding in the neurodegenerative disorders: a review.

    PubMed

    Bolshette, N B; Thakur, K K; Bidkar, A P; Trandafir, C; Kumar, P; Gogoi, R

    2014-03-01

    Protein misfolding is an intrinsic aspect of normal folding within the complex cellular environment. Its effects are minimized in living system by the action of a range of protective mechanisms including molecular chaperones and quality control systems. According to the current growing research, protein misfolding is a recognized key feature of most neurodegenerative diseases. Extensive biochemical, neuropathological, and genetic evidence suggest that the cerebral accumulation of amyloid fibrils is the central event in the pathogenesis of neurodegenerative disorders. In the first part of this review we have discussed the general course of action of folding and misfolding of the proteins. Later part of this review gives an outline regarding the role of protein misfolding in the molecular and cellular mechanisms in the pathogenesis of Alzheimer's and Parkinson along with their treatment possibilities. Finally, we have mentioned about the recent findings in neurodegenerative diseases. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  20. Single-domain protein folding: a multi-faceted problem

    NASA Astrophysics Data System (ADS)

    Junier, Ivan; Ritort, Felix

    2006-08-01

    We review theoretical approaches, experiments and numerical simulations that have been recently proposed to investigate the folding problem in single-domain proteins. From a theoretical point of view, we emphasize the energy landscape approach. As far as experiments are concerned, we focus on the recent development of single-molecule techniques. In particular, we compare the results obtained with two main techniques: single protein force measurements with optical tweezers and single-molecule fluorescence in studies on the same protein (RNase H). This allows us to point out some controversial issues such as the nature of the denatured and intermediate states and possible folding pathways. After reviewing the various numerical simulation techniques, we show that on-lattice protein-like models can help to understand many controversial issues.

  1. A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins.

    PubMed

    Tejeda-Dominguez, Farid; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia; Navarro-Garcia, Fernando

    2017-03-28

    The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia, require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens.IMPORTANCE The type 3 secretion system is essential for injection of virulence factors, which are delivered directly into the cytosol of the host cells for usurping and subverting host processes. Recent studies have shown that these effectors proteins indeed travel inside an "injectisome" conduit through a single step of translocation by connecting the bacterium and host cell cytoplasms. However, all findings are not compatible with this model. For example, both YopH, a protein detected on the surface of Yersinia, and EspC, an autotransporter protein secreted by enteropathogenic E. coli, require a functional T3

  2. Folding of Aggregated Proteins to Functionally Active Form

    DTIC Science & Technology

    2006-06-01

    detergent [31]. The detergent is then removed by washes with cyclodextrin . Because the pro- cedure can be carried out on gravity-flow columns, it can be...capitalizes on the ability of chemicals such as cyclodextrin to prevent the aggregation of renatured protein when denaturants and/or detergents are removed... cyclodextrin . The cyclodextrin is then removed by another solvent exchange and the properly folded protein released from the column. In a variation of this

  3. An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding.

    PubMed

    Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad

    2017-09-01

    The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Size and sequence and the volume change of protein folding.

    PubMed

    Rouget, Jean-Baptiste; Aksel, Tural; Roche, Julien; Saldana, Jean-Louis; Garcia, Angel E; Barrick, Doug; Royer, Catherine A

    2011-04-20

    The application of hydrostatic pressure generally leads to protein unfolding, implying, in accordance with Le Chatelier's principle, that the unfolded state has a smaller molar volume than the folded state. However, the origin of the volume change upon unfolding, ΔV(u), has yet to be determined. We have examined systematically the effects of protein size and sequence on the value of ΔV(u) using as a model system a series of deletion variants of the ankyrin repeat domain of the Notch receptor. The results provide strong evidence in support of the notion that the major contributing factor to pressure effects on proteins is their imperfect internal packing in the folded state. These packing defects appear to be specifically localized in the 3D structure, in contrast to the uniformly distributed effects of temperature and denaturants that depend upon hydration of exposed surface area upon unfolding. Given its local nature, the extent to which pressure globally affects protein structure can inform on the degree of cooperativity and long-range coupling intrinsic to the folded state. We also show that the energetics of the protein's conformations can significantly modulate their volumetric properties, providing further insight into protein stability.

  5. Folding propensity of intrinsically disordered proteins by osmotic stress

    DOE PAGES

    Mansouri, Amanda L.; Grese, Laura N.; Rowe, Erica L.; ...

    2016-10-11

    Proteins imparted with intrinsic disorder conduct a range of essential cellular functions. To better understand the folding and hydration properties of intrinsically disordered proteins (IDPs), we used osmotic stress to induce conformational changes in nuclear co-activator binding domain (NCBD) and activator for thyroid hormone and retinoid receptor (ACTR). Osmotic stress was applied by the addition of small and polymeric osmolytes, where we discovered that water contributions to NCBD folding always exceeded those for ACTR. Both NCBD and ACTR were found to gain a-helical structure with increasing osmotic stress, consistent with their folding upon NCBD/ACTR complex formation. Using small-angle neutron scatteringmore » (SANS), we further characterized NCBD structural changes with the osmolyte ethylene glycol. Here a large reduction in overall size initially occurred before substantial secondary structural change. In conclusion, by focusing on folding propensity, and linked hydration changes, we uncover new insights that may be important for how IDP folding contributes to binding.« less

  6. Folding propensity of intrinsically disordered proteins by osmotic stress†

    PubMed Central

    Mansouri, Amanda L.; Grese, Laura N.; Rowe, Erica L.; Pino, James C.; Chennubhotla, S. Chakra; Ramanathan, Arvind; O’Neill, Hugh M.; Berthelier, Valerie

    2017-01-01

    Proteins imparted with intrinsic disorder conduct a range of essential cellular functions. To better understand the folding and hydration properties of intrinsically disordered proteins (IDPs), we used osmotic stress to induce conformational changes in nuclear co-activator binding domain (NCBD) and activator for thyroid hormone and retinoid receptor (ACTR) separate from their mutual binding. Osmotic stress was applied by the addition of small and polymeric osmolytes, where we discovered that water contributions to NCBD folding always exceeded those for ACTR. Both NCBD and ACTR were found to gain α-helical structure with increasing osmotic stress, consistent with their folding upon NCBD/ACTR complex formation. Using small-angle neutron scattering (SANS), we further characterized NCBD structural changes with the osmolyte ethylene glycol. Here a large reduction in overall size initially occurred before substantial secondary structural change. By focusing on folding propensity, and linked hydration changes, we uncover new insights that may be important for how IDP folding contributes to binding. PMID:27752679

  7. MatureP: prediction of secreted proteins with exclusive information from their mature regions.

    PubMed

    Orfanoudaki, Georgia; Markaki, Maria; Chatzi, Katerina; Tsamardinos, Ioannis; Economou, Anastassios

    2017-06-12

    More than a third of the cellular proteome is non-cytoplasmic. Most secretory proteins use the Sec system for export and are targeted to membranes using signal peptides and mature domains. To specifically analyze bacterial mature domain features, we developed MatureP, a classifier that predicts secretory sequences through features exclusively computed from their mature domains. MatureP was trained using Just Add Data Bio, an automated machine learning tool. Mature domains are predicted efficiently with ~92% success, as measured by the Area Under the Receiver Operating Characteristic Curve (AUC). Predictions were validated using experimental datasets of mutated secretory proteins. The features selected by MatureP reveal prominent differences in amino acid content between secreted and cytoplasmic proteins. Amino-terminal mature domain sequences have enhanced disorder, more hydroxyl and polar residues and less hydrophobics. Cytoplasmic proteins have prominent amino-terminal hydrophobic stretches and charged regions downstream. Presumably, secretory mature domains comprise a distinct protein class. They balance properties that promote the necessary flexibility required for the maintenance of non-folded states during targeting and secretion with the ability of post-secretion folding. These findings provide novel insight in protein trafficking, sorting and folding mechanisms and may benefit protein secretion biotechnology.

  8. Visualization of Protein Folding Funnels in Lattice Models

    PubMed Central

    Oliveira, Antonio B.; Fatore, Francisco M.; Paulovich, Fernando V.; Oliveira, Osvaldo N.; Leite, Vitor B. P.

    2014-01-01

    Protein folding occurs in a very high dimensional phase space with an exponentially large number of states, and according to the energy landscape theory it exhibits a topology resembling a funnel. In this statistical approach, the folding mechanism is unveiled by describing the local minima in an effective one-dimensional representation. Other approaches based on potential energy landscapes address the hierarchical structure of local energy minima through disconnectivity graphs. In this paper, we introduce a metric to describe the distance between any two conformations, which also allows us to go beyond the one-dimensional representation and visualize the folding funnel in 2D and 3D. In this way it is possible to assess the folding process in detail, e.g., by identifying the connectivity between conformations and establishing the paths to reach the native state, in addition to regions where trapping may occur. Unlike the disconnectivity maps method, which is based on the kinetic connections between states, our methodology is based on structural similarities inferred from the new metric. The method was developed in a 27-mer protein lattice model, folded into a 3×3×3 cube. Five sequences were studied and distinct funnels were generated in an analysis restricted to conformations from the transition-state to the native configuration. Consistent with the expected results from the energy landscape theory, folding routes can be visualized to probe different regions of the phase space, as well as determine the difficulty in folding of the distinct sequences. Changes in the landscape due to mutations were visualized, with the comparison between wild and mutated local minima in a single map, which serves to identify different trapping regions. The extension of this approach to more realistic models and its use in combination with other approaches are discussed. PMID:25010343

  9. Combining Optimal Control Theory and Molecular Dynamics for Protein Folding

    PubMed Central

    Arkun, Yaman; Gur, Mert

    2012-01-01

    A new method to develop low-energy folding routes for proteins is presented. The novel aspect of the proposed approach is the synergistic use of optimal control theory with Molecular Dynamics (MD). In the first step of the method, optimal control theory is employed to compute the force field and the optimal folding trajectory for the atoms of a Coarse-Grained (CG) protein model. The solution of this CG optimization provides an harmonic approximation of the true potential energy surface around the native state. In the next step CG optimization guides the MD simulation by specifying the optimal target positions for the atoms. In turn, MD simulation provides an all-atom conformation whose positions match closely the reference target positions determined by CG optimization. This is accomplished by Targeted Molecular Dynamics (TMD) which uses a bias potential or harmonic restraint in addition to the usual MD potential. Folding is a dynamical process and as such residues make different contacts during the course of folding. Therefore CG optimization has to be reinitialized and repeated over time to accomodate these important changes. At each sampled folding time, the active contacts among the residues are recalculated based on the all-atom conformation obtained from MD. Using the new set of contacts, the CG potential is updated and the CG optimal trajectory for the atoms is recomputed. This is followed by MD. Implementation of this repetitive CG optimization - MD simulation cycle generates the folding trajectory. Simulations on a model protein Villin demonstrate the utility of the method. Since the method is founded on the general tools of optimal control theory and MD without any restrictions, it is widely applicable to other systems. It can be easily implemented with available MD software packages. PMID:22238629

  10. Combining optimal control theory and molecular dynamics for protein folding.

    PubMed

    Arkun, Yaman; Gur, Mert

    2012-01-01

    A new method to develop low-energy folding routes for proteins is presented. The novel aspect of the proposed approach is the synergistic use of optimal control theory with Molecular Dynamics (MD). In the first step of the method, optimal control theory is employed to compute the force field and the optimal folding trajectory for the Cα atoms of a Coarse-Grained (CG) protein model. The solution of this CG optimization provides an harmonic approximation of the true potential energy surface around the native state. In the next step CG optimization guides the MD simulation by specifying the optimal target positions for the Cα atoms. In turn, MD simulation provides an all-atom conformation whose Cα positions match closely the reference target positions determined by CG optimization. This is accomplished by Targeted Molecular Dynamics (TMD) which uses a bias potential or harmonic restraint in addition to the usual MD potential. Folding is a dynamical process and as such residues make different contacts during the course of folding. Therefore CG optimization has to be reinitialized and repeated over time to accomodate these important changes. At each sampled folding time, the active contacts among the residues are recalculated based on the all-atom conformation obtained from MD. Using the new set of contacts, the CG potential is updated and the CG optimal trajectory for the Cα atoms is recomputed. This is followed by MD. Implementation of this repetitive CG optimization-MD simulation cycle generates the folding trajectory. Simulations on a model protein Villin demonstrate the utility of the method. Since the method is founded on the general tools of optimal control theory and MD without any restrictions, it is widely applicable to other systems. It can be easily implemented with available MD software packages.

  11. Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii

    PubMed Central

    Ribeiro, Orquídea; Magalhães, Frederico; Aguiar, Tatiana Q; Wiebe, Marilyn G; Penttilä, Merja; Domingues, Lucília

    2013-01-01

    To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities.   A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants. PMID:23644277

  12. The oxidative protein folding machinery in plant cells.

    PubMed

    Aller, Isabel; Meyer, Andreas J

    2013-08-01

    Formation of intra-molecular disulfides and concomitant oxidative protein folding is essential for stability and catalytic function of many soluble and membrane-bound proteins in the endomembrane system, the mitochondrial inter-membrane space and the thylakoid lumen. Disulfide generation from free cysteines in nascent polypeptide chains is generally a catalysed process for which distinct pathways exist in all compartments. A high degree of similarities between highly diverse eukaryotic and bacterial systems for generation of protein disulfides indicates functional conservation of key processes throughout evolution. However, while many aspects about molecular function of enzymatic systems promoting disulfide formation have been demonstrated for bacterial and non-plant eukaryotic organisms, it is now clear that the plant machinery for oxidative protein folding displays distinct details, suggesting that the different pathways have been adapted to plant-specific requirements in terms of compartmentation, molecular function and regulation. Here, we aim to evaluate biological diversity by comparing the plant systems for oxidative protein folding to the respective systems from non-plant eukaryotes.

  13. The topomer-sampling model of protein folding

    PubMed Central

    Debe, Derek A.; Carlson, Matt J.; Goddard, William A.

    1999-01-01

    Clearly, a protein cannot sample all of its conformations (e.g., ≈3100 ≈ 1048 for a 100 residue protein) on an in vivo folding timescale (<1 s). To investigate how the conformational dynamics of a protein can accommodate subsecond folding time scales, we introduce the concept of the native topomer, which is the set of all structures similar to the native structure (obtainable from the native structure through local backbone coordinate transformations that do not disrupt the covalent bonding of the peptide backbone). We have developed a computational procedure for estimating the number of distinct topomers required to span all conformations (compact and semicompact) for a polypeptide of a given length. For 100 residues, we find ≈3 × 107 distinct topomers. Based on the distance calculated between different topomers, we estimate that a 100-residue polypeptide diffusively samples one topomer every ≈3 ns. Hence, a 100-residue protein can find its native topomer by random sampling in just ≈100 ms. These results suggest that subsecond folding of modest-sized, single-domain proteins can be accomplished by a two-stage process of (i) topomer diffusion: random, diffusive sampling of the 3 × 107 distinct topomers to find the native topomer (≈0.1 s), followed by (ii) intratopomer ordering: nonrandom, local conformational rearrangements within the native topomer to settle into the precise native state. PMID:10077555

  14. Protein folding, stability, and solvation structure in osmolyte solutions hydrophobicity

    NASA Astrophysics Data System (ADS)

    Montgomery Pettitt, B.

    2008-03-01

    The hydrophobic effect between solutes in aqueous solutions plays a central role in our understanding of recognition and folding of proteins and self assembly of lipids. Hydrophobicity induces nonideal solution behavior which plays a role in many aspects of biophysics. Work on the use of small biochemical compounds to crowd protein solutions indicates that a quantitative description of their non-ideal behavior is possible and straightforward. Here, we will show what the structural origin of this non-ideal solution behavior is from expression derived from a semi grand ensemble approach. We discuss the consequences of these findings regarding protein folding stability and solvation in crowded solutions through a structural analysis of the m-value or the change in free energy difference of a macromolecule in solution with respect to the concentration of a third component. This effect has recently been restudied and new mechanisms proposed for its origins in terms of transfer free energies and hydrophobicity.

  15. WeFold: A Coopetition for Protein Structure Prediction

    PubMed Central

    Khoury, George A.; Liwo, Adam; Khatib, Firas; Zhou, Hongyi; Chopra, Gaurav; Bacardit, Jaume; Bortot, Leandro O.; Faccioli, Rodrigo A.; Deng, Xin; He, Yi; Krupa, Pawel; Li, Jilong; Mozolewska, Magdalena A.; Sieradzan, Adam K.; Smadbeck, James; Wirecki, Tomasz; Cooper, Seth; Flatten, Jeff; Xu, Kefan; Baker, David; Cheng, Jianlin; Delbem, Alexandre C. B.; Floudas, Christodoulos A.; Keasar, Chen; Levitt, Michael; Popović, Zoran; Scheraga, Harold A.; Skolnick, Jeffrey; Crivelli, Silvia N.; Players, Foldit

    2014-01-01

    The protein structure prediction problem continues to elude scientists. Despite the introduction of many methods, only modest gains were made over the last decade for certain classes of prediction targets. To address this challenge, a social-media based worldwide collaborative effort, named WeFold, was undertaken by thirteen labs. During the collaboration, the labs were simultaneously competing with each other. Here, we present the first attempt at “coopetition” in scientific research applied to the protein structure prediction and refinement problems. The coopetition was possible by allowing the participating labs to contribute different components of their protein structure prediction pipelines and create new hybrid pipelines that they tested during CASP10. This manuscript describes both successes and areas needing improvement as identified throughout the first WeFold experiment and discusses the efforts that are underway to advance this initiative. A footprint of all contributions and structures are publicly accessible at http://www.wefold.org. PMID:24677212

  16. WeFold: a coopetition for protein structure prediction.

    PubMed

    Khoury, George A; Liwo, Adam; Khatib, Firas; Zhou, Hongyi; Chopra, Gaurav; Bacardit, Jaume; Bortot, Leandro O; Faccioli, Rodrigo A; Deng, Xin; He, Yi; Krupa, Pawel; Li, Jilong; Mozolewska, Magdalena A; Sieradzan, Adam K; Smadbeck, James; Wirecki, Tomasz; Cooper, Seth; Flatten, Jeff; Xu, Kefan; Baker, David; Cheng, Jianlin; Delbem, Alexandre C B; Floudas, Christodoulos A; Keasar, Chen; Levitt, Michael; Popović, Zoran; Scheraga, Harold A; Skolnick, Jeffrey; Crivelli, Silvia N

    2014-09-01

    The protein structure prediction problem continues to elude scientists. Despite the introduction of many methods, only modest gains were made over the last decade for certain classes of prediction targets. To address this challenge, a social-media based worldwide collaborative effort, named WeFold, was undertaken by 13 labs. During the collaboration, the laboratories were simultaneously competing with each other. Here, we present the first attempt at "coopetition" in scientific research applied to the protein structure prediction and refinement problems. The coopetition was possible by allowing the participating labs to contribute different components of their protein structure prediction pipelines and create new hybrid pipelines that they tested during CASP10. This manuscript describes both successes and areas needing improvement as identified throughout the first WeFold experiment and discusses the efforts that are underway to advance this initiative. A footprint of all contributions and structures are publicly accessible at http://www.wefold.org. © 2014 Wiley Periodicals, Inc.

  17. Invariant patterns in crystal lattices: Implications for protein folding algorithms

    SciTech Connect

    HART,WILLIAM E.; ISTRAIL,SORIN

    2000-06-01

    Crystal lattices are infinite periodic graphs that occur naturally in a variety of geometries and which are of fundamental importance in polymer science. Discrete models of protein folding use crystal lattices to define the space of protein conformations. Because various crystal lattices provide discretizations of the same physical phenomenon, it is reasonable to expect that there will exist invariants across lattices related to fundamental properties of the protein folding process. This paper considers whether performance-guaranteed approximability is such an invariant for HP lattice models. The authors define a master approximation algorithm that has provable performance guarantees provided that a specific sublattice exists within a given lattice. They describe a broad class of crystal lattices that are approximable, which further suggests that approximability is a general property of HP lattice models.

  18. Fold Recognition Using Sequence Fingerprints of Protein Local Substructures

    SciTech Connect

    Kryshtafovych, A A; Hvidsten, T; Komorowski, J; Fidelis, K

    2003-06-04

    A protein local substructure (descriptor) is a set of several short non-overlapping fragments of the polypeptide chain. Each descriptor describes local environment of a particular residue and includes only those segments that are located in the proximity of this residue. Similar descriptors from the representative set of proteins were analyzed to reveal links between the substructures and sequences of their segments. Using detected sequence-based fingerprints specific geometrical conformations are assigned to new sequences. The ability of the approach to recognize correct SCOP folds was tested on 273 sequences from the 49 most popular folds. Good predictions were obtained in 85% of cases. No performance drop was observed with decreasing sequence similarity between target sequences and sequences from the training set of proteins.

  19. Femtomole Mixer for Microsecond Kinetic Studies of Protein Folding

    PubMed Central

    Hertzog, David E.; Michalet, Xavier; Jäger, Marcus; Kong, Xiangxu; Santiago, Juan G.; Weiss, Shimon; Bakajin, Olgica

    2005-01-01

    We have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8 μs and sample consumption of femtomoles. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. In this paper, we discuss the design and optimization of the mixer using modeling of convective diffusion phenomena and a characterization of the mixer performance using microparticle image velocimetry, dye quenching, and Förster resonance energy-transfer (FRET) measurements of single-stranded DNA. We also demonstrate the feasibility of measuring fast protein folding kinetics using FRET with acyl-CoA binding protein. PMID:15595857

  20. Characterization of Nops, nodulation outer proteins, secreted via the type III secretion system of NGR234.

    PubMed

    Marie, Corinne; Deakin, William J; Viprey, Virginie; Kopciñska, Joanna; Golinowski, Wladyslaw; Krishnan, Hari B; Perret, Xavier; Broughton, William J

    2003-09-01

    The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.

  1. Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

    Treesearch

    Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

    2003-01-01

    Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

  2. Folding by Numbers: Primary Sequence Statistics and Their Use in Studying Protein Folding

    PubMed Central

    Wathen, Brent; Jia, Zongchao

    2009-01-01

    The exponential growth over the past several decades in the quantity of both primary sequence data available and the number of protein structures determined has provided a wealth of information describing the relationship between protein primary sequence and tertiary structure. This growing repository of data has served as a prime source for statistical analysis, where underlying relationships between patterns of amino acids and protein structure can be uncovered. Here, we survey the main statistical approaches that have been used for identifying patterns within protein sequences, and discuss sequence pattern research as it relates to both secondary and tertiary protein structure. Limitations to statistical analyses are discussed, and a context for their role within the field of protein folding is given. We conclude by describing a novel statistical study of residue patterning in β-strands, which finds that hydrophobic (i,i+2) pairing in β-strands occurs more often than expected at locations near strand termini. Interpretations involving β-sheet nucleation and growth are discussed. PMID:19468326

  3. Symmetric structures in the universe of protein folds.

    PubMed

    Guerler, Aysam; Wang, Connie; Knapp, Ernst-Walter

    2009-09-01

    Insights in structural biology can be gained by analyzing protein architectures and characterizing their structural similarities. Current computational approaches enable a comparison of a variety of structural and physicochemical properties in protein space. Here we describe the automated detection of rotational symmetries within a representative set of nearly 10,000 nonhomologous protein structures. To find structural symmetries in proteins initially, equivalent pairs of secondary structure elements (SSE), i.e., alpha-helices and beta-strands, are assigned. Thereby, we also allow SSE pairs to be assigned in reverse sequential order. The results highlight that the generation of symmetric, i.e., repetitive, protein structures is one of nature's major strategies to explore the universe of possible protein folds. This way structurally separated 'islands' of protein folds with a significant amount of symmetry were identified. The complete results of the present study are available at http://agknapp.chemie.fu-berlin.de/gplus, where symmetry analysis of new protein structures can also be performed.

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

    PubMed

    De Marothy, Minttu T; Elofsson, Arne

    2015-07-01

    Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices-implying a dynamic relationship between membrane proteins and their environment. © 2015 The Protein Society.

  5. How optimization of potential functions affects protein folding.

    PubMed Central

    Hao, M H; Scheraga, H A

    1996-01-01

    The relationship between the optimization of the potential function and the foldability of theoretical protein models is studied based on investigations of a 27-mer cubic-lattice protein model and a more realistic lattice model for the protein crambin. In both the simple and the more complicated systems, optimization of the energy parameters achieves significant improvements in the statistical-mechanical characteristics of the systems and leads to foldable protein models in simulation experiments. The foldability of the protein models is characterized by their statistical-mechanical properties--e.g., by the density of states and by Monte Carlo folding simulations of the models. With optimized energy parameters, a high level of consistency exists among different interactions in the native structures of the protein models, as revealed by a correlation function between the optimized energy parameters and the native structure of the model proteins. The results of this work are relevant to the design of a general potential function for folding proteins by theoretical simulations. PMID:8643516

  6. Energetics-Based Methods for Protein Folding and Stability Measurements

    NASA Astrophysics Data System (ADS)

    Geer, M. Ariel; Fitzgerald, Michael C.

    2014-06-01

    Over the past 15 years, a series of energetics-based techniques have been developed for the thermodynamic analysis of protein folding and stability. These techniques include Stability of Unpurified Proteins from Rates of amide H/D Exchange (SUPREX), pulse proteolysis, Stability of Proteins from Rates of Oxidation (SPROX), slow histidine H/D exchange, lysine amidination, and quantitative cysteine reactivity (QCR). The above techniques, which are the subject of this review, all utilize chemical or enzymatic modification reactions to probe the chemical denaturant- or temperature-induced equilibrium unfolding properties of proteins and protein-ligand complexes. They employ various mass spectrometry-, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-, and optical spectroscopy-based readouts that are particularly advantageous for high-throughput and in some cases multiplexed analyses. This has created the opportunity to use protein folding and stability measurements in new applications such as in high-throughput screening projects to identify novel protein ligands and in mode-of-action studies to identify protein targets of a particular ligand.

  7. Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

    PubMed Central

    Delic, Marizela; Graf, Alexandra B.; Koellensperger, Gunda; Haberhauer-Troyer, Christina; Hann, Stephan; Mattanovich, Diethard; Gasser, Brigitte

    2014-01-01

    Oxidative folding of secretory proteins in the endoplasmic reticulum (ER) is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS) caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG) in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant) proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity. PMID:28357216

  8. Modeling chain folding in protein-constrained circular DNA.

    PubMed Central

    Martino, J A; Olson, W K

    1998-01-01

    An efficient method for sampling equilibrium configurations of DNA chains binding one or more DNA-bending proteins is presented. The technique is applied to obtain the tertiary structures of minimal bending energy for a selection of dinucleosomal minichromosomes that differ in degree of protein-DNA interaction, protein spacing along the DNA chain contour, and ring size. The protein-bound portions of the DNA chains are represented by tight, left-handed supercoils of fixed geometry. The protein-free regions are modeled individually as elastic rods. For each random spatial arrangement of the two nucleosomes assumed during a stochastic search for the global minimum, the paths of the flexible connecting DNA segments are determined through a numerical solution of the equations of equilibrium for torsionally relaxed elastic rods. The minimal energy forms reveal how protein binding and spacing and plasmid size differentially affect folding and offer new insights into experimental minichromosome systems. PMID:9591675

  9. LMAN1 (ERGIC-53) is a potential carrier protein for matrix metalloproteinase-9 glycoprotein secretion

    PubMed Central

    Duellman, Tyler; Burnett, John; Shin, Alice; Yang, Jay

    2015-01-01

    Matrix metalloproteinase-9 (MMP-9) is a secreted glycoprotein with a major role in shaping the extra-cellular matrix and a detailed understanding of the secretory mechanism could help identify methods to correct diseases resulting from dysregulation of secretion. MMP-9 appears to follow a canonical secretory pathway through a quality control cycle in the endoplasmic reticulum (ER) before transport of the properly folded protein to the Golgi apparatus and beyond for secretion. Through a complementation assay, we determined that LMAN1, a well-studied lectin-carrier protein, interacts with a secretion-competent N-glycosylated MMP-9 in the ER while N-glycosylation-deficient secretion-compromised MMP-9 does not. In contrast, co-immunoprecipitation demonstrated protein interaction between LMAN1 and secretion-compromised N-glycosylation-deficient MMP-9. MMP-9 secretion was reduced in the LMAN1 knockout cell line compared to control cells confirming the functional role of LMAN1. These observations support the role of LMAN1 as a lectin-carrier protein mediating efficient MMP-9 secretion. PMID:26150355

  10. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Parekh, R N; Wittrup, K D

    1997-01-01

    The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture.

  11. LMAN1 (ERGIC-53) is a potential carrier protein for matrix metalloproteinase-9 glycoprotein secretion.

    PubMed

    Duellman, Tyler; Burnett, John; Shin, Alice; Yang, Jay

    2015-08-28

    Matrix metalloproteinase-9 (MMP-9) is a secreted glycoprotein with a major role in shaping the extracellular matrix and a detailed understanding of the secretory mechanism could help identify methods to correct diseases resulting from dysregulation of secretion. MMP-9 appears to follow a canonical secretory pathway through a quality control cycle in the endoplasmic reticulum (ER) before transport of the properly folded protein to the Golgi apparatus and beyond for secretion. Through a complementation assay, we determined that LMAN1, a well-studied lectin-carrier protein, interacts with a secretion-competent N-glycosylated MMP-9 in the ER while N-glycosylation-deficient secretion-compromised MMP-9 does not. In contrast, co-immunoprecipitation demonstrated protein interaction between LMAN1 and secretion-compromised N-glycosylation-deficient MMP-9. MMP-9 secretion was reduced in the LMAN1 knockout cell line compared to control cells confirming the functional role of LMAN1. These observations support the role of LMAN1 as a lectin-carrier protein mediating efficient MMP-9 secretion.

  12. Work done by titin protein folding assists muscle contraction

    PubMed Central

    Popa, Ionel; Kosuri, Pallav; Linke, Wolfgang A.; Fernández, Julio M.

    2016-01-01

    Current theories of muscle contraction propose that the power stroke of a myosin motor is the sole source of mechanical energy driving the sliding filaments of a contracting muscle. These models exclude titin, the largest protein in the human body, which determines the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin Ig domains occurs in the elastic I band region of intact myofibrils at physiological sarcomere lengths and forces of 6-8 pN. We use single molecule techniques to demonstrate that unfolded titin Ig domains undergo a spontaneous stepwise folding contraction at forces below 10 pN, delivering up to 105 zJ of additional contractile energy, which is larger than the mechanical energy delivered by the power stroke of a myosin motor. Thus, it appears inescapable that folding of titin Ig domains is an important, but so far unrecognized contributor to the force generated by a contracting muscle. PMID:26854230

  13. Work Done by Titin Protein Folding Assists Muscle Contraction.

    PubMed

    Rivas-Pardo, Jaime Andrés; Eckels, Edward C; Popa, Ionel; Kosuri, Pallav; Linke, Wolfgang A; Fernández, Julio M

    2016-02-16

    Current theories of muscle contraction propose that the power stroke of a myosin motor is the sole source of mechanical energy driving the sliding filaments of a contracting muscle. These models exclude titin, the largest protein in the human body, which determines the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin immunoglobulin (Ig) domains occurs in the elastic I band region of intact myofibrils at physiological sarcomere lengths and forces of 6-8 pN. We use single-molecule techniques to demonstrate that unfolded titin Ig domains undergo a spontaneous stepwise folding contraction at forces below 10 pN, delivering up to 105 zJ of additional contractile energy, which is larger than the mechanical energy delivered by the power stroke of a myosin motor. Thus, it appears inescapable that folding of titin Ig domains is an important, but as yet unrecognized, contributor to the force generated by a contracting muscle.

  14. A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins

    PubMed Central

    Tejeda-Dominguez, Farid; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia

    2017-01-01

    ABSTRACT The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia, require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens. PMID:28351918

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

    PubMed Central

    De Marothy, Minttu T; Elofsson, Arne

    2015-01-01

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

  16. Twin-arginine-dependent translocation of folded proteins.

    PubMed

    Fröbel, Julia; Rose, Patrick; Müller, Matthias

    2012-04-19

    Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF.

  17. Twin-arginine-dependent translocation of folded proteins

    PubMed Central

    Fröbel, Julia; Rose, Patrick; Müller, Matthias

    2012-01-01

    Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF. PMID:22411976

  18. On the Role of Entropy in the Protein Folding Process

    NASA Astrophysics Data System (ADS)

    Hoppe, Travis

    2011-12-01

    A protein's ultimate function and activity is determined by the unique three-dimensional structure taken by the folding process. Protein malfunction due to misfolding is the culprit of many clinical disorders, such as abnormal protein aggregations. This leads to neurodegenerative disorders like Huntington's and Alzheimer's disease. We focus on a subset of the folding problem, exploring the role and effects of entropy on the process of protein folding. Four major concepts and models are developed and each pertains to a specific aspect of the folding process: entropic forces, conformational states under crowding, aggregation, and macrostate kinetics from microstate trajectories. The exclusive focus on entropy is well-suited for crowding studies, as many interactions are nonspecific. We show how a stabilizing entropic force can arise purely from the motion of crowders in solution. In addition we are able to make a a quantitative prediction of the crowding effect with an implicit crowding approximation using an aspherical scaled-particle theory. In order to investigate the effects of aggregation, we derive a new operator expansion method to solve the Ising/Potts model with external fields over an arbitrary graph. Here the external fields are representative of the entropic forces. We show that this method reduces the problem of calculating the partition function to the solution of recursion relations. Many of the methods employed are coarse-grained approximations. As such, it is useful to have a viable method for extracting macrostate information from time series data. We develop a method to cluster the microstates into physically meaningful macrostates by grouping similar relaxation times from a transition matrix. Overall, the studied topics allow us to understand deeper the complicated process involving proteins.

  19. High-Yield Secretion of Multiple Client Proteins in Aspergillus

    SciTech Connect

    Segato, F.; Damasio, A. R. L.; Goncalves, T. A.; de Lucas, R. C.; Squina, F. M.; Decker, S. R.; Prade, R. A.

    2012-07-15

    Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.

  20. High-yield secretion of multiple client proteins in Aspergillus.

    PubMed

    Segato, Fernando; Damásio, André R L; Gonçalves, Thiago A; de Lucas, Rosymar C; Squina, Fabio M; Decker, Stephen R; Prade, Rolf A

    2012-07-15

    Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Type II secretion system: a magic beanstalk or a protein escalator.

    PubMed

    Nivaskumar, Mangayarkarasi; Francetic, Olivera

    2014-08-01

    Type II protein secretion systems (T2SS) are molecular machines that promote specific transport of folded periplasmic proteins in Gram-negative bacteria, across a dedicated channel in the outer membrane. Secreted substrates, released to the milieu or displayed on the cell surface, contribute to bacterial adaptation to a range of habitats, from deep-sea waters to animal and plant tissues. The past decade has seen remarkable progress in structural, biochemical and functional analysis of T2SS and related systems, bringing new mechanistic insights into these dynamic complexes. This review focuses on recent advances in the field, and discusses open questions regarding the secretion mechanism. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Statistical mechanics of simple models of protein folding and design.

    PubMed Central

    Pande, V S; Grosberg, A Y; Tanaka, T

    1997-01-01

    It is now believed that the primary equilibrium aspects of simple models of protein folding are understood theoretically. However, current theories often resort to rather heavy mathematics to overcome some technical difficulties inherent in the problem or start from a phenomenological model. To this end, we take a new approach in this pedagogical review of the statistical mechanics of protein folding. The benefit of our approach is a drastic mathematical simplification of the theory, without resort to any new approximations or phenomenological prescriptions. Indeed, the results we obtain agree precisely with previous calculations. Because of this simplification, we are able to present here a thorough and self contained treatment of the problem. Topics discussed include the statistical mechanics of the random energy model (REM), tests of the validity of REM as a model for heteropolymer freezing, freezing transition of random sequences, phase diagram of designed ("minimally frustrated") sequences, and the degree to which errors in the interactions employed in simulations of either folding and design can still lead to correct folding behavior. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 6 PMID:9414231

  3. Saddles of the energy landscape and folding of model proteins

    NASA Astrophysics Data System (ADS)

    Angelani, L.; Ruocco, G.

    2009-07-01

    We numerically investigate the Potential Energy Landscape of an off-lattice β-sheet model protein, looking at saddles and minima probed by the system during the folding process. G {\\bar o} - like (with native-state-dependent force field and funnel-like landscape) and non-G {\\bar o} -like models are considered. In the G {\\bar o} -like case, on varying the temperature, we observe: i) a pronounced peak at the collapse/folding temperature T θsimeTf in the energy elevation of visited saddles from underlying minima, ii) a crossover at the same point of the saddle order. Saddles-based quantities seem then to be good candidates as indicators of the funneled shape of the landscape in protein models.

  4. Nucleation-based prediction of the protein folding rate and its correlation with the folding nucleus size.

    PubMed

    Galzitskaya, Oxana V; Glyakina, Anna V

    2012-12-01

    The problem of protein self-organization is in the focus of current molecular biology studies. Although the general principles are understood, many details remain unclear. Specifically, protein folding rates are of interest because they dictate the rate of protein aggregation which underlies many human diseases. Here we offer predictions of protein folding rates and their correlation with folding nucleus sizes. We calculated free energies of the transition state and sizes of folding nuclei for 84 proteins and peptides whose other parameters were measured at the point of thermodynamic equilibrium between their unfolded and native states. We used the dynamic programming method where each residue was considered to be either as folded as in its native state or completely disordered. The calculated and measured folding rates showed a good correlation at the temperature mid-transition point (the correlation coefficient was 0.75). Also, we pioneered in demonstrating a moderate (-0.57) correlation coefficient between the calculated sizes of folding nuclei and the folding rates. Predictions made by different methods were compared. The established good correlation between the estimated free energy barrier and the experimentally found folding rate of each studied protein/peptide indicates that our model gives reliable results for the considered data set. Copyright © 2012 Wiley Periodicals, Inc.

  5. Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris

    PubMed Central

    2011-01-01

    Background The increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers. Results Based on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER) has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p), we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold) without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion. Conclusions The current study presents a systems biotechnology-based strategy for the engineering of the

  6. Identification of secreted bacterial proteins by noncanonical amino acid tagging.

    PubMed

    Mahdavi, Alborz; Szychowski, Janek; Ngo, John T; Sweredoski, Michael J; Graham, Robert L J; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K; Tirrell, David A

    2014-01-07

    Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.

  7. Substrate recognition by the Yersinia type III protein secretion machinery.

    PubMed

    Ramamurthi, Kumaran S; Schneewind, Olaf

    2003-11-01

    Type III secretion is the designation given to those protein secretion pathways, primarily in pathogenic Gram-negative bacteria, whose secretion machinery components share an amino acid sequence homology to components of the flagellar basal body. In Yersinia spp., these secretion machineries inject virulence proteins called Yops into the cytosol of target macrophages in an effort to evade phagocytic killing. To date, a clear mechanism by which Yops are recognized by the type III secretion machinery has not been elucidated. Unlike most, if not all, previously characterized protein sorting pathways, the information that identifies Yops as substrates for secretion seems not to be wholly encoded within the Yop peptide sequence. In fact, it appears that at least some of this information is contained within yop mRNAs. This review summarizes recent observations that have been made in this unusual field and proposes models by which proteins may be initiated into this pathway.

  8. Combined approach to the inverse protein folding problem. Final report

    SciTech Connect

    Ruben A. Abagyan

    2000-06-01

    The main scientific contribution of the project ''Combined approach to the inverse protein folding problem'' submitted in 1996 and funded by the Department of Energy in 1997 is the formulation and development of the idea of the multilink recognition method for identification of functional and structural homologues of newly discovered genes. This idea became very popular after they first announced it and used it in prediction of the threading targets for the CASP2 competition (Critical Assessment of Structure Prediction).

  9. Protein folding on the ribosome studied using NMR spectroscopy

    PubMed Central

    Waudby, Christopher A.; Launay, Hélène; Cabrita, Lisa D.; Christodoulou, John

    2013-01-01

    NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome–nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity. PMID:24083462

  10. Common fold in helix-hairpin-helix proteins.

    PubMed

    Shao, X; Grishin, N V

    2000-07-15

    Helix-hairpin-helix (HhH) is a widespread motif involved in non-sequence-specific DNA binding. The majority of HhH motifs function as DNA-binding modules, however, some of them are used to mediate protein-protein interactions or have acquired enzymatic activity by incorporating catalytic residues (DNA glycosylases). From sequence and structural analysis of HhH-containing proteins we conclude that most HhH motifs are integrated as a part of a five-helical domain, termed (HhH)(2) domain here. It typically consists of two consecutive HhH motifs that are linked by a connector helix and displays pseudo-2-fold symmetry. (HhH)(2) domains show clear structural integrity and a conserved hydrophobic core composed of seven residues, one residue from each alpha-helix and each hairpin, and deserves recognition as a distinct protein fold. In addition to known HhH in the structures of RuvA, RadA, MutY and DNA-polymerases, we have detected new HhH motifs in sterile alpha motif and barrier-to-autointegration factor domains, the alpha-subunit of Escherichia coli RNA-polymerase, DNA-helicase PcrA and DNA glycosylases. Statistically significant sequence similarity of HhH motifs and pronounced structural conservation argue for homology between (HhH)(2) domains in different protein families. Our analysis helps to clarify how non-symmetric protein motifs bind to the double helix of DNA through the formation of a pseudo-2-fold symmetric (HhH)(2) functional unit.

  11. Kinetics of chain motions within a protein-folding intermediate

    PubMed Central

    Neuweiler, Hannes; Banachewicz, Wiktor; Fersht, Alan R.

    2010-01-01

    Small proteins can fold remarkably rapidly, even in μs. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 μs, but the final docking of preformed helix1 in I requires approximately 20 μs. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-μs time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-μs and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-μs phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion. PMID:21135210

  12. Protein Folding, Stability, and Solvation Structure in Osmolyte Solutions

    PubMed Central

    Rösgen, Jörg; Pettitt, B. Montgomery; Bolen, David Wayne

    2005-01-01

    An understanding of the impact of the crowded conditions in the cytoplasm on its biomolecules is of clear importance to biochemical, medical, and pharmaceutical science. Our previous work on the use of small biochemical compounds to crowd protein solutions indicates that a quantitative description of their nonideal behavior is possible and straightforward. Here, we show the structural origin of the nonideal solution behavior. We discuss the consequences of these findings regarding protein folding stability and solvation in crowded solutions through a structural analysis of the m-value or the change in free-energy difference of a macromolecule in solution with respect to the concentration of a third component. PMID:16113118

  13. Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

    PubMed Central

    2012-01-01

    Background The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase). Results HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress. Conclusions Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases. PMID:22380681

  14. An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

    PubMed

    Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K

    2017-01-01

    Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently

  15. Sphingolipid transfer proteins defined by the GLTP-fold

    PubMed Central

    Malinina, Lucy; Simanshu, Dhirendra K.; Zhai, Xiuhong; Samygina, Valeria R.; Kamlekar, RaviKanth; Kenoth, Roopa; Ochoa-Lizarralde, Borja; Malakhova, Margarita L.; Molotkovsky, Julian G.; Patel, Dinshaw J.; Brown, Rhoderick E.

    2015-01-01

    Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer ‘sandwich’ architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation. A new understanding of the structural versatility and evolutionary premium placed on the GLTP motif has emerged. Human GLTP-motifs have evolved to function not only as glucosylceramide binding/transferring domains for phosphoinositol 4-phosphate adaptor protein-2 during glycosphingolipid biosynthesis but also as selective binding/transfer proteins for ceramide-1-phosphate. The latter, known as ceramide-l-phosphate transfer protein, recently has been shown to form GLTP-fold while critically regulating Group-IV cytoplasmic phospholipase A2 activity and pro-inflammatory eicosanoid production. PMID:25797198

  16. Redox-Assisted Protein Folding Systems in Eukaryotic Parasites

    PubMed Central

    Haque, Saikh Jaharul; Majumdar, Tanmay

    2012-01-01

    Abstract Significance: The cysteine (Cys) residues of proteins play two fundamentally important roles. They serve as sites of post-translational redox modifications as well as influence the conformation of the protein through the formation of disulfide bonds. Recent Advances: Redox-related and redox-associated protein folding in protozoan parasites has been found to be a major mode of regulation, affecting myriad aspects of the parasitic life cycle, host-parasite interactions, and the disease pathology. Available genome sequences of various parasites have begun to complement the classical biochemical and enzymological studies of these processes. In this article, we summarize the reversible Cys disulfide (S-S) bond formation in various classes of strategically important parasitic proteins, and its structural consequence and functional relevance. Critical Issues: Molecular mechanisms of folding remain under-studied and often disconnected from functional relevance. Future Directions: The clinical benefit of redox research will require a comprehensive characterization of the various isoforms and paralogs of the redox enzymes and their concerted effect on the structure and function of the specific parasitic client proteins. Antioxid. Redox Signal. 17, 674–683. PMID:22122448

  17. The protein folding problem: global optimization of the force fields.

    PubMed

    Scheraga, H A; Liwo, A; Oldziej, S; Czaplewski, C; Pillardy, J; Ripoll, D R; Vila, J A; Kazmierkiewicz, R; Saunders, J A; Arnautova, Y A; Jagielska, A; Chinchio, M; Nanias, M

    2004-09-01

    The evolutionary development of a theoretical approach to the protein folding problem, in our laboratory, is traced. The theoretical foundations and the development of a suitable empirical all-atom potential energy function and a global optimization search are examined. Whereas the all-atom approach has thus far succeeded for relatively small molecules and for alpha-helical proteins containing up to 46 residues, it has been necessary to develop a hierarchical approach to treat larger proteins. In the hierarchical approach to single- and multiple-chain proteins, global optimization is carried out for a simplified united residue (UNRES) description of a polypeptide chain to locate the region in which the global minimum lies. Conversion of the UNRES structures in this region to all-atom structures is followed by a local search in this region. The performance of this approach in successive CASP blind tests for predicting protein structure by an ab initio physics-based method is described. Finally, a recent attempt to compute a folding pathway is discussed.

  18. Secreted and immunogenic proteins produced by the honeybee bacterial pathogen, Paenibacillus larvae.

    PubMed

    Antúnez, Karina; Anido, Matilde; Evans, Jay D; Zunino, Pablo

    2010-03-24

    American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae during larval infection. The aim of the present work was to perform a first approach to the identification and characterization of P. larvae secretome. P. larvae secreted proteins were analyzed by SDS-PAGE and identified by MALDI-TOF. Protein toxicity was evaluated using an experimental model based on feeding of A. mellifera larvae and immunogenicity was evaluated by Western blot, using an antiserum raised against cells and spores of P. larvae. Ten different proteins were identified among P. larvae secreted proteins, including proteins involved in transcription, metabolism, translation, cell envelope, transport, protein folding, degradation of polysaccharides and motility. Although most of these proteins are cytosolic, many of them have been previously detected in the extracellular medium of different Bacillus spp. cultures and have been related to virulence. The secreted proteins resulted highly toxic and immunogenic when larvae were exposed using an experimental model. This is the first description of proteins secreted by the honeybee pathogen P. larvae. This information may be relevant for the elucidation of bacterial pathogenesis mechanisms. Copyright 2009 Elsevier B.V. All rights reserved.

  19. Hydrophobicity – Shake Flasks, Protein Folding and Drug Discovery

    PubMed Central

    Sarkar, Aurijit; Kellogg, Glen E.

    2009-01-01

    Hydrophobic interactions are some of the most important interactions in nature. They are the primary driving force in a number of phenomena. This is mostly an entropic effect and can account for a number of biophysical events such as protein-protein or protein-ligand binding that are of immense importance in drug design. The earliest studies on this phenomenon can be dated back to the end of the 19th century when Meyer and Overton independently correlated the hydrophobic nature of gases to their anesthetic potency. Since then, significant progress has been made in this realm of science. This review briefly traces the history of hydrophobicity research along with the theoretical estimation of partition coefficients. Finally, the application of hydrophobicity estimation methods in the field of drug design and protein folding is discussed. PMID:19929828

  20. Folding a protein by discretizing its backbone torsional dynamics

    NASA Astrophysics Data System (ADS)

    Fernández, Ariel

    1999-05-01

    The aim of this work is to provide a coarse codification of local conformational constraints associated with each folding motif of a peptide chain in order to obtain a rough solution to the protein folding problem. This is accomplished by implementing a discretized version of the soft-mode dynamics on a personal computer (PC). Our algorithm mimics a parallel process as it evaluates concurrent folding possibilities by pattern recognition. It may be implemented in a PC as a sequence of perturbation-translation-renormalization (p-t-r) cycles performed on a matrix of local topological constraints (LTM). This requires suitable representational tools and a periodic quenching of the dynamics required for renormalization. We introduce a description of the peptide chain based on a local discrete variable the values of which label the basins of attraction of the Ramachandran map for each residue. Thus, the local variable indicates the basin in which the torsional coordinates of each residue lie at a given time. In addition, a coding of local topological constraints associated with each secondary and tertiary structural motif is introduced. Our treatment enables us to adopt a computation time step of 81 ps, a value far larger than hydrodynamic drag time scales. Folding pathways are resolved as transitions between patterns of locally encoded structural signals that change within the 10 μs-100 ms time scale range. These coarse folding pathways are generated by the periodic search for structural patterns in the time-evolving LTM. Each pattern is recorded as a contact matrix, an operation subject to a renormalization feedback loop. The validity of our approach is tested vis-a-vis experimentally-probed folding pathways eventually generating tertiary interactions in proteins which recover their active structure under in vitro renaturation conditions. As an illustration, we focus on determining significant folding intermediates and late kinetic bottlenecks that occur within the

  1. Protein GB1 Folding and Assembly from Structural Elements

    PubMed Central

    Bauer, Mikael C.; Xue, Wei-Feng; Linse, Sara

    2009-01-01

    Folding of the Protein G B1 domain (PGB1) shifts with increasing salt concentration from a cooperative assembly of inherently unstructured subdomains to an assembly of partly pre-folded structures. The salt-dependence of pre-folding contributes to the stability minimum observed at physiological salt conditions. Our conclusions are based on a study in which the reconstitution of PGB1 from two fragments was studied as a function of salt concentrations and temperature using circular dichroism spectroscopy. Salt was found to induce an increase in β-hairpin structure for the C-terminal fragment (residues 41 – 56), whereas no major salt effect on structure was observed for the isolated N-terminal fragment (residues 1 – 41). In line with the increasing evidence on the interrelation between fragment complementation and stability of the corresponding intact protein, we also find that salt effects on reconstitution can be predicted from salt dependence of the stability of the intact protein. Our data show that our variant (which has the mutations T2Q, N8D, N37D and reconstitutes in a manner similar to the wild type) displays the lowest equilibrium association constant around physiological salt concentration, with higher affinity observed both at lower and higher salt concentration. This corroborates the salt effects on the stability towards denaturation of the intact protein, for which the stability at physiological salt is lower compared to both lower and higher salt concentrations. Hence we conclude that reconstitution reports on molecular factors that govern the native states of proteins. PMID:19468325

  2. Common fold in helix–hairpin–helix proteins

    PubMed Central

    Shao, Xuguang; Grishin, Nick V.

    2000-01-01

    Helix–hairpin–helix (HhH) is a widespread motif involved in non-sequence-specific DNA binding. The majority of HhH motifs function as DNA-binding modules, however, some of them are used to mediate protein–protein interactions or have acquired enzymatic activity by incorporating catalytic residues (DNA glycosylases). From sequence and structural analysis of HhH-containing proteins we conclude that most HhH motifs are integrated as a part of a five-helical domain, termed (HhH)2 domain here. It typically consists of two consecutive HhH motifs that are linked by a connector helix and displays pseudo-2-fold symmetry. (HhH)2 domains show clear structural integrity and a conserved hydrophobic core composed of seven residues, one residue from each α-helix and each hairpin, and deserves recognition as a distinct protein fold. In addition to known HhH in the structures of RuvA, RadA, MutY and DNA-polymerases, we have detected new HhH motifs in sterile alpha motif and barrier-to-autointegration factor domains, the α-subunit of Escherichia coli RNA-polymerase, DNA-helicase PcrA and DNA glyco­s­y­lases. Statistically significant sequence similarity of HhH motifs and pronounced structural conservation argue for homology between (HhH)2 domains in different protein families. Our analysis helps to clarify how non-symmetric protein motifs bind to the double helix of DNA through the formation of a pseudo-2-fold symmetric (HhH)2 functional unit. PMID:10908318

  3. Energetic Frustrations in Protein Folding at Residue Resolution: A Homologous Simulation Study of Im9 Proteins

    PubMed Central

    Sun, Yunxiang; Ming, Dengming

    2014-01-01

    Energetic frustration is becoming an important topic for understanding the mechanisms of protein folding, which is a long-standing big biological problem usually investigated by the free energy landscape theory. Despite the significant advances in probing the effects of folding frustrations on the overall features of protein folding pathways and folding intermediates, detailed characterizations of folding frustrations at an atomic or residue level are still lacking. In addition, how and to what extent folding frustrations interact with protein topology in determining folding mechanisms remains unclear. In this paper, we tried to understand energetic frustrations in the context of protein topology structures or native-contact networks by comparing the energetic frustrations of five homologous Im9 alpha-helix proteins that share very similar topology structures but have a single hydrophilic-to-hydrophobic mutual mutation. The folding simulations were performed using a coarse-grained Gō-like model, while non-native hydrophobic interactions were introduced as energetic frustrations using a Lennard-Jones potential function. Energetic frustrations were then examined at residue level based on φ-value analyses of the transition state ensemble structures and mapped back to native-contact networks. Our calculations show that energetic frustrations have highly heterogeneous influences on the folding of the four helices of the examined structures depending on the local environment of the frustration centers. Also, the closer the introduced frustration is to the center of the native-contact network, the larger the changes in the protein folding. Our findings add a new dimension to the understanding of protein folding the topology determination in that energetic frustrations works closely with native-contact networks to affect the protein folding. PMID:24498176

  4. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  5. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  6. Coarse semiempirical solution to the protein folding problem

    NASA Astrophysics Data System (ADS)

    Fernández, Ariel; Colubri, Andrés; Appignanesi, Gustavo; Burastero, Teresita

    2001-04-01

    We introduce a semiempirical theory leading to the ab initio prediction of conducive folding pathways and coarsely resolved native backbone geometries of proteins suddenly exposed to in vitro renaturation conditions. The underlying model incorporates a discrete codification of local steric hindrances of the peptide backbone. We first determine a time-evolving finite set of local torsional constraints upon which large-scale organization is built. Thus, the torsional state of the chain is topologically represented by viewing the ( Φ, Ψ)-state of each residue modulo the basin of attraction to which it belongs in the Ramachandran plot. A grammar to combine such coarsely defined torsional states (topologies) and translate them into meaningful patterns of long-range interactions is developed. An algorithm for structure prediction is shown to emerge once this grammar is combined with prescriptions for the time evolution of topological patterns. This algorithm is rooted in the fact that local contributions to the potential energy may be subsumed into time-evolving conformational constraints coarsely defining sets of restricted backbone geometries responsible for framing the patterns of nonbonded interactions. The predictive power of the algorithm is established by obtaining stable topologies of small proteins, which prove to be compatible with their native folds, and computing ab-initio folding pathways for mammalian ubiquitin that ultimately yield a stable structural pattern reproducing its native features.

  7. Coupled Protein Diffusion and Folding in the Cell

    PubMed Central

    Guo, Minghao; Gelman, Hannah; Gruebele, Martin

    2014-01-01

    When a protein unfolds in the cell, its diffusion coefficient is affected by its increased hydrodynamic radius and by interactions of exposed hydrophobic residues with the cytoplasmic matrix, including chaperones. We characterize protein diffusion by photobleaching whole cells at a single point, and imaging the concentration change of fluorescent-labeled protein throughout the cell as a function of time. As a folded reference protein we use green fluorescent protein. The resulting region-dependent anomalous diffusion is well characterized by 2-D or 3-D diffusion equations coupled to a clustering algorithm that accounts for position-dependent diffusion. Then we study diffusion of a destabilized mutant of the enzyme phosphoglycerate kinase (PGK) and of its stable control inside the cell. Unlike the green fluorescent protein control's diffusion coefficient, PGK's diffusion coefficient is a non-monotonic function of temperature, signaling ‘sticking’ of the protein in the cytosol as it begins to unfold. The temperature-dependent increase and subsequent decrease of the PGK diffusion coefficient in the cytosol is greater than a simple size-scaling model suggests. Chaperone binding of the unfolding protein inside the cell is one plausible candidate for even slower diffusion of PGK, and we test the plausibility of this hypothesis experimentally, although we do not rule out other candidates. PMID:25436502

  8. 1.6 A crystal structure of the secreted chorismate mutase from Mycobacterium tuberculosis: novel fold topology revealed.

    PubMed

    Okvist, Mats; Dey, Raja; Sasso, Severin; Grahn, Elin; Kast, Peter; Krengel, Ute

    2006-04-14

    The presence of exported chorismate mutases produced by certain organisms such as Mycobacterium tuberculosis has been shown to correlate with their pathogenicity. As such, these proteins comprise a new group of promising selective drug targets. Here, we report the high-resolution crystal structure of the secreted dimeric chorismate mutase from M. tuberculosis (*MtCM; encoded by Rv1885c), which represents the first 3D-structure of a member of this chorismate mutase family, termed the AroQ(gamma) subclass. Structures are presented both for the unliganded enzyme and for a complex with a transition state analog. The protomer fold resembles the structurally characterized (dimeric) Escherichia coli chorismate mutase domain, but exhibits a new topology, with helix H4 of *MtCM carrying the catalytic site residue missing in the shortened helix H1. Furthermore, the structure of each *MtCM protomer is significantly more compact and only harbors one active site pocket, which is formed entirely by one polypeptide chain. Apart from the structural model, we present evidence as to how the substrate may enter the active site.

  9. Predictive energy landscapes for folding membrane protein assemblies

    NASA Astrophysics Data System (ADS)

    Truong, Ha H.; Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.

    2015-12-01

    We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na+-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

  10. Asparagine-linked glycosylation of human chymotrypsin C (CTRC) is required for folding and secretion but not for enzyme activity

    PubMed Central

    Bence, Melinda; Sahin-Tóth, Miklós

    2011-01-01

    SUMMARY Human chymotrypsin C (CTRC) plays a protective role in the pancreas by mitigating premature trypsinogen activation through degradation. Mutations that abolish activity or secretion of CTRC increase the risk for chronic pancreatitis. The aim of the present study was to determine whether human CTRC undergoes asparagine-linked (N-linked) glycosylation and to examine the role of this modification in CTRC folding and function. We abolished potential sites of N-linked glycosylation (Asn-Xaa-Ser/Thr) in human CTRC by mutating the Asn residues to Ser individually or in combination, expressed the CTRC mutants in HEK 293T cells and determined their glycosylation state using PNGase F and endo H digestion. We found that human CTRC contains a single N-linked glycan on Asn52. Elimination of N-glycosylation by mutation of Asn52 (N52S) reduced CTRC secretion about 10-fold from HEK 293T cells but had no effect on CTRC activity or inhibitor binding. Overexpression of the N52S CTRC mutant elicited endoplasmic reticulum stress in AR42J acinar cells, indicating that N-glycosylation is required for folding of human CTRC. Despite its important role, Asn52 is poorly conserved in other mammalian CTRC orthologs, including the rat which is monoglycosylated on Asn90. Introduction of the Asn90 site in a non-glycosylated human CTRC mutant restored full glycosylation but only partially rescued the secretion defect. We conclude that N-linked glycosylation of human CTRC is required for efficient folding and secretion, however, the N-linked glycan is unimportant for enzyme activity or inhibitor binding. The position of the N-linked glycan is critical for optimal folding, and it may vary among the otherwise highly homologous mammalian CTRC sequences. PMID:21920023

  11. A Secretion-Amplification Role for Salmonella enterica Translocon Protein SipD.

    PubMed

    Glasgow, Anum Azam; Wong, Han Teng; Tullman-Ercek, Danielle

    2017-03-16

    The bacterial type III secretion system (T3SS) is an important target for enabling high-titer production of proteins of biotechnological interest as well as for synthetic biology applications that rely on protein delivery to host cells. The T3SS forms a membrane-embedded needle complex that is capped by the translocon proteins and extends into the extracellular space. The needle tip complex in Salmonella enterica consists of three translocon proteins: SipB, SipC, and SipD. It is known that knocking out sipD disrupts T3SS regulation to cause constitutive secretion of native proteins. However, we discovered that complementation of SipD in trans via exogenous addition to T3SS-expressing cultures further improves heterologous protein secretion titers, suggesting a previously unknown but important role for this protein. Building on this knowledge, we have engineered a hyper-secreting strain of S. enterica for a greater than 100-fold improvement in the production of a variety of biotechnologically valuable heterologous proteins that are challenging to produce, such as toxic antimicrobial peptides and proteolysis-prone biopolymer proteins. We determined that transcription by several T3SS promoters is upregulated with the addition of SipD, that the N-terminal domain of SipD is sufficient to observe the increased secretion phenotype, and that the effect is post-transcriptional and post-translational. These results lend support to the use of bacterial secretion as a powerful protein production strategy, and the hypothesis that translocon proteins contribute to type III secretion regulation.

  12. Consistency in structural energetics of protein folding and peptide recognition.

    PubMed Central

    Zhang, C.; Cornette, J. L.; Delisi, C.

    1997-01-01

    We report a new free energy decomposition that includes structure-derived atomic contact energies for the desolvation component, and show that it applies equally well to the analysis of single-domain protein folding and to the binding of flexible peptides to proteins. Specifically, we selected the 17 single-domain proteins for which the three-dimensional structures and thermodynamic unfolding free energies are available. By calculating all terms except the backbone conformational entropy change and comparing the result to the experimentally measured free energy, we estimated that the mean entropy gain by the backbone chain upon unfolding (delta Sbb) is 5.3 cal/K per mole of residue, and that the average backbone entropy for glycine is 6.7 cal/K. Both numbers are in close agreement with recent estimates made by entirely different methods, suggesting a promising degree of consistency between data obtained from disparate sources. In addition, a quantitative analysis of the folding free energy indicates that the unfavorable backbone entropy for each of the proteins is balanced predominantly by favorable backbone interactions. Finally, because the binding of flexible peptides to receptors is physically similar to folding, the free energy function should, in principle, be equally applicable to flexible docking. By combining atomic contact energies, electrostatics, and sequence-dependent backbone entropy, we calculated a priori the free energy changes associated with the binding of four different peptides to HLA-A2, 1 MHC molecule and found agreement with experiment to within 10% without parameter adjustment. PMID:9144777

  13. Sec-Secretion and Sortase-Mediated Anchoring of Proteins in Gram-Postive Bacteria

    PubMed Central

    Schneewind, Olaf; Missiakas, Dominique

    2014-01-01

    Signal peptide-driven secretion of precursor proteins directs polypeptides across the plasma membrane of bacteria. Two pathways, Sec- and SRP-dependent, converge at the SecYEG translocon to thread unfolded precursor proteins across the membrane, whereas folded preproteins are routed via the Tat secretion pathway. Gram-positive bacteria lack an outer membrane and are surrounded by a rigid layer of peptidoglycan. Interactions with their environment are mediated by proteins that are retained in the cell wall, often through covalent attachment to the peptidoglycan. In this review, we describe the mechanisms for both Sec-dependent secretion and sortase-dependent assembly of proteins in the envelope of Gram-positive bacteria. PMID:24269844

  14. Effect of protein backbone folding on the stability of protein-ligand complexes.

    PubMed

    Estrada, Ernesto; Uriarte, Eugenio; Vilar, Santiago

    2006-01-01

    The role played by the degree of folding of protein backbones in explaining the binding energetics of protein-ligand interactions has been studied. We analyzed the protein/peptide interactions in the RNase-S system in which amino acids at two positions of the peptide S have been mutated. The global degree of folding of the protein S correlates in a significant way with the free energy and enthalpy of the protein-peptide interactions. A much better correlation is found with the local contribution to the degree of folding of one amino acid residue: Thr36. This residue is shown to have a destabilizing interaction with Lys41, which interacts directly with peptide S. Another system, consisting of the interactions of small organic molecules with HIV-1 protease was also studied. In this case, the global change in the degree of folding of the protease backbone does not explain the binding energetics of protein-ligand interactions. However, a significant correlation is observed between the free energy of binding and the contribution of two amino acid residues in the HVI-1 protease: Gly49 and Ile66. In general, it was observed that the changes in the degree of folding are not restricted to the binding site of the protein chain but are distributed along the whole protein backbone. This study provides a basis for further consideration of the degree of folding as a parameter for empirical structural parametrizations of the binding energetics of protein folding and binding.

  15. Accurate prediction of cellular co-translational folding indicates proteins can switch from post- to co-translational folding

    PubMed Central

    Nissley, Daniel A.; Sharma, Ajeet K.; Ahmed, Nabeel; Friedrich, Ulrike A.; Kramer, Günter; Bukau, Bernd; O'Brien, Edward P.

    2016-01-01

    The rates at which domains fold and codons are translated are important factors in determining whether a nascent protein will co-translationally fold and function or misfold and malfunction. Here we develop a chemical kinetic model that calculates a protein domain's co-translational folding curve during synthesis using only the domain's bulk folding and unfolding rates and codon translation rates. We show that this model accurately predicts the course of co-translational folding measured in vivo for four different protein molecules. We then make predictions for a number of different proteins in yeast and find that synonymous codon substitutions, which change translation-elongation rates, can switch some protein domains from folding post-translationally to folding co-translationally—a result consistent with previous experimental studies. Our approach explains essential features of co-translational folding curves and predicts how varying the translation rate at different codon positions along a transcript's coding sequence affects this self-assembly process. PMID:26887592

  16. Mapping fast protein folding with multiple-site fluorescent probes.

    PubMed

    Prigozhin, Maxim B; Chao, Shu-Han; Sukenik, Shahar; Pogorelov, Taras V; Gruebele, Martin

    2015-06-30

    Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.

  17. Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

    DOEpatents

    Waldo, Geoffrey S.

    2007-09-18

    The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

  18. Exploring energy landscapes of protein folding and aggregation.

    PubMed

    Mousseau, Normand; Derreumaux, Philippe

    2008-05-01

    Human diseases, such as Alzheimer's and Creutzfeldt-Jakob's are associated with misfolding and aggregation of specific proteins into amyloid fibrils sharing a generic cross-beta structure. The self-assembly process is complex, but once a nucleus is formed, rapid fibril formation occurs. Insight into the structures of the oligomers during the lag phase, varying between hours and days, is very difficult experimentally because these species are transient, and numerically using all-atom molecular dynamics because the time scale explored is on the order of 10-100 ns. It is therefore important to develop simplified protein models and alternative methods to sample more efficiently the conformational space. In the past few years, we have developed the activation-relaxation technique (ART nouveau) coupled to the OPEP coarse-grained force field. This review reports the application of ART-OPEP on protein folding and aggregation.

  19. Dynamic Complexes in the Chaperonin-Mediated Protein Folding Cycle

    PubMed Central

    Weiss, Celeste; Jebara, Fady; Nisemblat, Shahar; Azem, Abdussalam

    2016-01-01

    The GroEL–GroES chaperonin system is probably one of the most studied chaperone systems at the level of the molecular mechanism. Since the first reports of a bacterial gene involved in phage morphogenesis in 1972, these proteins have stimulated intensive research for over 40 years. During this time, detailed structural and functional studies have yielded constantly evolving concepts of the chaperonin mechanism of action. Despite of almost three decades of research on this oligomeric protein, certain aspects of its function remain controversial. In this review, we highlight one central aspect of its function, namely, the active intermediates of its reaction cycle, and present how research to this day continues to change our understanding of chaperonin-mediated protein folding. PMID:28008398

  20. Identification and characterization of secreted proteins in Eimeria tenella

    NASA Astrophysics Data System (ADS)

    Ramlee, Intan Azlinda; Firdaus-Raih, Mohd; Wan, Kiew-Lian

    2015-09-01

    Eimeria tenella is a protozoan parasite that causes coccidiosis, an economically important disease in the poultry industry. The characterization of proteins that are secreted by parasites have been shown to play important roles in parasite invasion and are considered to be potential control agents. In this study, 775 proteins potentially secreted by E. tenella were identified. These proteins were further filtered to remove mitochondrial proteins. Out of 763 putative secreted proteins, 259 proteins possess transmembrane domains while another 150 proteins have GPI (Glycosylphosphatidylinositol) anchors. Homology search revealed that 315 and 448 proteins have matches with known and hypothetical proteins in the database, respectively. Within this data set, previously characterized secretory proteins such as micronemes, rhoptry kinases and dense granules were detected.

  1. Type V Protein Secretion Pathway: the Autotransporter Story

    PubMed Central

    Henderson, Ian R.; Navarro-Garcia, Fernando; Desvaux, Mickaël; Fernandez, Rachel C.; Ala'Aldeen, Dlawer

    2004-01-01

    Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function. PMID:15590781

  2. Protein folding in the cell envelope of Escherichia coli.

    PubMed

    De Geyter, Jozefien; Tsirigotaki, Alexandra; Orfanoudaki, Georgia; Zorzini, Valentina; Economou, Anastassios; Karamanou, Spyridoula

    2016-07-26

    While the entire proteome is synthesized on cytoplasmic ribosomes, almost half associates with, localizes in or crosses the bacterial cell envelope. In Escherichia coli a variety of mechanisms are important for taking these polypeptides into or across the plasma membrane, maintaining them in soluble form, trafficking them to their correct cell envelope locations and then folding them into the right structures. The fidelity of these processes must be maintained under various environmental conditions including during stress; if this fails, proteases are called in to degrade mislocalized or aggregated proteins. Various soluble, diffusible chaperones (acting as holdases, foldases or pilotins) and folding catalysts are also utilized to restore proteostasis. These responses can be general, dealing with multiple polypeptides, with functional overlaps and operating within redundant networks. Other chaperones are specialized factors, dealing only with a few exported proteins. Several complex machineries have evolved to deal with binding to, integration in and crossing of the outer membrane. This complex protein network is responsible for fundamental cellular processes such as cell wall biogenesis; cell division; the export, uptake and degradation of molecules; and resistance against exogenous toxic factors. The underlying processes, contributing to our fundamental understanding of proteostasis, are a treasure trove for the development of novel antibiotics, biopharmaceuticals and vaccines.

  3. Nonequilibrium single molecule protein folding in a coaxial mixer.

    PubMed

    Hamadani, Kambiz M; Weiss, Shimon

    2008-07-01

    We have developed a continuous-flow mixing device suitable for monitoring bioconformational reactions at the single-molecule level with a response time of approximately 10 ms under single-molecule flow conditions. Its coaxial geometry allows three-dimensional hydrodynamic focusing of sample fluids to diffraction-limited dimensions where diffusional mixing is rapid and efficient. The capillary-based design enables rapid in-lab construction of mixers without the need for expensive lithography-based microfabrication facilities. In-line filtering of sample fluids using granulated silica particles virtually eliminates clogging and extends the lifetime of each device to many months. In this article, to determine both the distance-to-time transfer function and the instrument response function of the device we characterize its fluid flow and mixing properties using both fluorescence cross-correlation spectroscopy velocimetry and finite element fluid dynamics simulations. We then apply the mixer to single molecule FRET protein folding studies of Chymotrypsin Inhibitor protein 2. By transiently populating the unfolded state of Chymotrypsin Inhibitor Protein 2 (CI2) under nonequilibrium in vitro refolding conditions, we spatially and temporally resolve the denaturant-dependent nonspecific collapse of the unfolded state from the barrier-limited folding transition of CI2. Our results are consistent with previous CI2 mixing results that found evidence for a heterogeneous unfolded state consisting of cis- and trans-proline conformers.

  4. Two-Partner Secretion: Combining Efficiency and Simplicity in the Secretion of Large Proteins for Bacteria-Host and Bacteria-Bacteria Interactions.

    PubMed

    Guérin, Jeremy; Bigot, Sarah; Schneider, Robert; Buchanan, Susan K; Jacob-Dubuisson, Françoise

    2017-01-01

    Initially identified in pathogenic Gram-negative bacteria, the two-partner secretion (TPS) pathway, also known as Type Vb secretion, mediates the translocation across the outer membrane of large effector proteins involved in interactions between these pathogens and their hosts. More recently, distinct TPS systems have been shown to secrete toxic effector domains that participate in inter-bacterial competition or cooperation. The effects of these systems are based on kin vs. non-kin molecular recognition mediated by specific immunity proteins. With these new toxin-antitoxin systems, the range of TPS effector functions has thus been extended from cytolysis, adhesion, and iron acquisition, to genome maintenance, inter-bacterial killing and inter-bacterial signaling. Basically, a TPS system is made up of two proteins, the secreted TpsA effector protein and its TpsB partner transporter, with possible additional factors such as immunity proteins for protection against cognate toxic effectors. Structural studies have indicated that TpsA proteins mainly form elongated β helices that may be followed by specific functional domains. TpsB proteins belong to the Omp85 superfamily. Open questions remain on the mechanism of protein secretion in the absence of ATP or an electrochemical gradient across the outer membrane. The remarkable dynamics of the TpsB transporters and the progressive folding of their TpsA partners at the bacterial surface in the course of translocation are thought to be key elements driving the secretion process.

  5. Mapping the Geometric Evolution of Protein Folding Motor.

    PubMed

    Jerath, Gaurav; Hazam, Prakash Kishore; Shekhar, Shashi; Ramakrishnan, Vibin

    2016-01-01

    Polypeptide chain has an invariant main-chain and a variant side-chain sequence. How the side-chain sequence determines fold in terms of its chemical constitution has been scrutinized extensively and verified periodically. However, a focussed investigation on the directive effect of side-chain geometry may provide important insights supplementing existing algorithms in mapping the geometrical evolution of protein chains and its structural preferences. Geometrically, folding of protein structure may be envisaged as the evolution of its geometric variables: ϕ, and ψ dihedral angles of polypeptide main-chain directed by χ1, and χ2 of side chain. In this work, protein molecule is metaphorically modelled as a machine with 4 rotors ϕ, ψ, χ1 and χ2, with its evolution to the functional fold is directed by combinations of its rotor directions. We observe that differential rotor motions lead to different secondary structure formations and the combinatorial pattern is unique and consistent for particular secondary structure type. Further, we found that combination of rotor geometries of each amino acid is unique which partly explains how different amino acid sequence combinations have unique structural evolution and functional adaptation. Quantification of these amino acid rotor preferences, resulted in the generation of 3 substitution matrices, which later on plugged in the BLAST tool, for evaluating their efficiency in aligning sequences. We have employed BLOSUM62 and PAM30 as standard for primary evaluation. Generation of substitution matrices is a logical extension of the conceptual framework we attempted to build during the development of this work. Optimization of matrices following the conventional routines and possible application with biologically relevant data sets are beyond the scope of this manuscript, though it is a part of the larger project design.

  6. Mapping the Geometric Evolution of Protein Folding Motor

    PubMed Central

    Hazam, Prakash Kishore; Shekhar, Shashi

    2016-01-01

    Polypeptide chain has an invariant main-chain and a variant side-chain sequence. How the side-chain sequence determines fold in terms of its chemical constitution has been scrutinized extensively and verified periodically. However, a focussed investigation on the directive effect of side-chain geometry may provide important insights supplementing existing algorithms in mapping the geometrical evolution of protein chains and its structural preferences. Geometrically, folding of protein structure may be envisaged as the evolution of its geometric variables: ϕ, and ψ dihedral angles of polypeptide main-chain directed by χ1, and χ2 of side chain. In this work, protein molecule is metaphorically modelled as a machine with 4 rotors ϕ, ψ, χ1 and χ2, with its evolution to the functional fold is directed by combinations of its rotor directions. We observe that differential rotor motions lead to different secondary structure formations and the combinatorial pattern is unique and consistent for particular secondary structure type. Further, we found that combination of rotor geometries of each amino acid is unique which partly explains how different amino acid sequence combinations have unique structural evolution and functional adaptation. Quantification of these amino acid rotor preferences, resulted in the generation of 3 substitution matrices, which later on plugged in the BLAST tool, for evaluating their efficiency in aligning sequences. We have employed BLOSUM62 and PAM30 as standard for primary evaluation. Generation of substitution matrices is a logical extension of the conceptual framework we attempted to build during the development of this work. Optimization of matrices following the conventional routines and possible application with biologically relevant data sets are beyond the scope of this manuscript, though it is a part of the larger project design. PMID:27716851

  7. An intrinsic mechanism of secreted protein aging and turnover.

    PubMed

    Yang, Won Ho; Aziz, Peter V; Heithoff, Douglas M; Mahan, Michael J; Smith, Jeffrey W; Marth, Jamey D

    2015-11-03

    The composition and functions of the secreted proteome are controlled by the life spans of different proteins. However, unlike intracellular protein fate, intrinsic factors determining secreted protein aging and turnover have not been identified and characterized. Almost all secreted proteins are posttranslationally modified with the covalent attachment of N-glycans. We have discovered an intrinsic mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosaminidase B (HexB), possess hydrolytic activities that temporally remodel N-glycan structures, progressively exposing different saccharides with increased protein age. Subsequently, endocytic lectins with distinct binding specificities, including the Ashwell-Morell receptor, integrin αM, and macrophage mannose receptor, are engaged in N-glycan ligand recognition and the turnover of secreted proteins. Glycosidase inhibition and lectin deficiencies increased protein life spans and abundance, and the basal rate of N-glycan remodeling varied among distinct proteins, accounting for differences in their life spans. This intrinsic multifactorial mechanism of secreted protein aging and turnover contributes to health and the outcomes of disease.

  8. An intrinsic mechanism of secreted protein aging and turnover

    PubMed Central

    Yang, Won Ho; Aziz, Peter V.; Heithoff, Douglas M.; Mahan, Michael J.; Smith, Jeffrey W.; Marth, Jamey D.

    2015-01-01

    The composition and functions of the secreted proteome are controlled by the life spans of different proteins. However, unlike intracellular protein fate, intrinsic factors determining secreted protein aging and turnover have not been identified and characterized. Almost all secreted proteins are posttranslationally modified with the covalent attachment of N-glycans. We have discovered an intrinsic mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosaminidase B (HexB), possess hydrolytic activities that temporally remodel N-glycan structures, progressively exposing different saccharides with increased protein age. Subsequently, endocytic lectins with distinct binding specificities, including the Ashwell–Morell receptor, integrin αM, and macrophage mannose receptor, are engaged in N-glycan ligand recognition and the turnover of secreted proteins. Glycosidase inhibition and lectin deficiencies increased protein life spans and abundance, and the basal rate of N-glycan remodeling varied among distinct proteins, accounting for differences in their life spans. This intrinsic multifactorial mechanism of secreted protein aging and turnover contributes to health and the outcomes of disease. PMID:26489654

  9. Microsecond Microfluidic Mixing for Investigation of Protein Folding Kinetics

    SciTech Connect

    Hertzog, D E; Santiago, J G; Bakajin, O

    2005-02-10

    We have developed and characterized a mixer to study the reaction kinetics of protein folding on a microsecond timescale. The mixer uses hydrodynamic focusing of pressure-driven flow in a microfluidic channel to reduce diffusion times as first demonstrated by Knight et al.[1]. Features of the mixer include 1 {micro}s mixing times, sample consumptions of order 1 nl/s, loading sample volumes on the order of microliters, and the ability to manufacture in fused silica for compatibility with most spectroscopic methods.

  10. Microsecond Microfluidic Mixing for Investigation of Protein Folding Kinetics

    SciTech Connect

    Hertzog, D E; Santiago, J G; Bakajin, O

    2003-06-25

    We have developed and characterized a mixer to study the reaction kinetics of protein folding on a microsecond timescale. The mixer uses hydrodynamic focusing of pressure-driven flow in a microfluidic channel to reduce diffusion times as first demonstrated by Knight et al.[1]. Features of the mixer include 1 {micro}s mixing times, sample consumptions of order 1 nl/s, loading sample volumes on the order of microliters, and the ability to manufacture in fused silica for compatibility with most spectroscopic methods.

  11. Heuristic algorithm for off-lattice protein folding problem*

    PubMed Central

    Chen, Mao; Huang, Wen-qi

    2006-01-01

    Enlightened by the law of interactions among objects in the physical world, we propose a heuristic algorithm for solving the three-dimensional (3D) off-lattice protein folding problem. Based on a physical model, the problem is converted from a nonlinear constraint-satisfied problem to an unconstrained optimization problem which can be solved by the well-known gradient method. To improve the efficiency of our algorithm, a strategy was introduced to generate initial configuration. Computational results showed that this algorithm could find states with lower energy than previously proposed ground states obtained by nPERM algorithm for all chains with length ranging from 13 to 55. PMID:16365919

  12. Dynamic Folding Pathway Models of the Trp-Cage Protein

    PubMed Central

    Kim, Seung-Yeon

    2013-01-01

    Using action-derived molecular dynamics (ADMD), we study the dynamic folding pathway models of the Trp-cage protein by providing its sequential conformational changes from its initial disordered structure to the final native structure at atomic details. We find that the numbers of native contacts and native hydrogen bonds are highly correlated, implying that the native structure of Trp-cage is achieved through the concurrent formations of native contacts and native hydrogen bonds. In early stage, an unfolded state appears with partially formed native contacts (~40%) and native hydrogen bonds (~30%). Afterward, the folding is initiated by the contact of the side chain of Tyr3 with that of Trp6, together with the formation of the N-terminal α-helix. Then, the C-terminal polyproline structure docks onto the Trp6 and Tyr3 rings, resulting in the formations of the hydrophobic core of Trp-cage and its near-native state. Finally, the slow adjustment processes of the near-native states into the native structure are dominant in later stage. The ADMD results are in agreement with those of the experimental folding studies on Trp-cage and consistent with most of other computational studies. PMID:23865078

  13. Exploration of twin‐arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

    PubMed Central

    Fisher, Adam C.; Kim, Jae‐Young; Perez‐Rodriguez, Ritsdeliz; Tullman‐Ercek, Danielle; Fish, Wallace R.; Henderson, Lee A.; DeLisa, Matthew P.

    2008-01-01

    Summary Historically, the general secretory (Sec) pathway of Gram‐negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin‐arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N‐termini upon reaching the periplasm and (iii) proteins fused to maltose‐binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well‐folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step. PMID:21261860

  14. Water mediation in protein folding and molecular recognition.

    PubMed

    Levy, Yaakov; Onuchic, José N

    2006-01-01

    Water is essential for life in many ways, and without it biomolecules might no longer truly be biomolecules. In particular, water is important to the structure, stability, dynamics, and function of biological macromolecules. In protein folding, water mediates the collapse of the chain and the search for the native topology through a funneled energy landscape. Water actively participates in molecular recognition by mediating the interactions between binding partners and contributes to either enthalpic or entropic stabilization. Accordingly, water must be included in recognition and structure prediction codes to capture specificity. Thus water should not be treated as an inert environment, but rather as an integral and active component of biomolecular systems, where it has both dynamic and structural roles. Focusing on water sheds light on the physics and function of biological machinery and self-assembly and may advance our understanding of the natural design of proteins and nucleic acids.

  15. Energy optimization for off-lattice protein folding.

    PubMed

    Huang, Wenqi; Chen, Mao; Lü, Zhipeng

    2006-10-01

    Two three-dimensional AB off-lattice protein models consisting of hydrophobic and hydrophilic monomers are studied in this paper. By incorporating an extra energy contribution into the original energy function, the protein folding is converted from a constraint optimization problem into an unconstrained one which can be solved by the well-known gradient method. From the initial configurations randomly generated by the heuristic strategy proposed in this paper, our algorithm can find better results than those by nPERM for the four Fibonacci sequences. Based on the initial configurations obtained by energy landscape paving (ELP) routine, some of our results for the lowest energies are better than the best values reported in the literature.

  16. Species-specific protein sequence and fold optimizations

    PubMed Central

    Dumontier, Michel; Michalickova, Katerina; Hogue, Christopher WV

    2002-01-01

    Background An organism's ability to adapt to its particular environmental niche is of fundamental importance to its survival and proliferation. In the largest study of its kind, we sought to identify and exploit the amino-acid signatures that make species-specific protein adaptation possible across 100 complete genomes. Results Environmental niche was determined to be a significant factor in variability from correspondence analysis using the amino acid composition of over 360,000 predicted open reading frames (ORFs) from 17 archae, 76 bacteria and 7 eukaryote complete genomes. Additionally, we found clusters of phylogenetically unrelated archae and bacteria that share similar environments by amino acid composition clustering. Composition analyses of conservative, domain-based homology modeling suggested an enrichment of small hydrophobic residues Ala, Gly, Val and charged residues Asp, Glu, His and Arg across all genomes. However, larger aromatic residues Phe, Trp and Tyr are reduced in folds, and these results were not affected by low complexity biases. We derived two simple log-odds scoring functions from ORFs (CG) and folds (CF) for each of the complete genomes. CF achieved an average cross-validation success rate of 85 ± 8% whereas the CG detected 73 ± 9% species-specific sequences when competing against all other non-redundant CG. Continuously updated results are available at . Conclusion Our analysis of amino acid compositions from the complete genomes provides stronger evidence for species-specific and environmental residue preferences in genomic sequences as well as in folds. Scoring functions derived from this work will be useful in future protein engineering experiments and possibly in identifying horizontal transfer events. PMID:12487631

  17. Thermodynamics of folding and association of lattice-model proteins

    NASA Astrophysics Data System (ADS)

    Cellmer, Troy; Bratko, Dusan; Prausnitz, John M.; Blanch, Harvey

    2005-05-01

    Closely related to the "protein folding problem" is the issue of protein misfolding and aggregation. Protein aggregation has been associated with the pathologies of nearly 20 human diseases and presents serious difficulties during the manufacture of pharmaceutical proteins. Computational studies of multiprotein systems have recently emerged as a powerful complement to experimental efforts aimed at understanding the mechanisms of protein aggregation. We describe the thermodynamics of systems containing two lattice-model 64-mers. A parallel tempering algorithm abates problems associated with glassy systems and the weighted histogram analysis method improves statistical quality. The presence of a second chain has a substantial effect on single-chain conformational preferences. The melting temperature is substantially reduced, and the increase in the population of unfolded states is correlated with an increase in interactions between chains. The transition from two native chains to a non-native aggregate is entropically favorable. Non-native aggregates receive ˜25% of their stabilizing energy from intraprotein contacts not found in the lowest-energy structure. Contact maps show that for non-native dimers, nearly 50% of the most probable interprotein contacts involve pairs of residues that form native contacts, suggesting that a domain-swapping mechanism is involved in self-association.

  18. Methyl Transfer by Substrate Signaling from a Knotted Protein Fold

    PubMed Central

    Christian, Thomas; Sakaguchi, Reiko; Perlinska, Agata P.; Lahoud, Georges; Ito, Takuhiro; Taylor, Erika A.; Yokoyama, Shigeyuki; Sulkowska, Joanna I.; Hou, Ya-Ming

    2017-01-01

    Proteins with knotted configurations are restricted in conformational space relative to unknotted proteins. Little is known if knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. In bacteria, TrmD is a methyl transferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product m1G37-tRNA is essential for life as a determinant to maintain protein synthesis reading-frame. Using an integrated approach of structure, kinetic, and computational analysis, we show here that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot has complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding to stabilize tRNA binding and to assemble the active site. This work demonstrates new principles of knots as an organized structure that captures the free energies of substrate binding to facilitate catalysis. PMID:27571175

  19. Expanding the proteome: disordered and alternatively folded proteins.

    PubMed

    Dyson, H Jane

    2011-11-01

    Proteins provide much of the scaffolding for life, as well as undertaking a variety of essential catalytic reactions. These characteristic functions have led us to presuppose that proteins are in general functional only when well structured and correctly folded. As we begin to explore the repertoire of possible protein sequences inherent in the human and other genomes, two stark facts that belie this supposition become clear: firstly, the number of apparent open reading frames in the human genome is significantly smaller than appears to be necessary to code for all of the diverse proteins in higher organisms, and secondly that a significant proportion of the protein sequences that would be coded by the genome would not be expected to form stable three-dimensional (3D) structures. Clearly the genome must include coding for a multitude of alternative forms of proteins, some of which may be partly or fully disordered or incompletely structured in their functional states. At the same time as this likelihood was recognized, experimental studies also began to uncover examples of important protein molecules and domains that were incompletely structured or completely disordered in solution, yet remained perfectly functional. In the ensuing years, we have seen an explosion of experimental and genome-annotation studies that have mapped the extent of the intrinsic disorder phenomenon and explored the possible biological rationales for its widespread occurrence. Answers to the question 'why would a particular domain need to be unstructured?' are as varied as the systems where such domains are found. This review provides a survey of recent new directions in this field, and includes an evaluation of the role not only of intrinsically disordered proteins but also of partially structured and highly dynamic members of the disorder-order continuum.

  20. Expanding the proteome: disordered and alternatively-folded proteins

    PubMed Central

    Dyson, H. Jane

    2011-01-01

    Proteins provide much of the scaffolding for life, as well as undertaking a variety of essential catalytic reactions. These characteristic functions have led us to presuppose that proteins are in general functional only when well-structured and correctly folded. As we begin to explore the repertoire of possible protein sequences inherent in the human and other genomes, two stark facts that belie this supposition become clear: firstly, the number of apparent open reading frames in the human genome is significantly smaller than appears to be necessary to code for all of the diverse proteins in higher organisms, and secondly that a significant proportion of the protein sequences that would be coded by the genome would not be expected to form stable three-dimensional structures. Clearly the genome must include coding for a multitude of alternative forms of proteins, some of which may be partly or fully disordered or incompletely structured in their functional states. At the same time as this likelihood was recognized, experimental studies also began to uncover examples of important protein molecules and domains that were incompletely structured or completely disordered in solution, yet remained perfectly functional. In the ensuing years, we have seen an explosion of experimental and genome-annotation studies that have mapped the extent of the intrinsic disorder phenomenon and explored the possible biological rationales for its widespread occurrence. Answers to the question “why would a particular domain need to be unstructured?” are as varied as the systems where such domains are found. This review provides a survey of recent new directions in this field, and includes an evaluation of the role not only of intrinsically disordered proteins but of partially structured and highly dynamic members of the disorder-order continuum. PMID:21729349

  1. Efficient fold-change detection based on protein-protein interactions.

    PubMed

    Buijsman, W; Sheinman, M

    2014-02-01

    Various biological sensory systems exhibit a response to a relative change of the stimulus, often referred to as fold-change detection. In the past few years, fold-change detecting mechanisms, based on transcriptional networks, have been proposed. Here we present a fold-change detecting mechanism, based on protein-protein interactions, consisting of two interacting proteins. This mechanism does not consume chemical energy and is not subject to transcriptional and translational noise, in contrast to previously proposed mechanisms. We show by analytical and numerical calculations that the mechanism is robust and can have a fast, precise, and efficient response for parameters that are relevant to eukaryotic cells.

  2. Efficient fold-change detection based on protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Buijsman, W.; Sheinman, M.

    2014-02-01

    Various biological sensory systems exhibit a response to a relative change of the stimulus, often referred to as fold-change detection. In the past few years, fold-change detecting mechanisms, based on transcriptional networks, have been proposed. Here we present a fold-change detecting mechanism, based on protein-protein interactions, consisting of two interacting proteins. This mechanism does not consume chemical energy and is not subject to transcriptional and translational noise, in contrast to previously proposed mechanisms. We show by analytical and numerical calculations that the mechanism is robust and can have a fast, precise, and efficient response for parameters that are relevant to eukaryotic cells.

  3. Protein knotting through concatenation significantly reduces folding stability

    PubMed Central

    Hsu, Shang-Te Danny

    2016-01-01

    Concatenation by covalent linkage of two protomers of an intertwined all-helical HP0242 homodimer from Helicobacter pylori results in the first example of an engineered knotted protein. While concatenation does not affect the native structure according to X-ray crystallography, the folding kinetics is substantially slower compared to the parent homodimer. Using NMR hydrogen-deuterium exchange analysis, we showed here that concatenation destabilises significantly the knotted structure in solution, with some regions close to the covalent linkage being destabilised by as much as 5 kcal mol−1. Structural mapping of chemical shift perturbations induced by concatenation revealed a pattern that is similar to the effect induced by concentrated chaotrophic agent. Our results suggested that the design strategy of protein knotting by concatenation may be thermodynamically unfavourable due to covalent constrains imposed on the flexible fraying ends of the template structure, leading to rugged free energy landscape with increased propensity to form off-pathway folding intermediates. PMID:27982106

  4. Prediction of protein structure: the problem of fold multiplicity.

    PubMed

    Lomize, A L; Pogozheva, I D; Mosberg, H I

    1999-01-01

    Three-dimensional (3D) models of four CASP3 targets were calculated using a simple modeling procedure that includes prediction of regular secondary structure, analysis of possible beta-sheet topologies, assembly of amphiphilic helices and beta-sheets to bury their nonpolar surfaces, and adjustment of side-chain conformers and loops to provide close packing and saturation of the "hydrogen bond potential" (exposure of all polar groups to water or their involvement in intramolecular hydrogen bonds). It has been found that this approach allows construction of 3D models that, in some cases, properly reproduce the structural class of the protein (such as beta-barrel or beta-sandwich of definite shape and size) and details of tertiary structure (such as pairing of beta-strands), although all four models were more or less incorrect. Remarkably, some models had fewer water-exposed nonpolar side-chains, more hydrogen bonds, and smaller holes than the corresponding native structures (although the models had a larger water-accessible nonpolar surface). The results obtained indicate that hydrophobicity patterns do not unequivocally determine protein folds, and that any ab initio or fold recognition methods that operate with imprecise potential energy functions, or use crude geometrical approximations of the peptide chain, will probably produce many different nonnative structures.

  5. Fold homology detection using sequence fragment composition profiles of proteins.

    PubMed

    Solis, Armando D; Rackovsky, Shalom R

    2010-10-01

    The effectiveness of sequence alignment in detecting structural homology among protein sequences decreases markedly when pairwise sequence identity is low (the so-called "twilight zone" problem of sequence alignment). Alternative sequence comparison strategies able to detect structural kinship among highly divergent sequences are necessary to address this need. Among them are alignment-free methods, which use global sequence properties (such as amino acid composition) to identify structural homology in a rapid and straightforward way. We explore the viability of using tetramer sequence fragment composition profiles in finding structural relationships that lie undetected by traditional alignment. We establish a strategy to recast any given protein sequence into a tetramer sequence fragment composition profile, using a series of amino acid clustering steps that have been optimized for mutual information. Our method has the effect of compressing the set of 160,000 unique tetramers (if using the 20-letter amino acid alphabet) into a more tractable number of reduced tetramers (approximately 15-30), so that a meaningful tetramer composition profile can be constructed. We test remote homology detection at the topology and fold superfamily levels using a comprehensive set of fold homologs, culled from the CATH database that share low pairwise sequence similarity. Using the receiver-operating characteristic measure, we demonstrate potentially significant improvement in using information-optimized reduced tetramer composition, over methods relying only on the raw amino acid composition or on traditional sequence alignment, in homology detection at or below the "twilight zone". 2010 Wiley-Liss, Inc.

  6. Fold Homology Detection Using Sequence Fragment Composition Profiles of Proteins

    PubMed Central

    Solis, Armando D.; Rackovsky, Shalom R.

    2010-01-01

    The effectiveness of sequence alignment in detecting structural homology among protein sequences decreases markedly when pairwise sequence identity is low (the so-called “twilight zone” problem of sequence alignment). Alternative sequence comparison strategies able to detect structural kinship among highly divergent sequences are necessary to address this need. Among them are alignment-free methods, which use global sequence properties (such as amino acid composition) to identify structural homology in a rapid and straightforward way. We explore the viability of using tetramer sequence fragment composition profiles in finding structural relationships that lie undetected by traditional alignment. We establish a strategy to recast any given protein sequence into a tetramer sequence fragment composition profile, using a series of amino acid clustering steps that have been optimized for mutual information. Our method has the effect of compressing the set of 160,000 unique tetramers (if using the 20-letter amino acid alphabet) into a more tractable number of reduced tetramers (around 15 to 30), so that a meaningful tetramer composition profile can be constructed. We test remote homology detection at the topology and fold superfamily levels using a comprehensive set of fold homologs, culled from the CATH database, that share low pairwise sequence similarity. Using the receiver operating characteristic (ROC) measure, we demonstrate potentially significant improvement in using information-optimized reduced tetramer composition, over methods relying only on the raw amino acid composition or on traditional sequence alignment, in homology detection at or below the “twilight zone”. PMID:20635424

  7. Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

    SciTech Connect

    Lee, Je Min; Lee, Sang-Jik; Rose, Jocelyn K.C.; Yeam, Inhwa; Kim, Byung-Dong

    2014-04-18

    Highlights: • Yeast secretion trap (YST) is a valuable tool for mining secretome. • A total of 80 secreted proteins are newly identified via YST in pepper fruits. • The secreted proteins are differentially regulated during pepper development and ripening. • Transient GFP-fusion assay and in planta secretion trap can effectively validate the secretion of proteins. - Abstract: Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function in fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.

  8. When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches

    PubMed Central

    Muñoz, Victor; Cerminara, Michele

    2016-01-01

    Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico. All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. PMID:27574021

  9. When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches.

    PubMed

    Muñoz, Victor; Cerminara, Michele

    2016-09-01

    Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats.

  10. Structural Conservation of the Myoviridae Phage Tail Sheath Protein Fold

    SciTech Connect

    Aksyuk, Anastasia A.; Kurochkina, Lidia P.; Fokine, Andrei; Forouhar, Farhad; Mesyanzhinov, Vadim V.; Tong, Liang; Rossmann, Michael G.

    2012-02-21

    Bacteriophage phiKZ is a giant phage that infects Pseudomonas aeruginosa, a human pathogen. The phiKZ virion consists of a 1450 {angstrom} diameter icosahedral head and a 2000 {angstrom}-long contractile tail. The structure of the whole virus was previously reported, showing that its tail organization in the extended state is similar to the well-studied Myovirus bacteriophage T4 tail. The crystal structure of a tail sheath protein fragment of phiKZ was determined to 2.4 {angstrom} resolution. Furthermore, crystal structures of two prophage tail sheath proteins were determined to 1.9 and 3.3 {angstrom} resolution. Despite low sequence identity between these proteins, all of these structures have a similar fold. The crystal structure of the phiKZ tail sheath protein has been fitted into cryo-electron-microscopy reconstructions of the extended tail sheath and of a polysheath. The structural rearrangement of the phiKZ tail sheath contraction was found to be similar to that of phage T4.

  11. Protein Motions and Folding Investigated by NMR Spectroscopy

    NASA Astrophysics Data System (ADS)

    Palmer, Arthur

    2002-03-01

    NMR spin relaxation spectroscopy is a powerful experimental approach for globally characterizing conformational dynamics of proteins in solution. Laboratory frame relaxation measurements are sensitive to overall rotational diffusion and internal motions on picosecond-nanosecond time scales, while rotating frame relaxation measurements are sensitive to chemical exchange processes on microsecond-millisecond time scales. The former approach is illustrated by ^15N laboratory-frame relaxation experiments as a function of temperature for the helical subdomain HP36 of the F-actin-binding headpiece domain of chicken villin. The data are analyzed using the model-free formalism to characterize order parameters and effective correlation times for intramolecular motions of individual ^15N sites. The latter approach is illustrated by ^13C Carr-Purcell-Meiboom-Gill relaxation measurements for the de novo designed α_2D protein and by ^15N rotating-frame relaxation measurements for the peripheral subunit-binding domain (PSBD) from the dihydrolopoamide acetyltransferase component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus. These experiments are used to determine the folding and unfolding kinetic rate constants for the two proteins. The results for HP36, α_2D, and PSBD illustrate the capability of current NMR methods for characterizing dynamic processes on multiple time scales in proteins.

  12. Protein folding kinetics: barrier effects in chemical and thermal denaturation experiments

    PubMed Central

    Naganathan, Athi N.; Doshi, Urmi; Muñoz, Victor

    2008-01-01

    Recent experimental work on fast protein folding brings about an intriguing paradox. Microsecond-folding proteins are supposed to fold near or at the folding speed limit (downhill folding), but yet their folding behavior seems to comply with classical two-state analyses, which imply the crossing of high free energy barriers. However, close inspection of chemical and thermal denaturation kinetic experiments in fast-folding proteins reveals systematic deviations from two-state behavior. Using a simple one-dimensional free energy surface approach we find that such deviations are indeed diagnostic of marginal folding barriers. Furthermore, the quantitative analysis of available fast-kinetic data indicates that many microsecond-folding proteins fold downhill in native conditions. All of these proteins are then promising candidates for an atom-by-atom analysis of protein folding using nuclear magnetic resonance1. We also find that the diffusion coefficient for protein folding is strongly temperature dependent, corresponding to an activation energy of ~1 kJ.mol−1 per protein residue. As a consequence, the folding speed limit at room temperature is about an order of magnitude slower than the ~ 1μs estimates from high temperature T-jump experiments. Our analysis is quantitatively consistent with the available thermodynamic and kinetic data on two-state folding proteins, and provides a straightforward explanation for the apparent fast-folding paradox. PMID:17419630

  13. A New Scaffold of an Old Protein Fold Ensures Binding to the Bisintercalator Thiocoraline

    SciTech Connect

    Biswas, Tapan; Zolova, Olga E.; Lombó, Felipe; de la Calle, Fernando; Salas, Jose A.; Tsodikov, Oleg V.; Garneau-Tsodikova, Sylvie

    2010-09-02

    Thiocoraline is a thiodepsipeptide with potent antitumor activity. TioX, a protein with an unidentified function, is encoded by a gene of the thiocoraline biosynthetic gene cluster. The crystal structure of the full-length TioX protein at 2.15 {angstrom} resolution reveals that TioX protomer shares an ancient {beta}{alpha}{beta}{beta}{beta} fold motif with glyoxalase I and bleomycin resistance protein families, despite a very low sequence homology. Intriguingly, four TioX monomers form a unique 2-fold symmetric tetrameric assembly that is stabilized by four intermolecular disulfide bonds formed cyclically between Cys60 and Cys66 of adjacent monomers. The arrangement of two of the four monomers in the TioX tetramer is analogous to that in dimeric bleomycin resistance proteins. This analogy indicates that this novel higher-order structural scaffold of TioX may have evolved to bind thiocoraline. Our equilibrium titration studies demonstrate the binding of a thiocoraline chromophore analog, quinaldic acid, to TioX, thereby substantiating this model. Furthermore, a strain of Streptomyces albus containing an exogenous thiocoraline gene cluster devoid of functional tioX maintains thiocoraline production, albeit with a lower yield. Taken together, these observations rule out a direct enzymatic function of TioX and suggest that TioX is involved in thiocoraline resistance or secretion.

  14. Molecular chaperones and protein-folding catalysts as intercellular signaling regulators in immunity and inflammation.

    PubMed

    Henderson, Brian; Pockley, A Graham

    2010-09-01

    This review critically examines the hypothesis that molecular chaperones and protein-folding catalysts from prokaryotes and eukaryotes can be secreted by cells and function as intercellular signals, principally but not exclusively, for leukocytes. A growing number of molecular chaperones have been reported to function as ligands for selected receptors and/or receptors for specific ligands. Molecular chaperones initially appeared to act primarily as stimulatory signals for leukocytes and thus, were seen as proinflammatory mediators. However, evidence is now emerging that molecular chaperones can have anti-inflammatory actions or, depending on the protein and concentration, anti- and proinflammatory functions. Recasting the original hypothesis, we propose that molecular chaperones and protein-folding catalysts are "moonlighting" proteins that function as homeostatic immune regulators but may also under certain circumstances, contribute to tissue pathology. One of the key issues in the field of molecular chaperone biology relates to the role of microbial contaminants in their signaling activity; this too will be evaluated critically. The most fascinating aspect of molecular chaperones probably relates to evidence for their therapeutic potential in human disease, and ongoing studies are evaluating this potential in a range of clinical settings.

  15. Overview of the regulation of disulfide bond formation in Peptide and protein folding.

    PubMed

    Hidaka, Yuji

    2014-04-01

    Disulfide bonds play a critical role in the maintenance of the native conformation of proteins under thermodynamic control. In general, disulfide bond formation is associated with protein folding, and this restricts the formation of folding intermediates such as misbridged disulfide isomers or kinetically trapped conformations, which provide important information related to how proteins fold into their native conformation. Therefore, numerous studies have focused on the structural analysis of folding intermediates in vitro. However, isolating or trapping folding intermediates, as well as the entire proteins, including mutant proteins, is not an easy task. Several chemical methods have recently been developed for examining peptide and protein folding and for producing, e.g., intact, post-translationally modified, or kinetically trapped proteins, or proteins with misbridged disulfide bonds. This overview introduces chemical methods for regulating the formation of disulfide bonds of peptides and proteins in the context of the thermodynamic and kinetic control of peptide and protein folding.

  16. Iron-nucleated Folding of a Metalloprotein in High Urea: Resolution of Metal Binding and Protein Folding Events†

    PubMed Central

    Morleo, Anna; Bonomi, Francesco; Iametti, Stefania; Huang, Victor W.; Kurtz, Donald M.

    2010-01-01

    Addition of iron salts to chaotrope-denatured aporubredoxin (apoRd) leads to nearly quantitative recovery of its single Fe(SCys)4 site and native protein structure without significant dilution of the chaotrope. This “high chaotrope” approach was used to examine iron binding and protein folding events using stopped-flow UV/vis absorption and CD spectroscopies. At 100-fold molar excess ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFeIIIRd structure was recovered in >90% yield with t1/2 < 10 msec. More modest excesses of iron also gave nearly quantitative holoRd formation in 5 M urea but with chronological resolution of iron binding and protein folding events. The results indicate structural recovery in 5 M urea consists of the minimal sequence: (1) binding of ferrous iron to the unfolded apoRd, (2) rapid formation of a near-native ferrous Fe(SCys)4 site within a protein having no detectable secondary structure, (3) recovery of the ferrous Fe(SCys)4 site chiral environment nearly concomitantly with (4) recovery of the native protein secondary structure. The rate of step 2 (and by inference, step 1) was not saturated even at 100-fold molar excess of iron. Analogous results obtained on Cys→Ser iron ligand variants support formation of an unfolded-Fe(SCys)3 complex between steps 1 and 2, which we propose is the key nucleation event that pulls distal regions of the protein chain together. These results show that folding of chaotrope-denatured apoRd is iron-nucleated and driven by extraordinarily rapid formation of the Fe(SCys)4 site from an essentially random coil apoprotein. This high chaotrope, multi-spectroscopic approach could clarify folding pathways of other [M(SCys)3 or 4]-containing proteins. PMID:20614892

  17. Proteolytic events in the processing of secreted proteins in fungi.

    PubMed

    Calmels, T P; Martin, F; Durand, H; Tiraby, G

    1991-01-01

    Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.

  18. Correlation of secretion of retinol and protein by the lacrimal gland

    SciTech Connect

    Ubels, J.L.; Rismondo, V.

    1986-03-01

    Retinol, which is present in tears, is secreted by the lacrimal gland. Retinol secretion is stimulated by cholinergic drugs and vasoactive intestinal peptide with characteristics very similar to the exocytotic secretion of protein by the lacrimal gland, suggesting that retinol and protein are secreted by similar mechanisms. The authors investigated this by cannulating the lacrimal gland ducts of rabbits and collecting lacrimal gland fluid (LGF) under conditions of maximal flow stimulated by IV injection of pilocarpine (400 ..mu..g/kg) every 20 min for 4.5 hr. Over this period LGF protein concentration decreased 36.4% from 22.8 +/- 1.94 mg/ml to 8.29 1.86 mg/ml while retinol decreased 37% from 55.1 +/- 16.2 ng/ml to 20.4 +/- 6.5 ng/ml. The retinol/protein ratio remained constant at 2.88 ng/mg. This demonstrates a strong correlation between retinol and protein secretion, suggesting that retinol may be protein bound. To investigate binding of retinol to LGF protein, LGF was incubated with /sup 3/H-retinol. The bound and unbound retinol were separated on a Lipidex 1000 column. Retinol binding was linear over a range of 1.25-200 nM /sup 3/H-retinol. Binding was not inhibited by PCMBS or addition of a 100-fold excess of unlabeled retinol and was not increased by prior extraction of endogenous retinol from the LGF. This indicates that the binding of retinol to LGF protein is non-specific. Retinol therefore appears to be secreted by the lacrimal gland cells in non-specific association with protein.

  19. Unexpected fold in the circumsporozoite protein target of malaria vaccines

    PubMed Central

    Doud, Michael B.; Koksal, Adem C.; Mi, Li-Zhi; Song, Gaojie; Lu, Chafen; Springer, Timothy A.

    2012-01-01

    Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an “αTSR” domain. The αTSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but αTSR does not. Interestingly, polymorphic T-cell epitopes map to specialized αTSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket. PMID:22547819

  20. Unexpected fold in the circumsporozoite protein target of malaria vaccines

    SciTech Connect

    Doud, Michael B.; Koksal, Adem C.; Mi, Li-Zhi; Song, Gaojie; Lu, Chafen; Springer, Timothy A.

    2012-10-09

    Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an '{alpha}TSR' domain. The {alpha}TSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but {alpha}TSR does not. Interestingly, polymorphic T-cell epitopes map to specialized {alpha}TSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket.

  1. Initial assembly steps of a translocase for folded proteins

    PubMed Central

    Blümmel, Anne-Sophie; Haag, Laura A.; Eimer, Ekaterina; Müller, Matthias; Fröbel, Julia

    2015-01-01

    The so-called Tat (twin-arginine translocation) system transports completely folded proteins across cellular membranes of archaea, prokaryotes and plant chloroplasts. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures. We have now elucidated the fine architecture of TatBC oligomers assembled to form closed intramembrane substrate-binding cavities. The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers. We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors. PMID:26068441

  2. Initial assembly steps of a translocase for folded proteins.

    PubMed

    Blümmel, Anne-Sophie; Haag, Laura A; Eimer, Ekaterina; Müller, Matthias; Fröbel, Julia

    2015-06-11

    The so-called Tat (twin-arginine translocation) system transports completely folded proteins across cellular membranes of archaea, prokaryotes and plant chloroplasts. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures. We have now elucidated the fine architecture of TatBC oligomers assembled to form closed intramembrane substrate-binding cavities. The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers. We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors.

  3. Highly Diverse Protein Library Based on the Ubiquitous (β/α)8 Enzyme Fold Yields Well-Structured Proteins Through In Vitro Folding Selection

    PubMed Central

    Golynskiy, Misha V.; Haugner, John C.

    2013-01-01

    Proper protein folding is a prerequisite for protein stability and enzymatic activity. While directed evolution can be a powerful tool to investigate enzymatic function and to isolate novel activities, well-designed libraries of folded proteins are essential. In vitro selection methods are particularly capable of searching for enzymatic activities in libraries of trillions of protein variants, yet high-quality libraries of well-folded enzymes with such high diversity are lacking. We describe the construction and detailed characterization of a folding-enriched protein library based on the ubiquitous (β/α)8 barrel fold found in five of the six enzyme classes. We introduced seven randomized loops on the catalytic face of the monomeric, thermostable (β/α)8 barrel of glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima. We employed an in vitro folding selection based on protease digestion to enrich intermediate libraries containing three to four randomized loops for folded variants and then combined them to assemble the final library (1014 DNA sequences). The resulting library was analyzed using the in vitro protease assay and an in vivo GFP-folding assay and contains ~1012 soluble monomeric protein variants. We isolated six library members and demonstrated that these proteins are soluble, monomeric and show (β/α)8 barrel fold-like secondary and tertiary structure. The quality of the folding-enriched library improved up to 50-fold compared to a control library that was assembled without the folding selection. To the best of our knowledge, this work is the first example of combining the ultra-high throughput method mRNA display with a selection for folding. The resulting (β/α)8 barrel libraries provide a valuable starting point to study the unique catalytic capabilities of the (β/α)8 fold, and to isolate novel enzymes. PMID:23956201

  4. Misplaced helix slows down ultrafast pressure-jump protein folding

    PubMed Central

    Prigozhin, Maxim B.; Liu, Yanxin; Wirth, Anna Jean; Kapoor, Shobhna; Winter, Roland; Schulten, Klaus; Gruebele, Martin

    2013-01-01

    Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6–85, from 3 μs to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coil-rich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jump–induced refolding. To complement the experiments, we performed over 50 μs of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced α-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of λ*YA contains mainly residual helix and little β-sheet; following a fast P-drop, at least some λ*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct. PMID:23620522

  5. A Simple and Effective Protein Folding Activity Suitable for Large Lectures

    ERIC Educational Resources Information Center

    White, Brian

    2006-01-01

    This article describes a simple and inexpensive hands-on simulation of protein folding suitable for use in large lecture classes. This activity uses a minimum of parts, tools, and skill to simulate some of the fundamental principles of protein folding. The major concepts targeted are that proteins begin as linear polypeptides and fold to…

  6. A Simple and Effective Protein Folding Activity Suitable for Large Lectures

    ERIC Educational Resources Information Center

    White, Brian

    2006-01-01

    This article describes a simple and inexpensive hands-on simulation of protein folding suitable for use in large lecture classes. This activity uses a minimum of parts, tools, and skill to simulate some of the fundamental principles of protein folding. The major concepts targeted are that proteins begin as linear polypeptides and fold to…

  7. Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

    PubMed Central

    Kang’ethe, Wanyoike; Bernstein, Harris D.

    2013-01-01

    Autotransporters are a large class of virulence proteins produced by Gram-negative bacteria. They contain an N-terminal extracellular (“passenger”) domain that folds into a β-helical structure and a C-terminal β-barrel (“β”) domain that anchors the protein to the outer membrane. Because the periplasm lacks ATP, the source of energy that drives passenger domain secretion is unknown. The prevailing model postulates that vectorial folding of the β-helix in the extracellular space facilitates unidirectional secretion of the passenger domain. In this study we used a chimeric protein composed of the 675-residue receptor-binding domain (RD) of the Bordetella pertussis adenylate cyclase toxin CyaA fused to the C terminus of the Escherichia coli O157:H7 autotransporter EspP to test this hypothesis. The RD is a highly acidic, repetitive polypeptide that is intrinsically disordered in the absence of calcium. Surprisingly, we found that the RD moiety was efficiently secreted when it remained in an unfolded conformation. Furthermore, we found that neutralizing or reversing the charge of acidic amino acid clusters stalled translocation in the vicinity of the altered residues. These results challenge the vectorial folding model and, together with the finding that naturally occurring passenger domains are predominantly acidic, provide evidence that a net negative charge plays a significant role in driving the translocation reaction. PMID:24145447

  8. A Hooke׳s law-based approach to protein folding rate.

    PubMed

    Ruiz-Blanco, Yasser B; Marrero-Ponce, Yovani; Prieto, Pablo J; Salgado, Jesús; García, Yamila; Sotomayor-Torres, Clivia M

    2015-01-07

    Kinetics is a key aspect of the renowned protein folding problem. Here, we propose a comprehensive approach to folding kinetics where a polypeptide chain is assumed to behave as an elastic material described by the Hooke׳s law. A novel parameter called elastic-folding constant results from our model and is suggested to distinguish between protein with two-state and multi-state folding pathways. A contact-free descriptor, named folding degree, is introduced as a suitable structural feature to study protein-folding kinetics. This approach generalizes the observed correlations between varieties of structural descriptors with the folding rate constant. Additionally several comparisons among structural classes and folding mechanisms were carried out showing the good performance of our model with proteins of different types. The present model constitutes a simple rationale for the structural and energetic factors involved in protein folding kinetics.

  9. Efficient conformational space exploration in ab initio protein folding simulation

    PubMed Central

    Ullah, Ahammed; Ahmed, Nasif; Pappu, Subrata Dey; Shatabda, Swakkhar; Ullah, A. Z. M. Dayem; Rahman, M. Sohel

    2015-01-01

    Ab initio protein folding simulation largely depends on knowledge-based energy functions that are derived from known protein structures using statistical methods. These knowledge-based energy functions provide us with a good approximation of real protein energetics. However, these energy functions are not very informative for search algorithms and fail to distinguish the types of amino acid interactions that contribute largely to the energy function from those that do not. As a result, search algorithms frequently get trapped into the local minima. On the other hand, the hydrophobic–polar (HP) model considers hydrophobic interactions only. The simplified nature of HP energy function makes it limited only to a low-resolution model. In this paper, we present a strategy to derive a non-uniform scaled version of the real 20×20 pairwise energy function. The non-uniform scaling helps tackle the difficulty faced by a real energy function, whereas the integration of 20×20 pairwise information overcomes the limitations faced by the HP energy function. Here, we have applied a derived energy function with a genetic algorithm on discrete lattices. On a standard set of benchmark protein sequences, our approach significantly outperforms the state-of-the-art methods for similar models. Our approach has been able to explore regions of the conformational space which all the previous methods have failed to explore. Effectiveness of the derived energy function is presented by showing qualitative differences and similarities of the sampled structures to the native structures. Number of objective function evaluation in a single run of the algorithm is used as a comparison metric to demonstrate efficiency. PMID:26361554

  10. Efficient conformational space exploration in ab initio protein folding simulation.

    PubMed

    Ullah, Ahammed; Ahmed, Nasif; Pappu, Subrata Dey; Shatabda, Swakkhar; Ullah, A Z M Dayem; Rahman, M Sohel

    2015-08-01

    Ab initio protein folding simulation largely depends on knowledge-based energy functions that are derived from known protein structures using statistical methods. These knowledge-based energy functions provide us with a good approximation of real protein energetics. However, these energy functions are not very informative for search algorithms and fail to distinguish the types of amino acid interactions that contribute largely to the energy function from those that do not. As a result, search algorithms frequently get trapped into the local minima. On the other hand, the hydrophobic-polar (HP) model considers hydrophobic interactions only. The simplified nature of HP energy function makes it limited only to a low-resolution model. In this paper, we present a strategy to derive a non-uniform scaled version of the real 20×20 pairwise energy function. The non-uniform scaling helps tackle the difficulty faced by a real energy function, whereas the integration of 20×20 pairwise information overcomes the limitations faced by the HP energy function. Here, we have applied a derived energy function with a genetic algorithm on discrete lattices. On a standard set of benchmark protein sequences, our approach significantly outperforms the state-of-the-art methods for similar models. Our approach has been able to explore regions of the conformational space which all the previous methods have failed to explore. Effectiveness of the derived energy function is presented by showing qualitative differences and similarities of the sampled structures to the native structures. Number of objective function evaluation in a single run of the algorithm is used as a comparison metric to demonstrate efficiency.

  11. SecretEPDB: a comprehensive web-based resource for secreted effector proteins of the bacterial types III, IV and VI secretion systems

    PubMed Central

    An, Yi; Wang, Jiawei; Li, Chen; Revote, Jerico; Zhang, Yang; Naderer, Thomas; Hayashida, Morihiro; Akutsu, Tatsuya; Webb, Geoffrey I.; Lithgow, Trevor; Song, Jiangning

    2017-01-01

    Bacteria translocate effector molecules to host cells through highly evolved secretion systems. By definition, the function of these effector proteins is to manipulate host cell biology and the sequence, structural and functional annotations of these effector proteins will provide a better understanding of how bacterial secretion systems promote bacterial survival and virulence. Here we developed a knowledgebase, termed SecretEPDB (Bacterial Secreted Effector Protein DataBase), for effector proteins of type III secretion system (T3SS), type IV secretion system (T4SS) and type VI secretion system (T6SS). SecretEPDB provides enriched annotations of the aforementioned three classes of effector proteins by manually extracting and integrating structural and functional information from currently available databases and the literature. The database is conservative and strictly curated to ensure that every effector protein entry is supported by experimental evidence that demonstrates it is secreted by a T3SS, T4SS or T6SS. The annotations of effector proteins documented in SecretEPDB are provided in terms of protein characteristics, protein function, protein secondary structure, Pfam domains, metabolic pathway and evolutionary details. It is our hope that this integrated knowledgebase will serve as a useful resource for biological investigation and the generation of new hypotheses for research efforts aimed at bacterial secretion systems. PMID:28112271

  12. Chemical methods for producing disulfide bonds in peptides and proteins to study folding regulation.

    PubMed

    Okumura, Masaki; Shimamoto, Shigeru; Hidaka, Yuji

    2014-04-01

    Disulfide bonds play a critical role in the folding of secretory and membrane proteins. Oxidative folding reactions of disulfide bond-containing proteins typically require several hours or days, and numerous misbridged disulfide isomers are often observed as intermediates. The rate-determining step in refolding is thought to be the disulfide-exchange reaction from nonnative to native disulfide bonds in folding intermediates, which often precipitate during the refolding process because of their hydrophobic properties. To overcome this, chemical additives or a disulfide catalyst, protein disulfide isomerase (PDI), are generally used in refolding experiments to regulate disulfide-coupled peptide and protein folding. This unit describes such methods in the context of the thermodynamic and kinetic control of peptide and protein folding, including (1) regulation of disulfide-coupled peptides and protein folding assisted by chemical additives, (2) reductive unfolding of disulfide-containing peptides and proteins, and (3) regulation of disulfide-coupled peptide and protein folding using PDI.

  13. The ExPortal: an organelle dedicated to the biogenesis of secreted proteins in Streptococcus pyogenes.

    PubMed

    Rosch, Jason W; Caparon, Michael G

    2005-11-01

    The Gram-positive pathogen Streptococcus pyogenes secretes proteins through the ExPortal, a unique single microdomain of the cellular membrane specialized to contain the Sec translocons. It has been proposed that the ExPortal functions as an organelle to promote the biogenesis of secreted proteins by coordinating interactions between nascent unfolded secretory proteins and membrane-associated chaperones. In this study we provide evidence to support this model. It was found that HtrA (DegP), a surface anchored accessory factor required for maturation of the secreted SpeB cysteine protease, was localized exclusively to the ExPortal. Furthermore, the ATP synthase beta subunit was not localized to the ExPortal, suggesting that retention is likely restricted to a specific subset of exported proteins. Mutations that disrupted the anchoring, but not the protease activity, of HtrA, also altered the maturation kinetics of SpeB demonstrating that localization to the ExPortal was important for HtrA function. These data indicate that the ExPortal provides a mechanism by which Gram-positive bacteria can coordinate protein secretion and subsequent biogenesis in the absence of a specialized protein-folding compartment.

  14. A simple quantitative model of macromolecular crowding effects on protein folding: Application to the murine prion protein(121-231)

    NASA Astrophysics Data System (ADS)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2013-06-01

    A model of protein folding kinetics is applied to study the effects of macromolecular crowding on protein folding rate and stability. Macromolecular crowding is found to promote a decrease of the entropic cost of folding of proteins that produces an increase of both the stability and the folding rate. The acceleration of the folding rate due to macromolecular crowding is shown to be a topology-dependent effect. The model is applied to the folding dynamics of the murine prion protein (121-231). The differential effect of macromolecular crowding as a function of protein topology suffices to make non-native configurations relatively more accessible.

  15. How the folding rates of two- and multistate proteins depend on the amino acid properties.

    PubMed

    Huang, Jitao T; Huang, Wei; Huang, Shanran R; Li, Xin

    2014-10-01

    Proteins fold by either two-state or multistate kinetic mechanism. We observe that amino acids play different roles in different mechanism. Many residues that are easy to form regular secondary structures (α helices, β sheets and turns) can promote the two-state folding reactions of small proteins. Most of hydrophilic residues can speed up the multistate folding reactions of large proteins. Folding rates of large proteins are equally responsive to the flexibility of partial amino acids. Other properties of amino acids (including volume, polarity, accessible surface, exposure degree, isoelectric point, and phase transfer energy) have contributed little to folding kinetics of the proteins. Cysteine is a special residue, it triggers two-state folding reaction and but inhibits multistate folding reaction. These findings not only provide a new insight into protein structure prediction, but also could be used to direct the point mutations that can change folding rate. © 2014 Wiley Periodicals, Inc.

  16. Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers.

    PubMed

    Jiao, Junyi; Rebane, Aleksander A; Ma, Lu; Zhang, Yongli

    2017-01-01

    How proteins fold from linear chains of amino acids to delicate three-dimensional structures remains a fundamental biological problem. Single-molecule manipulation based on high-resolution optical tweezers (OT) provides a powerful approach to study protein folding with unprecedented spatiotemporal resolution. In this method, a single protein or protein complex is tethered between two beads confined in optical traps and pulled. Protein unfolding induced by the mechanical force is counteracted by the spontaneous folding of the protein, reaching a dynamic equilibrium at a characteristic force and rate. The transition is monitored by the accompanying extension change of the protein and used to derive conformations and energies of folding intermediates and their associated transition kinetics. Here, we provide general strategies and detailed protocols to study folding of proteins and protein complexes using optical tweezers, including sample preparation, DNA-protein conjugation and methods of data analysis to extract folding energies and rates from the single-molecule measurements.

  17. Identification of luminal and secreted proteins in bull epididymis.

    PubMed

    Belleannée, Clémence; Labas, Valérie; Teixeira-Gomes, Ana-Paula; Gatti, Jean Luc; Dacheux, Jean-Louis; Dacheux, Françoise

    2011-01-01

    The epididymis plays a major role in the acquisition of sperm fertility. In order to shed light on specific features of epididymal function in mammalian species, we characterized the luminal proteins (luminal proteome) and secreted proteins (secretome) in the bovine epididymis. We identified 172 different luminal proteins in 9 distinct epididymal regions. The concentration and secretory activity of luminal proteins were quantified throughout the epididymis. Among the most abundant secreted proteins, we found lipocalin 5, (LCN5), NADP(+)dependent prostaglandin dehydrogenase (PTGDS), Niemann-Pick disease type C2 protein (NPC2), glutathione peroxidase type 5 (GPX 5), clusterin (CLU), hexosaminidase B (HEXB) and galactosidase (GLB1), each of which is released in distinct epididymal regions. Gelsolin, (GSN) previously not described in mammalian epididymal fluid, appeared to be a major protein secreted exclusively in the distal region of the bovine epididymis, where fully mature spermatozoa are stored. Although the major epididymal proteins are conserved between mammalian species, this study highlights the specificity and mechanisms of protein processing of epididymal secretion in the bull. In addition, this study provides a major insight into the sequential changes occurring in the sperm environment while gaining fertilizing capacity and could provide new information for the future identification of potential fertility markers. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Protein folding and protein metallocluster studies using synchrotron small angler X-ray scattering

    SciTech Connect

    Eliezer, D.

    1994-06-01

    Proteins, biological macromolecules composed of amino-acid building blocks, possess unique three dimensional shapes or conformations which are intimately related to their biological function. All of the information necessary to determine this conformation is stored in a protein`s amino acid sequence. The problem of understanding the process by which nature maps protein amino-acid sequences to three-dimensional conformations is known as the protein folding problem, and is one of the central unsolved problems in biophysics today. The possible applications of a solution are broad, ranging from the elucidation of thousands of protein structures to the rational modification and design of protein-based drugs. The scattering of X-rays by matter has long been useful as a tool for the characterization of physical properties of materials, including biological samples. The high photon flux available at synchrotron X-ray sources allows for the measurement of scattering cross-sections of dilute and/or disordered samples. Such measurements do not yield the detailed geometrical information available from crystalline samples, but do allow for lower resolution studies of dynamical processes not observable in the crystalline state. The main focus of the work described here has been the study of the protein folding process using time-resolved small-angle x-ray scattering measurements. The original intention was to observe the decrease in overall size which must accompany the folding of a protein from an extended conformation to its compact native state. Although this process proved too fast for the current time-resolution of the technique, upper bounds were set on the probable compaction times of several small proteins. In addition, an interesting and unexpected process was detected, in which the folding protein passes through an intermediate state which shows a tendency to associate. This state is proposed to be a kinetic molten globule folding intermediate.

  19. Folding RaCe: a robust method for predicting changes in protein folding rates upon point mutations.

    PubMed

    Chaudhary, Priyashree; Naganathan, Athi N; Gromiha, M Michael

    2015-07-01

    Protein engineering methods are commonly employed to decipher the folding mechanism of proteins and enzymes. However, such experiments are exceedingly time and resource intensive. It would therefore be advantageous to develop a simple computational tool to predict changes in folding rates upon mutations. Such a method should be able to rapidly provide the sequence position and chemical nature to modulate through mutation, to effect a particular change in rate. This can be of importance in protein folding, function or mechanistic studies. We have developed a robust knowledge-based methodology to predict the changes in folding rates upon mutations formulated from amino and acid properties using multiple linear regression approach. We benchmarked this method against an experimental database of 790 point mutations from 26 two-state proteins. Mutants were first classified according to secondary structure, accessible surface area and position along the primary sequence. Three prime amino acid features eliciting the best relationship with folding rates change were then shortlisted for each class along with an optimized window length. We obtained a self-consistent mean absolute error of 0.36 s(-1) and a mean Pearson correlation coefficient (PCC) of 0.81. Jack-knife test resulted in a MAE of 0.42 s(-1) and a PCC of 0.73. Moreover, our method highlights the importance of outlier(s) detection and studying their implications in the folding mechanism. A web server 'Folding RaCe' has been developed and is available at http://www.iitm.ac.in/bioinfo/proteinfolding/foldingrace.html. gromiha@iitm.ac.in Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. Kinetic barriers and the role of topology in protein and RNA folding

    PubMed Central

    Sosnick, Tobin R.

    2008-01-01

    This review compares the folding behavior of proteins and RNAs. Topics covered include the role of topology in the determination of folding rates, major folding events including collapse, properties of denatured states, pathway heterogeneity, and the influence of the mode of initiation on the folding pathway. PMID:18502978

  1. Odorant-Binding Protein: Localization to Nasal Glands and Secretions

    NASA Astrophysics Data System (ADS)

    Pevsner, Jonathan; Sklar, Pamela B.; Snyder, Solomon H.

    1986-07-01

    An odorant-binding protein (OBP) was isolated from bovine olfactory and respiratory mucosa. We have produced polyclonal antisera to this protein and report its immunohistochemical localization to mucus-secreting glands of the olfactory and respiratory mucosa. Although OBP was originally isolated as a pyrazine binding protein, both rat and bovine OBP also bind the odorants [3H]methyldihydrojasmonate and 3,7-dimethyl-octan-1-ol as well as 2-isobutyl-3-[3H]methoxypyrazine. We detect substantial odorant-binding activity attributable to OBP in secreted rat nasal mucus and tears but not in saliva, suggesting a role for OBP in transporting or concentrating odorants.

  2. Key proteins involved in insulin vesicle exocytosis and secretion

    PubMed Central

    Xiong, Qian-Yin; Yu, Cui; Zhang, Yao; Ling, Liefeng; Wang, Lizhuo; Gao, Jia-Lin

    2017-01-01

    In vivo insulin secretion is predominantly affected by blood glucose concentration, blood concentration of amino acids, gastrointestinal hormones and free nerve functional status, in addition to other factors. Insulin is one of the most important hormones in the body, and its secretion is precisely controlled by nutrients, neurotransmitters and hormones. The insulin exocytosis process is similar to the neurotransmitter release mechanism. There are various types of proteins and lipids that participate in the insulin secretory vesicle fusion process, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, Ras-related proteins and vacuolar-type H+-ATPase (V-ATPase). Notably, the SNARE protein is the molecular basis of exocytotic activity. In the current review, the role of the vesicle membrane proteins (synaptobrevins, vesicle associated membrane proteins and target membrane proteins) and auxiliary proteins (Rab proteins and Munc-18 proteins) in vesicle fusion activity were summarized. A summary of these key proteins involved in insulin granule secretion will facilitate understanding of the pathogenesis of diabetes. PMID:28357064

  3. Is the unfolded state the Rosetta Stone of the protein folding problem?

    PubMed

    Hammarström, P; Carlsson, U

    2000-09-24

    Solving the protein folding problem is one of the most challenging tasks in the post genomic era. Identification of folding-initiation sites is very important in order to understand the protein folding mechanism. Detection of residual structure in unfolded proteins can yield important clues to the initiation sites in protein folding. A substantial number of studied proteins possess residual structure in hydrophobic regions clustered together in the protein core. These stable structures can work as seeds in the folding process. In addition, local preferences for secondary structure in the form of turns for beta-sheet initiation and helical turns for alpha-helix formation can guide the folding reaction. In this respect the unfolded states, studied at increasing structural resolution, can be the Rosetta Stone of the protein folding problem.

  4. Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers

    PubMed Central

    Jiao, Junyi; Rebane, Aleksander A.; Ma, Lu; Zhang, Yongli

    2017-01-01

    Summary How proteins fold from linear chains of amino acids to delicate three dimensional structures remains a fundamental biological problem. Single-molecule manipulation based on high-resolution optical tweezers (OT) provides a powerful approach to study protein folding with unprecedented spatiotemporal resolution. In this method, a single protein or protein complex is tethered between two beads confined in optical traps and pulled. Protein unfolding induced by the mechanical force is counteracted by the spontaneous folding of the protein, reaching a dynamic equilibrium at a characteristic force and rate. The transition is monitored by the accompanying extension change of the protein and used to derive conformations and energies of folding intermediates and their associated transition kinetics. Here, we provide general strategies and detailed protocols to study folding of proteins and protein complexes using optical tweezers, including methods of data analysis to extract folding energies and rates from the single-molecule measurements. PMID:27844436

  5. The effect of surface tethering on the folding of the src-SH3 protein domain

    NASA Astrophysics Data System (ADS)

    Zhuang, Zhuoyun; Jewett, Andrew I.; Soto, Patricia; Shea, Joan-Emma

    2009-03-01

    The effect of surface tethering on the folding mechanism of the src-SH3 protein domain was investigated using a coarse-grained Gō-type protein model. The protein was tethered at various locations along the protein chain and the thermodynamics and kinetics of folding were studied using replica exchange and constant temperature Langevin dynamics. Our simulations reveal that tethering in a structured part of the transition state can dramatically alter the folding mechanism, while tethering in an unstructured part leaves the folding mechanism unaltered as compared to bulk folding. Interestingly, there is only modest correlation between the tethering effect on the folding mechanism and its effect on thermodynamic stability and folding rates. We suggest locations on the protein at which tethering could be performed in single-molecule experiments so as to leave the folding mechanism unaltered from the bulk.

  6. Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

    NASA Astrophysics Data System (ADS)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2014-01-01

    A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.

  7. SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli

    PubMed Central

    Deng, Wanyin; Yu, Hong B.; Li, Yuling

    2015-01-01

    ABSTRACT The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. IMPORTANCE Many bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into

  8. Parameter Optimization for the Gaussian Model of Folded Proteins

    NASA Astrophysics Data System (ADS)

    Erman, Burak; Erkip, Albert

    2000-03-01

    Recently, we proposed an analytical model of protein folding (B. Erman, K. A. Dill, J. Chem. Phys, 112, 000, 2000) and showed that this model successfully approximates the known minimum energy configurations of two dimensional HP chains. All attractions (covalent and non-covalent) as well as repulsions were treated as if the monomer units interacted with each other through linear spring forces. Since the governing potential of the linear springs are derived from a Gaussian potential, the model is called the ''Gaussian Model''. The predicted conformations from the model for the hexamer and various 9mer sequences all lie on the square lattice, although the model does not contain information about the lattice structure. Results of predictions for chains with 20 or more monomers also agreed well with corresponding known minimum energy lattice structures. However, these predicted conformations did not lie exactly on the square lattice. In the present work, we treat the specific problem of optimizing the potentials (the strengths of the spring constants) so that the predictions are in better agreement with the known minimum energy structures.

  9. Folding and aggregation of export-defective mutants of the maltose-binding protein.

    PubMed

    Betton, Jean-Michel; Phichith, Denis; Hunke, Sabine

    2002-09-01

    We previously characterized a defective-folding variant of the periplasmic maltose-binding protein, MalE31. To examine the alternative folding pathways open to the MalE31 precursor, we have analyzed the cellular fates of this aggregation-prone protein carrying altered signal sequences. Our results are most easily interpreted by a kinetic competition between exportation, folding, and degradation.

  10. Sequence-Based Prediction of Type III Secreted Proteins

    PubMed Central

    Arnold, Roland; Brandmaier, Stefan; Kleine, Frederick; Tischler, Patrick; Heinz, Eva; Behrens, Sebastian; Niinikoski, Antti; Mewes, Hans-Werner; Horn, Matthias; Rattei, Thomas

    2009-01-01

    The type III secretion system (TTSS) is a key mechanism for host cell interaction used by a variety of bacterial pathogens and symbionts of plants and animals including humans. The TTSS represents a molecular syringe with which the bacteria deliver effector proteins directly into the host cell cytosol. Despite the importance of the TTSS for bacterial pathogenesis, recognition and targeting of type III secreted proteins has up until now been poorly understood. Several hypotheses are discussed, including an mRNA-based signal, a chaperon-mediated process, or an N-terminal signal peptide. In this study, we systematically analyzed the amino acid composition and secondary structure of N-termini of 100 experimentally verified effector proteins. Based on this, we developed a machine-learning approach for the prediction of TTSS effector proteins, taking into account N-terminal sequence features such as frequencies of amino acids, short peptides, or residues with certain physico-chemical properties. The resulting computational model revealed a strong type III secretion signal in the N-terminus that can be used to detect effectors with sensitivity of ∼71% and selectivity of ∼85%. This signal seems to be taxonomically universal and conserved among animal pathogens and plant symbionts, since we could successfully detect effector proteins if the respective group was excluded from training. The application of our prediction approach to 739 complete bacterial and archaeal genome sequences resulted in the identification of between 0% and 12% putative TTSS effector proteins. Comparison of effector proteins with orthologs that are not secreted by the TTSS showed no clear pattern of signal acquisition by fusion, suggesting convergent evolutionary processes shaping the type III secretion signal. The newly developed program EffectiveT3 (http://www.chlamydiaedb.org) is the first universal in silico prediction program for the identification of novel TTSS effectors. Our findings will

  11. Ethanol stress impairs protein folding in the endoplasmic reticulum and activates Ire1 in Saccharomyces cerevisiae.

    PubMed

    Miyagawa, Ken-Ichi; Ishiwata-Kimata, Yuki; Kohno, Kenji; Kimata, Yukio

    2014-01-01

    Impaired protein folding in the endoplasmic reticulum (ER) evokes the unfolded protein response (UPR), which is triggered in budding yeast, Saccharomyces cerevisiae, by the ER-located transmembrane protein Ire1. Here, we report that ethanol stress damages protein folding in the ER, causing activation of Ire1 in yeast cells. The UPR likely contributes to the ethanol tolerance of yeast cells.

  12. High yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

    PubMed

    Ramos-Martinez, E M; Fimognari, L; Sakuragi, Y

    2017-02-16

    Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability, and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. In order to increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n, wherein n=10 or 20]. The yields of the (SP)n-fused Venus were higher than Venus without the glycomodule by up to 12 folds, with the maximum yield of 15 mg L(-1) . Moreover, the presence of the glycomodules confererred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with Brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. This article is protected by copyright. All rights reserved.

  13. Distinct folding pathways of two homologous disulfide proteins: bovine pancreatic trypsin inhibitor and tick anticoagulant peptide.

    PubMed

    Chang, Jui-Yoa

    2011-01-01

    The folding pathways of disulfide proteins vary substantially (Arolas et al., Trends Biochem Sci 31: 292-301, 2006). The diversity is mainly manifested by (a) the extent of heterogeneity of folding intermediates, (b) the extent of presence of native-like intermediates, and (c) the variation of folding kinetics. Even among structurally similar proteins, the difference can be enormous. This is demonstrated in this concise review with two structurally homologous kunitz-type protease inhibitors, bovine pancreatic trypsin inhibitor and tick anticoagulant peptide, as well as a group of cystine knot proteins. The diversity of their folding mechanisms is illustrated with two different folding techniques: (a) the conventional method of disulfide oxidation (oxidative folding), and (b) the novel method of disulfide scrambling (Chang, J Biol Chem 277: 120-126, 2002). This review also highlights the convergence of folding models concluded form the conventional conformational folding and those obtained by oxidative folding.

  14. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

    PubMed

    Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

    2013-01-01

    The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  15. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

    PubMed Central

    Gasser, Brigitte; Saloheimo, Markku; Rinas, Ursula; Dragosits, Martin; Rodríguez-Carmona, Escarlata; Baumann, Kristin; Giuliani, Maria; Parrilli, Ermenegilda; Branduardi, Paola; Lang, Christine; Porro, Danilo; Ferrer, Pau; Tutino, Maria Luisa; Mattanovich, Diethard; Villaverde, Antonio

    2008-01-01

    Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes. PMID:18394160

  16. Important amino acid residues involved in folding and binding of protein-protein complexes.

    PubMed

    Kulandaisamy, A; Lathi, V; ViswaPoorani, K; Yugandhar, K; Gromiha, M Michael

    2017-01-01

    Protein-protein interactions perform diverse functions in living organism. The integrative analysis of binding and stabilizing residues will provide insights on the functions of protein-protein complexes. In this work, we constructed a non-redundant dataset of 261 protein-protein complexes and identified binding site residues, stabilizing residues and common to both binding and stabilizing, termed as "key residues". We found that 6.1% of residues are involved in binding and 6.8% of residues are important for folding and stability. Among them, only 2% are involved in both folding and binding, which shows the importance and specific roles played by these residues. The key residues have been analyzed based on protein function, binding affinity, rigid and flexible complexes, amino acid preference and structure based parameters. We found that high affinity complexes have more key residues than low affinity complexes. In addition, key residues are enriched with the combination of specific hydrophobic and charged/polar residues. Atomic contacts between interacting proteins have distinct preferences of polar-polar, nonpolar-nonpolar and polar-nonpolar contacts in different functional classes of protein-protein complexes. Further, the influence of sequence and structural parameters such as surrounding hydrophobicity, solvent accessibility, secondary structure, long-range order and conservation score has been discussed. The analysis can be used to comprehend the interplay between stability and binding in protein-protein complexes. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Proteomic analysis of secreted proteins from Arabidopsis thaliana seedlings: improved recovery following removal of phenolic compounds.

    PubMed

    Charmont, Stéphane; Jamet, Elisabeth; Pont-Lezica, Rafael; Canut, Hervé

    2005-02-01

    Arabidopsis thaliana seedlings grown in liquid culture were used to recover proteins secreted from the whole plant. The aim was to identify apoplastic proteins that may be lost during classical extraction procedures such as preparation of cell walls. The inclusion of polyvinyl-polypyrrolidone (PVPP) in the protocol of purification of secreted proteins allowed a more efficient identification of proteins after their separation by two-dimensional gel electrophoresis (2-DE) and mass spectrometry analyses. Improvement of identification was 4-fold. It is related to an increased number of detectable peaks on mass spectra increasing the percentage of sequence coverage, and the identification confidence. The role of PVPP was to trap phenolic compounds and to prevent their unspecific interactions with proteins. These experiments resulted in the identification of 44 secreted proteins, of which 70% were not identified in previous cell wall proteomic studies. This may be due to specific gene regulation in seedlings and/or to a better access to apoplastic proteins not bound to cell walls.

  18. Excreted/Secreted Proteins from Trypanosome Procyclic Strains

    PubMed Central

    Atyame Nten, Celestine Michelle; Sommerer, Nicolas; Rofidal, Valerie; Hirtz, Christophe; Rossignol, Michel; Cuny, Gerard; Peltier, Jean-Benoit; Geiger, Anne

    2010-01-01

    Trypanosoma secretome was shown to be involved in parasite virulence and is suspected of interfering in parasite life-cycle steps such as establishment in the Glossina midgut, metacyclogenesis. Therefore, we attempted to identify the proteins secreted by procyclic strains of T. brucei gambiense and T. brucei brucei, responsible for human and animal trypanosomiasis, respectively. Using mass spectrometry, 427 and 483 nonredundant proteins were characterized in T. brucei brucei and T. brucei gambiense secretomes, respectively; 35% and 42% of the corresponding secretome proteins were specifically secreted by T. brucei brucei and T. brucei gambiense, respectively, while 279 proteins were common to both subspecies. The proteins were assigned to 12 functional classes. Special attention was paid to the most abundant proteases (14 families) because of their potential implication in the infection process and nutrient supply. The presence of proteins usually secreted via an exosome pathway suggests that this type of process is involved in trypanosome ESP secretion. The overall results provide leads for further research to develop novel tools for blocking trypanosome transmission. PMID:20011064

  19. Can natural proteins designed with 'inverted' peptide sequences adopt native-like protein folds?

    PubMed

    Sridhar, Settu; Guruprasad, Kunchur

    2014-01-01

    We have carried out a systematic computational analysis on a representative dataset of proteins of known three-dimensional structure, in order to evaluate whether it would possible to 'swap' certain short peptide sequences in naturally occurring proteins with their corresponding 'inverted' peptides and generate 'artificial' proteins that are predicted to retain native-like protein fold. The analysis of 3,967 representative proteins from the Protein Data Bank revealed 102,677 unique identical inverted peptide sequence pairs that vary in sequence length between 5-12 and 18 amino acid residues. Our analysis illustrates with examples that such 'artificial' proteins may be generated by identifying peptides with 'similar structural environment' and by using comparative protein modeling and validation studies. Our analysis suggests that natural proteins may be tolerant to accommodating such peptides.

  20. Characterization of protein folding by a Φ-value calculation with a statistical-mechanical model

    PubMed Central

    Wako, Hiroshi; Abe, Haruo

    2016-01-01

    The Φ-value analysis approach provides information about transition-state structures along the folding pathway of a protein by measuring the effects of an amino acid mutation on folding kinetics. Here we compared the theoretically calculated Φ values of 27 proteins with their experimentally observed Φ values; the theoretical values were calculated using a simple statistical-mechanical model of protein folding. The theoretically calculated Φ values reflected the corresponding experimentally observed Φ values with reasonable accuracy for many of the proteins, but not for all. The correlation between the theoretically calculated and experimentally observed Φ values strongly depends on whether the protein-folding mechanism assumed in the model holds true in real proteins. In other words, the correlation coefficient can be expected to illuminate the folding mechanisms of proteins, providing the answer to the question of which model more accurately describes protein folding: the framework model or the nucleation-condensation model. In addition, we tried to characterize protein folding with respect to various properties of each protein apart from the size and fold class, such as the free-energy profile, contact-order profile, and sensitivity to the parameters used in the Φ-value calculation. The results showed that any one of these properties alone was not enough to explain protein folding, although each one played a significant role in it. We have confirmed the importance of characterizing protein folding from various perspectives. Our findings have also highlighted that protein folding is highly variable and unique across different proteins, and this should be considered while pursuing a unified theory of protein folding. PMID:28409079

  1. Fast Protein Translation Can Promote Co- and Posttranslational Folding of Misfolding-Prone Proteins.

    PubMed

    Trovato, Fabio; O'Brien, Edward P

    2017-05-09

    Chemical kinetic modeling has previously been used to predict that fast-translating codons can enhance cotranslational protein folding by helping to avoid misfolded intermediates. Consistent with this prediction, protein aggregation in yeast and worms was observed to increase when translation was globally slowed down, possibly due to increased cotranslational misfolding. Observation of similar behavior in molecular simulations would confirm predictions from the simpler chemical kinetic model and provide a molecular perspective on cotranslational folding, misfolding, and the impact of translation speed on these processes. All-atom simulations cannot reach the timescales relevant to protein synthesis, and most conventional structure-based coarse-grained models do not allow for nonnative structure formation. Here, we introduce a protocol to incorporate misfolding using the functional forms of publicly available force fields. With this model we create two artificial proteins that are capable of undergoing structural transitions between a native and a misfolded conformation and simulate their synthesis by the ribosome. Consistent with the chemical kinetic predictions, we find that rapid synthesis of misfolding-prone nascent-chain segments increases the fraction of folded proteins by kinetically partitioning more molecules through on-pathway intermediates, decreasing the likelihood of sampling misfolded conformations. Novel to this study, to our knowledge, we observe that differences in protein dynamics, arising from different translation-elongation schedules, can persist long after the nascent protein has been released from the ribosome, and that a sufficient level of energetic frustration is needed for fast-translating codons to be beneficial for folding. These results provide further evidence that fast-translating codons can be as biologically important as pause sites in coordinating cotranslational folding. Copyright © 2017 Biophysical Society. Published by Elsevier

  2. Secretion, delivery and function of oomycete effector proteins.

    PubMed

    Wawra, Stephan; Belmonte, Rodrigo; Löbach, Lars; Saraiva, Marcia; Willems, Ariane; van West, Pieter

    2012-12-01

    Oomycetes are responsible for multi-billion dollar damages in aquaculture, agriculture and forestry. One common strategy they share with most cellular disease agents is the secretion of effector proteins. Effectors are molecules that change host physiology by initiating and allowing an infection to develop. Oomycetes secrete both extracellular and intracellular effectors. Studying secretion, delivery and function of effectors will hopefully lead to alternative control measures, which is much needed as several chemicals to control plant and animal pathogenic oomycetes cannot be used anymore; due to resistance in the host, or because the control measures have been prohibited as a result of toxicity to the environment and/or consumers. Here the latest findings on oomycete effector secretion, delivery and function are discussed.

  3. Protein folding: Over half a century lasting quest. Comment on "There and back again: Two views on the protein folding puzzle" by Alexei V. Finkelstein et al.

    NASA Astrophysics Data System (ADS)

    Krokhotin, Andrey; Dokholyan, Nikolay V.

    2017-07-01

    Most proteins fold into unique three-dimensional (3D) structures that determine their biological functions, such as catalytic activity or macromolecular binding. Misfolded proteins can pose a threat through aberrant interactions with other proteins leading to a number of diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [1,2]. What does determine 3D structure of proteins? The first clue to this question came more than fifty years ago when Anfinsen demonstrated that unfolded proteins can spontaneously fold to their native 3D structures [3,4]. Anfinsen's experiments lead to the conclusion that proteins fold to unique native structure corresponding to the stable and kinetically accessible free energy minimum, and protein native structure is solely determined by its amino acid sequence. The question of how exactly proteins find their free energy minimum proved to be a difficult problem. One of the puzzles, initially pointed out by Levinthal, was an inconsistency between observed protein folding times and theoretical estimates. A self-avoiding polymer model of a globular protein of 100-residues length on a cubic lattice can sample at least 1047 states. Based on the assumption that conformational sampling occurs at the highest vibrational mode of proteins (∼picoseconds), predicted folding time by searching among all the possible conformations leads to ∼1027 years (much larger than the age of the universe) [5]. In contrast, observed protein folding time range from microseconds to minutes. Due to tremendous theoretical progress in protein folding field that has been achieved in past decades, the source of this inconsistency is currently understood that is thoroughly described in the review by Finkelstein et al. [6].

  4. The energy landscape of modular repeat proteins: topology determines folding mechanism in the ankyrin family.

    PubMed

    Ferreiro, Diego U; Cho, Samuel S; Komives, Elizabeth A; Wolynes, Peter G

    2005-12-02

    Proteins consisting of repeating amino acid motifs are abundant in all kingdoms of life, especially in higher eukaryotes. Repeat-containing proteins self-organize into elongated non-globular structures. Do the same general underlying principles that dictate the folding of globular domains apply also to these extended topologies? Using a simplified structure-based model capturing a perfectly funneled energy landscape, we surveyed the predicted mechanism of folding for ankyrin repeat containing proteins. The ankyrin family is one of the most extensively studied classes of non-globular folds. The model based only on native contacts reproduces most of the experimental observations on the folding of these proteins, including a folding mechanism that is reminiscent of a nucleation propagation growth. The confluence of simulation and experimental results suggests that the folding of non-globular proteins is accurately described by a funneled energy landscape, in which topology plays a determinant role in the folding mechanism.

  5. ROP2 from Toxoplasma gondii: a virulence factor with a protein-kinase fold and no enzymatic activity.

    PubMed

    Labesse, Gilles; Gelin, Muriel; Bessin, Yannick; Lebrun, Maryse; Papoin, Julien; Cerdan, Rachel; Arold, Stefan T; Dubremetz, Jean-François

    2009-01-14

    The ROP2 protein and its paralogs are important virulence factors secreted into the host cell by the parasite Toxoplasma gondii. Here we describe the crystal structure of a large and soluble domain of mature ROP2, representative of the ROP2-like protein family. This is a structure of a protein-kinase fold that is devoid of catalytic residues and does not bind ATP. Various structural extensions constitute a signature of this protein family and act to maintain the protein kinase in an open conformation. Our ROP2 structure rules out a previous structural model of attachment of ROP2-like proteins to the parasitophorous vacuole membrane. We propose an alternative mode of membrane attachment implicating basic and amphiphatic helices present in the flexible N terminus of ROP2.

  6. Understanding protein domain-swapping using structure-based models of protein folding.

    PubMed

    Mascarenhas, Nahren Manuel; Gosavi, Shachi

    2017-09-01

    In domain-swapping, two or more identical protein monomers exchange structural elements and fold into dimers or multimers whose units are structurally similar to the original monomer. Domain-swapping is of biotechnological interest because inhibiting domain-swapping can reduce disease-causing fibrillar protein aggregation. To achieve such inhibition, it is important to understand both the energetics that stabilize the domain-swapped structure and the protein dynamics that enable the swapping. Structure-based models (SBMs) encode the folded structure of the protein in their potential energy functions. SBMs have been successfully used to understand diverse aspects of monomer folding. Symmetrized SBMs model interactions between two identical protein chains using only intra-monomer interactions. Molecular dynamics simulations of such symmetrized SBMs have been used to correctly predict the domain-swapped structure and to understand the mechanism of domain-swapping. Here, we review such models and illustrate that monomer topology determines key aspects of domain-swapping. However, in some proteins, specifics of local energetic interactions modulate domain-swapping and these need to be added to the symmetrized SBMs. We then summarize some general principles of the mechanism of domain-swapping that emerge from the symmetrized SBM simulations. Finally, using our own results, we explore how symmetrized SBMs could be used to design domain-swapping in proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. A collaborative visual analytics suite for protein folding research.

    PubMed

    Harvey, William; Park, In-Hee; Rübel, Oliver; Pascucci, Valerio; Bremer, Peer-Timo; Li, Chenglong; Wang, Yusu

    2014-09-01

    Molecular dynamics (MD) simulation is a crucial tool for understanding principles behind important biochemical processes such as protein folding and molecular interaction. With the rapidly increasing power of modern computers, large-scale MD simulation experiments can be performed regularly, generating huge amounts of MD data. An important question is how to analyze and interpret such massive and complex data. One of the (many) challenges involved in analyzing MD simulation data computationally is the high-dimensionality of such data. Given a massive collection of molecular conformations, researchers typically need to rely on their expertise and prior domain knowledge in order to retrieve certain conformations of interest. It is not easy to make and test hypotheses as the data set as a whole is somewhat "invisible" due to its high dimensionality. In other words, it is hard to directly access and examine individual conformations from a sea of molecular structures, and to further explore the entire data set. There is also no easy and convenient way to obtain a global view of the data or its various modalities of biochemical information. To this end, we present an interactive, collaborative visual analytics tool for exploring massive, high-dimensional molecular dynamics simulation data sets. The most important utility of our tool is to provide a platform where researchers can easily and effectively navigate through the otherwise "invisible" simulation data sets, exploring and examining molecular conformations both as a whole and at individual levels. The visualization is based on the concept of a topological landscape, which is a 2D terrain metaphor preserving certain topological and geometric properties of the high dimensional protein energy landscape. In addition to facilitating easy exploration of conformations, this 2D terrain metaphor also provides a platform where researchers can visualize and analyze various properties (such as contact density) overlayed on the

  8. Energy landscape and multiroute folding of topologically complex proteins adenylate kinase and 2ouf-knot

    PubMed Central

    Li, Wenfei; Terakawa, Tsuyoshi; Wang, Wei; Takada, Shoji

    2012-01-01

    While fast folding of small proteins has been relatively well characterized by experiments and theories, much less is known for slow folding of larger proteins, for which recent experiments suggested quite complex and rich folding behaviors. Here, we address how the energy landscape theory can be applied to these slow folding reactions. Combining the perfect-funnel approximation with a multiscale method, we first extended our previous atomic-interaction based coarse grained (AICG) model to take into account local flexibility of protein molecules. Using this model, we then investigated the energy landscapes and folding routes of two proteins with complex topologies: a multidomain protein adenylate kinase (AKE) and a knotted protein 2ouf-knot. In the AKE folding, consistent with experimental results, the kinetic free energy surface showed several substates between the fully unfolded and native states. We characterized the structural features of these substates and transitions among them, finding temperature-dependent multiroute folding. For protein 2ouf-knot, we found that the improved atomic-interaction based coarse-grained model can spontaneously tie a knot and fold the protein with a probability up to 96%. The computed folding rate of the knotted protein was much slower than that of its unknotted counterpart, in agreement with experimental findings. Similar to the AKE case, the 2ouf-knot folding exhibited several substates and transitions among them. Interestingly, we found a dead-end substate that lacks the knot, thus suggesting backtracking mechanisms. PMID:22753508

  9. Estimation of protein folding rate from Monte Carlo simulations and entropy capacity.

    PubMed

    Galzitskaya, Oxana V

    2010-11-01

    The problem of protein self-organization is one of the most important problems of molecular biology nowadays. Despite the recent success in the understanding of general principles of protein folding, details of this process are yet to be elucidated. Moreover, the prediction of protein folding rates has its own practical value due to the fact that aggregation directly depends on the rate of protein folding. The time of folding has been calculated for 67 proteins with known experimental data at the point of thermodynamic equilibrium between unfolded and native states using a Monte Carlo model where each residue is considered to be either folded as in the native state or completely disordered. The times of folding for 67 proteins which reach the native state within the limit of 10(8) Monte Carlo steps are in a good correlation with the experimentally measured folding rate at the mid-transition point (the correlation coefficient is -0.82). Theoretical consideration of a capillarity model for the process of protein folding demonstrates that the difference in the folding rate for proteins sharing more spherical and less spherical folds is the result of differences in the conformational entropy due to a larger surface of the boundary between folded and unfolded phases in the transition state for proteins with more spherical fold. The capillarity model allows us to predict the folding rate at the same level of correlation as by Monte Carlo simulations. The calculated model entropy capacity (conformational entropy per residue divided by the average contact energy per residue) for 67 proteins correlates by about 78% with the experimentally measured folding rate at the mid-transition point.

  10. The Folding of a Family of Three-Helix Bundle Proteins: Spectrin R15 Has a Robust Folding Nucleus, Unlike Its Homologous Neighbours☆

    PubMed Central

    Kwa, Lee Gyan; Wensley, Beth G.; Alexander, Crispin G.; Browning, Stuart J.; Lichman, Benjamin R.; Clarke, Jane

    2014-01-01

    Three homologous spectrin domains have remarkably different folding characteristics. We have previously shown that the slow-folding R16 and R17 spectrin domains can be altered to resemble the fast folding R15, in terms of speed of folding (and unfolding), landscape roughness and folding mechanism, simply by substituting five residues in the core. Here we show that, by contrast, R15 cannot be engineered to resemble R16 and R17. It is possible to engineer a slow-folding version of R15, but our analysis shows that this protein neither has a rougher energy landscape nor does change its folding mechanism. Quite remarkably, R15 appears to be a rare example of a protein with a folding nucleus that does not change in position or in size when its folding nucleus is disrupted. Thus, while two members of this protein family are remarkably plastic, the third has apparently a restricted folding landscape. PMID:24373753

  11. The folding of a family of three-helix bundle proteins: spectrin R15 has a robust folding nucleus, unlike its homologous neighbours.

    PubMed

    Kwa, Lee Gyan; Wensley, Beth G; Alexander, Crispin G; Browning, Stuart J; Lichman, Benjamin R; Clarke, Jane

    2014-04-03

    Three homologous spectrin domains have remarkably different folding characteristics. We have previously shown that the slow-folding R16 and R17 spectrin domains can be altered to resemble the fast folding R15, in terms of speed of folding (and unfolding), landscape roughness and folding mechanism, simply by substituting five residues in the core. Here we show that, by contrast, R15 cannot be engineered to resemble R16 and R17. It is possible to engineer a slow-folding version of R15, but our analysis shows that this protein neither has a rougher energy landscape nor does change its folding mechanism. Quite remarkably, R15 appears to be a rare example of a protein with a folding nucleus that does not change in position or in size when its folding nucleus is disrupted. Thus, while two members of this protein family are remarkably plastic, the third has apparently a restricted folding landscape.

  12. Thermodynamics of downhill folding: multi-probe analysis of PDD, a protein that folds over a marginal free energy barrier.

    PubMed

    Naganathan, Athi N; Muñoz, Victor

    2014-07-31

    Downhill folding proteins fold in microseconds by crossing a very low or no free energy barrier (<3 RT), and exhibit a complex unfolding behavior in equilibrium. Such unfolding complexity is due to the weak thermodynamic coupling that exists between the various structural segments of these proteins, and it is manifested in unfolding curves that differ depending on the structural probe employed to monitor the process. Probe-dependent unfolding has important practical implications because it permits one to investigate the folding energy landscape in detail using multiprobe thermodynamic experiments. This type of thermodynamic behavior has been investigated in depth on the protein BBL, an example of extreme (one-state) downhill folding in which there is no free energy barrier at any condition, including the denaturation midpoint. However, an open question is, to what extent is such thermodynamic behavior observed on less extreme downhill folders? Here we perform a multiprobe spectroscopic characterization of the microsecond folder PDD, a structural and functional homologue of BBL that folds within the downhill regime, but is not an example of one-state downhill folding; rather at the denaturation midpoint PDD folds by crossing an incipient free energy barrier. Model-free analysis of the unfolding curves from four different spectroscopic probes together with differential scanning calorimetry reveals a dispersion of ∼9 K in the apparent melting temperature and also marked differences in unfolding broadness (from ∼50 to ∼130 kJ mol(-1) when analyzed with a two-state model), confirming that such properties are also observed on less extreme downhill folders. We subsequently perform a global quantitative analysis of the unfolding data of PDD using the same ME statistical mechanical model that was used before for the BBL domain. The analysis shows that this simple model captures all of the features observed on the unfolding of PDD (i.e., the intensity and temperature

  13. Residual ordered structure in denatured proteins and the problem of protein folding.

    PubMed

    Basharov, Mahmud A

    2012-02-01

    Structural characteristics of numerous globular proteins in the denatured state have been reviewed using literature data. Recent more precise experiments show that in contrast to the conventional standpoint, proteins under strongly denaturing conditions do not unfold completely and adopt a random coil state, but contain significant residual ordered structure. These results cast doubt on the basis of the conventional approach representing the process of protein folding as a spontaneous transition of a polypeptide chain from the random coil state to the unique globular structure. The denaturation of proteins is explained in terms of the physical properties of proteins such as stability, conformational change, elasticity, irreversible denaturation, etc. The spontaneous renaturation of some denatured proteins most probably is merely the manifestation of the physical properties (e.g., the elasticity) of the proteins per se, caused by the residual structure present in the denatured state. The pieces of the ordered structure might be the centers of the initiation of renaturation, where the restoration of the initial native conformation of denatured proteins begins. Studies on the denaturation of proteins hardly clarify how the proteins fold into the native conformation during the successive residue-by-residue elongation of the polypeptide chain on the ribosome.

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

    SciTech Connect

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

    2014-07-18

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

  15. Design and structure of an equilibrium protein folding intermediate: a hint into dynamical regions of proteins.

    PubMed

    Ayuso-Tejedor, Sara; Angarica, Vladimir Espinosa; Bueno, Marta; Campos, Luis A; Abián, Olga; Bernadó, Pau; Sancho, Javier; Jiménez, M Angeles

    2010-07-23

    Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity.

  16. Disulphide bridge formation of proinsulin fusion proteins during secretion in Streptomyces.

    PubMed

    Vértesy, L; Tripier, D; Koller, K P; Riess, G

    1991-03-01

    To study disulphide bridge formation by Streptomyces lividans TK 24 in secreted single chain precursors of insulin a fusion protein (PTF 1) was investigated consisting of monkey proinsulin and the aminoterminal sequence Asp1 to Gly43 of the alpha-amylase inhibitor tendamistat from Streptomyces tendae. The purified soluble protein PTF 1 has a molecular mass of 14.4 kDa. The primary structure was elucidated after digestion with lysyl endopeptidase and fragment analysis. In this system, disulphide bond formation occurs in a way that the first cysteine in proinsulin is linked to the next following cysteine in the amino-acid chain resulting in a non-natural folding of the insulin part of the fusion protein. Re-folding of PTF 1 by reduction and re-oxidation followed by proteolytic digestions led to insulins which are identical to authentic material. The ease of correct disulphide bond formation in solution and incorrect processing during secretion suggests involvement of yet unknown factors leading to an unfavourable folding of proinsulin.

  17. Symbiotic implications of type III protein secretion machinery in Rhizobium.

    PubMed

    Viprey, V; Del Greco, A; Golinowski, W; Broughton, W J; Perret, X

    1998-06-01

    The symbiotic plasmid of Rhizobium sp. NGR234 carries a cluster of genes that encodes components of a bacterial type III secretion system (TTSS). In both animal and plant pathogens, the TTSS is an essential component of pathogenicity. Here, we show that secretion of at least two proteins (y4xL and NolX) is controlled by the TTSS of NGR234 and occurs after the induction with flavonoids. Polar mutations in two TTSS genes, rhcN and the nod-box controlled regulator of transcription y4xl, block the secretion of both proteins and strongly affect the ability of NGR234 to nodulate a variety of tropical legumes including Pachyrhizus tuberosus and Tephrosia vogelii.

  18. Protein folding occurs while bound to the ATP-independent chaperone Spy

    PubMed Central

    Humes, Julia R; Radford, Sheena E; Bardwell, James C A

    2016-01-01

    Chaperones assist the folding of many proteins in the cell. While the most well studied chaperones use cycles of ATP binding and hydrolysis to assist protein folding, a number of chaperones have been identified that promote protein folding in the absence of high-energy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we have characterized the kinetic mechanism of substrate folding by the small, ATP-independent chaperone, Spy. Spy rapidly associates with its substrate, Immunity protein 7 (Im7), eliminating its potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while remaining bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones can assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while continuously bound to a chaperone. PMID:26619265

  19. In-Situ Observation of Membrane Protein Folding during Cell-Free Expression

    PubMed Central

    Fitter, Jörg; Büldt, Georg; Heberle, Joachim; Schlesinger, Ramona; Ataka, Kenichi

    2016-01-01

    Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando) at molecular resolution. PMID:26978519

  20. Size and conformation limits to secretion of disulfide-bonded loops in autotransporter proteins.

    PubMed

    Leyton, Denisse L; Sevastsyanovich, Yanina R; Browning, Douglas F; Rossiter, Amanda E; Wells, Timothy J; Fitzpatrick, Rebecca E; Overduin, Michael; Cunningham, Adam F; Henderson, Ian R

    2011-12-09

    Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein. The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.

  1. Contact order revisited: Influence of protein size on the folding rate

    SciTech Connect

    Ivankov, Dmitry N.; Garbuzynskiy, Sergiy O.; Alm, Eric; Plaxco, Kevin W.; Baker, David; Finkelstein, Alexei V.

    2003-05-28

    Guided by the recent success of empirical model predicting the folding rates of small two-state folding proteins from the relative contact order (CO) of their native structures, by a theoretical model of protein folding that predicts that logarithm of the folding rate decreases with the protein chain length L as L2/3, and by the finding that the folding rates of multistate folding proteins strongly correlate with their sizes and have very bad correlation with CO, we reexamined the dependence of folding rate on CO and L in attempt to find a structural parameter that determines folding rates for the totality of proteins. We show that the Abs{sub CO} = CO x L, is able to predict rather accurately folding rates for both two-state and multistate folding proteins, as well as short peptides, and that this Abs{sub CO} scales with the protein chain length as L0.70 {+-} 0.07 for the totality of studied single-domain proteins and peptides.

  2. Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing*

    PubMed Central

    Mehler, Michaela; Eckert, Carl Elias; Busche, Alena; Kulhei, Jennifer; Michaelis, Jonas; Becker-Baldus, Johanna; Wachtveitl, Josef; Dötsch, Volker; Glaubitz, Clemens

    2015-01-01

    Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a 13C-labeled retinal cofactor and extensively 13C-15N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications. PMID:26405032

  3. Mathematics, Thermodynamics, and Modeling to Address Ten Common Misconceptions about Protein Structure, Folding, and Stability

    ERIC Educational Resources Information Center

    Robic, Srebrenka

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative…

  4. Mathematics, Thermodynamics, and Modeling to Address Ten Common Misconceptions about Protein Structure, Folding, and Stability

    ERIC Educational Resources Information Center

    Robic, Srebrenka

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative…

  5. Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    DOE PAGES

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-10-01

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed "type VII." How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here in this paper, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), whichmore » adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE-PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Furthermore, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.« less

  6. Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion.

    PubMed

    Ekiert, Damian C; Cox, Jeffery S

    2014-10-14

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed "type VII." How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE-PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.

  7. A spatio-temporal mining approach towards summarizing and analyzing protein folding trajectories.

    PubMed

    Yang, Hui; Parthasarathy, Srinivasan; Ucar, Duygu

    2007-04-04

    Understanding the protein folding mechanism remains a grand challenge in structural biology. In the past several years, computational theories in molecular dynamics have been employed to shed light on the folding process. Coupled with high computing power and large scale storage, researchers now can computationally simulate the protein folding process in atomistic details at femtosecond temporal resolution. Such simulation often produces a large number of folding trajectories, each consisting of a series of 3D conformations of the protein under study. As a result, effectively managing and analyzing such trajectories is becoming increasingly important. In this article, we present a spatio-temporal mining approach to analyze protein folding trajectories. It exploits the simplicity of contact maps, while also integrating 3D structural information in the analysis. It characterizes the dynamic folding process by first identifying spatio-temporal association patterns in contact maps, then studying how such patterns evolve along a folding trajectory. We demonstrate that such patterns can be leveraged to summarize folding trajectories, and to facilitate the detection and ordering of important folding events along a folding path. We also show that such patterns can be used to identify a consensus partial folding pathway across multiple folding trajectories. Furthermore, we argue that such patterns can capture both local and global structural topology in a 3D protein conformation, thereby facilitating effective structural comparison amongst conformations. We apply this approach to analyze the folding trajectories of two small synthetic proteins-BBA5 and GSGS (or Beta3S). We show that this approach is promising towards addressing the above issues, namely, folding trajectory summarization, folding events detection and ordering, and consensus partial folding pathway identification across trajectories.

  8. Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein.

    PubMed

    Pirchi, Menahem; Ziv, Guy; Riven, Inbal; Cohen, Sharona Sedghani; Zohar, Nir; Barak, Yoav; Haran, Gilad

    2011-10-11

    Proteins attain their function only after folding into a highly organized three-dimensional structure. Much remains to be learned about the mechanisms of folding of large multidomain proteins, which may populate metastable intermediate states on their energy landscapes. Here we introduce a novel method, based on high-throughput single-molecule fluorescence experiments, which is specifically geared towards tracing the dynamics of folding in the presence of a plethora of intermediates. We employ this method to characterize the folding reaction of a three-domain protein, adenylate kinase. Using thousands of single-molecule trajectories and hidden Markov modelling, we identify six metastable states on adenylate kinase's folding landscape. Remarkably, the connectivity of the intermediates depends on denaturant concentration; at low concentration, multiple intersecting folding pathways co-exist. We anticipate that the methodology introduced here will find broad applicability in the study of folding of large proteins, and will provide a more realistic scenario of their conformational dynamics.

  9. Single-molecule spectroscopy of protein folding in a chaperonin cage

    PubMed Central

    Hofmann, Hagen; Hillger, Frank; Pfeil, Shawn H.; Hoffmann, Armin; Streich, Daniel; Haenni, Dominik; Nettels, Daniel; Lipman, Everett A.; Schuler, Benjamin

    2010-01-01

    Molecular chaperones are known to be essential for avoiding protein aggregation in vivo, but it is still unclear how they affect protein folding mechanisms. We use single-molecule Förster resonance energy transfer to follow the folding of a protein inside the GroEL/GroES chaperonin cavity over a time range from milliseconds to hours. Our results show that confinement in the chaperonin decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. Microfluidic mixing experiments indicate that strong interactions of the substrate with the cavity walls impede the folding process, but the folding hierarchy is preserved. Our results imply that no universal chaperonin mechanism exists. Rather, a competition between intra- and intermolecular interactions determines the folding rates and mechanisms of a substrate inside the GroEL/GroES cage. PMID:20547872

  10. Secreted Protein Acidic and Rich in Cysteine in Ocular Tissue

    PubMed Central

    Scavelli, Kurt; Chatterjee, Ayan

    2015-01-01

    Abstract Secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is the prototypical matricellular protein. Matricellular proteins are nonstructural secreted proteins that provide an integration between cells and their surrounding extracellular matrix (ECM). Regulation of the ECM is important in maintaining the physiologic function of tissues. Elevated levels of SPARC have been identified in a variety of diseases involving pathologic tissue remodeling, such as hepatic fibrosis, systemic sclerosis, and certain carcinomas. Within the eye, SPARC has been identified in the trabecular meshwork, lens, and retina. Studies have begun to show the role of SPARC in these tissues and its possible role, specifically in primary open-angle glaucoma, cataracts, and proliferative vitreoretinopathy. SPARC may, therefore, be a therapeutic target in the treatment of certain ocular diseases. Further investigation into the mechanism of action of SPARC will be necessary in the development of SPARC-targeted therapy. PMID:26167673

  11. Recent Progress in Machine Learning-Based Methods for Protein Fold Recognition

    PubMed Central

    Wei, Leyi; Zou, Quan

    2016-01-01

    Knowledge on protein folding has a profound impact on understanding the heterogeneity and molecular function of proteins, further facilitating drug design. Predicting the 3D structure (fold) of a protein is a key problem in molecular biology. Determination of the fold of a protein mainly relies on molecular experimental methods. With the development of next-generation sequencing techniques, the discovery of new protein sequences has been rapidly increasing. With such a great number of proteins, the use of experimental techniques to determine protein folding is extremely difficult because these techniques are time consuming and expensive. Thus, developing computational prediction methods that can automatically, rapidly, and accurately classify unknown protein sequences into specific fold categories is urgently needed. Computational recognition of protein folds has been a recent research hotspot in bioinformatics and computational biology. Many computational efforts have been made, generating a variety of computational prediction methods. In this review, we conduct a comprehensive survey of recent computational methods, especially machine learning-based methods, for protein fold recognition. This review is anticipated to assist researchers in their pursuit to systematically understand the computational recognition of protein folds. PMID:27999256

  12. Secretion of sulfated and nonsulfated forms of parathyroid chromogranin A (secretory protein-I)

    SciTech Connect

    Gorr, S.U.; Cohn, D.V. )

    1990-02-25

    Chromogranin A (secretory protein-I) is an acidic, sulfated glycoprotein found in secretory granules of most endocrine cells but not in exocrine or epithelial cells. Parathyroid chromogranin A is sulfated on tyrosine residues, whereas adrenal chromogranin A appears to be sulfated mainly on oligosaccharide residues. Chromogranin B, on the other hand, is tyrosine-sulfated in the bovine adrenal whereas this protein is absent from the parathyroid. The role of this tissue- or species-specific sulfation of chromogranin is not known. Tyrosine sulfation is a common post-translational modification of proteins in the exocytotic pathway and has been suggested to play a role in the sorting or intracellular transport of secretory proteins. To test this, porcine parathyroid tissue slices were metabolically labeled with 35SO4 and (3H)Lys, and the tissue and incubation medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoprecipitation with chromogranin A-specific antiserum or by radioimmunoassay for parathormone. Secretion of total and 3H-labeled chromogranin A was about 3- and 7-fold higher, respectively, at 0.5 mM than at 3.0 mM Ca2+, and secretion of 35SO4-labeled chromogranin A was 67-fold higher. This indicates that either sulfated chromogranin A is directed primarily to the Ca2+-regulated pathway or that sulfation occurs following sorting to this pathway. Sodium chlorate (1-10 mM) inhibited sulfation in a dose-dependent manner by up to 95% but it had no effect on the onset or rate of chromogranin A secretion. These data indicate that regulated secretion of parathyroid chromogranin A does not require sulfation of tyrosine residues.

  13. Substrate protein folds while it is bound to the ATP-independent chaperone Spy.

    PubMed

    Stull, Frederick; Koldewey, Philipp; Humes, Julia R; Radford, Sheena E; Bardwell, James C A

    2016-01-01

    Chaperones assist in the folding of many proteins in the cell. Although the most well-studied chaperones use cycles of ATP binding and hydrolysis to assist in protein folding, a number of chaperones have been identified that promote folding in the absence of high-energy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we characterized the kinetic mechanism of substrate folding by the small ATP-independent chaperone Spy from Escherichia coli. Spy rapidly associates with its substrate, immunity protein 7 (Im7), thereby eliminating Im7's potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while it remains bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while being continuously bound to a chaperone.

  14. Coarse-grained models of protein folding: toy models or predictive tools?

    PubMed

    Clementi, Cecilia

    2008-02-01

    Coarse-grained models are emerging as a practical alternative to all-atom simulations for the characterization of protein folding mechanisms over long time scales. While a decade ago minimalist toy models were mainly designed to test general hypotheses on the principles regulating protein folding, the latest coarse-grained models are increasingly realistic and can be used to characterize quantitatively the detailed folding mechanism of specific proteins. The ability of such models to reproduce the essential features of folding dynamics suggests that each single atomic degree of freedom is not by itself particularly relevant to folding and supports a statistical mechanical approach to characterize folding transitions. When combined with more refined models and with experimental studies, the systematic investigation of protein systems and complexes using coarse-grained models can advance our theoretical understanding of the actual organizing principles that emerge from the complex network of interactions among protein atomic constituents.

  15. Por Secretion System-Dependent Secretion and Glycosylation of Porphyromonas gingivalis Hemin-Binding Protein 35

    PubMed Central

    Shoji, Mikio; Sato, Keiko; Yukitake, Hideharu; Kondo, Yoshio; Narita, Yuka; Kadowaki, Tomoko; Naito, Mariko; Nakayama, Koji

    2011-01-01

    The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study. PMID:21731719

  16. Alternative protein secretion: The Mam1 ABC transporter supports secretion of M-factor linked GFP in fission yeast

    SciTech Connect

    Kjaerulff, Soren

    2005-12-30

    To examine whether the fission yeast Mam1 ABC transporter can be used for secretion of heterologous proteins, thereby bypassing the classical secretion pathway, we have analyzed chimeric forms of the M-factor precursor. It was demonstrated that GFP can be exported when fused to both the amino-terminal prosequence from mfm1 and a CaaX motif. This secretion was dependent on the Mam1 transporter and not the classical secretion pathway. The secretion efficiency of GFP, however, was relatively low and most of the reporter protein was trapped in the vacuolar membranes. Our findings suggest that the Mam1 ABC protein is a promiscuous peptide transporter that can accommodate globular proteins of a relatively large size. Furthermore, our results help in defining the sequences required for processing and secretion of natural M-factor.

  17. Mutagenic dissection of the sequence determinants of protein folding, recognition, and machine function.

    PubMed

    Sauer, Robert T

    2013-11-01

    Understanding the relationship between the amino-acid sequence of a protein and its ability to fold and to function is one of the major challenges of protein science. Here, cases are reviewed in which mutagenesis, biochemistry, structure determination, protein engineering, and single-molecule biophysics have illuminated the sequence determinants of folding, binding specificity, and biological function for DNA-binding proteins and ATP-fueled machines that forcibly unfold native proteins as a prelude to degradation. In addition to structure-function relationships, these studies provide information about folding