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Sample records for protein folding secretion

  1. 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

  2. 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.

  3. Adiporedoxin, an upstream regulator of ER oxidative folding and protein secretion in adipocytes

    PubMed Central

    Jedrychowski, Mark P.; Liu, Libin; Laflamme, Collette J.; Karastergiou, Kalypso; Meshulam, Tova; Ding, Shi-Ying; Wu, Yuanyuan; Lee, Mi-Jeong; Gygi, Steven P.; Fried, Susan K.; Pilch, Paul F.

    2015-01-01

    Objective Adipocytes are robust protein secretors, most notably of adipokines, hormone-like polypeptides, which act in an endocrine and paracrine fashion to affect numerous physiological processes such as energy balance and insulin sensitivity. To understand how such proteins are assembled for secretion we describe the function of a novel endoplasmic reticulum oxidoreductase, adiporedoxin (Adrx). Methods Adrx knockdown and overexpressing 3T3-L1 murine adipocyte cell lines and a knockout mouse model were used to assess the influence of Adrx on secreted proteins as well as the redox state of ER resident chaperones. The metabolic phenotypes of Adrx null mice were characterized and compared to WT mice. The correlation of Adrx levels BMI, adiponectin levels, and other inflammatory markers from adipose tissue of human subjects was also studied. Results Adiporedoxin functions via a CXXC active site, and is upstream of protein disulfide isomerase whose direct function is disulfide bond formation, and ultimately protein secretion. Over and under expression of Adrx in vitro enhances and reduces, respectively, the secretion of the disulfide-bonded proteins including adiponectin and collagen isoforms. On a chow diet, Adrx null mice have normal body weights, and glucose tolerance, are moderately hyperinsulinemic, have reduced levels of circulating adiponectin and are virtually free of adipocyte fibrosis resulting in a complex phenotype tending towards insulin resistance. Adrx protein levels in human adipose tissue correlate positively with adiponectin levels and negatively with the inflammatory marker phospho-Jun kinase. Conclusion These data support the notion that Adrx plays a critical role in adipocyte biology and in the regulation of mouse and human metabolism via its modulation of adipocyte protein secretion. PMID:26629401

  4. Structural models of intrinsically disordered and calcium-bound folded states of a protein adapted for secretion

    PubMed Central

    O’Brien, Darragh P.; Hernandez, Belen; Durand, Dominique; Hourdel, Véronique; Sotomayor-Pérez, Ana-Cristina; Vachette, Patrice; Ghomi, Mahmoud; Chamot-Rooke, Julia; Ladant, Daniel; Brier, Sébastien; Chenal, Alexandre

    2015-01-01

    Many Gram-negative bacteria use Type I secretion systems, T1SS, to secrete virulence factors that contain calcium-binding Repeat-in-ToXin (RTX) motifs. Here, we present structural models of an RTX protein, RD, in both its intrinsically disordered calcium-free Apo-state and its folded calcium-bound Holo-state. Apo-RD behaves as a disordered polymer chain comprising several statistical elements that exhibit local rigidity with residual secondary structure. Holo-RD is a folded multi-domain protein with an anisometric shape. RTX motifs thus appear remarkably adapted to the structural and mechanistic constraints of the secretion process. In the low calcium environment of the bacterial cytosol, Apo-RD is an elongated disordered coil appropriately sized for transport through the narrow secretion machinery. The progressive folding of Holo-RD in the extracellular calcium-rich environment as it emerges form the T1SS may then favor its unidirectional export through the secretory channel. This process is relevant for hundreds of bacterial species producing virulent RTX proteins. PMID:26374675

  5. 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

  6. 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

    2016-04-01

    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.

  7. 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

    2016-04-01

    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. PMID:27058787

  8. Protein folding in the ER.

    SciTech Connect

    Stevens, F. J.; Argon, Y.; Biosciences Division; Univ. of Chicago

    1999-10-01

    The endoplasmic reticulum (ER) is a major protein folding compartment for secreted, plasma membrane and organelle proteins. Each of these newly-synthesized polypeptides folds in a deterministic process, affected by the unique conditions that exist in the ER. An understanding of protein folding in the ER is a fundamental biomolecular challenge at two levels. The first level addresses how the amino acid sequence programs that polypeptide to efficiently arrive at a particular fold out of a multitude of alternatives, and how different sequences obtain similar folds. At the second level are the issues introduced by folding not in the cytosol, but in the ER, including the risk of aggregation in a molecularly crowded environment, accommodation of post-translational modifications and the compatibility with subsequent intracellular trafficking. This review discusses both the physicochemical and cell biological constraints of folding, which are the challenges that the ER molecular chaperones help overcome.

  9. 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

  10. 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

  11. Evolutionary optimization of protein folding.

    PubMed

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

    2013-01-01

    Nature has shaped the make up of proteins since their appearance, [Formula: see text]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 [Formula: see text]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

  12. Multidimensional theory of protein folding

    NASA Astrophysics Data System (ADS)

    Itoh, Kazuhito; Sasai, Masaki

    2009-04-01

    Theory of multidimensional representation of free energy surface of protein folding is developed by adopting structural order parameters of multiple regions in protein as multiple coordinates. Various scenarios of folding are classified in terms of cooperativity within individual regions and interactions among multiple regions and thus obtained classification is used to analyze the folding process of several example proteins. Ribosomal protein S6, src-SH3 domain, CheY, barnase, and BBL domain are analyzed with the two-dimensional representation by using a structure-based Hamiltonian model. The extension to the higher dimensional representation leads to the finer description of the folding process. Barnase, NtrC, and an ankyrin repeat protein are examined with the three-dimensional representation. The multidimensional representation allows us to directly address questions on folding pathways, intermediates, and transition states.

  13. 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)

  14. Fast events in protein folding

    SciTech Connect

    Woodruff, W.; Callender, R.; Causgrove, T.; Dyer, R.; Williams, S.

    1996-04-01

    The primary objective of this work was to develop a molecular understanding of how proteins achieve their native three-dimensional (folded) structures. This requires the identification and characterization of intermediates in the protein folding process on all relevant timescales, from picoseconds to seconds. The short timescale events in protein folding have been entirely unknown. Prior to this work, state-of-the-art experimental approaches were limited to milliseconds or longer, when much of the folding process is already over. The gap between theory and experiment is enormous: current theoretical and computational methods cannot realistically model folding processes with lifetimes longer than one nanosecond. This unique approach to employ laser pump-probe techniques that combine novel methods of laser flash photolysis with time-resolved vibrational spectroscopic probes of protein transients. In this scheme, a short (picosecond to nanosecond) laser photolysis pulse was used to produce an instantaneous pH or temperature jump, thereby initiating a protein folding or unfolding reaction. Structure-specific, time-resolved vibrational probes were then used to identify and characterize protein folding intermediates.

  15. 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.

  16. 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.

  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.

  18. Quantum theory on protein folding

    NASA Astrophysics Data System (ADS)

    Luo, LiaoFu

    2014-03-01

    The conformational change of biological macromolecule is investigated from the point of quantum transition. A quantum theory on protein folding is proposed. Compared with other dynamical variables such as mobile electrons, chemical bonds and stretching-bending vibrations the molecular torsion has the lowest energy and can be looked as the slow variable of the system. Simultaneously, from the multi-minima property of torsion potential the local conformational states are well defined. Following the idea that the slow variables slave the fast ones and using the nonadiabaticity operator method we deduce the Hamiltonian describing conformational change. It is shown that the influence of fast variables on the macromolecule can fully be taken into account through a phase transformation of slow variable wave function. Starting from the conformation-transition Hamiltonian the nonradiative matrix element was calculated and a general formulas for protein folding rate was deduced. The analytical form of the formula was utilized to study the temperature dependence of protein folding rate and the curious non-Arrhenius temperature relation was interpreted. By using temperature dependence data the multi-torsion correlation was studied. The decoherence time of quantum torsion state is estimated. The proposed folding rate formula gives a unifying approach for the study of a large class problems of biological conformational change.

  19. Semiempirical prediction of protein folds

    NASA Astrophysics Data System (ADS)

    Fernández, Ariel; Colubri, Andrés; Appignanesi, Gustavo

    2001-08-01

    We introduce a semiempirical approach to predict ab initio expeditious pathways and native backbone geometries of proteins that fold under in vitro renaturation conditions. The algorithm is engineered to incorporate a discrete codification of local steric hindrances that constrain the movements of the peptide backbone throughout the folding process. Thus, the torsional state of the chain is assumed to be conditioned by the fact that hopping from one basin of attraction to another in the Ramachandran map (local potential energy surface) of each residue is energetically more costly than the search for a specific (Φ, Ψ) torsional state within a single basin. A combinatorial procedure is introduced to evaluate coarsely defined torsional states of the chain defined ``modulo basins'' and translate them into meaningful patterns of long range interactions. Thus, an algorithm for structure prediction is designed based on the fact that local contributions to the potential energy may be subsumed into time-evolving conformational constraints defining sets of restricted backbone geometries whereupon the patterns of nonbonded interactions are constructed. The predictive power of the algorithm is assessed by (a) computing ab initio folding pathways for mammalian ubiquitin that ultimately yield a stable structural pattern reproducing all of its native features, (b) determining the nucleating event that triggers the hydrophobic collapse of the chain, and (c) comparing coarse predictions of the stable folds of moderately large proteins (N~100) with structural information extracted from the protein data bank.

  20. 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

  1. Learning Protein Folding Energy Functions

    PubMed Central

    Guan, Wei; Ozakin, Arkadas; Gray, Alexander; Borreguero, Jose; Pandit, Shashi; Jagielska, Anna; Wroblewska, Liliana; Skolnick, Jeffrey

    2014-01-01

    A critical open problem in ab initio protein folding is protein energy function design, which pertains to defining the energy of protein conformations in a way that makes folding most efficient and reliable. In this paper, we address this issue as a weight optimization problem and utilize a machine learning approach, learning-to-rank, to solve this problem. We investigate the ranking-via-classification approach, especially the RankingSVM method and compare it with the state-of-the-art approach to the problem using the MINUIT optimization package. To maintain the physicality of the results, we impose non-negativity constraints on the weights. For this we develop two efficient non-negative support vector machine (NNSVM) methods, derived from L2-norm SVM and L1-norm SVMs, respectively. We demonstrate an energy function which maintains the correct ordering with respect to structure dissimilarity to the native state more often, is more efficient and reliable for learning on large protein sets, and is qualitatively superior to the current state-of-the-art energy function. PMID:25311546

  2. 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

  3. 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.

  4. 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

  5. Unconventional Protein Secretion in Plants.

    PubMed

    Davis, Destiny J; Kang, Byung-Ho; Heringer, Angelo S; Wilkop, Thomas E; Drakakaki, Georgia

    2016-01-01

    Unconventional protein secretion (UPS) describes secretion pathways that bypass one or several of the canonical secretion pit-stops on the way to the plasma membrane, and/or involve the secretion of leaderless proteins. So far, alternatives to conventional secretion were primarily observed and studied in yeast and animal cells. The sessile lifestyle of plants brings with it unique restraints on how they adapt to adverse conditions and environmental challenges. Recently, attention towards unconventional secretion pathways in plant cells has substantially increased, with the large number of leaderless proteins identified through proteomic studies. While UPS pathways in plants are certainly not yet exhaustively researched, an emerging notion is that induction of UPS pathways is correlated with pathogenesis and stress responses. Given the multitude UPS events observed, comprehensively organizing the routes proteins take to the apoplast in defined UPS categories is challenging. With the establishment of a larger collection of studied plant proteins taking these UPS pathways, a clearer picture of endomembrane trafficking as a whole will emerge. There are several novel enabling technologies, such as vesicle proteomics and chemical genomics, with great potential for dissecting secretion pathways, providing information about the cargo that travels along them and the conditions that induce them. PMID:27665550

  6. Reduced alphabet for protein folding prediction.

    PubMed

    Huang, Jitao T; Wang, Titi; Huang, Shanran R; Li, Xin

    2015-04-01

    What are the key building blocks that would have been needed to construct complex protein folds? This is an important issue for understanding protein folding mechanism and guiding de novo protein design. Twenty naturally occurring amino acids and eight secondary structures consist of a 28-letter alphabet to determine folding kinetics and mechanism. Here we predict folding kinetic rates of proteins from many reduced alphabets. We find that a reduced alphabet of 10 letters achieves good correlation with folding rates, close to the one achieved by full 28-letter alphabet. Many other reduced alphabets are not significantly correlated to folding rates. The finding suggests that not all amino acids and secondary structures are equally important for protein folding. The foldable sequence of a protein could be designed using at least 10 folding units, which can either promote or inhibit protein folding. Reducing alphabet cardinality without losing key folding kinetic information opens the door to potentially faster machine learning and data mining applications in protein structure prediction, sequence alignment and protein design.

  7. Protein secretion in Pichia pastoris and advances in protein production.

    PubMed

    Damasceno, Leonardo M; Huang, Chung-Jr; Batt, Carl A

    2012-01-01

    Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P. pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production. PMID:22057543

  8. On the universe of protein folds.

    PubMed

    Kolodny, Rachel; Pereyaslavets, Leonid; Samson, Abraham O; Levitt, Michael

    2013-01-01

    In the fifty years since the first atomic structure of a protein was revealed, tens of thousands of additional structures have been solved. Like all objects in biology, proteins structures show common patterns that seem to define family relationships. Classification of proteins structures, which started in the 1970s with about a dozen structures, has continued with increasing enthusiasm, leading to two main fold classifications, SCOP and CATH, as well as many additional databases. Classification is complicated by deciding what constitutes a domain, the fundamental unit of structure. Also difficult is deciding when two given structures are similar. Like all of biology, fold classification is beset by exceptions to all rules. Thus, the perspectives of protein fold space that the fold classifications offer differ from each other. In spite of these ambiguities, fold classifications are useful for prediction of structure and function. Studying the characteristics of fold space can shed light on protein evolution and the physical laws that govern protein behavior.

  9. Monster Mash: Protein Folding Gone Wrong

    MedlinePlus

    ... Articles | Inside Life Science Home Page Monster Mash: Protein Folding Gone Wrong By Joseph Piergrossi Posted October 31, 2013 In this image, globs of misfolded proteins called amyloid plaques (blobs) are found outside neurons ( ...

  10. 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.

  11. Visualizing chaperone-assisted protein folding.

    PubMed

    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

    2016-07-01

    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. 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. PMID:27239796

  12. 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

  13. Protein Folding and Misfolding on Surfaces

    PubMed Central

    Stefani, Massimo

    2008-01-01

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

  14. 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

  15. 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

  16. Protein Vivisection Reveals Elusive Intermediates in Folding

    SciTech Connect

    Zheng, Zhongzhou; Sosnick, Tobin R.

    2010-05-25

    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 {yields} Glu{sup -}) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the {beta}5-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.

  17. Protein vivisection reveals elusive intermediates in folding.

    PubMed

    Zheng, Zhongzhou; Sosnick, Tobin R

    2010-04-01

    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 applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the beta5-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.

  18. 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

  19. Network measures for protein folding state discrimination.

    PubMed

    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. Similarities between protein folding and granular jamming

    PubMed Central

    Jose, Prasanth P; Andricioaei, Ioan

    2012-01-01

    Grains and glasses, widely different materials, arrest their motions upon decreasing temperature and external load, respectively, in common ways, leading to a universal jamming phase diagram conjecture. However, unified theories are lacking, mainly because of the disparate nature of the particle interactions. Here we demonstrate that folded proteins exhibit signatures common to both glassiness and jamming by using temperature- and force-unfolding molecular dynamics simulations. Upon folding, proteins develop a peak in the interatomic force distributions that falls on a universal curve with experimentally measured forces on jammed grains and droplets. Dynamical signatures are found as a dramatic slowdown of stress relaxation upon folding. Together with granular similarities, folding is tied not just to the jamming transition, but a more nuanced picture of anisotropy, preparation protocol and internal interactions emerges. Results have implications for designing stable polymers and can open avenues to link protein folding to jamming theory. PMID:23093180

  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.

  2. 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

  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. Protein folding using contact maps.

    PubMed

    Vendruscolo, M; Domany, E

    2000-01-01

    We discuss the problem of representations of protein structure and give the definition of contact maps. We present a method to obtain a three-dimensional polypeptide conformation from a contact map. We also explain how to deal with the case of nonphysical contact maps. We describe a stochastic method to perform dynamics in contact map space. We explain how the motion is restricted to physical regions of the space. First, we introduce the exact free energy of a contact map and discuss two simple approximations to it. Second, we present a method to derive energy parameters based on perception learning. We prove in an extensive number of situations that the pairwise contact approximation both when alone and when supplemented with a hydrophobic term is unsuitable for stabilizing proteins' native states. PMID:10668399

  5. Cellular Mechanisms of Membrane Protein Folding

    PubMed Central

    Skach, William R.

    2010-01-01

    The membrane protein folding problem can be articulated by two central questions. How is protein topology established by selective peptide transport to opposite sides of the cellular membrane? And how are transmembrane segments inserted, integrated and folded within the lipid bilayer? In eukaryotes, this process usually takes place in the endoplasmic reticulum (ER) coincident with protein synthesis, and is facilitated by the translating Ribosome and the Sec61 Translocon Complex (RTC). At its core, the RTC forms a dynamic pathway through which the elongating nascent polypeptide moves as it is delivered into cytosolic, lumenal and lipid compartments. This perspective will focus on emerging evidence that the RTC functions as a protein folding machine that restricts conformational space by establishing transmembrane topology and yet provides a permissive environment that enables nascent transmembrane domains to efficiently progress down their folding energy landscape. PMID:19491932

  6. [Protein structure: Folding and prions].

    PubMed

    Rey-Gayo, Antonio; Calbo Torrecilla, Francisco

    2002-04-01

    Transmissible spongiform encephalopathies have become a subject of prime social concern in recent years because of its relation to "mad cow disease" and their potential for transmission to humans. Among the most important scientific aspects of these diseases are the peculiar characteristics of the agent involved in their transmission. In this article we briefly describe the outstanding features of prions, the most widely accepted hypothesis for these diseases. We focus on the molecular characteristics of this protein, coded in the genome of the affected host, and describe the conformational alterations in the protein's tertiary structure that have been blamed for its pathologic activity. Our aim is to summarize the state-of-the-art knowledge on prions, the hypotheses proposed to explain mechanisms of disease transmission without agents containing genetic material, and some specific peculiarities of this new infectious agent. The links between this knowledge and possible therapeutic strategies to overcome the disease justify, once again, close interaction among chemistry, molecular biology, and medicine. PMID:11996702

  7. Nonlinear Models for Protein Folding and Function

    NASA Astrophysics Data System (ADS)

    Cruzeiro, L.

    Earlier a specific kinetic process for reproducible protein folding was proposed according to which the nascent chain is helical and the first step in in vivo protein folding is the bending of the initial helix at specific amino acid sites. Here the theoretical feasibility of this kinetic process is tested. To that end, two proteins, one belonging to the mainly α class and the other belonging to the α/β class, are selected and targeted molecular dynamics is applied to generate folding pathways for those two proteins, starting from two well defined initial conformations: a fully extended and a α-helical conformation. Not only are the native states closer to an initial helical structure for both proteins but also the pathways from the α-helical initial conformation to the native state have lower potential energy than the pathways that start from the fully extended conformation. For the α/β protein, 30% (40%) of the pathways from an initial α-helix (fully extended) structure lead to unentangled native folds, a success rate that can be increased to 85% by the introduction of a putative intermediate structure. These results lend support to the kinetic process proposed and open up a new direction in which to look for a solution to the protein folding problem. The chapter ends with a section that emphasizes the formal similarities between the dynamics quantum vibrational excited states in proteins and electrons in nonlinear lattices.

  8. 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

  9. Towards a systematic classification of protein folds

    NASA Astrophysics Data System (ADS)

    Lindgård, Per-Anker; Bohr, Henrik

    1997-10-01

    A lattice model Hamiltonian is suggested for protein structures that can explain the division into structural fold classes during the folding process. Proteins are described by chains of secondary structure elements, with the hinges in between being the important degrees of freedom. The protein structures are given a unique name, which simultaneously represent a linear string of physical coupling constants describing hinge spin interactions. We have defined a metric and a precise distance measure between the fold classes. An automated procedure is constructed in which any protein structure in the usual protein data base coordinate format can be transformed into the proposed chain representation. Taking into account hydrophobic forces we have found a mechanism for the formation of domains with a unique fold containing predicted magic numbers \\{4,6,9,12,16,18,...\\} of secondary structures and multiples of these domains. It is shown that the same magic numbers are robust and occur as well for packing on other nonclosed packed lattices. We have performed a statistical analysis of available protein structures and found agreement with the predicted preferred abundances of proteins with a predicted magic number of secondary structures. Thermodynamic arguments for the increased abundance and a phase diagram for the folding scenario are given. This includes an intermediate high symmetry phase, the parent structures, between the molten globule and the native states. We have made an exhaustive enumeration of dense lattice animals on a cubic lattice for acceptance number Z=4 and Z=5 up to 36 vertices.

  10. Structural Characteristics of Novel Protein Folds

    PubMed Central

    Fernandez-Fuentes, Narcis; Dybas, Joseph M.; Fiser, Andras

    2010-01-01

    Folds are the basic building blocks of protein structures. Understanding the emergence of novel protein folds is an important step towards understanding the rules governing the evolution of protein structure and function and for developing tools for protein structure modeling and design. We explored the frequency of occurrences of an exhaustively classified library of supersecondary structural elements (Smotifs), in protein structures, in order to identify features that would define a fold as novel compared to previously known structures. We found that a surprisingly small set of Smotifs is sufficient to describe all known folds. Furthermore, novel folds do not require novel Smotifs, but rather are a new combination of existing ones. Novel folds can be typified by the inclusion of a relatively higher number of rarely occurring Smotifs in their structures and, to a lesser extent, by a novel topological combination of commonly occurring Smotifs. When investigating the structural features of Smotifs, we found that the top 10% of most frequent ones have a higher fraction of internal contacts, while some of the most rare motifs are larger, and contain a longer loop region. PMID:20421995

  11. Experimental investigation of protein folding and misfolding.

    PubMed

    Dobson, Christopher M

    2004-09-01

    Newly synthesised proteins need to fold, often to intricate and close-packed structures, in order to function. The underlying mechanism by which this complex process takes place both in vitro and in vivo is now becoming understood, at least in general terms, as a result of the application of a wide range of biophysical and computational methods used in combination with the techniques of biochemistry and protein engineering. It is increasingly apparent, however, that folding is not only crucial for generating biological activity, but that it is also coupled to a wide range of processes within the cell, ranging from the trafficking of proteins to specific organelles to the regulation of cell growth and differentiation. Not surprisingly, therefore, the failure of proteins to fold appropriately, or to remain correctly folded, is associated with a large number of cellular malfunctions that give rise to disease. Misfolding, and its consequences such as aggregation, can be investigated by extending the types of techniques used to study the normal folding process. Application of these techniques is enabling the development of a unified description of the interconversion and regulation of the different conformational states available to proteins in living systems. Such a description proves a generic basis for understanding the fundamental links between protein misfolding and its associated clinical disorders, such as Alzheimer's disease and Type II diabetes, and for exploring novel therapeutic strategies directed at their prevention and treatment on a rational basis.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. Kinetic Analysis of Protein Folding Lattice Models

    NASA Astrophysics Data System (ADS)

    Chen, Hu; Zhou, Xin; Liaw, Chih Young; Koh, Chan Ghee

    Based on two-dimensional square lattice models of proteins, the relation between folding time and temperature is studied by Monte Carlo simulation. The results can be represented by a kinetic model with three states — random coil, molten globule, and native state. The folding process is composed of nonspecific collapse and final searching for the native state. At high temperature, it is easy to escape from local traps in the folding process. With decreasing temperature, because of the trapping in local traps, the final searching speed decreases. Then the folding shows chevron rollover. Through the analysis of the fitted parameters of the kinetic model, it is found that the main difference between the energy landscapes of the HP model and the Go model is that the number of local minima of the Go model is less than that of the HP model.

  18. Computational and theoretical methods for protein folding.

    PubMed

    Compiani, Mario; Capriotti, Emidio

    2013-12-01

    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.

  19. Effects of Knots on Protein Folding Properties

    PubMed Central

    Soler, Miguel A.; Faísca, Patrícia F. N.

    2013-01-01

    This work explores the impact of knots, knot depth and motif of the threading terminus in protein folding properties (kinetics, thermodynamics and mechanism) via extensive Monte Carlo simulations of lattice models. A knotted backbone has no effect on protein thermodynamic stability but it may affect key aspects of folding kinetics. In this regard, we found clear evidence for a functional advantage of knots: knots enhance kinetic stability because a knotted protein unfolds at a distinctively slower rate than its unknotted counterpart. However, an increase in knot deepness does not necessarily lead to more effective changes in folding properties. In this regard, a terminus with a non-trivial conformation (e.g. hairpin) can have a more dramatic effect in enhancing kinetic stability than knot depth. Nevertheless, our results suggest that the probability of the denatured ensemble to keep knotted is higher for proteins with deeper knots, indicating that knot depth plays a role in determining the topology of the denatured state. Refolding simulations starting from denatured knotted conformations show that not every knot is able to nucleate folding and further indicate that the formation of the knotting loop is a key event in the folding of knotted trefoils. They also show that there are specific native contacts within the knotted core that are crucial to keep a native knotting loop in denatured conformations which otherwise have no detectable structure. The study of the knotting mechanism reveals that the threading of the knotting loop generally occurs towards late folding in conformations that exhibit a significant degree of structural consolidation. PMID:24023962

  20. 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

  1. 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

  2. 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.

  3. Foldons as independently folding units of proteins

    NASA Astrophysics Data System (ADS)

    Panchenko, Anna R.; Luthey-Schulten, Zaida; Wolynes, Peter G.

    1997-02-01

    Independently folding units of proteins, foldons, have been identified by maxima in a scan of the ratio of an energetic stability gap to the energy variance of that segment's molten globule states, reflecting the requirement of minimal frustration. Foldon boundaries, unlike structural domains, depend on the sequence of the protein. Therefore, domains defined by purely structural criteria and the foldons of a given protein may differ in size and structure. The predicted foldons have been compared to the exons and structural modules. Statistical analysis indicates a strong correlation between the energetically determined foldons and Go's geometrically defined structural modules. There is only a weak correlation of foldons to exons.

  4. Plant secretome: unlocking secrets of the secreted proteins.

    PubMed

    Agrawal, Ganesh Kumar; Jwa, Nam-Soo; Lebrun, Marc-Henri; Job, Dominique; Rakwal, Randeep

    2010-02-01

    Plant secretomics is a newly emerging area of the plant proteomics field. It basically describes the global study of secreted proteins into the extracellular space of plant cell or tissue at any given time and under certain conditions through various secretory mechanisms. A combination of biochemical, proteomics and bioinformatics approaches has been developed to isolate, identify and profile secreted proteins using complementary in vitro suspension-cultured cells and in planta systems. Developed inventories of secreted proteins under normal, biotic and abiotic conditions revealed several different types of novel secreted proteins, including the leaderless secretory proteins (LSPs). On average, LSPs can account for more than 50% of the total identified secretome, supporting, as in other eukaryotes, the existence of novel secretory mechanisms independent of the classical endoplasmic reticulum-Golgi secretory pathway, and suggesting that this non-classical mechanism of protein expression is, for as yet unknown reasons, more massively used than in other eukaryotic systems. Plants LSPs, which seem to be potentially involved in the defense/stress responses, might have dual (extracellular and/or intracellular) roles as most of them have established intracellular functions, yet presently unknown extracellular functions. Evidence is emerging on the role of glycosylation in the apical sorting and trafficking of secretory proteins. These initial secretome studies in plants have considerably advanced our understanding on secretion of different types of proteins and their underlying mechanisms, and opened a door for comparative analyses of plant secretomes with those of other organisms. In this first review on plant secretomics, we summarize and discuss the secretome definition, the applied approaches for unlocking secrets of the secreted proteins in the extracellular fluid, the possible functional significance and secretory mechanisms of LSPs, as well as glycosylation of

  5. [Characterization of Schizosaccharomyces pombe secreted proteins].

    PubMed

    Liu, Yu-Ling; Liu, Yun-Fan; Xie, Jian-Ping

    2007-02-01

    Secreted proteins play a vital physiological role. Schizosaccharomyces pombe is an important model organism for cell cycle study and a potential useful drug screen model. Secreted proteins also initiate the mating. However, few global studies concerning the secreted proteins of S. pombe was reported. To address this issue, bioinformatics were used to reveal the global secreted proteins of S. pombe. The 4997 proteins deduced from the S. pombe genome were analyzed by combined several programs. One hundred and sixty proteins were identified carrying an NH2-terminal secretory signal peptide by signalP3.0. Among them, 117 proteins are integral membrane proteins (TMpred), 13 proteins are lipoproteins (PrositeS-can), and 66 proteins are secreted proteins. The location of the secreted proteins was also predicted by Target P. Some of the secreted proteins are involved in the nutrition, reproduction, as well as the communication between cells and environment. The global information of the secreted proteins of S. pombe will benefit further studies in drug screening model and host searching for heterologous gene expression.

  6. Simulating protein folding in different environmental conditions.

    PubMed

    Homouz, Dirar

    2014-01-01

    Molecular dynamics simulations have become an invaluable tool in investigating the dynamics of protein folding. However, most computational studies of protein folding assume dilute aqueous simulation conditions in order to reduce the complexity of the system under study and enhance the efficiency. Nowadays, it is evident that environmental conditions encountered in vivo (or even in vitro) play a major role in regulating the dynamics of protein folding especially when one considers the highly condensed environment in the cellular cytoplasm. In order to factor in these conditions, we can utilize the high efficiency of well-designed low resolution (coarse-grained) simulation models to reduce the complexity of these added protein-milieu interactions involving different time and length scales. The goal of this chapter is to describe some recently developed coarse-grained simulation techniques that are specifically designed to go beyond traditional aqueous solvent conditions. The chapter also gives the reader a flavor of the things that we can study using such "smart" low resolution models.

  7. 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

  8. A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host

    PubMed Central

    Lyngsø, Christina; Kjaerulff, Søren; Müller, Sven; Bratt, Tomas; Mortensen, Uffe H; Dal Degan, Florence

    2010-01-01

    Recombinant expression of native or modified eukaryotic proteins is pivotal for structural and functional studies and for industrial and pharmaceutical production of proteins. However, it is often impeded by the lack of proper folding. Here, we present a stringent and broadly applicable eukaryotic in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C-terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality-control systems to retain misfolded proteins in the ER and redirect them for cytosolic degradation, thereby only allowing folded proteins to reach the cell surface. Accordingly, the folding potential of the tested protein determines the ability of autotrophic colony growth. This system was successfully demonstrated using a complex insertion mutant library of TNF-α, from which different folding competent mutant proteins were uncovered. PMID:20082307

  9. 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. PMID:24150425

  10. Protein Folding Stages and Universal Exponents

    NASA Astrophysics Data System (ADS)

    Huang, Kerson

    We propose three stages in protein folding, based on physical arguements involving the interplay between the hydrophobic effect and hydrogen bonding, and computer simulations using the CSAW (conditioned self-avoiding walk) model. These stages are characterized by universal exponents ν = 3/5, 3/7, 2/5 in the power law R ~ Nν, where R is the radius of gyration and N is the number of residues. They correspond to the experimentally observed stages: unfolded, preglobule, molten globule.

  11. Protein Folding Stages and Universal Exponents

    NASA Astrophysics Data System (ADS)

    Huang, Kerson

    2011-11-01

    We propose three stages in protein folding, based on physical arguements involving the interplay between the hydrophobic effect and hydrogen bonding, and computer simulations using the CSAW (conditioned self-avoiding walk) model. These stages are characterized by universal exponents ν = 3/5, 3/7, 2/5 in the power law R ˜ Nν, where R is the radius of gyration and N is the number of residues. They correspond to the experimentally observed stages: unfolded, preglobule, molten globule.

  12. Protein folding at atomic resolution: analysis of autonomously folding supersecondary structure motifs by nuclear magnetic resonance.

    PubMed

    Sborgi, Lorenzo; Verma, Abhinav; Sadqi, Mourad; de Alba, Eva; Muñoz, Victor

    2013-01-01

    The study of protein folding has been conventionally hampered by the assumption that all single-domain proteins fold by an all-or-none process (two-state folding) that makes it impossible to resolve folding mechanisms experimentally. Here we describe an experimental method for the thermodynamic analysis of protein folding at atomic resolution using nuclear magnetic resonance (NMR). The method is specifically developed for the study of small proteins that fold autonomously into basic supersecondary structure motifs, and that do so in the sub-millisecond timescale (folding archetypes). From the NMR experiments we obtain hundreds of atomic unfolding curves that are subsequently analyzed leading to the determination of the characteristic network of folding interactions. The application of this approach to a comprehensive catalog of elementary folding archetypes holds the promise of becoming the first experimental approach capable of unraveling the basic rules connecting protein structure and folding mechanism. PMID:22987355

  13. Domains in folding of model proteins.

    PubMed Central

    Abkevich, V. I.; Gutin, A. M.; Shakhnovich, E. I.

    1995-01-01

    By means of Monte Carlo simulation, we investigated the equilibrium between folded and unfolded states of lattice model proteins. The amino acid sequences were designed to have pronounced energy minimum target conformations of different length and shape. For short fully compact (36-mer) proteins, the all-or-none transition from the unfolded state to the native state was observed. This was not always the case for longer proteins. Among 12 designed sequences with the native structure of a fully compact 48-mer, a simple all-or-none transition was observed in only three cases. For the other nine sequences, three states of behavior-the native, denatured, and intermediate states-were found. The contiguous part of the native structure (domain) was conserved in the intermediate state, whereas the remaining part was completely unfolded and structureless. These parts melted separately from each other. PMID:7549881

  14. An Alternative Approach to Protein Folding

    PubMed Central

    Kang, Yeona; Fortmann, Charles M.

    2013-01-01

    A diffusion theory-based, all-physical ab initio protein folding simulation is described and applied. The model is based upon the drift and diffusion of protein substructures relative to one another in the multiple energy fields present. Without templates or statistical inputs, the simulations were run at physiologic and ambient temperatures (including pH). Around 100 protein secondary structures were surveyed, and twenty tertiary structures were determined. Greater than 70% of the secondary core structures with over 80% alpha helices were correctly identified on protein ranging from 30 to 200 amino-acid sequence. The drift-diffusion model predicted tertiary structures with RMSD values in the 3–5 Angstroms range for proteins ranging 30 to 150 amino acids. These predictions are among the best for an all ab initio protein simulation. Simulations could be run entirely on a desktop computer in minutes; however, more accurate tertiary structures were obtained using molecular dynamic energy relaxation. The drift-diffusion model generated realistic energy versus time traces. Rapid secondary structures followed by a slow compacting towards lower energy tertiary structures occurred after an initial incubation period in agreement with observations. PMID:24078920

  15. 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

  16. 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.

  17. Understanding the Folding-Function Tradeoff in Proteins

    PubMed Central

    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. PMID:23593437

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

    PubMed Central

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

    2016-01-01

    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. PMID:27112569

  19. Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae

    PubMed Central

    Krehenbrink, Martin; Downie, J Allan

    2008-01-01

    Background Proteins secreted by bacteria play an important role in infection of eukaryotic hosts. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. Proteins secreted during the infection process by some rhizobial strains can influence infection and modify the plant defence signalling pathways. The aim of this study was to systematically analyse protein secretion in the recently sequenced strain Rhizobium leguminosarum bv. viciae 3841. Results Similarity searches using defined protein secretion systems from other Gram-negative bacteria as query sequences revealed that R. l. bv. viciae 3841 has ten putative protein secretion systems. These are the general export pathway (GEP), a twin-arginine translocase (TAT) secretion system, four separate Type I systems, one putative Type IV system and three Type V autotransporters. Mutations in genes encoding each of these (except the GEP) were generated, but only mutations affecting the PrsDE (Type I) and TAT systems were observed to affect the growth phenotype and the profile of proteins in the culture supernatant. Bioinformatic analysis and mass fingerprinting of tryptic fragments of culture supernatant proteins identified 14 putative Type I substrates, 12 of which are secreted via the PrsDE, secretion system. The TAT mutant was defective for the symbiosis, forming nodules incapable of nitrogen fixation. Conclusion None of the R. l. bv. viciae 3841 protein secretion systems putatively involved in the secretion of proteins to the extracellular space (Type I, Type IV, Type V) is required for establishing the symbiosis with legumes. The PrsDE (Type I) system was shown to be the major route of protein secretion in non-symbiotic cells and to secrete proteins of widely varied size and predicted function. This is in contrast to many Type I systems from other bacteria, which typically secrete specific substrates encoded by genes often localised in close proximity to the genes encoding the

  20. 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.

  1. Dynamics of an Ultrafast Folding Subdomain in the Context of a Larger Protein Fold

    PubMed Central

    Davis, Caitlin M.; Dyer, R. Brian

    2014-01-01

    Small fast folding subdomains with low contact order have been postulated to facilitate the folding of larger proteins. We have tested this idea by determining how the fastest folding linear β-hairpin, CLN025, which folds on the nanosecond time scale, folds within the context of a two-hairpin WW domain system, which folds on the microsecond time scale. The folding of the wild type FBP28 WW domain was compared to constructs in which each of the loops was replaced by CLN025. A combination of FTIR spectroscopy and laser-induced temperature-jump coupled with infrared spectroscopy was used to probe changes in the peptide backbone. The relaxation dynamics of the β-sheets and β-turn were measured independently by probing the corresponding bands assigned in the amide I region. The folding rate of the CLN025 β-hairpin is unchanged within the larger protein. Insertion of the β-hairpin into the second loop results in an overall stabilization of the WW domain and a relaxation lifetime five times faster than the parent WW domain. In both mutants, folding is initiated in the turns and the β-sheets form last. These results demonstrate that fast folding subdomains can be used to speed the folding of more complex proteins, and that the folding dynamics of the subdomain is unchanged within the context of the larger protein. PMID:24320936

  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. Biochemical Methods to Analyze Wnt Protein Secretion.

    PubMed

    Glaeser, Kathrin; Boutros, Michael; Gross, Julia Christina

    2016-01-01

    Wnt proteins act as potent morphogens in various aspects of embryonic development and adult tissue homeostasis. However, in addition to its physiological importance, aberrant Wnt signaling has been linked to the onset and progression of different types of cancer. On the cellular level, the secretion of Wnt proteins involves trafficking of lipid-modified Wnts from the endoplasmic reticulum (ER) to Golgi and further compartments via the Wnt cargo receptor evenness interrupted. Others and we have recently shown that Wnt proteins are secreted on extracellular vesicles (EVs) such as microvesicles and exosomes. Although more details about specific regulation of Wnt secretion steps are emerging, it remains largely unknown how Wnt proteins are channeled into different release pathways such as lipoprotein particles, EVs and cytonemes. Here, we describe protocols to purify and quantify Wnts from the supernatant of cells by either assessing total Wnt proteins in the supernatant or monitoring Wnt proteins on EVs. Purified Wnts from the supernatant as well as total cellular protein content can be investigated by immunoblotting. Additionally, the relative activity of canonical Wnts in the supernatant can be assessed by a dual-luciferase Wnt reporter assay. Quantifying the amount of secreted Wnt proteins and their activity in the supernatant of cells allows the investigation of intracellular trafficking events that regulate Wnt secretion and the role of extracellular modulators of Wnt spreading. PMID:27590148

  4. The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion*

    PubMed Central

    Chong, Brandi M.; Russell, Tanya D.; Schaack, Jerome; Orlicky, David J.; Reigan, Philip; Ladinsky, Mark; McManaman, James L.

    2011-01-01

    Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion. PMID:21383012

  5. Improving Protein Fold Recognition by Deep Learning Networks

    PubMed Central

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

    2015-01-01

    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. PMID:26634993

  6. Improving Protein Fold Recognition by Deep Learning Networks.

    PubMed

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

    2015-12-04

    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.

  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. Improving Protein Fold Recognition by Deep Learning Networks.

    PubMed

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

    2015-01-01

    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. PMID:26634993

  9. Prions and protein-folding diseases.

    PubMed

    Norrby, E

    2011-07-01

    Prions represent a group of proteins with a unique capacity to fold into different conformations. One isoform is rich in beta-pleated sheets and can aggregate into amyloid that may be pathogenic. This abnormal form propagates itself by imposing its confirmation on the homologous normal host cell protein. Pathogenic prions have been shown to cause lethal neurodegenerative diseases in humans and animals. These diseases are sometimes infectious and hence referred to as transmissible spongiform encephalopathies. In the present review, the remarkable evolution of the heterodox prion concept is summarized. The origin of this phenomenon is based on information transfer between homologous proteins, without the involvement of nucleic acid-encoded mechanisms. Historically, kuru and Creutzfeldt-Jakob disease (CJD) were the first infectious prion diseases to be identified in man. It was their relationship to scrapie in sheep and experimental rodents that allowed an unravelling of the particular molecular mechanism that underlie the disease process. Transmission between humans has been documented to have occurred in particular contexts, including ritual cannibalism, iatrogenic transmission because of pituitary gland-derived growth hormone or the use in neurosurgical procedures of dura mater from cadavers, and the temporary use of a prion-contaminated protein-rich feed for cows. The latter caused a major outbreak of bovine spongiform encephalopathy, which spread to man by human consumption of contaminated meat, causing approximately 200 cases of variant CJD. All these epidemics now appear to be over because of measures taken to curtail further spread of prions. Recent studies have shown that the mechanism of protein aggregation may apply to a wider range of diseases in and possibly also outside the brain, some of which are relatively common such as Alzheimer's and Parkinson's diseases. Furthermore, it has become apparent that the phenomenon of prion aggregation may have a wider

  10. A global representation of the protein fold space.

    PubMed

    Hou, Jingtong; Sims, Gregory E; Zhang, Chao; Kim, Sung-Hou

    2003-03-01

    One of the principal goals of the structural genomics initiative is to identify the total repertoire of protein folds and obtain a global view of the "protein structure universe." Here, we present a 3D map of the protein fold space in which structurally related folds are represented by spatially adjacent points. Such a representation reveals a high-level organization of the fold space that is intuitively interpretable. The shape of the fold space and the overall distribution of the folds are defined by three dominant trends: secondary structure class, chain topology, and protein domain size. Random coil-like structures of small proteins and peptides are mapped to a region where the three trends converge, offering an interesting perspective on both the demography of fold space and the evolution of protein structures. PMID:12606708

  11. 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

  12. Mechanism of Action of Secreted Newt Anterior Gradient Protein.

    PubMed

    Grassme, Kathrin S; Garza-Garcia, Acely; Delgado, Jean-Paul; Godwin, James W; Kumar, Anoop; Gates, Phillip B; Driscoll, Paul C; 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

  13. Molecular Simulations of Cotranslational Protein Folding: Fragment Stabilities, Folding Cooperativity, and Trapping in the Ribosome

    PubMed Central

    Elcock, Adrian H

    2006-01-01

    Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these

  14. Sorting sweet sorting. Protein secretion.

    PubMed

    Ponnambalam, S; Banting, G

    1996-09-01

    Membrane-spanning, lectin-like proteins in the eukaryotic secretory pathway seem to operate quality-control checkpoints by fine tuning protein exit or retention within each subcompartment. PMID:8805362

  15. 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.

  16. Elastic energy of proteins and the stages of protein folding

    NASA Astrophysics Data System (ADS)

    Lei, J.; Huang, K.

    2009-12-01

    We propose a universal elastic energy for proteins, which depends only on the radius of gyration Rg and the residue number N. It is constructed using physical arguments based on the hydrophobic effect and hydrogen bonding. Adjustable parameters are fitted to data from the computer simulation of the folding of a set of proteins using the CSAW (conditioned self-avoiding walk) model. The elastic energy gives rise to scaling relations of the form Rg~Nν in different regions. It shows three folding stages characterized by the progression with exponents ν=3/5, 3/7, 2/5, which we identify as the unfolded stage, pre-globule, and molten globule, respectively. The pre-globule goes over to the molten globule via a break in behavior akin to a first-order phase transition, which is initiated by a sudden acceleration of hydrogen bonding.

  17. Dynamics of protein secretion during adipocyte differentiation.

    PubMed

    Ojima, Koichi; Oe, Mika; Nakajima, Ikuyo; Muroya, Susumu; Nishimura, Takanori

    2016-08-01

    The major functions of adipocytes include both lipid storage and the production of secretory factors. However, the type of proteins released from mouse 3T3-L1 cells during adipocyte differentiation remains poorly understood. We examined the dynamics of secreted proteins during adipocyte differentiation using mass spectrometry (MS) combined with an iTRAQ (®) labeling method that enables the simultaneous analysis of relative protein expression levels. A total of 215 proteins were identified and quantified from approximately 10 000 MS/MS spectra. Of these, approximately 38% were categorized as secreted proteins based on gene ontology classification. Adipokine secretion levels were increased with the progression of differentiation. By contrast, levels of fibril collagen components, such as subunits of type I and III collagens, were decreased during differentiation. Basement membrane components attained their peak levels at day 4 when small lipid droplets accumulated in differentiated 3T3-L1 cells. Simultaneously, peak levels of collagen microfibril components that comprise type V and VI collagen subunits were also observed. Our data demonstrated that extracellular matrix components were predominantly released during the early and middle stages of adipocyte differentiation, with a subsequent increase in the secretion of adipokines. This suggests that 3T3-L1 cells secrete adipokines after their ECM is constructed during adipocyte differentiation. PMID:27516960

  18. Distribution of protein folds in the three superkingdoms of life.

    PubMed

    Wolf, Y I; Brenner, S E; Bash, P A; Koonin, E V

    1999-01-01

    A sensitive protein-fold recognition procedure was developed on the basis of iterative database search using the PSI-BLAST program. A collection of 1193 position-dependent weight matrices that can be used as fold identifiers was produced. In the completely sequenced genomes, folds could be automatically identified for 20%-30% of the proteins, with 3%-6% more detectable by additional analysis of conserved motifs. The distribution of the most common folds is very similar in bacteria and archaea but distinct in eukaryotes. Within the bacteria, this distribution differs between parasitic and free-living species. In all analyzed genomes, the P-loop NTPases are the most abundant fold. In bacteria and archaea, the next most common folds are ferredoxin-like domains, TIM-barrels, and methyltransferases, whereas in eukaryotes, the second to fourth places belong to protein kinases, beta-propellers and TIM-barrels. The observed diversity of protein folds in different proteomes is approximately twice as high as it would be expected from a simple stochastic model describing a proteome as a finite sample from an infinite pool of proteins with an exponential distribution of the fold fractions. Distribution of the number of domains with different folds in one protein fits the geometric model, which is compatible with the evolution of multidomain proteins by random combination of domains. [Fold predictions for proteins from 14 proteomes are available on the World Wide Web at. The FIDs are available by anonymous ftp at the same location.

  19. 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.

  20. Cotranslational protein folding on the ribosome monitored in real time.

    PubMed

    Holtkamp, Wolf; Kokic, Goran; Jäger, Marcus; Mittelstaet, Joerg; Komar, Anton A; Rodnina, Marina V

    2015-11-27

    Protein domains can fold into stable tertiary structures while they are synthesized on the ribosome. We used a high-performance, reconstituted in vitro translation system to investigate the folding of a small five-helix protein domain-the N-terminal domain of Escherichia coli N5-glutamine methyltransferase HemK-in real time. Our observations show that cotranslational folding of the protein, which folds autonomously and rapidly in solution, proceeds through a compact, non-native conformation that forms within the peptide tunnel of the ribosome. The compact state rearranges into a native-like structure immediately after the full domain sequence has emerged from the ribosome. Both folding transitions are rate-limited by translation, allowing for quasi-equilibrium sampling of the conformational space restricted by the ribosome. Cotranslational folding may be typical of small, intrinsically rapidly folding protein domains. PMID:26612953

  1. 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

  2. A hierarchical protein folding scheme based on the building block folding model.

    PubMed

    Haspel, Nurit; Wainreb, Gilad; Inbar, Yuval; Tsai, Hui-Hsu; Tsai, Chung-Jung; Wolfson, Haim J; Nussinov, Ruth

    2007-01-01

    The building block protein folding model states that the native protein structure is the product of a combinatorial assembly of relatively structurally independent contiguous parts of the protein that possess a hydrophobic core, i.e., building blocks (BBs). According to this model, our group proposed a three-stage scheme for a feasible time-wise semi ab-intio protein structure prediction. Given a protein sequence, at the first stage of the prediction scheme, we propose cutting the sequence into structurally assigned BBs. Next, we perform a combinatorial assembly and attempt to predict the relative three-dimensional arrangement of the BBs. In the third stage, we refine and rank the assemblies. The scheme has proven to be very promising in reducing the complexity of the protein folding problem and gaining insight into the protein folding process. In this chapter, we describe the different stages of the scheme and discuss a possible application of the model to protein design. PMID:16957324

  3. Targeted Secretion Inhibitors—Innovative Protein Therapeutics

    PubMed Central

    Foster, Keith; Chaddock, John

    2010-01-01

    Botulinum neurotoxins are highly effective therapeutic products. Their therapeutic success results from highly specific and potent inhibition of neurotransmitter release with a duration of action measured in months. These same properties, however, make the botulinum neurotoxins the most potent acute lethal toxins known. Their toxicity and restricted target cell activity severely limits their clinical utility. Understanding the structure-function relationship of the neurotoxins has enabled the development of recombinant proteins selectively incorporating specific aspects of their pharmacology. The resulting proteins are not neurotoxins, but a new class of biopharmaceuticals, Targeted Secretion Inhibitors (TSI), suitable for the treatment of a wide range of diseases where secretion plays a major role. TSI proteins inhibit secretion for a prolonged period following a single application, making them particularly suited to the treatment of chronic diseases. A TSI for the treatment of chronic pain is in clinical development. PMID:22069575

  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. Folding of β-barrel membrane proteins in lipid bilayers - Unassisted and assisted folding and insertion.

    PubMed

    Kleinschmidt, Jörg H

    2015-09-01

    In cells, β-barrel membrane proteins are transported in unfolded form to an outer membrane into which they fold and insert. Model systems have been established to investigate the mechanisms of insertion and folding of these versatile proteins into detergent micelles, lipid bilayers and even synthetic amphipathic polymers. In these experiments, insertion into lipid membranes is initiated from unfolded forms that do not display residual β-sheet secondary structure. These studies therefore have allowed the investigation of membrane protein folding and insertion in great detail. Folding of β-barrel membrane proteins into lipid bilayers has been monitored from unfolded forms by dilution of chaotropic denaturants that keep the protein unfolded as well as from unfolded forms present in complexes with molecular chaperones from cells. This review is aimed to provide an overview of the principles and mechanisms observed for the folding of β-barrel transmembrane proteins into lipid bilayers, the importance of lipid-protein interactions and the function of molecular chaperones and folding assistants. This article is part of a Special Issue entitled: Lipid-protein interactions.

  6. 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. PMID:25107533

  7. Small protein domains fold inside the ribosome exit tunnel.

    PubMed

    Marino, Jacopo; von Heijne, Gunnar; Beckmann, Roland

    2016-03-01

    Cotranslational folding of small protein domains within the ribosome exit tunnel may be an important cellular strategy to avoid protein misfolding. However, the pathway of cotranslational folding has so far been described only for a few proteins, and therefore, it is unclear whether folding in the ribosome exit tunnel is a common feature for small protein domains. Here, we have analyzed nine small protein domains and determined at which point during translation their folding generates sufficient force on the nascent chain to release translational arrest by the SecM arrest peptide, both in vitro and in live E. coli cells. We find that all nine protein domains initiate folding while still located well within the ribosome exit tunnel. PMID:26879042

  8. 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.

  9. 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.

  10. 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

  11. Protein folding: Turbo-charged crosslinking

    NASA Astrophysics Data System (ADS)

    Craik, David J.

    2012-08-01

    The efficient production of stable bioactive proteins often requires the selective formation of several disulfide crosslinks. Two recent studies have now shown that replacing cysteine with selenocysteine in the unfolded protein can autocatalyse the formation of the desired crosslinks.

  12. 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.

  13. Impact of structure space continuity on protein fold classification

    PubMed Central

    Xu, Jinrui; Zhang, Jianzhi

    2016-01-01

    Protein structure classification hierarchically clusters domain structures based on structure and/or sequence similarities and plays important roles in the study of protein structure-function relationship and protein evolution. Among many classifications, SCOP and CATH are widely viewed as the gold standards. Fold classification is of special interest because this is the lowest level of classification that does not depend on protein sequence similarity. The current fold classifications such as those in SCOP and CATH are controversial because they implicitly assume that folds are discrete islands in the structure space, whereas increasing evidence suggests significant similarities among folds and supports a continuous fold space. Although this problem is widely recognized, its impact on fold classification has not been quantitatively evaluated. Here we develop a likelihood method to classify a domain into the existing folds of CATH or SCOP using both query-fold structure similarities and within-fold structure heterogeneities. The new classification differs from the original classification for 3.4–12% of domains, depending on factors such as the structure similarity score and original classification scheme used. Because these factors differ for different biological purposes, our results indicate that the importance of considering structure space continuity in fold classification depends on the specific question asked. PMID:27006112

  14. Protein folding under confinement: A role for solvent

    PubMed Central

    Lucent, Del; Vishal, V.; Pande, Vijay S.

    2007-01-01

    Although most experimental and theoretical studies of protein folding involve proteins in vitro, the effects of spatial confinement may complicate protein folding in vivo. In this study, we examine the folding dynamics of villin (a small fast folding protein) with explicit solvent confined to an inert nanopore. We have calculated the probability of folding before unfolding (Pfold) under various confinement regimes. Using Pfold correlation techniques, we observed two competing effects. Confining protein alone promotes folding by destabilizing the unfolded state. In contrast, confining both protein and solvent gives rise to a solvent-mediated effect that destabilizes the native state. When both protein and solvent are confined we see unfolding to a compact unfolded state different from the unfolded state seen in bulk. Thus, we demonstrate that the confinement of solvent has a significant impact on protein kinetics and thermodynamics. We conclude with a discussion of the implications of these results for folding in confined environments such as the chaperonin cavity in vivo. PMID:17563390

  15. Active Wnt proteins are secreted on exosomes.

    PubMed

    Gross, Julia Christina; Chaudhary, Varun; Bartscherer, Kerstin; Boutros, Michael

    2012-10-01

    Wnt signalling has important roles during development and in many diseases. As morphogens, hydrophobic Wnt proteins exert their function over a distance to induce patterning and cell differentiation decisions. Recent studies have identified several factors that are required for the secretion of Wnt proteins; however, how Wnts travel in the extracellular space remains a largely unresolved question. Here we show that Wnts are secreted on exosomes both during Drosophila development and in human cells. We demonstrate that exosomes carry Wnts on their surface to induce Wnt signalling activity in target cells. Together with the cargo receptor Evi/WIs, Wnts are transported through endosomal compartments onto exosomes, a process that requires the R-SNARE Ykt6. Our study demonstrates an evolutionarily conserved functional role of extracellular vesicular transport of Wnt proteins.

  16. Unconventional Protein Secretion in Animal Cells.

    PubMed

    Ng, Fanny; Tang, Bor Luen

    2016-01-01

    All eukaryotic cells secrete a range of proteins in a constitutive or regulated manner through the conventional or canonical exocytic/secretory pathway characterized by vesicular traffic from the endoplasmic reticulum, through the Golgi apparatus, and towards the plasma membrane. However, a number of proteins are secreted in an unconventional manner, which are insensitive to inhibitors of conventional exocytosis and use a route that bypasses the Golgi apparatus. These include cytosolic proteins such as fibroblast growth factor 2 (FGF2) and interleukin-1β (IL-1β), and membrane proteins that are known to also traverse to the plasma membrane by a conventional process of exocytosis, such as α integrin and the cystic fibrosis transmembrane conductor (CFTR). Mechanisms underlying unconventional protein secretion (UPS) are actively being analyzed and deciphered, and these range from an unusual form of plasma membrane translocation to vesicular processes involving the generation of exosomes and other extracellular microvesicles. In this chapter, we provide an overview on what is currently known about UPS in animal cells. PMID:27665549

  17. 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.

  18. 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.

  19. 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. PMID:23056480

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

    PubMed

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

    2013-09-28

    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

  1. 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

  2. Structure-Based Prediction of Protein-Folding Transition Paths.

    PubMed

    Jacobs, William M; Shakhnovich, Eugene I

    2016-09-01

    We propose a general theory to describe the distribution of protein-folding transition paths. We show that transition paths follow a predictable sequence of high-free-energy transient states that are separated by free-energy barriers. Each transient state corresponds to the assembly of one or more discrete, cooperative units, which are determined directly from the native structure. We show that the transition state on a folding pathway is reached when a small number of critical contacts are formed between a specific set of substructures, after which folding proceeds downhill in free energy. This approach suggests a natural resolution for distinguishing parallel folding pathways and provides a simple means to predict the rate-limiting step in a folding reaction. Our theory identifies a common folding mechanism for proteins with diverse native structures and establishes general principles for the self-assembly of polymers with specific interactions. PMID:27602721

  3. Structure-Based Prediction of Protein-Folding Transition Paths

    NASA Astrophysics Data System (ADS)

    Jacobs, William M.; Shakhnovich, Eugene I.

    2016-09-01

    We propose a general theory to describe the distribution of protein-folding transition paths. We show that transition paths follow a predictable sequence of high-free-energy transient states that are separated by free-energy barriers. Each transient state corresponds to the assembly of one or more discrete, cooperative units, which are determined directly from the native structure. We show that the transition state on a folding pathway is reached when a small number of critical contacts are formed between a specific set of substructures, after which folding proceeds downhill in free energy. This approach suggests a natural resolution for distinguishing parallel folding pathways and provides a simple means to predict the rate-limiting step in a folding reaction. Our theory identifies a common folding mechanism for proteins with diverse native structures and establishes general principles for the self-assembly of polymers with specific interactions.

  4. 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

  5. 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

  6. Structural origin of slow diffusion in protein folding.

    PubMed

    Chung, Hoi Sung; Piana-Agostinetti, Stefano; Shaw, David E; Eaton, William A

    2015-09-25

    Experimental, theoretical, and computational studies of small proteins suggest that interresidue contacts not present in the folded structure play little or no role in the self-assembly mechanism. Non-native contacts can, however, influence folding kinetics by introducing additional local minima that slow diffusion over the global free-energy barrier between folded and unfolded states. Here, we combine single-molecule fluorescence with all-atom molecular dynamics simulations to discover the structural origin for the slow diffusion that markedly decreases the folding rate for a designed α-helical protein. Our experimental determination of transition path times and our analysis of the simulations point to non-native salt bridges between helices as the source, which provides a quantitative glimpse of how specific intramolecular interactions influence protein folding rates by altering dynamics and not activation free energies.

  7. Statistical mechanics of kinetic proofreading in protein folding in vivo.

    PubMed Central

    Gulukota, K; Wolynes, P G

    1994-01-01

    The statistical energy landscape picture of protein folding has led to the understanding that the energy landscape must have guiding forces leading to a protein folding funnel in order to avoid the Levinthal paradox in vitro. Since folding in vivo often requires the action of chaperone molecules and ATP hydrolysis, we must ask whether folding in a system maintained away from equilibrium can avoid the Levinthal paradox in other ways. We describe a model of the action of chaperone molecules in protein folding in vivo on the basis of a repetitive cycle of binding and unbinding, allowing the possibility of kinetic proofreading. We also study models in which chaperone binding is locally biased, depending on the similarity of the conformation to the native one. We show that while kinetic proofreading can modestly facilitate folding, it is insufficient by itself to overcome the Levinthal paradox. On the other hand, such kinetic proofreading with biasing can provide the nonequilibrium analog of a folding funnel and greatly enhance folding yields and speed up folding. PMID:7937758

  8. Molecular dynamics studies of protein folding and aggregation

    NASA Astrophysics Data System (ADS)

    Ding, Feng

    This thesis applies molecular dynamics simulations and statistical mechanics to study: (i) protein folding; and (ii) protein aggregation. Most small proteins fold into their native states via a first-order-like phase transition with a major free energy barrier between the folded and unfolded states. A set of protein conformations corresponding to the free energy barrier, Delta G >> kBT, are the folding transition state ensemble (TSE). Due to their evasive nature, TSE conformations are hard to capture (probability ∝ exp(-DeltaG/k BT)) and characterize. A coarse-grained discrete molecular dynamics model with realistic steric constraints is constructed to reproduce the experimentally observed two-state folding thermodynamics. A kinetic approach is proposed to identify the folding TSE. A specific set of contacts, common to the TSE conformations, is identified as the folding nuclei which are necessary to be formed in order for the protein to fold. Interestingly, the amino acids at the site of the identified folding nuclei are highly conserved for homologous proteins sharing the same structures. Such conservation suggests that amino acids that are important for folding kinetics are under selective pressure to be preserved during the course of molecular evolution. In addition, studies of the conformations close to the transition states uncover the importance of topology in the construction of order parameter for protein folding transition. Misfolded proteins often form insoluble aggregates, amyloid fibrils, that deposit in the extracellular space and lead to a type of disease known as amyloidosis. Due to its insoluble and non-crystalline nature, the aggregation structure and, thus the aggregation mechanism, has yet to be uncovered. Discrete molecular dynamics studies reveal an aggregate structure with the same structural signatures as in experimental observations and show a nucleation aggregation scenario. The simulations also suggest a generic aggregation mechanism

  9. In vivo aspects of protein folding and quality control.

    PubMed

    Balchin, David; Hayer-Hartl, Manajit; Hartl, F Ulrich

    2016-07-01

    Most proteins must fold into unique three-dimensional structures to perform their biological functions. In the crowded cellular environment, newly synthesized proteins are at risk of misfolding and forming toxic aggregate species. To ensure efficient folding, different classes of molecular chaperones receive the nascent protein chain emerging from the ribosome and guide it along a productive folding pathway. Because proteins are structurally dynamic, constant surveillance of the proteome by an integrated network of chaperones and protein degradation machineries is required to maintain protein homeostasis (proteostasis). The capacity of this proteostasis network declines during aging, facilitating neurodegeneration and other chronic diseases associated with protein aggregation. Understanding the proteostasis network holds the promise of identifying targets for pharmacological intervention in these pathologies. PMID:27365453

  10. Folding and escape of nascent proteins at ribosomal exit tunnel

    NASA Astrophysics Data System (ADS)

    Bui, Phuong Thuy; Hoang, Trinh Xuan

    2016-03-01

    We investigate the interplay between post-translational folding and escape of two small single-domain proteins at the ribosomal exit tunnel by using Langevin dynamics with coarse-grained models. It is shown that at temperatures lower or near the temperature of the fastest folding, folding proceeds concomitantly with the escape process, resulting in vectorial folding and enhancement of foldability of nascent proteins. The concomitance between the two processes, however, deteriorates as temperature increases. Our folding simulations as well as free energy calculation by using umbrella sampling show that, at low temperatures, folding at the tunnel follows one or two specific pathways without kinetic traps. It is shown that the escape time can be mapped to a one-dimensional diffusion model with two different regimes for temperatures above and below the folding transition temperature. Attractive interactions between amino acids and attractive sites on the tunnel wall lead to a free energy barrier along the escape route of the protein. It is suggested that this barrier slows down the escape process and consequently promotes correct folding of the released nascent protein.

  11. Metal ion coupled protein folding and allosteric motions

    NASA Astrophysics Data System (ADS)

    Wang, Wei

    2014-03-01

    Many proteins need the help of cofactors for their successful folding and functioning. Metal ions, i.e., Zn2+, Ca2+, and Mg2+ etc., are typical biological cofactors. Binding of metal ions can reshape the energy landscapes of proteins, thereby modifying the folding and allosteric motions. For example, such binding may make the intrinsically disordered proteins have funneled energy landscapes, consequently, ensures their spontaneous folding. In addition, the binding may activate certain biological processes by inducing related conformational changes of regulation proteins. However, how the local interactions involving the metal ion binding can induce the global conformational motions of proteins remains elusive. Investigating such question requires multiple models with different details, including quantum mechanics, atomistic models, and coarse grained models. In our recent work, we have been developing such multiscale methods which can reasonably model the metal ion binding induced charge transfer, protonation/deprotonation, and large conformational motions of proteins. With such multiscale model, we elucidated the zinc-binding induced folding mechanism of classical zinc finger and the calcium-binding induced dynamic symmetry breaking in the allosteric motions of calmodulin. In addition, we studied the coupling of folding, calcium binding and allosteric motions of calmodulin domains. In this talk, I will introduce the above progresses on the metal ion coupled protein folding and allosteric motions. We thank the finacial support from NSFC and the 973 project.

  12. 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.

  13. Exploring one-state downhill protein folding in single molecules

    PubMed Central

    Liu, Jianwei; Campos, Luis A.; Cerminara, Michele; Wang, Xiang; Ramanathan, Ravishankar; English, Douglas S.; Muñoz, Victor

    2012-01-01

    A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (ca. 106 s-1) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out free-diffusion single-molecule FRET experiments with 50-μs resolution and maximal photoprotection using a recently developed Trolox-cysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRET efficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the one-state downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics. PMID:22184219

  14. 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

  15. 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…

  16. 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

  17. What makes a protein fold amenable to functional innovation? Fold polarity and stability trade-offs.

    PubMed

    Dellus-Gur, Eynat; Toth-Petroczy, Agnes; Elias, Mikael; Tawfik, Dan S

    2013-07-24

    Protein evolvability includes two elements--robustness (or neutrality, mutations having no effect) and innovability (mutations readily inducing new functions). How are these two conflicting demands bridged? Does the ability to bridge them relate to the observation that certain folds, such as TIM barrels, accommodate numerous functions, whereas other folds support only one? Here, we hypothesize that the key to innovability is polarity--an active site composed of flexible, loosely packed loops alongside a well-separated, highly ordered scaffold. We show that highly stabilized variants of TEM-1 β-lactamase exhibit selective rigidification of the enzyme's scaffold while the active-site loops maintained their conformational plasticity. Polarity therefore results in stabilizing, compensatory mutations not trading off, but instead promoting the acquisition of new activities. Indeed, computational analysis indicates that in folds that accommodate only one function throughout evolution, for example, dihydrofolate reductase, ≥ 60% of the active-site residues belong to the scaffold. In contrast, folds associated with multiple functions such as the TIM barrel show high scaffold-active-site polarity (~20% of the active site comprises scaffold residues) and >2-fold higher rates of sequence divergence at active-site positions. Our work suggests structural measures of fold polarity that appear to be correlated with innovability, thereby providing new insights regarding protein evolution, design, and engineering.

  18. Mechanisms of protein-folding diseases at a glance

    PubMed Central

    Valastyan, Julie S.; Lindquist, Susan

    2014-01-01

    For a protein to function appropriately, it must first achieve its proper conformation and location within the crowded environment inside the cell. Multiple chaperone systems are required to fold proteins correctly. In addition, degradation pathways participate by destroying improperly folded proteins. The intricacy of this multisystem process provides many opportunities for error. Furthermore, mutations cause misfolded, nonfunctional forms of proteins to accumulate. As a result, many pathological conditions are fundamentally rooted in the protein-folding problem that all cells must solve to maintain their function and integrity. Here, to illustrate the breadth of this phenomenon, we describe five examples of protein-misfolding events that can lead to disease: improper degradation, mislocalization, dominant-negative mutations, structural alterations that establish novel toxic functions, and amyloid accumulation. In each case, we will highlight current therapeutic options for battling such diseases. PMID:24396149

  19. Microwave-enhanced folding and denaturation of globular proteins

    NASA Astrophysics Data System (ADS)

    Bohr, Henrik; Bohr, Jakob

    2000-04-01

    It is shown that microwave irradiation can affect the kinetics of the folding process of some globular proteins, especially β-lactoglobulin. At low temperature the folding from the cold denatured phase of the protein is enhanced, while at a higher temperature the denaturation of the protein from its folded state is enhanced. In the latter case, a negative temperature gradient is needed for the denaturation process, suggesting that the effects of the microwaves are nonthermal. This supports the notion that coherent topological excitations can exist in proteins. The application of microwaves hold promises for a wide range of biotechnological applications, such as protein synthesis, protein aggregation, etc., and may have implications for biological systems as well.

  20. 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.

  1. An overview of protein-folding techniques: issues and perspectives.

    PubMed

    Sikder, Abdur Rahman; Zomaya, Albert Y

    2005-01-01

    The importance of protein folding has been recognised for many years. Almost a half century ago, Linus Pauling discovered two quite simple, regular arrangements of amino acids--the alpha-helix and the beta-sheet that are found in almost every protein. In the early 1960s, Christian Anfinsen showed that the proteins actually "tie" themselves: If proteins become unfolded, they fold back into proper shape of their own accord; no shaper or folder is needed. The nature of the unfolded state plays a great role in understanding proteins. Alzheimer's disease, cystic fibrosis, mad cow disease, and many cancers are inherited emphysema. Recent discoveries show that all these apparently unrelated diseases result from protein folding gone wrong. Theoretical and computational studies have recently achieved noticeable success in reproducing various features of the folding mechanism of several small to medium-sized fast-folding proteins. This survey presents the state-of-the-art in protein structure prediction methods from a computer scientist perspective. PMID:18048125

  2. 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

  3. 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.

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

    PubMed

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

    2016-04-26

    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.

  5. 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

  6. 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.

  7. Assessment of optimized Markov models in protein fold classification.

    PubMed

    Lampros, Christos; Simos, Thomas; Exarchos, Themis P; Exarchos, Konstantinos P; Papaloukas, Costas; Fotiadis, Dimitrios I

    2014-08-01

    Protein fold classification is a challenging task strongly associated with the determination of proteins' structure. In this work, we tested an optimization strategy on a Markov chain and a recently introduced Hidden Markov Model (HMM) with reduced state-space topology. The proteins with unknown structure were scored against both these models. Then the derived scores were optimized following a local optimization method. The Protein Data Bank (PDB) and the annotation of the Structural Classification of Proteins (SCOP) database were used for the evaluation of the proposed methodology. The results demonstrated that the fold classification accuracy of the optimized HMM was substantially higher compared to that of the Markov chain or the reduced state-space HMM approaches. The proposed methodology achieved an accuracy of 41.4% on fold classification, while Sequence Alignment and Modeling (SAM), which was used for comparison, reached an accuracy of 38%. PMID:25152041

  8. 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.

  9. Imipramine and citalopram facilitate amyloid precursor protein secretion in vitro.

    PubMed

    Pákáski, Magdolna; Bjelik, Annamária; Hugyecz, Marietta; Kása, Péter; Janka, Zoltán; Kálmán, János

    2005-08-01

    Comorbid depression of Alzheimer's disease (AD) is a common mood disorder in the elderly and a broad spectrum of antidepressants have been used for its treatment. Abeta peptides and other derivatives of the amyloid precursor protein (APP) have been implicated as central to the pathogenesis of AD. However, the functional relationship of APP and its proteolytic derivatives to antidepressant therapy is not known. In this study, Western blotting was used to test the ability of the tricyclic antidepressant (TCA) imipramine or the selective serotonin reuptake inhibitor (SSRI) citalopram to change the release of APP and the protein kinase C (PKC) content. Both antidepressants increased APP secretion in primary rat neuronal cultures. Imipramine or citalopram enhanced the level of secreted APP by 3.2- or 3.4-fold, respectively. Increases in PKC level were observed only after imipramine treatment. These in vitro data suggest that both TCA and SSRI are able to interfere with the APP metabolism. Imipramine promotes the non-amyloidogenic route of APP processing via stimulatory effects on PKC. We propose that PKC is not involved in the mechanism underlying the effects of citalopram on the APP metabolism. Since the secreted APP is not further available for the pathological cleavage of beta- and gamma-secretases, antidepressant medication might be beneficial in AD therapy. PMID:15955598

  10. Novel protein folds and their nonsequential structural analogs

    PubMed Central

    Guerler, Aysam; Knapp, Ernst-Walter

    2008-01-01

    Newly determined protein structures are classified to belong to a new fold, if the structures are sufficiently dissimilar from all other so far known protein structures. To analyze structural similarities of proteins, structure alignment tools are used. We demonstrate that the usage of nonsequential structure alignment tools, which neglect the polypeptide chain connectivity, can yield structure alignments with significant similarities between proteins of known three-dimensional structure and newly determined protein structures that possess a new fold. The recently introduced protein structure alignment tool, GANGSTA, is specialized to perform nonsequential alignments with proper assignment of the secondary structure types by focusing on helices and strands only. In the new version, GANGSTA+, the underlying algorithms were completely redesigned, yielding enhanced quality of structure alignments, offering alignment against a larger database of protein structures, and being more efficient. We applied DaliLite, TM-align, and GANGSTA+ on three protein crystal structures considered to be novel folds. Applying GANGSTA+ to these novel folds, we find proteins in the ASTRAL40 database, which possess significant structural similarities, albeit the alignments are nonsequential and in some cases involve secondary structure elements aligned in reverse orientation. A web server is available at http://agknapp.chemie.fu-berlin.de/gplus for pairwise alignment, visualization, and database comparison. PMID:18583523

  11. 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.

  12. 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

  13. 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

  14. 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

  15. Direct molecular dynamics observation of protein folding transition state ensemble.

    PubMed Central

    Ding, Feng; Dokholyan, Nikolay V; Buldyrev, Sergey V; Stanley, H Eugene; Shakhnovich, Eugene I

    2002-01-01

    The concept of the protein transition state ensemble (TSE), a collection of the conformations that have 50% probability to convert rapidly to the folded state and 50% chance to rapidly unfold, constitutes the basis of the modern interpretation of protein engineering experiments. It has been conjectured that conformations constituting the TSE in many proteins are the expanded and distorted forms of the native state built around a specific folding nucleus. This view has been supported by a number of on-lattice and off-lattice simulations. Here we report a direct observation and characterization of the TSE by molecular dynamic folding simulations of the C-Src SH3 domain, a small protein that has been extensively studied experimentally. Our analysis reveals a set of key interactions between residues, conserved by evolution, that must be formed to enter the kinetic basin of attraction of the native state. PMID:12496119

  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. Generating folded protein structures with a lattice chain growth algorithm

    NASA Astrophysics Data System (ADS)

    Gan, Hin Hark; Tropsha, Alexander; Schlick, Tamar

    2000-10-01

    We present a new application of the chain growth algorithm to lattice generation of protein structure and thermodynamics. Given the difficulty of ab initio protein structure prediction, this approach provides an alternative to current folding algorithms. The chain growth algorithm, unlike Metropolis folding algorithms, generates independent protein structures to achieve rapid and efficient exploration of configurational space. It is a modified version of the Rosenbluth algorithm where the chain growth transition probability is a normalized Boltzmann factor; it was previously applied only to simple polymers and protein models with two residue types. The independent protein configurations, generated segment-by-segment on a refined cubic lattice, are based on a single interaction site for each amino acid and a statistical interaction energy derived by Miyazawa and Jernigan. We examine for several proteins the algorithm's ability to produce nativelike folds and its effectiveness for calculating protein thermodynamics. Thermal transition profiles associated with the internal energy, entropy, and radius of gyration show characteristic folding/unfolding transitions and provide evidence for unfolding via partially unfolded (molten-globule) states. From the configurational ensembles, the protein structures with the lowest distance root-mean-square deviations (dRMSD) vary between 2.2 to 3.8 Å, a range comparable to results of an exhaustive enumeration search. Though the ensemble-averaged dRMSD values are about 1.5 to 2 Å larger, the lowest dRMSD structures have similar overall folds to the native proteins. These results demonstrate that the chain growth algorithm is a viable alternative to protein simulations using the whole chain.

  18. Folding of a large protein at high structural resolution

    PubMed Central

    Walters, Benjamin T.; Mayne, Leland; Hinshaw, James R.; Sosnick, Tobin R.; Englander, S. Walter

    2013-01-01

    Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways. PMID:24191053

  19. Protein Stability, Folding and Misfolding in Human PGK1 Deficiency.

    PubMed

    Valentini, Giovanna; Maggi, Maristella; Pey, Angel L

    2013-01-01

    Conformational diseases are often caused by mutations, altering protein folding and stability in vivo. We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein folding and misfolding. Expression analyses and in vitro biophysical studies indicate that disease-causing mutations enhance protein aggregation propensity. We found a strong correlation among protein aggregation propensity, thermodynamic stability, cooperativity and dynamics. Comparison of folding and unfolding properties with previous reports in PGKs from other species suggests that hPGK1 is very sensitive to mutations leading to enhance protein aggregation through changes in protein folding cooperativity and the structure of the relevant denaturation transition state for aggregation. Overall, we provide a mechanistic framework for protein misfolding of hPGK1, which is insightful to develop new therapeutic strategies aimed to target native state stability and foldability in hPGK1 deficient patients. PMID:24970202

  20. Folding Myoglobin within a Sol-Gel Glass: Protein Folding Constrained to a Small Volume

    PubMed Central

    Peterson, Eric S.; Leonard, Emma F.; Foulke, Jocelyn A.; Oliff, Matthew C.; Salisbury, Rosanne D.; Kim, David Y.

    2008-01-01

    The unfolding and refolding reaction of myoglobin was examined in solution and within a porous silica sol-gel glass. The sol-gel pores constrain the protein to a volume that is the same size and shape as the folded native state accompanied by a few layers of water solvation. Denaturants such as low pH buffers can be diffused through the gel pores to the protein to initiate unfolding and refolding. Acid-induced unfolding was hindered by the steric constraints imposed by the gel pores such that more denaturing conditions were required within the gel than in solution to create the unfolded state. No new folding intermediates were observed. Refolding of myoglobin was not complete in millimolar pH 7 buffer alone. Addition of 25% glycerol to the pH 7 buffer resulted in nearly complete refolding, and the use of 1 M phosphate buffer resulted in complete refolding. The role of this cosolvent and salt in disrupting the ordered water surrounding the protein within the gel is discussed in light of the Hofmeister series and entropic trapping via a diminished hydrophobic effect within the gel. These results are consistent with the premises of folding models in which secondary and tertiary structures are considered to form within a compact conformation of the protein backbone. PMID:18339762

  1. 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.

  2. Thermodynamically important contacts in folding of model proteins.

    PubMed

    Scala, A; Dokholyan, N V; Buldyrev, S V; Stanley, H E

    2001-03-01

    We introduce a quantity, the entropic susceptibility, that measures the thermodynamic importance-for the folding transition-of the contacts between amino acids in model proteins. Using this quantity, we find that only one equilibrium run of a computer simulation of a model protein is sufficient to select a subset of contacts that give rise to the peak in the specific heat observed at the folding transition. To illustrate the method, we identify thermodynamically important contacts in a model 46-mer. We show that only about 50% of all contacts present in the protein native state are responsible for the sharp peak in the specific heat at the folding transition temperature, while the remaining 50% of contacts do not affect the specific heat.

  3. Peptide folding in the presence of interacting protein crowders

    NASA Astrophysics Data System (ADS)

    Bille, Anna; Mohanty, Sandipan; Irbäck, Anders

    2016-05-01

    Using Monte Carlo methods, we explore and compare the effects of two protein crowders, BPTI and GB1, on the folding thermodynamics of two peptides, the compact helical trp-cage and the β-hairpin-forming GB1m3. The thermally highly stable crowder proteins are modeled using a fixed backbone and rotatable side-chains, whereas the peptides are free to fold and unfold. In the simulations, the crowder proteins tend to distort the trp-cage fold, while having a stabilizing effect on GB1m3. The extent of the effects on a given peptide depends on the crowder type. Due to a sticky patch on its surface, BPTI causes larger changes than GB1 in the melting properties of the peptides. The observed effects on the peptides stem largely from attractive and specific interactions with the crowder surfaces, and differ from those seen in reference simulations with purely steric crowder particles.

  4. The primary dynamics in protein folding: the earliest kinetic steps.

    NASA Astrophysics Data System (ADS)

    Callender, Robert

    1996-03-01

    A novel laser-induced temperature jump (T-jump) of 20 C or more is used to initiate the unfolding process of peptides and proteins on the picosecond time scale, and amide I time-resolved infrared absorbance transients are used to characterize the resulting kinetics. We have used this method to study the kinetics of folding and unfolding of a small 21 residue alanine based peptide and molten globule and native states of apomyoglobin, models for the helix which is an basic motif found in proteins. An essential result of our study is that the folding kinetics of a short length of peptide can occur within a few tens of nanoseconds which is much shorter than the time scale of the formation of intramolecular tertiary contacts from one point of a polypeptide chain to another. Furthermore, we observed that helices stabilized by tertiary contact formation unfold slower than helices surrounded by solvent by three orders of magnitude. These results bear directly on the protein folding problem, that is how do proteins fold from a large number of heterogeneous unfolded states to find the specific biologically active folded state on biologically relevent time scales, by suggesting that secondary structure forms first followed by tertiary structure. This work is a collaborative effort with R. GILMANSHIN at City College and S. WILLIAMS, R. B. DYER, and W. H. WOODRUFF at CST-4, Los Alamos National Laboratory, Los Alamos, NM 87545.

  5. Disulfide bonds in ER protein folding and homeostasis

    PubMed Central

    Feige, Matthias J.; Hendershot, Linda M.

    2010-01-01

    Proteins that are expressed outside the cell must be synthesized, folded and assembled in a way that ensures they can function in their designate location. Accordingly these proteins are primarily synthesized in the endoplasmic reticulum (ER), which has developed a chemical environment more similar to that outside the cell. This organelle is equipped with a variety of molecular chaperones and folding enzymes that both assist the folding process, while at the same time exerting tight quality control measures that are largely absent outside the cell. A major post-translational modification of ER-synthesized proteins is disulfide bridge formation, which is catalyzed by the family of protein disulfide isomerases. As this covalent modification provides unique structural advantages to extracellular proteins, multiple pathways to their formation have evolved. However, the advantages that disulfide bonds impart to these proteins come at a high cost to the cell. Very recent reports have shed light on how the cell can deal with or even exploit the side reactions of disulfide bond formation to maintain homeostasis of the ER and its folding machinery. PMID:21144725

  6. Globular Protein Folding In Vitro and In Vivo.

    PubMed

    Gruebele, Martin; Dave, Kapil; Sukenik, Shahar

    2016-07-01

    In vitro, computational, and theoretical studies of protein folding have converged to paint a rich and complex energy landscape. This landscape is sensitively modulated by environmental conditions and subject to evolutionary pressure on protein function. Of these environments, none is more complex than the cell itself, where proteins function in the cytosol, in membranes, and in different compartments. A wide variety of kinetic and thermodynamics experiments, ranging from single-molecule studies to jump kinetics and from nuclear magnetic resonance to imaging on the microscope, have elucidated how protein energy landscapes facilitate folding and how they are subject to evolutionary constraints and environmental perturbation. Here we review some recent developments in the field and refer the reader to some original work and additional reviews that cover this broad topic in protein science. PMID:27391927

  7. Macromolecular crowding fails to fold a globular protein in cells.

    PubMed

    Schlesinger, Alexander P; Wang, Yaqiang; Tadeo, Xavier; Millet, Oscar; Pielak, Gary J

    2011-06-01

    Proteins perform their functions in cells where macromolecular solutes reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules stabilizes globular proteins because the native state occupies less space than the denatured state. Theory predicts that crowding can increase the ratio of folded to unfolded protein by a factor of 100, amounting to 3 kcal/mol of stabilization at room temperature. We tested the idea that volume exclusion dominates the crowding effect in cells using a variant of protein L, a 7 kDa globular protein with seven lysine residues replaced by glutamic acids; 84% of the variant molecules populate the denatured state in dilute buffer at room temperature, compared with 0.1% for the wild-type protein. We then used in-cell NMR spectroscopy to show that the cytoplasm of Escherichia coli does not overcome even this modest (∼1 kcal/mol) free-energy deficit. The data are consistent with the idea that nonspecific interactions between cytoplasmic components can overcome the excluded-volume effect. Evidence for these interactions is provided by the observations that adding simple salts folds the variant in dilute solution but increasing the salt concentration inside E. coli does not fold the protein. Our data are consistent with the results of other studies of protein stability in cells and suggest that stabilizing excluded-volume effects, which must be present under crowded conditions, can be ameliorated by nonspecific interactions between cytoplasmic components.

  8. 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.

  9. Periodic and stochastic thermal modulation of protein folding kinetics

    NASA Astrophysics Data System (ADS)

    Platkov, Max; Gruebele, Martin

    2014-07-01

    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.

  10. 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.

  11. Criteria for folding in structure-based models of proteins

    NASA Astrophysics Data System (ADS)

    Wołek, Karol; Cieplak, Marek

    2016-05-01

    In structure-based models of proteins, one often assumes that folding is accomplished when all contacts are established. This assumption may frequently lead to a conceptual problem that folding takes place in a temperature region of very low thermodynamic stability, especially when the contact map used is too sparse. We consider six different structure-based models and show that allowing for a small, but model-dependent, percentage of the native contacts not being established boosts the folding temperature substantially while affecting the time scales of folding only in a minor way. We also compare other properties of the six models. We show that the choice of the description of the backbone stiffness has a substantial effect on the values of characteristic temperatures that relate both to equilibrium and kinetic properties. Models without any backbone stiffness (like the self-organized polymer) are found to perform similar to those with the stiffness, including in the studies of stretching.

  12. 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

  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. Protein fold recognition using geometric kernel data fusion

    PubMed Central

    Zakeri, Pooya; Jeuris, Ben; Vandebril, Raf; Moreau, Yves

    2014-01-01

    Motivation: Various approaches based on features extracted from protein sequences and often machine learning methods have been used in the prediction of protein folds. Finding an efficient technique for integrating these different protein features has received increasing attention. In particular, kernel methods are an interesting class of techniques for integrating heterogeneous data. Various methods have been proposed to fuse multiple kernels. Most techniques for multiple kernel learning focus on learning a convex linear combination of base kernels. In addition to the limitation of linear combinations, working with such approaches could cause a loss of potentially useful information. Results: We design several techniques to combine kernel matrices by taking more involved, geometry inspired means of these matrices instead of convex linear combinations. We consider various sequence-based protein features including information extracted directly from position-specific scoring matrices and local sequence alignment. We evaluate our methods for classification on the SCOP PDB-40D benchmark dataset for protein fold recognition. The best overall accuracy on the protein fold recognition test set obtained by our methods is ∼86.7%. This is an improvement over the results of the best existing approach. Moreover, our computational model has been developed by incorporating the functional domain composition of proteins through a hybridization model. It is observed that by using our proposed hybridization model, the protein fold recognition accuracy is further improved to 89.30%. Furthermore, we investigate the performance of our approach on the protein remote homology detection problem by fusing multiple string kernels. Availability and implementation: The MATLAB code used for our proposed geometric kernel fusion frameworks are publicly available at http://people.cs.kuleuven.be/∼raf.vandebril/homepage/software/geomean.php?menu=5/ Contact: pooyapaydar@gmail.com or yves

  15. Laboratory evolution of fast-folding green fluorescent protein using secretory pathway quality control.

    PubMed

    Fisher, Adam C; DeLisa, Matthew P

    2008-01-01

    Green fluorescent protein (GFP) has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2), superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar 'superfolded' proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues. PMID:18545653

  16. 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. PMID:9104035

  17. 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

  18. 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. PMID:21446709

  19. Folding analysis of the most complex Stevedore's protein knot.

    PubMed

    Wang, Iren; Chen, Szu-Yu; Hsu, Shang-Te Danny

    2016-01-01

    DehI is a homodimeric haloacid dehalogenase from Pseudomonas putida that contains the most complex 61 Stevedore's protein knot within its folding topology. To examine how DehI attains such an intricate knotted topology we combined far-UV circular dichroism (CD), intrinsic fluorescence spectroscopy and small angle X-ray scattering (SAXS) to investigate its folding mechanism. Equilibrium unfolding of DehI by chemical denaturation indicated the presence of two highly populated folding intermediates, I and I'. While the two intermediates vary in secondary structure contents and tertiary packing according to CD and intrinsic fluorescence, respectively, their overall dimension and compactness are similar according to SAXS. Three single-tryptophan variants (W34, W53, and W196) were generated to probe non-cooperative unfolding events localized around the three fluorophores. Kinetic fluorescence measurements indicated that the transition from the intermediate I' to the unfolded state is rate limiting. Our multiparametric folding analyses suggest that DehI unfolds through a linear folding pathway with two distinct folding intermediates by initial hydrophobic collapse followed by nucleation condensation, and that knotting precedes the formation of secondary structures. PMID:27527519

  20. 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.

  1. Understanding Protein Folding from Advances of Fluorescence Energy Transfer.

    NASA Astrophysics Data System (ADS)

    Tcherkasskaya, Olga; Gronenborn, Angela M.

    2001-03-01

    A multi-site fluorescence energy transfer method was developed for the study of protein folding. Technique uses "tyrosine-phenylalanine" substitution mutagenesis to place the "tyrosine-guest" into positions of interest into the protein structure. Tetranitromethane modification of the tyrosine-guest renders this amino acid an acceptor of the tryptophan fluorescence. This approach can be applied to any protein system, and, most importantly, does not require single- or double-labeling of the protein molecule by a donor and/or an acceptor fluorophore. It is equally suited for equilibrium as well as kinetic studies of folding. We tested the methodology to monitor the equilibrium (un)folding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) induced by guanidine hydrochloride. Wild-type GB1 contains three tyrosines located at positions 3, 33, 45 and a single tryptophan residue at position 43. Two of the three tyrosines were replaced in turn, thereby allowing us to measure the energy transfer from Trp43 to each particular tyrosine. Overall, multi-parametrical experiments on GB1 including circular dichroism, steady state and time-resolved fluorescence, as well as fluorescence energy transfer revealed the existence of highly stable unfolded intermediates, which precede the formation of the rigid (native) secondary structure.

  2. 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

  3. Dynameomics: A Consensus View of the Protein Unfolding/Folding Transition State Ensemble across a Diverse Set of Protein Folds

    PubMed Central

    Jonsson, Amanda L.; Scott, Kathryn A.; Daggett, Valerie

    2009-01-01

    Abstract The Dynameomics project aims to simulate a representative sample of all globular protein metafolds under both native and unfolding conditions. We have identified protein unfolding transition state (TS) ensembles from multiple molecular dynamics simulations of high-temperature unfolding in 183 structurally distinct proteins. These data can be used to study individual proteins and individual protein metafolds and to mine for TS structural features common across all proteins. Separating the TS structures into four different fold classes (all proteins, all-α, all-β, and mixed α/β and α + β) resulted in no significant difference in the overall protein properties. The residues with the most contacts in the native state lost the most contacts in the TS ensemble. On average, residues beginning in an α-helix maintained more structure in the TS ensemble than did residues starting in β-strands or any other conformation. The metafolds studied here represent 67% of all known protein structures, and this is, to our knowledge, the largest, most comprehensive study of the protein folding/unfolding TS ensemble to date. One might have expected broad distributions in the average global properties of the TS relative to the native state, indicating variability in the amount of structure present in the TS. Instead, the average global properties converged with low standard deviations across metafolds, suggesting that there are general rules governing the structure and properties of the TS. PMID:19948125

  4. 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.

  5. 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

  6. 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.

  7. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24347637

  9. Characterization of extracellular matrix proteins during wound healing in the lamina propria of vocal fold in a canine model: a long-term and consecutive study.

    PubMed

    Hu, Rong; Xu, Wen; Ling, Wei; Wang, Qi; Wu, Yan; Han, Demin

    2014-06-01

    The characterization of vocal fold wound healing can be reflected by the changes of extracellular matrix (ECM) proteins in the lamina propria. Although the expression of ECM proteins after vocal fold injury has been widely studied, such observations have lacked time continuity and integrity of marker proteins. In this study, we observed the morphology of injured vocal folds in a canine model. We used immunofluorescence staining to evaluate the expression and distribution of ECM proteins, such as collagen, elastin, hyaluronic acid, decorin and fibronectin, from 15 days to 6 months after injury. The results showed that large amounts of ECM proteins were secreted 15-40 days after injury. Collagen and fibronectin secretion increased significantly, and were disorderly deposited. The secretion of decorin and elastin increased slightly, while hyaluronic acid decreased. The 15-40 day post-injury period may be the critical intervention stage in wound healing of vocal folds. From 3 to 6 months after injury, the secretion of ECM proteins declined. However, collagen and fibronectin secretion were still significantly higher than normal with irregular arrangement, while the secretion of elastin, hyaluronic acid and decorin decreased significantly at 6 months. This led to vocal fold inelasticity and stiffness, which required effective long-term interventions to treat scar formation.

  10. 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

  11. 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

  12. Protein folds and families: sequence and structure alignments.

    PubMed

    Holm, L; Sander, C

    1999-01-01

    Dali and HSSP are derived databases organizing protein space in the structurally known regions. We use an automatic structure alignment program (Dali) for the classification of all known 3D structures based on all-against-all comparison of 3D structures in the Protein Data Bank. The HSSP database associates 1D sequences with known 3D structures using a position-weighted dynamic programming method for sequence profile alignment (MaxHom). As a result, the HSSP database not only provides aligned sequence families, but also implies secondary and tertiary structures covering 36% of all sequences in Swiss-Prot. The structure classification by Dali and the sequence families in HSSP can be browsed jointly from a web interface providing a rich network of links between neighbours in fold space, between domains and proteins, and between structures and sequences. In particular, this results in a database of explicit multiple alignments of protein families in the twilight zone of sequence similarity. The organization of protein structures and families provides a map of the currently known regions of the protein universe that is useful for the analysis of folding principles, for the evolutionary unification of protein families and for maximizing the information return from experimental structure determination. The databases are available from http://www.embl-ebi.ac.uk/dali/

  13. Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL.

    PubMed

    Pastor, Ashutosh; Singh, Amit K; Fisher, Mark T; Chaudhuri, Tapan K

    2016-08-01

    Protein folding has been extensively studied for the past six decades by employing solution-based methods such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR, and HD exchange. However, for rapid analysis of the folding process, solution-based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work, we demonstrate that a bio-layer interferometry (BLI) chaperonin detection system can identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high-affinity nucleotide-free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form, so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on bio-layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation-prone protein maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the bio-layer interferometry biosensor surface were reproduced on Ni-NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution. PMID:27367928

  14. 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.

  15. 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

  16. Molten globules, entropy-driven conformational change and protein folding.

    PubMed

    Baldwin, Robert L; Rose, George D

    2013-02-01

    The exquisite side chain close-packing in the protein core and at binding interfaces has prompted a conviction that packing selectivity is the primary mechanism for molecular recognition in folding and/or binding reactions. Contrary to this view, molten globule proteins can adopt native topology and bind targets tightly and specifically in the absence of side chain close-packing. The molten globule is a highly dynamic form with native-like secondary structure and a loose protein core that admits solvent. The related (but still controversial) dry molten globule is an expanded form of the native protein with largely intact topology but a tighter protein core that excludes solvent. Neither form retains side chain close-packing, and therefore both structure and function must result from other factors, assuming that the reality of the dry molten globule is accepted. This simplifying realization calls for a re-evaluation of established models. PMID:23237704

  17. Protein folding and amyloid formation in various environments

    NASA Astrophysics Data System (ADS)

    O'Brien, Edward P.

    Understanding and predicting the effect of various environments that differ in terms of pH and the presence of cosolutes and macromolecules on protein properties is a formidable challenge. Yet this knowledge is crucial in understanding the effect of cellular environments on a protein. By combining thermodynamic theories of solution condition effects with statistical mechanics and computer simulations we develop a molecular perspective of protein folding and amyloid formation that was previously unobtainable. The resulting Molecular Transfer Model offers, in some instances, quantitatively accurate predictions of cosolute and pH effects on various protein properties. We show that protein denatured state properties can change significantly with osmolyte concentration, and that residual structure can persist at high denaturant concentrations. We study the single molecule mechanical unfolding of proteins at various pH values and varying osmolyte and denaturant concentrations. We find that the the effect of varying solution conditions on a protein under tension can be understood and qualitatively predicted based on knowledge of that protein's behavior in the absence of force. We test the accuracy of FRET inferred denatured state properties and find that currently, only qualitative estimates of denatured state properties can be obtained with these experimental methods. We also explore the factors governing helix formation in peptides confined to carbon nanotubes. We find that the interplay of the peptide's sequence and dimensions, the nanotube's diameter, hydrophobicity and chemical heterogeneity, lead to a rich diversity of behavior in helix formation. We determine the structural and thermodynamic basis for the dock-lock mechanism of peptide deposition to a mature amyloid fibril. We find multiple basins of attraction on the free energy surface associated with structural transitions of the adding monomer. The models we introduce offer a better understanding of protein

  18. 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).

  19. 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

  20. Secretion of human interleukin-2 fused with green fluorescent protein in recombinant Pichia pastoris.

    PubMed

    Cha, Hyung Joon; Dalal, Nimish N; Bentley, William E

    2005-07-01

    Methylotrophic yeast Pichia pastoris is convenient for the expression of eukaryotic foreign proteins owing to its potential for posttranslational modifications, protein folding, and facile culturing. In this work, human interleukin (hIL)-2 was successfully produced as a secreted fusion form in recombinant P. pastoris. By employing green fluorescent protein (GFP) as a monitoring fusion partner, clear identification of fusion protein expression and quantification of intracellular hIL-2 were possible even though there was no correlation between culture supernatant fluorescence and secreted hIL-2 owing to high media interference. Importantly, by the addition of casamino acids in basal medium, we were able to enhance threefold amount of secreted hIL-2, which was present both as a fusion and as a clipped fragment. PMID:16014994

  1. 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

  2. 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

  3. Protein folding on the ribosome studied using NMR spectroscopy.

    PubMed

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

    2013-10-01

    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.

  4. Protein Folding and Quality Control in the ER

    PubMed Central

    Araki, Kazutaka; Nagata, Kazuhiro

    2011-01-01

    The endoplasmic reticulum (ER) uses an elaborate surveillance system called the ER quality control (ERQC) system. The ERQC facilitates folding and modification of secretory and membrane proteins and eliminates terminally misfolded polypeptides through ER-associated degradation (ERAD) or autophagic degradation. This mechanism of ER protein surveillance is closely linked to redox and calcium homeostasis in the ER, whose balance is presumed to be regulated by a specific cellular compartment. The potential to modulate proteostasis and metabolism with chemical compounds or targeted siRNAs may offer an ideal option for the treatment of disease. PMID:21875985

  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. Energetic frustrations in protein folding at residue resolution: a homologous simulation study of Im9 proteins.

    PubMed

    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.

  7. 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

  8. ER to Golgi-Dependent Protein Secretion: The Conventional Pathway.

    PubMed

    Viotti, Corrado

    2016-01-01

    Secretion is the cellular process present in every organism that delivers soluble proteins and cargoes to the extracellular space. In eukaryotes, conventional protein secretion (CPS) is the trafficking route that secretory proteins undertake when are transported from the endoplasmic reticulum (ER) to the Golgi apparatus (GA), and subsequently to the plasma membrane (PM) via secretory vesicles or secretory granules. This book chapter recalls the fundamental steps in cell biology research contributing to the elucidation of CPS; it describes the most prominent examples of conventionally secreted proteins in eukaryotic cells and the molecular mechanisms necessary to regulate each step of this process. PMID:27665548

  9. 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.

  10. Macromolecular crowding increases structural content of folded proteins.

    PubMed

    Perham, Michael; Stagg, Loren; Wittung-Stafshede, Pernilla

    2007-10-30

    Here we show that increased amount of secondary structure is acquired in the folded states of two structurally-different proteins (alpha-helical VlsE and alpha/beta flavodoxin) in the presence of macromolecular crowding agents. The structural content of flavodoxin and VlsE is enhanced by 33% and 70%, respectively, in 400 mg/ml Ficoll 70 (pH 7, 20 degrees C) and correlates with higher protein-thermal stability. In the same Ficoll range, there are only small effects on the unfolded-state structures of the proteins. This is the first in vitro assessment of crowding effects on the native-state structures at physiological conditions. Our findings imply that for proteins with low intrinsic stability, the functional structures in vivo may differ from those observed in dilute buffers. PMID:17919600

  11. 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

  12. Redox proteins are constitutively secreted by skeletal muscle.

    PubMed

    Manabe, Yasuko; Takagi, Mayumi; Nakamura-Yamada, Mio; Goto-Inoue, Naoko; Taoka, Masato; Isobe, Toshiaki; Fujii, Nobuharu L

    2014-11-01

    Myokines are skeletal muscle-derived hormones. In this study, using a C2C12 myotube contraction system, we sought to determine whether the skeletal muscle secreted thioredoxin (TRX) and related redox proteins. Redox proteins such as TRXs, peroxiredoxins, and glutaredoxins were detected in the C2C12 myotube culture medium in the absence of any stimulation. The amounts of TRXs, peroxiredoxins, and glutaredoxins secreted by the C2C12 myotubes were not affected by the contraction, unless the myotubes were injured. Because TRX-1 was known to be a secreted protein that lacks a signal peptide, we examined whether this protein was secreted via exosome vesicles. The results indicated that TRX-1 was not secreted via exosome vesicles. We concluded that TRX-1 and related redox proteins are myokines that are constitutively secreted by the skeletal muscle cells. Although the mechanism of TRX-1 secretion remains unclear, our findings suggest that the skeletal muscle is an endocrine organ and the redox proteins that are constitutively secreted from the skeletal muscle may exert antioxidant and systemic health-promoting effects.

  13. 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

  14. 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

  15. Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast.

    PubMed

    Jacobson, Therese; Navarrete, Clara; Sharma, Sandeep K; Sideri, Theodora C; Ibstedt, Sebastian; Priya, Smriti; Grant, Chris M; Christen, Philipp; Goloubinoff, Pierre; Tamás, Markus J

    2012-11-01

    Several metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

  16. Autocrine regulation of milk secretion by a protein in milk.

    PubMed Central

    Wilde, C J; Addey, C V; Boddy, L M; Peaker, M

    1995-01-01

    Frequency or completeness of milk removal from the lactating mammary gland regulates the rate of milk secretion by a mechanism which is local, chemical and inhibitory in nature. Screening of goat's milk proteins in rabbit mammary explant cultures identified a single whey protein of M(r) 7600 able to inhibit synthesis of milk constituents. The active whey protein, which we term FIL (Feedback inhibitor of Lactation), also decreased milk secretion temporarily when introduced into a mammary gland of lactating goats. FIL was synthesized by primary cultures of goat mammary epithelial cells, and was secreted vectorially together with other milk proteins. N-terminal amino acid sequencing indicated that it is a hitherto unknown protein. The evidence indicates that local regulation of milk secretion by milk removal is through autocrine feedback inhibition by this milk protein. Images Figure 1 Figure 2 Figure 5 PMID:7826353

  17. Coupled protein diffusion and folding in the cell.

    PubMed

    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.

  18. Hsp70 biases the folding pathways of client proteins.

    PubMed

    Sekhar, Ashok; Rosenzweig, Rina; Bouvignies, Guillaume; Kay, Lewis E

    2016-05-17

    The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a (1)H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein. PMID:27140645

  19. 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.

  20. 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.

  1. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    PubMed

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  2. Protein Secretion Systems in Pseudomonas aeruginosa: An Essay on Diversity, Evolution, and Function.

    PubMed

    Filloux, Alain

    2011-01-01

    Protein secretion systems are molecular nanomachines used by Gram-negative bacteria to thrive within their environment. They are used to release enzymes that hydrolyze complex carbon sources into usable compounds, or to release proteins that capture essential ions such as iron. They are also used to colonize and survive within eukaryotic hosts, causing acute or chronic infections, subverting the host cell response and escaping the immune system. In this article, the opportunistic human pathogen Pseudomonas aeruginosa is used as a model to review the diversity of secretion systems that bacteria have evolved to achieve these goals. This diversity may result from a progressive transformation of cell envelope complexes that initially may not have been dedicated to secretion. The striking similarities between secretion systems and type IV pili, flagella, bacteriophage tail, or efflux pumps is a nice illustration of this evolution. Differences are also needed since various secretion configurations call for diversity. For example, some proteins are released in the extracellular medium while others are directly injected into the cytosol of eukaryotic cells. Some proteins are folded before being released and transit into the periplasm. Other proteins cross the whole cell envelope at once in an unfolded state. However, the secretion system requires conserved basic elements or features. For example, there is a need for an energy source or for an outer membrane channel. The structure of this review is thus quite unconventional. Instead of listing secretion types one after each other, it presents a melting pot of concepts indicating that secretion types are in constant evolution and use basic principles. In other words, emergence of new secretion systems could be predicted the way Mendeleïev had anticipated characteristics of yet unknown elements. PMID:21811488

  3. 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

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

    PubMed

    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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26080403

  6. 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. PMID:26080403

  7. A deeply knotted protein structure and how it might fold

    NASA Astrophysics Data System (ADS)

    Taylor, William R.

    2000-08-01

    The search for knots in protein has uncovered little that would cause Alexander the Great to reach for his sword. Excluding knots formed by post-translational crosslinking, the few proteins considered to be knotted form simple trefoil knots with one end of the chain extending through a loop by only a few residues, ten in the `best' example. A knot in an open chain (as distinct from a closed circle) is not rigorously defined and many weak protein knots disappear if the structure is viewed from a different angle. Here I describe a computer algorithm to detect knots in open chains that is not sensitive to viewpoint and that can define the region of the chain giving rise to the knot. It characterizes knots in proteins by the number of residues that must be removed from each end to abolish the knot. I applied this algorithm to the protein structure database and discovered a deep, figure-of-eight knot in the plant protein acetohydroxy acid isomeroreductase. I propose a protein folding pathway that may explain how such a knot is formed.

  8. A deeply knotted protein structure and how it might fold.

    PubMed

    Taylor, W R

    2000-08-24

    The search for knots in protein has uncovered little that would cause Alexander the Great to reach for his sword. Excluding knots formed by post-translational crosslinking, the few proteins considered to be knotted form simple trefoil knots with one end of the chain extending through a loop by only a few residues, ten in the 'best' example. A knot in an open chain (as distinct from a closed circle) is not rigorously defined and many weak protein knots disappear if the structure is viewed from a different angle. Here I describe a computer algorithm to detect knots in open chains that is not sensitive to viewpoint and that can define the region of the chain giving rise to the knot. It characterizes knots in proteins by the number of residues that must be removed from each end to abolish the knot. I applied this algorithm to the protein structure database and discovered a deep, figure-of-eight knot in the plant protein acetohydroxy acid isomeroreductase. I propose a protein folding pathway that may explain how such a knot is formed.

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

    PubMed

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

    2016-04-19

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

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

    PubMed

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

    2016-01-01

    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. PMID:27573113

  11. 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

  12. 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.

  13. 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.

  14. Determination of essential ingredients for protein folding and design

    NASA Astrophysics Data System (ADS)

    Dima, Ruxandra Ioana

    This thesis describes computational studies of some essential ingredients for making progress in the problems of protein folding and design. First, using an observed parallel between heteropolymers and proteins, we outline a strategy for obtaining the free energy of a typical designed heteropolymer and we show its effectiveness on some exactly enumerable lattice models of proteins. Next, we analyze the various existing procedures for building scoring functions in proteins again using a lattice model. We find that the best method is likely to be one based on energy considerations but yet would not require the knowledge of viable alternative conformations which compete significantly with the native conformation in housing a given sequence. Finally, starting from this observation, we introduce a novel method for the extraction of the interaction potentials between the various types of amino acids present in real proteins. We validate this method on a lattice model and we then apply it to real proteins. In both cases, we obtain excellent results on tests of the quality of the extracted parameters.

  15. 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.

  16. Quantifying hub-like behavior in protein folding networks

    PubMed Central

    Dickson, Alex

    2013-01-01

    The free energy landscape of a protein is a function of many interdependent degrees of freedom. For this reason, conceptual constructs (e.g., funnels) have been useful to visualize these landscapes. One relatively new construct is the idea of a hub-like native state that is the final destination of many non-interconverting folding pathways. This is in contrast to the idea of a single predominant folding pathway connecting the native state to a rapidly interconverting ensemble of unfolded states. The key quantity to distinguish between these two ideas is the connectivity of the unfolded ensemble. We present a metric to determine this connectivity for a given network, which can be calculated either from continuous folding trajectories, or a Markov model. The metric determines how often a region of space is used as an intermediate on transition paths that connect two other regions of space, and we use it here to determine how often two parts of the unfolded ensemble are connected directly, versus how often these transitions are mediated by the native state. PMID:24027492

  17. Expression and Targeting of Secreted Proteins from Chlamydia trachomatis

    PubMed Central

    Bauler, Laura D.

    2014-01-01

    Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a vacuole termed the inclusion. Many of the interactions of chlamydiae with the host cell are dependent upon bacterial protein synthesis and presumably exposure of these proteins to the cytosol. Because of the dearth of genetic tools for chlamydiae, previous studies examining secreted proteins required the use of heterologous bacterial systems. Recent advances in genetic manipulation of chlamydia now allow for transformation of the bacteria with plasmids. We describe here a shuttle vector system, pBOMB4, that permits expression of recombinant proteins under constitutive or conditional promoter control. We show that the inclusion membrane protein IncD is secreted in a type III-dependent manner from Yersinia pseudotuberculosis and also secreted from C. trachomatis in infected cells where it localizes appropriately to the inclusion membrane. IncD truncated of the first 30 amino acids containing the secretion signal is no longer secreted and is retained by the bacteria. Cytosolic exposure of secreted proteins can be confirmed by using CyaA, GSK, or microinjection assays. A protein predicted to be retained within the bacteria, NrdB is indeed localized to the chlamydia. In addition, we have shown that the chlamydial effector protein, CPAF, which is secreted into the host cell cytosol by a Sec-dependent pathway, also accesses the cytosol when expressed from this system. These assays should prove useful to assess the secretion of other chlamydial proteins that are potentially exposed to the cytosol of the host cell. PMID:24443531

  18. Chemical, physical, and theoretical kinetics of an ultrafast folding protein.

    PubMed

    Kubelka, Jan; Henry, Eric R; Cellmer, Troy; Hofrichter, James; Eaton, William A

    2008-12-01

    An extensive set of equilibrium and kinetic data is presented and analyzed for an ultrafast folding protein--the villin subdomain. The equilibrium data consist of the excess heat capacity, tryptophan fluorescence quantum yield, and natural circular-dichroism spectrum as a function of temperature, and the kinetic data consist of time courses of the quantum yield from nanosecond-laser temperature-jump experiments. The data are well fit with three kinds of models--a three-state chemical-kinetics model, a physical-kinetics model, and an Ising-like theoretical model that considers 10(5) possible conformations (microstates). In both the physical-kinetics and theoretical models, folding is described as diffusion on a one-dimensional free-energy surface. In the physical-kinetics model the reaction coordinate is unspecified, whereas in the theoretical model, order parameters, either the fraction of native contacts or the number of native residues, are used as reaction coordinates. The validity of these two reaction coordinates is demonstrated from calculation of the splitting probability from the rate matrix of the master equation for all 10(5) microstates. The analysis of the data on site-directed mutants using the chemical-kinetics model provides information on the structure of the transition-state ensemble; the physical-kinetics model allows an estimate of the height of the free-energy barrier separating the folded and unfolded states; and the theoretical model provides a detailed picture of the free-energy surface and a residue-by-residue description of the evolution of the folded structure, yet contains many fewer adjustable parameters than either the chemical- or physical-kinetics models.

  19. Initiation sites of protein folding by NMR analysis.

    PubMed Central

    Freund, S M; Wong, K B; Fersht, A R

    1996-01-01

    Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possible conformations along fast routes for folding. Nuclear magnetic resonance experiments based on the nuclear Overhauser effect (NOE) detect hydrogen atoms close in space and provide information about local structure. Here we present an NMR procedure that detects almost all sequential NOEs between amide hydrogen atoms (HN-HN NOE), including those in random coil regions in a protein, barnase, in urea solutions. A semi-quantitative analysis of these HN-HN NOEs identified partly structured regions that are in remarkable agreement with those found to form early on the reaction pathway. Our results strongly suggest that the folding of barnase initiates at the first helix and the beta-turn between the third and the fourth strands. This strategy of defining residual structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be generally applicable. Images Fig. 1 Fig. 3 PMID:8855224

  20. Temperature dependence of the hydrophobic interaction in protein folding.

    PubMed Central

    Baldwin, R L

    1986-01-01

    Accurate calorimetric data for the thermodynamics of transfer of six liquid hydrocarbons to water have been combined with solubility data to provide a model for the temperature dependence of the hydrophobic interaction in protein folding. The model applies at temperatures for which the change in heat capacity (delta Cp) is constant. The extrapolated value of the temperature (Ts) at which the entropy of transfer (delta S degrees) reaches zero is strikingly similar (Ts = 112.8 degrees C +/- 2.2 degrees C) for the six hydrocarbons. This finding provides an interpretation for the empirical relation discovered by Sturtevant: the ratio delta S degrees/delta Cp measured at 25 degrees C is constant for the transfer of nonpolar substances from nonaqueous media to water. Constancy of this ratio is equivalent to Ts = constant. When applied to protein folding, the hydrocarbon model gives estimates of the contributions of the hydrophobic interaction to the entropy and enthalpy changes on unfolding and, by difference, estimates of the residual contributions from other sources. The major share of the large enthalpy change observed on unfolding at high temperatures comes from the hydrophobic interaction. The hydrophobic interaction changes from being entropy-driven at 22 degrees C to being enthalpy-driven at 113 degrees C. Finally, the hydrocarbon model predicts that plots of the specific entropy change on unfolding versus temperature should nearly intersect close to 113 degrees C, as observed by Privalov. PMID:3464944

  1. 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.

  2. 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

  3. 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. PMID:27574021

  4. 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.

  5. 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.

  6. Effects of positively charged redox molecules on disulfide-coupled protein folding.

    PubMed

    Okumura, Masaki; Shimamoto, Shigeru; Nakanishi, Takeyoshi; Yoshida, Yu-ichiro; Konogami, Tadafumi; Maeda, Shogo; Hidaka, Yuji

    2012-11-01

    In vitro folding of disulfide-containing proteins is generally regulated by redox molecules, such as glutathione. However, the role of the cross-disulfide-linked species formed between the redox molecule and the protein as a folding intermediate in the folding mechanism is poorly understood. In the present study, we investigated the effect of the charge on a redox molecule on disulfide-coupled protein folding. Several types of aliphatic thiol compounds including glutathione were examined for the folding of disulfide-containing-proteins, such as lysozyme and prouroguanylin. The results indicate that the positive charge and its dispersion play a critical role in accelerating disulfide-coupled protein folding.

  7. A framework for describing topological frustration in models of protein folding.

    PubMed

    Norcross, Todd S; Yeates, Todd O

    2006-09-22

    In a natively folded protein of moderate or larger size, the protein backbone may weave through itself in complex ways, raising questions about what sequence of events might have to occur in order for the protein to reach its native configuration from the unfolded state. A mathematical framework is presented here for describing the notion of a topological folding barrier, which occurs when a protein chain must pass through a hole or opening, formed by other regions of the protein structure. Different folding pathways encounter different numbers of such barriers and therefore different degrees of frustration. A dynamic programming algorithm finds the optimal theoretical folding path and minimal degree of frustration for a protein based on its natively folded configuration. Calculations over a database of protein structures provide insights into questions such as whether the path of minimal frustration might tend to favor folding from one or from many sites of folding nucleation, or whether proteins favor folding around the N terminus, thereby providing support for the hypothesis that proteins fold co-translationally. The computational methods are applied to a multi-disulfide bonded protein, with computational findings that are consistent with the experimentally observed folding pathway. Attention is drawn to certain complex protein folds for which the computational method suggests there may be a preferred site of nucleation or where folding is likely to proceed through a relatively well-defined pathway or intermediate. The computational analyses lead to testable models for protein folding. PMID:16930616

  8. Secreted proteins as a fundamental source for biomarker discovery

    PubMed Central

    Stastna, Miroslava; Van Eyk, Jennifer E.

    2012-01-01

    The proteins secreted by various cells (the secretomes) are a potential rich source of biomarkers since they reflect various states of the cells at real time and at given conditions. To have accessible, sufficient and reliable protein markers is desirable since they mark various stages of disease development and their presence/absence can be used for diagnosis, prognosis, risk stratification and therapeutic monitoring. As direct analysis of blood/plasma, a common and noninvasive patient screening method, can be difficult for candidate protein biomarker identification, the alternative/complementary approaches are required, one of them is the analysis of secretomes in cell conditioned media in vitro. Since the proteins secreted by cells as a response to various stimuli are most likely secreted into blood/plasma, the identification and preselection of candidate protein biomarkers from cell secretomes with subsequent validation of their presence at higher levels in serum/plasma is a promising approach. In this review, we discuss the proteins secreted by three progenitor cell types (smooth muscle, endothelial and cardiac progenitor cells) and two adult cell types (neonatal rat ventrical myocytes and smooth muscle cells) which can be relevant to cardiovascular research and which have been recently published in the literature. We found, at least for secretome studies included in this review, that secretomes of progenitor and adult cells overlap by 48% but the secretomes are very distinct among progenitor cell themselves as well as between adult cells. In addition, we compared secreted proteins to protein identifications listed in the Human Plasma PeptideAtlas and in two reports with cardiovascular-related proteins and we performed the extensive literature search to find if any of these secreted proteins were identified in a biomarker study. As expected, many proteins have been identified as biomarkers in cancer but 18 proteins (out of 62) have been tested as biomarkers in

  9. 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.

  10. 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

  11. Iron-nucleated folding of a metalloprotein in high urea: resolution of metal binding and protein folding events.

    PubMed

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

    2010-08-10

    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. With a 100-fold molar excess of ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFe(III)Rd structure was recovered in >90% yield with a t(1/2) of <10 ms. 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, and (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 a 100-fold molar excess of iron. Analogous results obtained for 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 together distal regions of the protein chain. 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, multispectroscopy approach could clarify folding pathways of other [M(SCys)(3)]- or [M(SCys)(4)]-containing proteins.

  12. 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…

  13. 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.

  14. Misplaced helix slows down ultrafast pressure-jump protein folding.

    PubMed

    Prigozhin, Maxim B; Liu, Yanxin; Wirth, Anna Jean; Kapoor, Shobhna; Winter, Roland; Schulten, Klaus; Gruebele, Martin

    2013-05-14

    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

  15. 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

    2015-01-01

    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. PMID:25245368

  16. Characterization of the Unconventional Secretion of the Ebola Matrix Protein VP40.

    PubMed

    Reynard, Olivier; Mateo, Mathieu

    2016-01-01

    While most secreted proteins use the classical endoplasmic reticulum (ER)-Golgi secretion pathway to reach the extracellular medium, a few proteins are secreted through unconventional secretary pathways. Viral proteins can be secreted through unconventional secretion pathways. Here, we describe how we have recently demonstrated that the Ebola virus (EBOV) matrix protein VP40 is released from transfected and infected cells in a soluble form through an unconventional secretion pathway. PMID:27665561

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. Proteomic analysis of Taenia solium metacestode excretion-secretion proteins.

    PubMed

    Victor, Bjorn; Kanobana, Kirezi; Gabriël, Sarah; Polman, Katja; Deckers, Nynke; Dorny, Pierre; Deelder, André M; Palmblad, Magnus

    2012-06-01

    The metacestode larval stage of Taenia solium is the causal agent of a zoonotic disease called cysticercosis. The disease has an important impact on pork trade (due to porcine cysticercosis) and public health (due to human neurocysticercosis). In order to improve the current diagnostic tools and to get a better understanding of the interaction between T. solium metacestodes and their host, there is a need for more information about the proteins that are released by the parasite. In this study, we used protein sequences from different helminths, 1DE, reversed-phase LC, and MS/MS to analyze the excretion-secretion proteins produced by T. solium metacestodes from infected pigs. This is the first report of the T. solium metacestode excretion-secretion proteome. We report 76 proteins including 27 already described T. solium proteins, 17 host proteins and 32 proteins likely to be of T. solium origin, but identified using sequences from other helminths.

  4. 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.

  5. Deducing the Energetic Cost of Protein Folding in Zinc Finger Proteins Using Designed Metallopeptides

    SciTech Connect

    Reddi,A.; Guzman, T.; Breece, r.; Tierney, D.; Gibney, B.

    2007-01-01

    Zinc finger transcription factors represent the largest single class of metalloproteins in the human genome. Binding of Zn(II) to their canonical Cys4, Cys3His1, or Cys2His2 sites results in metal-induced protein folding events required to achieve their proper structure for biological activity. The thermodynamic contribution of Zn(II) in each of these coordination spheres toward protein folding is poorly understood because of the coupled nature of the metal-ligand and protein-protein interactions. Using an unstructured peptide scaffold, GGG, we have employed fluorimetry, potentiometry, and calorimetry to determine the thermodynamics of Zn(II) binding to the Cys4, Cys3His1, and Cys2His2 ligand sets with minimal interference from protein folding effects. The data show that Zn(II) complexation is entropy driven and modulated by proton release. The formation constants for Zn(II)-GGG with a Cys4, Cys3His1, or Cys2His2 site are 5.6 x 1016, 1.5 x 1015, or 2.5 x 1013 M-1, respectively. Thus, the Zn(II)-Cys4, Zn(II)-Cys3His1, and Zn(II)-Cys2His2 interactions can provide up to 22.8, 20.7, and 18.3 kcal/mol, respectively, in driving force for protein stabilization, folding, and/or assembly at pH values above the ligand pKa values. While the contributions from the three coordination motifs differ by 4.5 kcal/mol in Zn(II) affinity at pH 9.0, they are equivalent at physiological pH, ?G = -16.8 kcal/mol or a Ka = 2.0 x 1012 M-1. Calorimetric data show that this is due to proton-based enthalpy-entropy compensation between the favorable entropic term from proton release and the unfavorable enthalpic term due to thiol deprotonation. Since protein folding effects have been minimized in the GGG scaffold, these peptides possess nearly the tightest Zn(II) affinities possible for their coordination motifs. The Zn(II) affinities in each coordination motif are compared between the GGG scaffold and natural zinc finger proteins to determine the free energy required to fold the latter

  6. 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

  7. 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.

  8. Slow and Bimolecular Folding of a De Novo Designed Monomeric Protein DS119

    PubMed Central

    Zhu, Cheng; Dai, Ziwei; Liang, Huanhuan; Zhang, Tao; Gai, Feng; Lai, Luhua

    2013-01-01

    De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding. PMID:24209859

  9. Protein folding in the endoplasmic reticulum: lessons from the human chorionic gonadotropin beta subunit.

    PubMed Central

    Ruddon, R. W.; Sherman, S. A.; Bedows, E.

    1996-01-01

    There have been few studies of protein folding in the endoplasmic reticulum of intact mammalian cells. In the one case where the in vivo and in vitro folding pathways of a mammalian secretory protein have been compared, the folding of the human chorionic gonadotropin beta subunit (hCG-beta), the order of formation of the detected folding intermediates is the same. The rate and efficiency with which multidomain proteins such as hCG-beta fold to native structure in intact cells is higher than in vitro, although intracellular rates of folding of the beta subunit can be approached in vitro in the presence of an optimal redox potential and protein disulfide isomerase. Understanding how proteins fold in vivo may provide a new way to diagnose and treat human illnesses that occur due to folding defects. PMID:8844836

  10. 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. PMID:23075153

  11. 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.

  12. Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein.

    PubMed

    Folders, J; Algra, J; Roelofs, M S; van Loon, L C; Tommassen, J; Bitter, W

    2001-12-01

    The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.

  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. Structural classification of proteins and structural genomics: new insights into protein folding and evolution

    PubMed Central

    Andreeva, Antonina; Murzin, Alexey G.

    2010-01-01

    During the past decade, the Protein Structure Initiative (PSI) centres have become major contributors of new families, superfamilies and folds to the Structural Classification of Proteins (SCOP) database. The PSI results have increased the diversity of protein structural space and accelerated our understanding of it. This review article surveys a selection of protein structures determined by the Joint Center for Structural Genomics (JCSG). It presents previously undescribed β-sheet architectures such as the double barrel and spiral β-roll and discusses new examples of unusual topologies and peculiar structural features observed in proteins characterized by the JCSG and other Structural Genomics centres. PMID:20944210

  15. 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.

  16. Correlation between rate of folding, energy landscape, and topology in the folding of a model protein HP-36

    NASA Astrophysics Data System (ADS)

    Mukherjee, Arnab; Bagchi, Biman

    2003-03-01

    We explore the correlation between the energy landscape and topology in the folding of a model protein (chicken villin headpiece HP-36) by using a force-field which incorporates the effects of water through a hydropathy scale and the role of helical propensity of amino acids through a nonlocal harmonic potential. Each amino acid is represented by one side chain atom which is attached to the backbone Cα atom. Sizes and interactions of all the side chain residues are different and depend on the hydrophobicity of a particular amino acid, whereas helical propensities are incorporated in the interaction of Cα atoms. Simulations have been carried out by quenching from a fixed high temperature to two different low temperatures for many initial random configurations. The simulated structures resemble the real native state rather closely, with the root mean square deviation of the best structure being 4.5 Å. Moreover, the structure shows both the helices and bends at the appropriate positions of the model protein. The simplified model allows the study of energy landscape and also of the correlation between energy landscape with the dynamics of folding and topology. The initial part of folding is very fast, followed by two distinct slow stages, with the last stage being certainly the rate determining of the folding process. The initial fast dynamics is primarily due to hydrophobic collapse. The very slow last stage of folding is accompanied by a significant and sharp increase in the relative contact order parameter but relatively small decrease in energy. Analysis of the time dependence of the formation of the individual contact pairs show rich and complicated dynamics, where some contacts wait for a long time to form. This seems to suggest that the slow late stage folding is due to long range contact formation and also that the free energy barrier is entropic in origin. Results have been correlated with the theories of protein folding.

  17. 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

  18. 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.

  19. Comparing Protein Folding In vitro and In vivo: Foldability Meets the Fitness Challenge

    PubMed Central

    Hingorani, Karan S.; Gierasch, Lila M.

    2014-01-01

    In this review, we compare and contrast current knowledge about in-vitro and in-vivo protein folding. Major advances in understanding fundamental principles underlying protein folding in optimized in-vitro conditions have yielded detailed physicochemical principles of folding landscapes for small, single domain proteins. In addition, there has been increased research focusing on the key features of protein folding in the cell that differentiate it from in-vitro folding, such as co-translational folding, chaperone-facilitated folding, and folding in crowded conditions with many weak interactions. Yet these two research areas have not been bridged effectively in research carried out to date. This review points to gaps between the two that are ripe for future research. Moreover, we emphasize the biological selection pressures that impact protein folding in-vivo and how fitness drives the evolution of protein sequences in ways that may place foldability in tension with other requirements on a given protein. We suggest that viewing the physicochemical process of protein folding through the lens of evolution will unveil new insights and pose novel challenges about in-cell folding landscapes. PMID:24434632

  20. Quantifying Nonnative Interactions in the Protein-Folding Free-Energy Landscape.

    PubMed

    Mouro, Paulo Ricardo; de Godoi Contessoto, Vinícius; Chahine, Jorge; Junio de Oliveira, Ronaldo; Pereira Leite, Vitor Barbanti

    2016-07-26

    Protein folding is a central problem in biological physics. Energetic roughness is an important aspect that controls protein-folding stability and kinetics. The roughness is associated with conflicting interactions in the protein and is also known as frustration. Recent studies indicate that an addition of a small amount of energetic frustration may enhance folding speed for certain proteins. In this study, we have investigated the conditions under which frustration increases the folding rate. We used a Cα structure-based model to simulate a group of proteins. We found that the free-energy barrier at the transition state (ΔF) correlates with nonnative-contact variation (ΔA), and the simulated proteins are clustered according to their fold motifs. These findings are corroborated by the Clementi-Plotkin analytical model. As a consequence, the optimum frustration regime for protein folding can be predicted analytically. PMID:27463131

  1. Structure of a PE–PPE–EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    PubMed Central

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-01-01

    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, 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. PMID:25275011

  2. GRP94: an HSP90-like protein specialized for protein folding and quality control in the Endoplasmic Reticulum

    PubMed Central

    Marzec, Michal; Eletto, Davide; Argon, Yair

    2011-01-01

    Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94 shares many biochemical features with other HSP90 proteins, in particular its domain structure and ATPase activity, but also displays distinct activities, such as calcium binding, necessitates by the conditions in the endoplasmic reticulum. GRP94’s mode of action varies from the general HSP90 theme in the conformational changes induced by nucleotide binding, and in its interactions with co-chaperones, which are very different from known cytosolic co-chaperones. GRP94 is more selective than many of the ER chaperones and the basis for this selectivity remain obscure. Recent development of molecular tools and functional assays has expanded the spectrum of clients that rely on GRP94 activity, but it is still not clear how the chaperone binds them, or what aspect of folding it impacts. These mechanistic questions and the regulation of GRP94 activity by other proteins and by post-translational modification differences pose new questions and present future research avenues. PMID:22079671

  3. Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase.

    PubMed

    Inan, Mehmet; Aryasomayajula, Dinesh; Sinha, Jayanta; Meagher, Michael M

    2006-03-01

    A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins. PMID:16255058

  4. Differentially expressed protein markers in human submandibular and sublingual secretions.

    PubMed

    Hu, Shen; Denny, Patricia; Denny, Paul; Xie, Yongming; Loo, Joseph A; Wolinsky, Lawrence E; Li, Yang; McBride, Jim; Ogorzalek Loo, Rachel R; Navazesh, Mavash; Wong, David T

    2004-11-01

    Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

  5. Characterization of novel secreted proteins from Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa is a bacterium that causes disease of agriculturally important crops, including Pierce’s disease of grapevine. Little is known about virulence factors that are necessary for X. fastidiosa to grow and cause disease in the xylem vessels of a plant host. Any protein secreted by the b...

  6. 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.

  7. Connecting thermal and mechanical protein (un)folding landscapes.

    PubMed

    Sun, Li; Noel, Jeffrey K; Sulkowska, Joanna I; Levine, Herbert; Onuchic, José N

    2014-12-16

    Molecular dynamics simulations supplement single-molecule pulling experiments by providing the possibility of examining the full free energy landscape using many coordinates. Here, we use an all-atom structure-based model to study the force and temperature dependence of the unfolding of the protein filamin by applying force at both termini. The unfolding time-force relation τ(F) indicates that the force-induced unfolding behavior of filamin can be characterized into three regimes: barrier-limited low- and intermediate-force regimes, and a barrierless high-force regime. Slope changes of τ(F) separate the three regimes. We show that the behavior of τ(F) can be understood from a two-dimensional free energy landscape projected onto the extension X and the fraction of native contacts Q. In the low-force regime, the unfolding rate is roughly force-independent due to the small (even negative) separation in X between the native ensemble and transition state ensemble (TSE). In the intermediate-force regime, force sufficiently separates the TSE from the native ensemble such that τ(F) roughly follows an exponential relation. This regime is typically explored by pulling experiments. While X may fail to resolve the TSE due to overlap with the unfolded ensemble just below the folding temperature, the overlap is minimal at lower temperatures where experiments are likely to be conducted. The TSE becomes increasingly structured with force, whereas the average order of structural events during unfolding remains roughly unchanged. The high-force regime is characterized by barrierless unfolding, and the unfolding time approaches a limit of ∼10 μs for the highest forces we studied. Finally, a combination of X and Q is shown to be a good reaction coordinate for almost the entire force range. PMID:25517160

  8. Constitutive secretion of tau protein by an unconventional mechanism.

    PubMed

    Chai, Xiyun; Dage, Jeffrey L; Citron, Martin

    2012-12-01

    The microtubule-associated protein tau plays a critical role in the pathogenesis of Alzheimer's disease and several related disorders. In the disease tau aggregates into paired helical and straight filaments, which can form higher order neurofibrillary tangles in neurons and this pathology is associated with progressive neuronal loss and cognitive decline. Tau is a cytoplasmic protein and is thought to be released only from degenerating cells. In contrast, here we provide evidence that tau is constitutively secreted at a low level. We directly show tau release in cell culture model systems. In inducible transfected cell lines we observe that a small proportion of full-length tau is released from intact cells in a time dependent manner. We show that this tau is released by an unconventional secretion process, as the release is temperature dependent but not blocked by inhibitors of the conventional secretory pathway. We characterize the released tau as full length, not vesicle associated and containing Phospho-Tau (181P) proportional to its intracellular concentration. We demonstrate that tau secretion and its suppression by low temperature also occurs in human neurons differentiated from induced pluripotent stem cells. The constitutive tau secretion that we propose provides the most parsimonious explanation for the observed presence of tau in the CSF of healthy animals and human beings. If previously postulated pathogenic extracellular tau intermediates are released by this route, low level constitutive tau secretion could play a role in the spread of tau pathology in Alzheimer's disease and other human tauopathies. PMID:22668776

  9. 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

  10. Type I Signal Peptidase and Protein Secretion in Staphylococcus aureus

    PubMed Central

    Schallenberger, Mark A.; Niessen, Sherry; Shao, Changxia; Fowler, Bruce J.

    2012-01-01

    Staphylococcus aureus is an important human pathogen whose virulence relies on the secretion of many different proteins. In general, the secretion of most proteins in S. aureus, as well as other bacteria, is dependent on the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein to the general secretory pathway. The arylomycins are a class of natural product antibiotics that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. While wild-type S. aureus (NCTC 8325) is naturally resistant to the arylomycins, sensitivity is conferred via a point mutation in its SPase. Here, we use a synthetic arylomycin along with a sensitized strain of S. aureus and multidimensional protein identification technology (MudPIT) mass spectrometry to identify 46 proteins whose extracellular accumulation requires SPase activity. Forty-four possess identifiable Sec-type signal peptides and thus are likely canonically secreted proteins, while four also appear to possess cell wall retention signals. We also identified the soluble C-terminal domains of two transmembrane proteins, lipoteichoic acid synthase, LtaS, and O-acyteltransferase, OatA, both of which appear to have noncanonical, internal SPase cleavage sites. Lastly, we identified three proteins, HtrA, PrsA, and SAOUHSC_01761, whose secretion is induced by arylomycin treatment. In addition to elucidating fundamental aspects of the physiology and pathology of S. aureus, the data suggest that an arylomycin-based therapeutic would reduce virulence while simultaneously eradicating an infection. PMID:22447899

  11. 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

  12. Is caseinomacropeptide from milk proteins, an inhibitor of gastric secretion?

    PubMed

    Guilloteau, Paul; Romé, Véronique; Delaby, Luc; Mendy, François; Roger, Loic; Chayvialle, Jean Alain

    2010-01-01

    The aim of this work was to study, in vivo, the effect of the ingestion of not glycosylated caseinomacropeptide (CMP) on gastric secretion. In Experiments #1 and #2, 7 calves fitted with a gastric pouch received either a diet without CMP (C diet) or C diet in which CMP was introduced (equal to and 5 folds that of CMP quantity contained in cow milk, diets CMP1 and CMP5, respectively). In Experiment #3, 2 calves (with gastric pouch) were fed C diet followed by an "iv perfusion" of CMP. In Experiment #4, 25 calves fed either C, CMP1 or CMP5 diets were fitted with a blood catheter for sample collections. The quantities of daily gastric secretions seemed few modified by CMP ingestion but the profile of these secretions was changed along the day. The most important result is that CMP can inhibit gastric secretions (mainly hydrochloric acid) stimulated by the meal, but there was no dose-dependent response. No similar observations were obtained after perfusion of CMP in jugular vein. CMP was not detected in blood. Results obtained in our experiments are not in favor of its significant intestinal absorption. Gastrin, somatostatin and VIP could be implicated in the mechanisms of regulation.

  13. 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…

  14. A New Heuristic Algorithm for Protein Folding in the HP Model.

    PubMed

    Traykov, Metodi; Angelov, Slav; Yanev, Nicola

    2016-08-01

    This article presents an efficient heuristic for protein folding. The protein folding problem is to predict the compact three-dimensional structure of a protein based on its amino acid sequence. The focus is on an original integer programming model derived from a platform used for Contact Map Overlap problem.

  15. KDEL-Containing Auxin-Binding Protein Is Secreted to the Plasma Membrane and Cell Wall.

    PubMed Central

    Jones, A. M.; Herman, E. M.

    1993-01-01

    The auxin-binding protein ABP1 has been postulated to mediate auxin-induced cellular changes associated with cell expansion. This protein contains the endoplasmic reticulum (ER) retention signal, the tetrapeptide lysine-aspartic acid-glutamic acid-leucine (KDEL), at its carboxy terminus, consistent with previous subcellular fractionation data that indicated an ER location for ABP1. We used electron microscopic immunocytochemistry to identify the subcellular localization of ABP1. Using maize (Zea mays) coleoptile tissue and a black Mexican sweet (BMS) maize cell line, we found that ABP1 is located in the ER as expected, but is also on or closely associated with the plasma membrane and within the cell wall. Labeling of the Golgi apparatus suggests that the transport of ABP1 to the cell wall occurs via the secretory system. Inhibition of secretion of an ABP homolog into the medium of BMS cell cultures by brefeldin A, a drug that specifically blocks secretion, is consistent with this secretion pathway. The secreted protein was recognized by an anti-KDEL peptide antibody, strongly supporting the interpretation that movement of this protein out of the ER does not involve loss of the carboxy-terminal signal. Cells starved for 2,4-dichlorophenoxyacetic acid for 72 h retained less ABP in the cell and secreted more of it into the medium. The significance of our observations is 2-fold. We have identified a KDEL-containing protein that specifically escapes the ER retention system, and we provide an explanation for the apparent discrepancy that most of the ABP is located in the ER, whereas ABP and auxin act at the plasma membrane. PMID:12231715

  16. Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing.

    PubMed

    Mehler, Michaela; Eckert, Carl Elias; Busche, Alena; Kulhei, Jennifer; Michaelis, Jonas; Becker-Baldus, Johanna; Wachtveitl, Josef; Dötsch, Volker; Glaubitz, Clemens

    2015-11-13

    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 (13)C-labeled retinal cofactor and extensively (13)C-(15)N-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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. Substrate-induced activation of a trapped IMC-mediated protein folding intermediate.

    PubMed

    Inouye, M; Fu, X; Shinde, U

    2001-04-01

    While several unfolded proteins acquire native structures through distinct folding intermediates, the physiological relevance and importance of such states in the folding kinetics remain controversial. The intramolecular chaperone (IMC) of subtilisin was used to trap a partially folded, stable crosslinked intermediate conformer (CLIC) through a disulfide bond between mutated IMC and subtilisin. The trapped CLIC contains non-native interactions. Here we show that CLIC can be induced into a catalytically active form by incubating it with small peptide substrates. The structure and catalytic properties of the activated crosslinked intermediate conformer (A-CLIC) differ from those of the fully folded enzyme in that A-CLIC lacks any endopeptidase activity toward a large protein substrate. Our results show that a disulfide-linked partially folded protein can be induced to acquire catalytic activity with a substrate specificity that is different from completely folded subtilisin. These results also suggest that protein folding intermediates may also participate in catalytic reactions.

  2. Physicochemical bases for protein folding, dynamics, and protein-ligand binding.

    PubMed

    Li, HuiMin; Xie, YueHui; Liu, CiQuan; Liu, ShuQun

    2014-03-01

    Proteins are essential parts of living organisms and participate in virtually every process within cells. As the genomic sequences for increasing number of organisms are completed, research into how proteins can perform such a variety of functions has become much more intensive because the value of the genomic sequences relies on the accuracy of understanding the encoded gene products. Although the static three-dimensional structures of many proteins are known, the functions of proteins are ultimately governed by their dynamic characteristics, including the folding process, conformational fluctuations, molecular motions, and protein-ligand interactions. In this review, the physicochemical principles underlying these dynamic processes are discussed in depth based on the free energy landscape (FEL) theory. Questions of why and how proteins fold into their native conformational states, why proteins are inherently dynamic, and how their dynamic personalities govern protein functions are answered. This paper will contribute to the understanding of structure-function relationship of proteins in the post-genome era of life science research.

  3. Activation Barrier-Limited Folding and Conformational Sampling of a Dynamic Protein Domain.

    PubMed

    Dogan, Jakob; Toto, Angelo; Andersson, Eva; Gianni, Stefano; Jemth, Per

    2016-09-20

    Folding reaction mechanisms of globular protein domains have been extensively studied by both experiment and simulation and found to be highly concerted chemical reactions in which numerous noncovalent bonds form in an apparent two-state fashion. However, less is known regarding intrinsically disordered proteins because their folding can usually be studied only in conjunction with binding to a ligand. We have investigated by kinetics the folding mechanism of such a disordered protein domain, the nuclear coactivator-binding domain (NCBD) from CREB-binding protein. While a previous computational study suggested that NCBD folds without an activation free energy barrier, our experimental data demonstrate that NCBD, despite its highly dynamic structure, displays relatively slow folding (∼10 ms at 277 K) consistent with a barrier-limited process. Furthermore, the folding kinetics corroborate previous nuclear magnetic resonance data showing that NCBD exists in two folded conformations and one more denatured conformation at equilibrium and, thus, that the folding mechanism is a three-state mechanism. The refolding kinetics is limited by unfolding of the less populated folded conformation, suggesting that the major route for interconversion between the two folded states is via the denatured state. Because the two folded conformations have been suggested to bind distinct ligands, our results have mechanistic implications for conformational sampling in protein-protein interactions. PMID:27542287

  4. Total protein output during rapid reduction of bile salt secretion rates in man.

    PubMed Central

    Harvey, P R; Toth, J L; Upadhya, G A; Ilson, R G; Strasberg, S M

    1989-01-01

    An investigation was undertaken to study the effect of bile salt secretion on total biliary protein secretion in man. Bile was collected in eight patients from a tube in the bile duct. Collection was started after a meal and continued for six hours, in order to obtain bile salt secretion rates over the entire physiological range. Total protein secretion rates did not vary with change in bile salt secretion or bile flow. The protein pattern assessed by SDS-PAGE did not vary with bile salt secretion. The results indicate that bile salt secretion has little influence on biliary protein secretion under these conditions in man. Changes in bile salt secretion were associated with linear change in bile flow, but there was no relationship between bile flow and protein secretion rates. This argues against convective sieving of plasma proteins into bile. Images Fig. 4 PMID:2920916

  5. Proteomic identification of secreted proteins of Propionibacterium acnes

    PubMed Central

    2010-01-01

    Background The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. Results Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE) and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS). A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, β-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP) factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. Conclusions Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence properties of P. acnes isolates

  6. Multiple contact network is a key determinant to protein folding rates.

    PubMed

    Gromiha, M Michael

    2009-04-01

    Understanding the relationship between amino acid sequences and folding rates of proteins is an important task in computational and molecular biology. It has been shown that topological parameters, contact order, long-range order, and total contact distance relate well with protein folding rates. In this work, we have systematically analyzed the influence of amino acid residues that form multiple contacts in protein structures to folding rates of proteins. We observed an inverse relationship between the number of residues with multiple contacts and protein folding rates. Further analysis indicates that multiple contacts are influenced by hydrophobic residues, whereas the role is minimal between the residues that are capable of forming hydrogen bonds. The propensity of multiple contacts forming residues showed that aromatic and hydrophobic residues are dominant in two-state proteins, whereas the polar residues Ser and Thr are also preferred ones in three-state proteins. In addition, multiple contact forming residues are interconnected with each other through contact networks in protein structures. The comparison between slow and fast folding proteins demonstrated the presence of more multiple contact forming residues in slow folding proteins with a limit of 4-6 contacts/residue. These results have been reflected in amino acid sequences in the form of short-, medium-, and long-range contacts, which could discriminate slow and fast folding proteins with an accuracy of 96% using a 5-fold cross-validation method.

  7. Identification of protein secretion systems in bacterial genomes

    PubMed Central

    Abby, Sophie S.; Cury, Jean; Guglielmini, Julien; Néron, Bertrand; Touchon, Marie; Rocha, Eduardo P. C.

    2016-01-01

    Bacteria with two cell membranes (diderms) have evolved complex systems for protein secretion. These systems were extensively studied in some model bacteria, but the characterisation of their diversity has lagged behind due to lack of standard annotation tools. We built online and standalone computational tools to accurately predict protein secretion systems and related appendages in bacteria with LPS-containing outer membranes. They consist of models describing the systems’ components and genetic organization to be used with MacSyFinder to search for T1SS-T6SS, T9SS, flagella, Type IV pili and Tad pili. We identified ~10,000 candidate systems in bacterial genomes, where T1SS and T5SS were by far the most abundant and widespread. All these data are made available in a public database. The recently described T6SSiii and T9SS were restricted to Bacteroidetes, and T6SSii to Francisella. The T2SS, T3SS, and T4SS were frequently encoded in single-copy in one locus, whereas most T1SS were encoded in two loci. The secretion systems of diderm Firmicutes were similar to those found in other diderms. Novel systems may remain to be discovered, since some clades of environmental bacteria lacked all known protein secretion systems. Our models can be fully customized, which should facilitate the identification of novel systems. PMID:26979785

  8. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

    PubMed Central

    Iacovache, Ioan; De Carlo, Sacha; Cirauqui, Nuria; Dal Peraro, Matteo; van der Goot, F. Gisou; Zuber, Benoît

    2016-01-01

    Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry. PMID:27405240

  9. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process.

    PubMed

    Iacovache, Ioan; De Carlo, Sacha; Cirauqui, Nuria; Dal Peraro, Matteo; van der Goot, F Gisou; Zuber, Benoît

    2016-01-01

    Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry. PMID:27405240

  10. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process.

    PubMed

    Iacovache, Ioan; De Carlo, Sacha; Cirauqui, Nuria; Dal Peraro, Matteo; van der Goot, F Gisou; Zuber, Benoît

    2016-07-13

    Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

  11. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

    NASA Astrophysics Data System (ADS)

    Iacovache, Ioan; de Carlo, Sacha; Cirauqui, Nuria; Dal Peraro, Matteo; van der Goot, F. Gisou; Zuber, Benoît

    2016-07-01

    Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

  12. 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. PMID:26619265

  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. Protein folding in the cytosol: chaperonin-dependent and -independent mechanisms.

    PubMed

    Netzer, W J; Hartl, F U

    1998-02-01

    Recent findings suggest that a combination of chaperonin-assisted and unassisted mechanisms operate in protein folding in the cytosol. While nascent chain-binding chaperones, such as Hsp70, could have a general role in maintaining the folding competence of translating polypeptide chains, the contribution of the cylindrical chaperonin complexes to overall folding is limited to a subset of aggregation-sensitive polypeptides. The majority of bacterial proteins are relatively small and they are synthesized rapidly and folded independently of the chaperonin GroEL in a posttranslational manner. Eukaryotes have a proportionally larger number of multi-domain proteins than bacteria. The individual domains of these proteins can be folded cotranslationally and sequentially. The use of this mechanism explains how large proteins fold independently of a chaperonin and could have been crucial in the evolution of a wide array of modular polypeptides in eukaryotes.

  15. Secretion and extracellular space travel of Wnt proteins.

    PubMed

    Gross, Julia Christina; Boutros, Michael

    2013-08-01

    Wnt signaling pathways control many processes during development, stem cell maintenance and homeostasis, and their aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer. Wnts are hydrophobic proteins, however, quite paradoxically, they can travel over distances to induce cell-type specific responses. While there has been an initial focus on elucidating the intracellular signaling cascade, discoveries in the past few years have shed light on a highly complex, and regulated secretory process that guides Wnt proteins through the exocytic pathway. Wnt proteins are at least in portion packaged onto extracellular carriers such as exosomes. Similar to dysregulation of components in the Wnt receiving cell, failure to regulate Wnt secretion has been linked to cancer. Here, we review recent discoveries on factors and processes implicated in Wnt secretion.

  16. Production and secretion of recombinant proteins in Dictyostelium discoideum.

    PubMed

    Dittrich, W; Williams, K L; Slade, M B

    1994-06-01

    We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system. The cellular slime mold Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface associated D. discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme beta-glucuronidase (GUS) from Escherichia coli was cell associated. Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium. The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection. PMID:7764951

  17. Selection for Genes Encoding Secreted Proteins and Receptors

    NASA Astrophysics Data System (ADS)

    Klein, Robert D.; Gu, Qimin; Goddard, Audrey; Rosenthal, Arnon

    1996-07-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

  18. Chain length scaling of protein folding time: Beta sheet structures

    NASA Astrophysics Data System (ADS)

    Dimitrievski, K.; Kasemo, B.; Zhdanov, V. P.

    2000-07-01

    We present comprehensive 3D lattice Monte Carlo simulations of the folding kinetics of two-turn antiparallel β sheets. The model employed takes into account isotropic nonspecific interactions as in previous flexible heteropolymer models and also orientation-dependent monomer-monomer interactions, mimicking the formation of hydrogen bonds and chain rigidity. The chain length is varied from N=15 to 33. For each chain length, we calculate the fastest folding temperature, Tfast, folding temperature, Tfold, and glass-transition temperature, Tg. The time-averaged occupation probability of the native state is found to be nearly independent of N at all temperatures. The dependence of Tfast and Tfold on N is accordingly relatively weak. The temperature interval where the folding is fast rapidly decreases with increasing N. For the chain lengths chosen, Tfold slightly exceeds Tg. The dependence of the folding time τf on N is well fitted by using the power law, τf∝Nλ. The exponent λ is found to depend on temperature and on the distribution of nonspecific interactions in the chain. In particular, λ=2.7-4.0 at T=Tfast and 5.2 at T slightly below Tfold. Evaluating τf in real units at T near Tfold yields physically reasonable results.

  19. Folding and Purification of Insoluble (Inclusion Body) Proteins from Escherichia coli.

    PubMed

    Wingfield, Paul T; Palmer, Ira; Liang, Shu-Mei

    2014-01-01

    Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed, (2) the cell wall and outer membrane components are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine·HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an "oxido-shuffling" buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein, which is then partially purified by gel filtration before folding; the protein is then folded and oxidized by simple dialysis against water. Thirdly, folding and purification of a fusion protein using metal-chelate affinity chromatography are described. PMID:25367010

  20. Smoothing a rugged protein folding landscape by sequence-based redesign

    PubMed Central

    Porebski, Benjamin T.; Keleher, Shani; Hollins, Jeffrey J.; Nickson, Adrian A.; Marijanovic, Emilia M.; Borg, Natalie A.; Costa, Mauricio G. S.; Pearce, Mary A.; Dai, Weiwen; Zhu, Liguang; Irving, James A.; Hoke, David E.; Kass, Itamar; Whisstock, James C.; Bottomley, Stephen P.; Webb, Geoffrey I.; McGowan, Sheena; Buckle, Ashley M.

    2016-01-01

    The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics. PMID:27667094

  1. Smoothing a rugged protein folding landscape by sequence-based redesign

    NASA Astrophysics Data System (ADS)

    Porebski, Benjamin T.; Keleher, Shani; Hollins, Jeffrey J.; Nickson, Adrian A.; Marijanovic, Emilia M.; Borg, Natalie A.; Costa, Mauricio G. S.; Pearce, Mary A.; Dai, Weiwen; Zhu, Liguang; Irving, James A.; Hoke, David E.; Kass, Itamar; Whisstock, James C.; Bottomley, Stephen P.; Webb, Geoffrey I.; McGowan, Sheena; Buckle, Ashley M.

    2016-09-01

    The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.

  2. What protein folding teaches us about biological function and molecular machines.

    PubMed

    Whitford, Paul C; Onuchic, José N

    2015-02-01

    Protein folding was the first area of molecular biology for which a systematic statistical-mechanical analysis of dynamics was developed. As a result, folding is described as a process by which a disordered protein chain diffuses across a high-dimensional energy landscape and finally reaches the folded ensemble. Folding studies have produced countless theoretical concepts that are generalizable to other biomolecular processes, such as the functional dynamics of molecular assemblies. Common themes in folding and function include the dominant role of excluded volume, that a balance between energetic roughness and geometrical effects guides dynamics, and that folding/functional landscapes are relatively smooth. Here, we discuss how insights into protein folding have been applied to investigate the functional dynamics of biomolecular assemblies.

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

    PubMed

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

    2014-04-18

    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.

  4. The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65.

    PubMed

    Kucknoor, Ashwini S; Mundodi, Vasanthakrishna; Alderete, John F

    2007-11-01

    We showed recently that contact of human vaginal epithelial cells (VECs) by Trichomonas vaginalis and incubation with trichomonad proteins in conditioned medium induced expression of VEC genes. We performed 2-D SDS-PAGE followed by MALDI-TOF to identify the major secreted proteins. Based on protein abundance and separation of spots in 2-D gels, 32 major secreted proteins were examined, which gave 19 proteins with accession numbers. These proteins included known secreted cysteine proteinases. In addition, other secreted proteins were enzymes of carbohydrate metabolism, adhesin protein AP65, heat shock proteins, thioredoxin reductase and coronins. We confirmed that the secreted trichomonad proteins induced expression of VEC genes, including interleukin 8 (IL-8), COX-2 and fibronectin. Purified AP65 added to VECs had a pronounced effect only on IL-8 gene expression, which was inhibited in the presence of 12G4 monoclonal antibody to AP65. Moreover, AP65 expressed episomally within epithelial cells was found to enhance the expression of IL-8 and COX-2. This may be the first report of analysis of the secreted proteins of T. vaginalis and of the host epithelial cell response to these proteins and to the prominent adhesin AP65. PMID:17590165

  5. The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65

    PubMed Central

    Kucknoor, Ashwini S.; Mundodi, Vasanthakrishna; Alderete, John F.

    2007-01-01

    Summary We showed recently that contact of human vaginal epithelial cells (VECs) by Trichomonas vaginalis and incubation with trichomonad proteins in conditioned medium induced expression of VEC genes. We performed 2-D SDS-PAGE followed by MALDI-TOF to identify the major secreted proteins. Based on protein abundance and separation of spots in 2-D gels, 32 major secreted proteins were examined, which gave 19 proteins with accession numbers. These proteins included known secreted cysteine proteinases. In addition, other secreted proteins were enzymes of carbohydrate metabolism, adhesin protein AP65, heat shock proteins, thioredoxin reductase and coronins. We confirmed that the secreted trichomonad proteins induced expression of VEC genes, including interleukin 8 (IL-8), COX-2 and fibronectin. Purified AP65 added to VECs had a pronounced effect only on IL-8 gene expression, which was inhibited in the presence of 12G4 monoclonal antibody to AP65. Moreover, AP65 expressed episomally within epithelial cells was found to enhance the expression of IL-8 and COX-2. This may be the first report of analysis of the secreted proteins of T. vaginalis and of the host epithelial cell response to these proteins and to the prominent adhesin AP65. PMID:17590165

  6. Cadmium impairs protein folding in the endoplasmic reticulum and induces the unfolded protein response.

    PubMed

    Le, Quynh Giang; Ishiwata-Kimata, Yuki; Kohno, Kenji; Kimata, Yukio

    2016-08-01

    Cellular exposure to cadmium is known to strongly induce the unfolded protein response (UPR), which suggests that the endoplasmic reticulum (ER) is preferentially damaged by cadmium. According to recent reports, the UPR is induced both dependent on and independently of accumulation of unfolded proteins in the ER. In order to understand the toxic mechanism of cadmium, here we investigated how cadmium exposure leads to Ire1 activation, which triggers the UPR, using yeast Saccharomyces cerevisiae as a model organism. Cadmium poorly induced the UPR when Ire1 carried a mutation that impairs its ability to recognize unfolded proteins. Ire1 activation by cadmium was also attenuated by the chemical chaperone 4-phenylbutyrate. Cadmium caused sedimentation of BiP, the molecular chaperone in the ER, which suggests the ER accumulation of unfolded proteins. A green fluorescent protein-based reporter assay also indicated that cadmium damages the oxidative protein folding in the ER. We also found that an excess concentration of extracellular calcium attenuates the Ire1 activation by cadmium. Taken together, we propose that cadmium exposure leads to the UPR induction through impairment of protein folding in the ER. PMID:27298227

  7. The earliest events in protein folding: Helix dynamics in proteins and model peptides

    SciTech Connect

    Dyer, R.B.; Williams, S.; Woodruff, W.H.

    1996-12-31

    The earliest events in protein folding are critically important in determining the folding pathway, but have proved difficult to study by conventional approaches. We have developed new rapid initiation methods and structure-specific probes to interrogate the earliest events of protein folding. Our focus is the pathways. Folding or unfolding reactions are initiated on a fast timescale (10 ns) using a laser induced temperature jump (15 C) and probed with time-resolved infrared spectroscopy. We obtained the kinetics of the helix-coil transition for a model 21-residue peptide. The observed rate constant k{sub obs} = k{sub f} + k{sub u} for reversible kinetics; from the observed rate (6 x 10{sup 6} s{sup -1}) and the equilibrium constant favoring folding of 7.5 at 27 C, we calculate a folding lifetime of 180 ns and an unfolding lifetime of 1.4 {mu}s. The {open_quotes}molten globule{close_quotes} form of apomyoglobin (horse, pH*3, 0.15M NaCl) shows similar kinetics for helix that is unconstrained by tertiary structure (helix with an unusually low Amide I frequency, near 1633 cm{sup -1}). In {open_quotes}native{close_quotes} apomyoglobin (horse, pH*5.3, 10 mM NaCl) two very different rates (45 ns and 70 {mu}s) are observed and we infer that a third occurs on a timescales inaccessible to our experiment (> 1 ms). We suggest that the slower processes are due to helix formation that is rate-limited by the formation of tertiary structure.

  8. Anticytolytic screen identifies inhibitors of mycobacterial virulence protein secretion.

    PubMed

    Rybniker, Jan; Chen, Jeffrey M; Sala, Claudia; Hartkoorn, Ruben C; Vocat, Anthony; Benjak, Andrej; Boy-Röttger, Stefanie; Zhang, Ming; Székely, Rita; Greff, Zoltán; Orfi, László; Szabadkai, István; Pató, János; Kéri, György; Cole, Stewart T

    2014-10-01

    Mycobacterium tuberculosis (Mtb) requires protein secretion systems like ESX-1 for intracellular survival and virulence. The major virulence determinant and ESX-1 substrate, EsxA, arrests phagosome maturation and lyses cell membranes, resulting in tissue damage and necrosis that promotes pathogen spread. To identify inhibitors of Mtb protein secretion, we developed a fibroblast survival assay exploiting this phenotype and selected molecules that protect host cells from Mtb-induced lysis without being bactericidal in vitro. Hit compounds blocked EsxA secretion and promoted phagosome maturation in macrophages, thus reducing bacterial loads. Target identification studies led to the discovery of BTP15, a benzothiophene inhibitor of the histidine kinase MprB that indirectly regulates ESX-1, and BBH7, a benzyloxybenzylidene-hydrazine compound. BBH7 affects Mtb metal-ion homeostasis and revealed zinc stress as an activating signal for EsxA secretion. This screening approach extends the target spectrum of small molecule libraries and will help tackle the mounting problem of antibiotic-resistant mycobacteria.

  9. Folding rates and low-entropy-loss routes of two-state proteins.

    PubMed

    Weikl, Thomas R; Dill, Ken A

    2003-06-01

    We develop a simple model for computing the rates and routes of folding of two-state proteins from the contact maps of their native structures. The model is based on the graph-theoretical concept of effective contact order (ECO). The model predicts that proteins fold by "zipping up" in a sequence of small-loop-closure events, depending on the native chain fold. Using a simple equation, with a few physical rate parameters, we obtain a good correlation with the folding rates of 24 two-state folding proteins. The model rationalizes data from Phi-value analysis that have been interpreted in terms of delocalized or polarized transition states. This model indicates how much of protein folding may take place in parallel, not along a single reaction coordinate or with a single transition state.

  10. Cooperative Protein Folding by Two Protein Thiol Disulfide Oxidoreductases and ERO1 in Soybean1[OPEN

    PubMed Central

    Okuda, Aya; Masuda, Taro; Koishihara, Katsunori; Mita, Ryuta; Iwasaki, Kensuke; Hara, Kumiko; Naruo, Yurika; Hirose, Akiho; Tsuchi, Yuichiro

    2016-01-01

    Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (Ero1s) supply oxidizing equivalent to the active centers of PDI. In this study, we expressed recombinant soybean Ero1 (GmERO1a) and found that GmERO1a oxidized multiple soybean ER oxidoreductases, in contrast to mammalian Ero1s having a high specificity for PDI. One of these ER oxidoreductases, GmPDIM, associated in vivo and in vitro with GmPDIL-2, was unable to be oxidized by GmERO1a. We therefore pursued the possible cooperative oxidative folding by GmPDIM, GmERO1a, and GmPDIL-2 in vitro and found that GmPDIL-2 synergistically accelerated oxidative refolding. In this process, GmERO1a preferentially oxidized the active center in the a′ domain among the a, a′, and b domains of GmPDIM. A disulfide bond introduced into the active center of the a′ domain of GmPDIM was shown to be transferred to the active center of the a domain of GmPDIM and the a domain of GmPDIM directly oxidized the active centers of both the a or a′ domain of GmPDIL-2. Therefore, we propose that the relay of an oxidizing equivalent from one ER oxidoreductase to another may play an essential role in cooperative oxidative folding by multiple ER oxidoreductases in plants. PMID:26645455

  11. Secreted proteins of Avibacterium paragallinarum are lethal for chicken embryo.

    PubMed

    Pérez-Márquez, Víctor; Pérez-Méndez, Alma; Ibarra-Caballero, Jorge; Gómez-Lugo, Gabriela; Vázquez-Cruz, Candelario; Vaca, Sergio; Negrete-Abascal, Erasmo

    2008-12-01

    Avibacterium paragallinarum causes infectious coryza in chickens. This bacterium secretes proteins of 110 kDa (a putative RTX protein) and 120 kDa. Expression of these proteins increases by the addition of CaCl(2), MgSO(4), MnSO(4), or ferric ammonium citrate and diminishes with CuSO(4) or ZnCl(2). Protein expression is optimal at 37 degrees C and pH 7.5. Mortality (90-100%) of chicken embryos was observed when secreted proteins (SPs) from A. paragallinarum reference or field isolates (serogroup A or C) were inoculated via yolk sac and was not observed when SPs from A. avium, a chicken respiratory tract indigenous bacterium, were inoculated. A. paragallinarum SPs could contain toxins responsible for the embryo deaths. Indeed, presence of the putative RTX protein of 110 kDa was confirmed by Western blotting with antibodies against the Actinobacillus pleuropneumoniae RTX ApxI, a closely related RTX protein.

  12. Using hydroxyl radical footprinting to explore the free energy landscape of protein folding

    PubMed Central

    Calabrese, Antonio N.; Ault, James R.; Radford, Sheena E.; Ashcroft, Alison E.

    2015-01-01

    Characterisation of the conformational states adopted during protein folding, including globally unfolded/disordered structures and partially folded intermediate species, is vital to gain fundamental insights into how a protein folds. In this work we employ fast photochemical oxidation of proteins (FPOP) to map the structural changes that occur in the folding of the four-helical bacterial immunity protein, Im7. Oxidative footprinting coupled with mass spectrometry (MS) is used to probe changes in the solvent accessibility of amino acid side-chains concurrent with the folding process, by quantifying the degree of oxidation experienced by the wild-type protein relative to a kinetically trapped, three-helical folding intermediate and an unfolded variant that lacks secondary structure. Analysis of the unfolded variant by FPOP–MS shows oxidative modifications consistent with the species adopting a solution conformation with a high degree of solvent accessibility. The folding intermediate, by contrast, experiences increased levels of oxidation relative to the wild-type, native protein only in regions destabilised by the amino acid substitutions introduced. The results demonstrate the utility of FPOP–MS to characterise protein variants in different conformational states and to provide insights into protein folding mechanisms that are complementary to measurements such as hydrogen/deuterium exchange labelling and Φ-value analysis. PMID:25746386

  13. Identification of Anaplasma marginale Type IV Secretion System Effector Proteins

    PubMed Central

    Brayton, Kelly A.; Beare, Paul A.; Brown, Wendy C.; Heinzen, Robert A.; Broschat, Shira L.

    2011-01-01

    Background Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now. Results By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system. Conclusions The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work. PMID:22140462

  14. Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

    SciTech Connect

    Sudhakaran, P.R.; Stamatoglou, S.C.; Hughes, R.C.

    1986-12-01

    Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and section of ..cap alpha..-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as ..cap alpha..-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.

  15. A growing toolbox of techniques for studying β-barrel outer membrane protein folding and biogenesis

    PubMed Central

    Horne, Jim E.; Radford, Sheena E.

    2016-01-01

    Great strides into understanding protein folding have been made since the seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of β-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed. PMID:27284045

  16. Metrnl: a secreted protein with new emerging functions

    PubMed Central

    Zheng, Si-li; Li, Zhi-yong; Song, Jie; Liu, Jian-min; Miao, Chao-yu

    2016-01-01

    Secreted proteins play critical roles in physiological and pathological processes and can be used as biomarkers and therapies for aging and disease. Metrnl is a novel secreted protein homologous to the neurotrophin Metrn. But this protein, unlike Metrn that is mainly expressed in the brain, shows a relatively wider distribution in the body with high levels of expression in white adipose tissue and barrier tissues. This protein plays important roles in neural development, white adipose browning and insulin sensitization. Based on its expression and distinct functions, this protein is also called Cometin, Subfatin and Interleukin 39, which refer to its neurotrophic effect, adipokine function and the possible action as a cytokine, respectively. The spectrum of Metrnl functions remains to be determined, and the mechanisms of Metrnl action need to be elucidated. In this review, we focus on the discovery, structural characteristics, expression pattern and physiological functions of Metrnl, which will assist in developing this protein as a new therapeutic target or agent. PMID:27063217

  17. Pollen tube growth and guidance: roles of small, secreted proteins

    PubMed Central

    Chae, Keun; Lord, Elizabeth M.

    2011-01-01

    Background Pollination is a crucial step in angiosperm (flowering plant) reproduction. Highly orchestrated pollen–pistil interactions and signalling events enable plant species to avoid inbreeding and outcrossing as a species-specific barrier. In compatible pollination, pollen tubes carrying two sperm cells grow through the pistil transmitting tract and are precisely guided to the ovules, discharging the sperm cells to the embryo sac for fertilization. Scope In Lilium longiflorum pollination, growing pollen tubes utilize two critical mechanisms, adhesion and chemotropism, for directional growth to the ovules. Among several molecular factors discovered in the past decade, two small, secreted cysteine-rich proteins have been shown to play major roles in pollen tube adhesion and reorientation bioassays: stigma/style cysteine-rich adhesin (SCA, approx. 9·3 kDa) and chemocyanin (approx. 9·8 kDa). SCA, a lipid transfer protein (LTP) secreted from the stylar transmitting tract epidermis, functions in lily pollen tube tip growth as well as in forming the adhesive pectin matrix at the growing pollen tube wall back from the tip. Lily chemocyanin is a plantacyanin family member and acts as a directional cue for reorienting pollen tubes. Recent consecutive studies revealed that Arabidopsis thaliana homologues for SCA and chemocyanin play pivotal roles in tip polarity and directionality of pollen tube growth, respectively. This review outlines the biological roles of various secreted proteins in angiosperm pollination, focusing on plant LTPs and chemocyanin. PMID:21307038

  18. Role of proteins in insulin secretion and glycemic control.

    PubMed

    Ranawana, Viren; Kaur, Bhupinder

    2013-01-01

    Dietary proteins are essential for the life of all animals and humans at all stages of the life cycle. They serve many structural and biochemical functions and have significant effects on health and wellbeing. Dietary protein consumption has shown an upward trend in developed countries in the past two decades primarily due to greater supply and affordability. Consumption is also on the rise in developing countries as affluence is increasing. Research shows that proteins have a notable impact on glucose homeostasis mechanisms, predominantly through their effects on insulin, incretins, gluconeogenesis, and gastric emptying. Since higher protein consumption and impaired glucose tolerance can be commonly seen in the same population demographics, a thorough understanding of the former's role in glucose homeostasis is crucial both toward the prevention and management of the latter. This chapter reviews the current state of the art on proteins, amino acids, and their effects on blood glucose and insulin secretion. PMID:23722093

  19. Nature’s favorite building block: Deciphering folding and capsid assembly of proteins with the HK97-fold

    PubMed Central

    Suhanovsky, Margaret M.; Teschke, Carolyn M.

    2015-01-01

    Summary For many (if not all) bacterial and archaeal tailed viruses and eukaryotic Herpesvirdae the HK97-fold serves as the major architectural element in icosahedral capsid formation while still enabling the conformational flexibility required during assembly and maturation. Auxiliary proteins or Δ-domains strictly control assembly of multiple, identical, HK97-like subunits into procapsids with specific icosahedral symmetries, rather than aberrant non-icosahedral structures. Procapsids are precursor structures that mature into capsids in a process involving release of auxiliary proteins (or cleavage of Δ-domains), dsDNA packaging, and conformational rearrangement of the HK97-like subunits. Some coat proteins built on the ubiquitous HK97-fold also have accessory domains or loops that impart specific functions, such as increased monomer, procapsid, or capsid stability. In this review, we analyze the numerous HK97-like coat protein structures that are emerging in the literature (over 40 at time of writing) by comparing their topology, additional domains, and their assembly and misassembly reactions. PMID:25864106

  20. Chemical, physical, and theoretical kinetics of an ultrafast folding protein

    PubMed Central

    Kubelka, Jan; Henry, Eric R.; Cellmer, Troy; Hofrichter, James; Eaton, William A.

    2008-01-01

    An extensive set of equilibrium and kinetic data is presented and analyzed for an ultrafast folding protein—the villin subdomain. The equilibrium data consist of the excess heat capacity, tryptophan fluorescence quantum yield, and natural circular-dichroism spectrum as a function of temperature, and the kinetic data consist of time courses of the quantum yield from nanosecond-laser temperature-jump experiments. The data are well fit with three kinds of models—a three-state chemical-kinetics model, a physical-kinetics model, and an Ising-like theoretical model that considers 105 possible conformations (microstates). In both the physical-kinetics and theoretical models, folding is described as diffusion on a one-dimensional free-energy surface. In the physical-kinetics model the reaction coordinate is unspecified, whereas in the theoretical model, order parameters, either the fraction of native contacts or the number of native residues, are used as reaction coordinates. The validity of these two reaction coordinates is demonstrated from calculation of the splitting probability from the rate matrix of the master equation for all 105 microstates. The analysis of the data on site-directed mutants using the chemical-kinetics model provides information on the structure of the transition-state ensemble; the physical-kinetics model allows an estimate of the height of the free-energy barrier separating the folded and unfolded states; and the theoretical model provides a detailed picture of the free-energy surface and a residue-by-residue description of the evolution of the folded structure, yet contains many fewer adjustable parameters than either the chemical- or physical-kinetics models. PMID:19033473

  1. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    NASA Astrophysics Data System (ADS)

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors and , and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein-protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.

  2. Fold of the conserved DTC domain in deltex proteins

    SciTech Connect

    Obiero, Josiah; Walker, John R.; Dhe-Paganon, Sirano

    2012-04-30

    Human Deltex 3-like (DTX3L) is a member of the Deltex family of proteins. Initially identified as a B-lymphoma and BAL-associated protein, DTX3L is an E3 ligase that regulates subcellular localization of its partner protein, BAL, by a dynamic nucleocytoplasmic trafficking mechanism. Unlike other members of the Deltex family of proteins, DTX3L lacks the highly basic N-terminal motif and the central proline-rich motif present in other Deltex proteins, and instead contains other unique N-terminal domains. The C-terminal domains are, however, homologous with other members of the Deltex family of proteins; these include a RING domain and a previously unidentified C-terminal domain. In this study, we report the high-resolution crystal structure of this previously uncharacterized C-terminal domain of human DTX3L, which we term the Deltex C-terminal domain.

  3. Legionella pneumophila secretes a mitochondrial carrier protein during infection.

    PubMed

    Dolezal, Pavel; Aili, Margareta; Tong, Janette; Jiang, Jhih-Hang; Marobbio, Carlo M T; Marobbio, Carlo M; Lee, Sau Fung; Schuelein, Ralf; Belluzzo, Simon; Binova, Eva; Mousnier, Aurelie; Frankel, Gad; Giannuzzi, Giulia; Palmieri, Ferdinando; Gabriel, Kipros; Naderer, Thomas; Hartland, Elizabeth L; Lithgow, Trevor

    2012-01-01

    The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

  4. An antifreeze protein folds with an interior network of more than 400 semi-clathrate waters.

    PubMed

    Sun, Tianjun; Lin, Feng-Hsu; Campbell, Robert L; Allingham, John S; Davies, Peter L

    2014-02-14

    When polypeptide chains fold into a protein, hydrophobic groups are compacted in the center with exclusion of water. We report the crystal structure of an alanine-rich antifreeze protein that retains ~400 waters in its core. The putative ice-binding residues of this dimeric, four-helix bundle protein point inwards and coordinate the interior waters into two intersecting polypentagonal networks. The bundle makes minimal protein contacts between helices, but is stabilized by anchoring to the semi-clathrate water monolayers through backbone carbonyl groups in the protein interior. The ordered waters extend outwards to the protein surface and likely are involved in ice binding. This protein fold supports both the anchored-clathrate water mechanism of antifreeze protein adsorption to ice and the water-expulsion mechanism of protein folding.

  5. The Skp chaperone helps fold soluble proteins in vitro by inhibiting aggregation*

    PubMed Central

    Entzminger, Kevin C.; Chang, Christine; Myhre, Ryan O.; McCallum, Katie C.; Maynard, Jennifer A.

    2013-01-01

    The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp co-expression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression, and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp co-expression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence. PMID:22650963

  6. Translocation boost protein-folding efficiency of double-barreled chaperonins.

    PubMed

    Coluzza, Ivan; van der Vies, Saskia M; Frenkel, Daan

    2006-05-15

    Incorrect folding of proteins in living cells may lead to malfunctioning of the cell machinery. To prevent such cellular disasters from happening, all cells contain molecular chaperones that assist nonnative proteins in folding into the correct native structure. One of the most studied chaperone complexes is the GroEL-GroES complex. The GroEL part has a "double-barrel" structure, which consists of two cylindrical chambers joined at the bottom in a symmetrical fashion. The hydrophobic rim of one of the GroEL chambers captures nonnative proteins. The GroES part acts as a lid that temporarily closes the filled chamber during the folding process. Several capture-folding-release cycles are required before the nonnative protein reaches its native state. Here we report molecular simulations that suggest that translocation of the nonnative protein through the equatorial plane of the complex boosts the efficiency of the chaperonin action. If the target protein is correctly folded after translocation, it is released. However, if it is still nonnative, it is likely to remain trapped in the second chamber, which then closes to start a reverse translocation process. This shuttling back and forth continues until the protein is correctly folded. Our model provides a natural explanation for the prevalence of double-barreled chaperonins. Moreover, we argue that internal folding is both more efficient and safer than a scenario where partially refolded proteins escape from the complex before being recaptured.

  7. From local structure to a global framework: recognition of protein folds

    PubMed Central

    Joseph, Agnel Praveen; de Brevern, Alexandre G.

    2014-01-01

    Protein folding has been a major area of research for many years. Nonetheless, the mechanisms leading to the formation of an active biological fold are still not fully apprehended. The huge amount of available sequence and structural information provides hints to identify the putative fold for a given sequence. Indeed, protein structures prefer a limited number of local backbone conformations, some being characterized by preferences for certain amino acids. These preferences largely depend on the local structural environment. The prediction of local backbone conformations has become an important factor to correctly identifying the global protein fold. Here, we review the developments in the field of local structure prediction and especially their implication in protein fold recognition. PMID:24740960

  8. Peroxin Puzzles and Folded Freight: Peroxisomal Protein Import in Review

    NASA Astrophysics Data System (ADS)

    Crookes, Wendy J.; Olsen, Laura J.

    Peroxisomes are organelles that perform a variety of functions, including the metabolism of hydrogen peroxide and the oxidation of fatty acids. Peroxisomes do not possess organellar DNA; all peroxisomal matrix proteins are posttranslationally translocated into the organelle. The mechanism of peroxisomal protein translocation has been the subject of vigorous research in the past decade. Many of the proteins (peroxins, abbreviated Pex) that play critical roles in peroxisome biogenesis have been identified through functional complementation of yeast strains and of Chinese hamster ovary cell lines that are defective in peroxisome biogenesis. Researchers are now turning towards biochemical and genetic analyses of these peroxins to define their roles in peroxisome biogenesis and to discover interacting protein partners. Evidence suggests that some of the interacting partners include molecular chaperones. Several current models for peroxisomal protein import are presented.

  9. Hydrophobic effect in protein folding and other noncovalent processes involving proteins.

    PubMed Central

    Spolar, R S; Ha, J H; Record, M T

    1989-01-01

    Large negative standard heat capacity changes (delta CP degree much less than 0) are the hallmark of processes that remove nonpolar surface from water, including the transfer of nonpolar solutes from water to a nonaqueous phase and the folding, aggregation/association, and ligand-binding reactions of proteins [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. USA 74, 2236-2240]. More recently, Baldwin [Baldwin, R. L. (1986) Proc. Natl. Acad. Sci. USA 83, 8069-8072] proposed that the delta CP degree of protein folding could be used to quantify the contribution of the burial of nonpolar surface (the hydrophobic effect) to the stability of a globular protein. We demonstrate that identical correlations between the delta CP degree and the change in water-accessible nonpolar surface area (delta Anp) are obtained for both the transfer of nonpolar solutes from water to the pure liquid phase and the folding of small globular proteins: delta CP degree/delta Anp = -(0.28 +/- 0.05) (where delta Anp is expressed in A2 and delta CP degree is expressed in cal.mol-1.K-1; 1 cal = 4.184 J). The fact that these correlations are identical validates the proposals by both Sturtevant and Baldwin that the hydrophobic effect is in general the dominant contributor to delta CP degree and provides a straightforward means of estimating the contribution of the hydrophobic driving force (delta Ghyd degree) to the standard free energy change of a noncovalent process characterized by a large negative delta CP degree in the physiological temperature range: delta Ghyd degree congruent to (80 +/- 10)delta CP degree. PMID:2813394

  10. Influence of fluorination on the thermodynamics of protein folding.

    PubMed

    Buer, Benjamin C; Levin, Benjamin J; Marsh, E Neil G

    2012-08-01

    The introduction of highly fluorinated analogues of hydrophobic amino acid residues into proteins has proved an effective and general strategy for increasing protein stability toward both chemical denaturants and heat. However, the thermodynamic basis for this stabilizing effect, whether enthalpic or entropic in nature, has not been extensively investigated. Here we describe studies in which the values of ΔH°, ΔS°, and ΔCp° have been determined for the unfolding of a series of 12 small, de novo-designed proteins in which the hydrophobic core is packed with various combinations of fluorinated and non-fluorinated amino acid residues. The increase in the free energy of unfolding with increasing fluorine content is associated with increasingly unfavorable entropies of unfolding and correlates well with calculated changes in apolar solvent-accessible surface area. ΔCp° for unfolding is positive for all the proteins and, similarly, correlates with changes in apolar solvent-accessible surface area. ΔH° for unfolding shows no correlation with either fluorine content or changes in apolar solvent-accessible surface area. We conclude that conventional hydrophobic effects adequately explain the enhanced stabilities of most highly fluorinated proteins. The extremely high thermal stability of these proteins results, in part, from their very low per-residue ΔCp°, as has been observed for natural thermostable proteins.

  11. Machine Learning: How Much Does It Tell about Protein Folding Rates?

    PubMed Central

    Chen, Heng-Chang; Bogatyreva, Natalya S.; Filion, Guillaume J.; Ivankov, Dmitry N.

    2015-01-01

    The prediction of protein folding rates is a necessary step towards understanding the principles of protein folding. Due to the increasing amount of experimental data, numerous protein folding models and predictors of protein folding rates have been developed in the last decade. The problem has also attracted the attention of scientists from computational fields, which led to the publication of several machine learning-based models to predict the rate of protein folding. Some of them claim to predict the logarithm of protein folding rate with an accuracy greater than 90%. However, there are reasons to believe that such claims are exaggerated due to large fluctuations and overfitting of the estimates. When we confronted three selected published models with new data, we found a much lower predictive power than reported in the original publications. Overly optimistic predictive powers appear from violations of the basic principles of machine-learning. We highlight common misconceptions in the studies claiming excessive predictive power and propose to use learning curves as a safeguard against those mistakes. As an example, we show that the current amount of experimental data is insufficient to build a linear predictor of logarithms of folding rates based on protein amino acid composition. PMID:26606303

  12. Quantification of the physiochemical constraints on the export of spider silk proteins by Salmonella type III secretion

    PubMed Central

    2010-01-01

    Background The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in Salmonella Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein. Results To test how the timing and level of protein expression affects secretion, we designed a hybrid promoter that combines an IPTG-inducible system with a natural genetic circuit that controls effector expression in Salmonella (psicA). LacO operators are placed in various locations in the psicA promoter and the optimal induction occurs when a single operator is placed at the +5nt (234-fold) and a lower basal level of expression is achieved when a second operator is placed at -63nt to take advantage of DNA looping. Using this tool, we find that the secretion efficiency (protein secreted divided by total expressed) is constant as a function of total expressed. We also demonstrate that the secretion flux peaks at 8 hours. We then use whole gene DNA synthesis to construct codon optimized spider silk genes for full-length (3129 amino acids) Latrodectus hesperus dragline silk, Bombyx mori cocoon silk, and Nephila clavipes flagelliform silk and PCR is used to create eight truncations of these genes. These proteins are all unfolded polypeptides and they encompass a variety of length, charge, and amino acid compositions. We find those proteins fewer than 550 amino acids reliably secrete and the probability declines significantly after ~700 amino acids. There also is a charge optimum at -2.4, and secretion efficiency declines for very positively or negatively charged proteins. There is no significant correlation with hydrophobicity. Conclusions We show that

  13. Probing Protein Fluctuations, Folding and Misfolding at Single-molecule Resolution

    NASA Astrophysics Data System (ADS)

    Deniz, Ashok

    2010-03-01

    The conformational fluctuations and folding of proteins are key for their function in cells and organisms. Conversely, misfolding and aggregation can cause disease, although amyloids with functional significance are also being identified. To better understand these aspects of protein biophysics, we utilize single-molecule fluorescence and complementary methods to directly study complex protein dynamics, structural distributions, and conformational transitions. In one example, we used these methods to investigate disorder and disorder-to-order transitions in intrinsically disordered proteins (IDPs). IDPs are an interesting class of proteins which are relatively unstructured in isolation, but can often fold by interacting with binding partners. These complex systems are increasingly found to play major roles in biology and disease. In one case, we used a combination of single-molecule FRET (smFRET), coincidence and correlation analyses to probe the native structural features of a yeast protein Sup35, whose amyloid state is believed to be used in a beneficial context in yeast. We find that the monomeric protein populates a compact and rapidly fluctuating ensemble of conformations. In another case, we studied the binding-coupled folding of the IDP alpha-synuclein, whose misfolding and aggregation have been linked to Parkinson's disease. Single-molecule measurements directly revealed a complex multi-state folding landscape for this protein. Observations of a transient folding intermediate using microfluidic mixing, and links to misfolding and aggregation will also be discussed. Our results highlight single-molecule methodology that is broadly applicable to map protein folding and misfolding landscapes.

  14. Processing of cholinesterase-like α/β-hydrolase fold proteins: alterations associated with congenital disorders.

    PubMed

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

    2012-02-01

    The α/β hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/β hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/β-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/β-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/β-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/β-hydrolase fold.

  15. Aphids evolved novel secreted proteins for symbiosis with bacterial endosymbiont.

    PubMed

    Shigenobu, Shuji; Stern, David L

    2013-01-01

    Aphids evolved novel cells, called bacteriocytes, that differentiate specifically to harbour the obligatory mutualistic endosymbiotic bacteria Buchnera aphidicola. The genome of the host aphid Acyrthosiphon pisum contains many orphan genes that display no similarity with genes found in other sequenced organisms, prompting us to hypothesize that some of these orphan genes are related to lineage-specific traits, such as symbiosis. We conducted deep sequencing of bacteriocytes mRNA followed by whole mount in situ hybridizations of over-represented transcripts encoding aphid-specific orphan proteins. We identified a novel class of genes that encode small proteins with signal peptides, which are often cysteine-rich, that are over-represented in bacteriocytes. These genes are first expressed at a developmental time point coincident with the incorporation of symbionts strictly in the cells that contribute to the bacteriocyte and this bacteriocyte-specific expression is maintained throughout the aphid's life. The expression pattern suggests that recently evolved secretion proteins act within bacteriocytes, perhaps to mediate the symbiosis with beneficial bacterial partners, which is reminiscent of the evolution of novel cysteine-rich secreted proteins of leguminous plants that regulate nitrogen-fixing endosymbionts.

  16. Local rules for protein folding on a triangular lattice and generalized hydrophobicity in the HP model

    SciTech Connect

    Agarwala, R.; Batzoglou, S.; Dancik, V.

    1997-06-01

    We consider the problem of determining the three-dimensional folding of a protein given its one-dimensional amino acid sequence. We use the HP model for protein folding proposed by Dill, which models protein as a chain of amino acid residues that are either hydrophobic or polar, and hydrophobic interactions are the dominant initial driving force for the protein folding. Hart and Istrail gave approximation algorithms for folding proteins on the cubic lattice under HP model. In this paper, we examine the choice of a lattice by considering its algorithmic and geometric implications and argue that triangular lattice is a more reasonable choice. We present a set of folding rules for a triangular lattice and analyze the approximation ratio which they achieve. In addition, we introduce a generalization of the HP model to account for residues having different levels of hydrophobicity. After describing the biological foundation for this generalization, we show that in the new model we are able to achieve similar constant factor approximation guarantees on the triangular lattice as were achieved in the standard HP model. While the structures derived from our folding rules are probably still far from biological reality, we hope that having a set of folding rules with different properties will yield more interesting folds when combined.

  17. Strategies for folding of affinity tagged proteins using GroEL and osmolytes

    PubMed Central

    Katayama, Hiroo; McGill, Mitchell; Kearns, Andrew; Brzozowski, Marek; Degner, Nicholas; Harnett, Bliss; Kornilayev, Boris; Matkovic-Calogovic, Dubravka; Holyoak, Todd; Calvet, James P.; Gogol, Edward P.; Seed, John; Fisher, Mark T.

    2012-01-01

    Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide 1) if further stabilization of the folded product is required and 2) if the protein construct in question will ever be competent to fold with osmolytes. PMID:19082872

  18. Strategies for folding of affinity tagged proteins using GroEL and osmolytes.

    PubMed

    Katayama, Hiroo; McGill, Mitchell; Kearns, Andrew; Brzozowski, Marek; Degner, Nicholas; Harnett, Bliss; Kornilayev, Boris; Matković-Calogović, Dubravka; Holyoak, Todd; Calvet, James P; Gogol, Edward P; Seed, John; Fisher, Mark T

    2009-03-01

    Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes. PMID:19082872

  19. Type III secretion needle proteins induce cell signaling and cytokine secretion via Toll-like receptors.

    PubMed

    Jessen, Danielle L; Osei-Owusu, Patrick; Toosky, Melody; Roughead, William; Bradley, David S; Nilles, Matthew L

    2014-06-01

    Pathogens are recognized by hosts by use of various receptors, including the Toll-like receptor (TLR) and Nod-like receptor (NLR) families. Ligands for these varied receptors, including bacterial products, are identified by the immune system, resulting in development of innate immune responses. Only a couple of components from type III secretion (T3S) systems are known to be recognized by TLR or NLR family members. Known T3S components that are detected by pattern recognition receptors (PRRs) are (i) flagellin, detected by TLR5 and NLRC4 (Ipaf); and (ii) T3S rod proteins (PrgJ and homologs) and needle proteins (PrgI and homologs), detected by NAIP and the NLRC4 inflammasome. In this report, we characterize the induction of proinflammatory responses through TLRs by the Yersinia pestis T3S needle protein, YscF, the Salmonella enterica needle proteins PrgI and SsaG, and the Shigella needle protein, MxiH. More specifically, we determine that the proinflammatory responses occur through TLR2 and -4. These data support the hypothesis that T3S needles have an unrecognized role in bacterial pathogenesis by modulating immune responses. PMID:24643544

  20. Type III Secretion Needle Proteins Induce Cell Signaling and Cytokine Secretion via Toll-Like Receptors

    PubMed Central

    Jessen, Danielle L.; Osei-Owusu, Patrick; Toosky, Melody; Roughead, William; Bradley, David S.

    2014-01-01

    Pathogens are recognized by hosts by use of various receptors, including the Toll-like receptor (TLR) and Nod-like receptor (NLR) families. Ligands for these varied receptors, including bacterial products, are identified by the immune system, resulting in development of innate immune responses. Only a couple of components from type III secretion (T3S) systems are known to be recognized by TLR or NLR family members. Known T3S components that are detected by pattern recognition receptors (PRRs) are (i) flagellin, detected by TLR5 and NLRC4 (Ipaf); and (ii) T3S rod proteins (PrgJ and homologs) and needle proteins (PrgI and homologs), detected by NAIP and the NLRC4 inflammasome. In this report, we characterize the induction of proinflammatory responses through TLRs by the Yersinia pestis T3S needle protein, YscF, the Salmonella enterica needle proteins PrgI and SsaG, and the Shigella needle protein, MxiH. More specifically, we determine that the proinflammatory responses occur through TLR2 and -4. These data support the hypothesis that T3S needles have an unrecognized role in bacterial pathogenesis by modulating immune responses. PMID:24643544

  1. Picosecond to nanosecond dynamics provide a source of conformational entropy for protein folding.

    PubMed

    Stadler, Andreas M; Demmel, Franz; Ollivier, Jacques; Seydel, Tilo

    2016-08-01

    Myoglobin can be trapped in fully folded structures, partially folded molten globules, and unfolded states under stable equilibrium conditions. Here, we report an experimental study on the conformational dynamics of different folded conformational states of apo- and holomyoglobin in solution. Global protein diffusion and internal molecular motions were probed by neutron time-of-flight and neutron backscattering spectroscopy on the picosecond and nanosecond time scales. Global protein diffusion was found to depend on the α-helical content of the protein suggesting that charges on the macromolecule increase the short-time diffusion of protein. With regard to the molten globules, a gel-like phase due to protein entanglement and interactions with neighbouring macromolecules was visible due to a reduction of the global diffusion coefficients on the nanosecond time scale. Diffusion coefficients, residence and relaxation times of internal protein dynamics and root mean square displacements of localised internal motions were determined for the investigated structural states. The difference in conformational entropy ΔSconf of the protein between the unfolded and the partially or fully folded conformations was extracted from the measured root mean square displacements. Using thermodynamic parameters from the literature and the experimentally determined ΔSconf values we could identify the entropic contribution of the hydration shell ΔShydr of the different folded states. Our results point out the relevance of conformational entropy of the protein and the hydration shell for stability and folding of myoglobin. PMID:27425443

  2. Signatures of the Protein Folding Pathway in Two-Dimensional Ultraviolet Spectroscopy

    PubMed Central

    2015-01-01

    The function of protein relies on their folding to assume the proper structure. Probing the structural variations during the folding process is crucial for understanding the underlying mechanism. We present a combined quantum mechanics/molecular dynamics simulation study that demonstrates how coherent resonant nonlinear ultraviolet spectra can be used to follow the fast folding dynamics of a mini-protein, Trp-cage. Two dimensional ultraviolet signals of the backbone transitions carry rich information of both local (secondary) and global (tertiary) structures. The complexity of signals decreases as the conformational entropy decreases in the course of the folding process. We show that the approximate entropy of the signals provides a quantitative marker of protein folding status, accessible by both theoretical calculations and experiments. PMID:24803996

  3. BiP Clustering Facilitates Protein Folding in the Endoplasmic Reticulum

    PubMed Central

    Robinson, Anne S.; Petzold, Linda

    2014-01-01

    The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER): translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as ‘clusters’). Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling. PMID:24991821

  4. BiP clustering facilitates protein folding in the endoplasmic reticulum.

    PubMed

    Griesemer, Marc; Young, Carissa; Robinson, Anne S; Petzold, Linda

    2014-07-01

    The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER): translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as 'clusters'). Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling. PMID:24991821

  5. BiP clustering facilitates protein folding in the endoplasmic reticulum.

    PubMed

    Griesemer, Marc; Young, Carissa; Robinson, Anne S; Petzold, Linda

    2014-07-01

    The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER): translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as 'clusters'). Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.

  6. Absence of repellents in Ustilago maydis induces genes encoding small secreted proteins.

    PubMed

    Teertstra, Wieke R; Krijgsheld, Pauline; Wösten, Han A B

    2011-08-01

    The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. A SG200 strain in which the rep1 gene is inactivated (∆rep1 strain) is affected in aerial hyphae formation. We here assessed changes in global gene expression as a consequence of the inactivation of the rep1 gene. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of ≥2. Twenty-two of these genes were up-regulated and half of them encode small secreted proteins (SSPs) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode proteins that can be classified as secreted cysteine-rich proteins (SCRPs). Interestingly, most of the SCRPs are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that inactivation of rep1 hardly affects the expression profile of U. maydis, despite the fact that the mutant strain has a strong reduced ability to form aerial hyphae.

  7. Systematic characterization of protein folding pathways using diffusion maps: Application to Trp-cage miniprotein

    SciTech Connect

    Kim, Sang Beom; Dsilva, Carmeline J.; Debenedetti, Pablo G.; Kevrekidis, Ioannis G.

    2015-02-28

    Understanding the mechanisms by which proteins fold from disordered amino-acid chains to spatially ordered structures remains an area of active inquiry. Molecular simulations can provide atomistic details of the folding dynamics which complement experimental findings. Conventional order parameters, such as root-mean-square deviation and radius of gyration, provide structural information but fail to capture the underlying dynamics of the protein folding process. It is therefore advantageous to adopt a method that can systematically analyze simulation data to extract relevant structural as well as dynamical information. The nonlinear dimensionality reduction technique known as diffusion maps automatically embeds the high-dimensional folding trajectories in a lower-dimensional space from which one can more easily visualize folding pathways, assuming the data lie approximately on a lower-dimensional manifold. The eigenvectors that parametrize the low-dimensional space, furthermore, are determined systematically, rather than chosen heuristically, as is done with phenomenological order parameters. We demonstrate that diffusion maps can effectively characterize the folding process of a Trp-cage miniprotein. By embedding molecular dynamics simulation trajectories of Trp-cage folding in diffusion maps space, we identify two folding pathways and intermediate structures that are consistent with the previous studies, demonstrating that this technique can be employed as an effective way of analyzing and constructing protein folding pathways from molecular simulations.

  8. The Principle of Stationary Action in Biophysics: Stability in Protein Folding

    PubMed Central

    Weiner, Joel L.

    2013-01-01

    We conceptualize protein folding as motion in a large dimensional dihedral angle space. We use Lagrangian mechanics and introduce an unspecified Lagrangian to study the motion. The fact that we have reliable folding leads us to conjecture the totality of paths forms caustics that can be recognized by the vanishing of the second variation of the action. There are two types of folding processes: stable against modest perturbations and unstable. We also conjecture that natural selection has picked out stable folds. More importantly, the presence of caustics leads naturally to the application of ideas from catastrophe theory and allows us to consider the question of stability for the folding process from that perspective. Powerful stability theorems from mathematics are then applicable to impose more order on the totality of motions. This leads to an immediate explanation for both the insensitivity of folding to solution perturbations and the fact that folding occurs using very little free energy. The theory of folding, based on the above conjectures, can also be used to explain the behavior of energy landscapes, the speed of folding similar to transition state theory, and the fact that random proteins do not fold. PMID:24454360

  9. Mathematics, thermodynamics, and modeling to address ten common misconceptions about protein structure, folding, and stability.

    PubMed

    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 phenomena in undergraduate classes. In the process of learning about these topics, students often form incorrect ideas. For example, by learning about protein folding in the context of protein synthesis, students may come to an incorrect conclusion that once synthesized on the ribosome, a protein spends its entire cellular life time in its fully folded native confirmation. This is clearly not true; proteins are dynamic structures that undergo both local fluctuations and global unfolding events. To prevent and address such misconceptions, basic concepts of protein science can be introduced in the context of simple mathematical models and hands-on explorations of publicly available data sets. Ten common misconceptions about proteins are presented, along with suggestions for using equations, models, sequence, structure, and thermodynamic data to help students gain a deeper understanding of basic concepts relating to protein structure, folding, and stability.

  10. Hands-on Force Spectroscopy: Weird Springs and Protein Folding

    ERIC Educational Resources Information Center

    Euler, Manfred

    2008-01-01

    A force spectroscopy model experiment is presented using a low-cost tensile apparatus described earlier. Force-extension measurements of twisted rubber bands are obtained. They exhibit a complex nonlinear elastic behaviour that resembles atomic force spectroscopy investigations of molecules of titin, a muscle protein. The model experiments open up…

  11. Redox regulation of protein folding in the mitochondrial intermembrane space

    PubMed Central

    Koehler, Carla M.; Tienson, Heather L.

    2014-01-01

    Protein translocation pathways to the mitochondrial matrix and inner membrane have been well characterized. However, translocation into the intermembrane space, which was thought to be simply a modification of the traditional translocation pathways, is complex. The mechanism by which a subset of intermembrane space proteins, those with disulfide bonds, are translocated has been largely unknown until recently. Specifically, the intermembrane space proteins with disulfide bonds are imported via the mitochondrial intermembrane space assembly (MIA) pathway. Substrates are imported via a disulfide exchange relay with two components Mia40 and Erv1. This new breakthrough has resulted in novel concepts for assembly of proteins in the intermembrane space, suggesting that this compartment may be similar to that of the endoplasmic reticulum and the prokaryotic periplasm. As a better understanding of this pathway emerges, new paradigms for thiol-disulfide exchange mechanisms may be developed. Given that the intermembrane space is important for disease processes including apoptosis and neurodegeneration, new roles in regulation by oxidation–reduction chemistry seem likely to be relevant. PMID:18761382

  12. Max Delbruck Prize in Biological Physics Lecture: Single-molecule protein folding and transition paths

    NASA Astrophysics Data System (ADS)

    Eaton, William

    2012-02-01

    The transition path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs by crossing the free energy barrier between two states. It is a uniquely single-molecule property, and has not yet been observed experimentally for any system in the condensed phase. The importance of the transition path in protein folding is that it contains all of the mechanistic information on how a protein folds. As a major step toward observing transition paths, we have determined the average transition-path time for a fast and a slow-folding protein from a photon-by-photon analysis of fluorescence trajectories in single-molecule FRET experiments. While the folding rate coefficients differ by 10,000-fold, surprisingly, the transition-path times differ by less than 5-fold, showing that a successful barrier crossing event takes almost the same time for a fast- and a slow-folding protein, i.e. almost the same time to fold when it actually happens.

  13. Amino Acid Distribution Rules Predict Protein Fold: Protein Grammar for Beta-Strand Sandwich-Like Structures

    PubMed Central

    Kister, Alexander

    2015-01-01

    We present an alternative approach to protein 3D folding prediction based on determination of rules that specify distribution of “favorable” residues, that are mainly responsible for a given fold formation, and “unfavorable” residues, that are incompatible with that fold, in polypeptide sequences. The process of determining favorable and unfavorable residues is iterative. The starting assumptions are based on the general principles of protein structure formation as well as structural features peculiar to a protein fold under investigation. The initial assumptions are tested one-by-one for a set of all known proteins with a given structure. The assumption is accepted as a “rule of amino acid distribution” for the protein fold if it holds true for all, or near all, structures. If the assumption is not accepted as a rule, it can be modified to better fit the data and then tested again in the next step of the iterative search algorithm, or rejected. We determined the set of amino acid distribution rules for a large group of beta sandwich-like proteins characterized by a specific arrangement of strands in two beta sheets. It was shown that this set of rules is highly sensitive (~90%) and very specific (~99%) for identifying sequences of proteins with specified beta sandwich fold structure. The advantage of the proposed approach is that it does not require that query proteins have a high degree of homology to proteins with known structure. So long as the query protein satisfies residue distribution rules, it can be confidently assigned to its respective protein fold. Another advantage of our approach is that it allows for a better understanding of which residues play an essential role in protein fold formation. It may, therefore, facilitate rational protein engineering design. PMID:25625198

  14. A dynamic study of protein secretion and aggregation in the secretory pathway.

    PubMed

    Mossuto, Maria Francesca; Sannino, Sara; Mazza, Davide; Fagioli, Claudio; Vitale, Milena; Yoboue, Edgar Djaha; Sitia, Roberto; Anelli, Tiziana

    2014-01-01

    Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC. PMID:25279560

  15. A Dynamic Study of Protein Secretion and Aggregation in the Secretory Pathway

    PubMed Central

    Mossuto, Maria Francesca; Sannino, Sara; Mazza, Davide; Fagioli, Claudio; Vitale, Milena; Yoboue, Edgar Djaha; Anelli, Tiziana

    2014-01-01

    Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC. PMID:25279560

  16. A hybrid MD-kMC algorithm for folding proteins in explicit solvent.

    PubMed

    Peter, Emanuel Karl; Shea, Joan-Emma

    2014-04-14

    We present a novel hybrid MD-kMC algorithm that is capable of efficiently folding proteins in explicit solvent. We apply this algorithm to the folding of a small protein, Trp-Cage. Different kMC move sets that capture different possible rate limiting steps are implemented. The first uses secondary structure formation as a relevant rate event (a combination of dihedral rotations and hydrogen-bonding formation and breakage). The second uses tertiary structure formation events through formation of contacts via translational moves. Both methods fold the protein, but via different mechanisms and with different folding kinetics. The first method leads to folding via a structured helical state, with kinetics fit by a single exponential. The second method leads to folding via a collapsed loop, with kinetics poorly fit by single or double exponentials. In both cases, folding times are faster than experimentally reported values, The secondary and tertiary move sets are integrated in a third MD-kMC implementation, which now leads to folding of the protein via both pathways, with single and double-exponential fits to the rates, and to folding rates in good agreement with experimental values. The competition between secondary and tertiary structure leads to a longer search for the helix-rich intermediate in the case of the first pathway, and to the emergence of a kinetically trapped long-lived molten-globule collapsed state in the case of the second pathway. The algorithm presented not only captures experimentally observed folding intermediates and kinetics, but yields insights into the relative roles of local and global interactions in determining folding mechanisms and rates. PMID:24499973

  17. Predicting protein folding rate change upon point mutation using residue-level coevolutionary information.

    PubMed

    Mallik, Saurav; Das, Smita; Kundu, Sudip

    2016-01-01

    Change in folding kinetics of globular proteins upon point mutation is crucial to a wide spectrum of biological research, such as protein misfolding, toxicity, and aggregations. Here we seek to address whether residue-level coevolutionary information of globular proteins can be informative to folding rate changes upon point mutations. Generating residue-level coevolutionary networks of globular proteins, we analyze three parameters: relative coevolution order (rCEO), network density (ND), and characteristic path length (CPL). A point mutation is considered to be equivalent to a node deletion of this network and respective percentage changes in rCEO, ND, CPL are found linearly correlated (0.84, 0.73, and -0.61, respectively) with experimental folding rate changes. The three parameters predict the folding rate change upon a point mutation with 0.031, 0.045, and 0.059 standard errors, respectively.

  18. Molecular origins of internal friction effects on protein-folding rates.

    PubMed

    de Sancho, David; Sirur, Anshul; Best, Robert B

    2014-07-02

    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.

  19. A highly stable protein chimera built from fragments of different folds.

    PubMed

    Shanmugaratnam, Sooruban; Eisenbeis, Simone; Höcker, Birte

    2012-11-01

    Proteins increased in complexity during the course of evolution. Domains as well as subdomain-sized fragments were recruited and adapted to form new proteins and novel folds. This concept can be used in engineering to construct new proteins. We previously reported the combination of fragments from two ancient protein folds, a flavodoxin-like and a (βα)₈-barrel protein. Here we report two further attempts at engineering a chimeric protein from fragments of these folds. While one of the constructs showed a high tendency to aggregate, the other turned out to be a highly stable, well-structured protein. In terms of stability against heat and chemical denaturation this chimera, named NarLHisF, is superior to the earlier presented CheYHisF. This is the second instance of a chimera build from two different protein folds, which demonstrates how easily recombination can lead to the development and diversification of new proteins--a mechanism that most likely occurred frequently in the course of evolution. Based on the results of the failed and the successful chimera, we discuss important considerations for a general design strategy for fold chimeras.

  20. Efficient traversal of beta-sheet protein folding pathways using ensemble models.

    PubMed

    Shenker, Solomon; O'Donnell, Charles W; Devadas, Srinivas; Berger, Bonnie; Waldispühl, Jérôme

    2011-11-01

    Molecular dynamics (MD) simulations can now predict ms-timescale folding processes of small proteins; however, this presently requires hundreds of thousands of CPU hours and is primarily applicable to short peptides with few long-range interactions. Larger and slower-folding proteins, such as many with extended β-sheet structure, would require orders of magnitude more time and computing resources. Furthermore, when the objective is to determine only which folding events are necessary and limiting, atomistic detail MD simulations can prove unnecessary. Here, we introduce the program tFolder as an efficient method for modelling the folding process of large β-sheet proteins using sequence data alone. To do so, we extend existing ensemble β-sheet prediction techniques, which permitted only a fixed anti-parallel β-barrel shape, with a method that predicts arbitrary β-strand/β-strand orientations and strand-order permutations. By accounting for all partial and final structural states, we can then model the transition from random coil to native state as a Markov process, using a master equation to simulate population dynamics of folding over time. Thus, all putative folding pathways can be energetically scored, including which transitions present the greatest barriers. Since correct folding pathway prediction is likely determined by the accuracy of contact prediction, we demonstrate the accuracy of tFolder to be comparable with state-of-the-art methods designed specifically for the contact prediction problem alone. We validate our method for dynamics prediction by applying it to the folding pathway of the well-studied Protein G. With relatively very little computation time, tFolder is able to reveal critical features of the folding pathways which were only previously observed through time-consuming MD simulations and experimental studies. Such a result greatly expands the number of proteins whose folding pathways can be studied, while the algorithmic integration of

  1. Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level.

    PubMed

    Alemany, Anna; Rey-Serra, Blanca; Frutos, Silvia; Cecconi, Ciro; Ritort, Felix

    2016-01-01

    The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale. PMID:26745410

  2. Angiogenic activity of an Onchocerca volvulus Ancylostoma secreted protein homologue.

    PubMed

    Higazi, Tarig B; Pearlman, Eric; Whikehart, David R; Unnasch, Thomas R

    2003-06-01

    Angiogenesis is an important step in the development of ocular onchocercaisis. In previous studies, it has been demonstrated that Onchocerca volvulus homologues of the Ancylostoma secreted protein family have pronounced angiogenic activity. The overall goal of the current study was to determine if this angiogenic effect is exerted through a direct or indirect mechanism. These studies focused on one member of this family, OvASP-2, as this protein is expressed in microfilaria, the stage of the parasite that causes ocular onchocercaisis. Clones encoding truncated and full length open reading frames were expressed as fusion proteins with Escherichia coli maltose binding protein (MBP), and angiogenic activity was compared in vitro and in vivo with MBP alone. Truncated constructs expressing only the first 105 amino acids of OvASP-2 were as active as the full length protein in inducing new blood vessel formation. The full length fusion protein did not stimulate proliferation or production of vascular endothelial growth factor in vascular endothelial cells in vitro, indicating that OvASP-2 does not directly stimulate angiogenesis. Sequence analysis demonstrated that the gene encoding OvASP-2 contained five introns. Sequence comparisons of the genomic loci from West African blinding and non-blinding strains of O. volvulus revealed that some polymorphism existed among the various isolates tested. However, none of these polymorphisms could be used to differentiate the parasite strains, suggesting that qualitative variation in OvASP-2 could not explain the difference in ocular pathogenic potential of the two parasite strains.

  3. The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes.

    PubMed

    Nickel, Walter

    2003-05-01

    Most of the examples of protein translocation across a membrane (such as the import of classical secretory proteins into the endoplasmic reticulum, import of proteins into mitochondria and peroxisomes, as well as protein import into and export from the nucleus), are understood in great detail. In striking contrast, the phenomenon of unconventional protein secretion (also known as nonclassical protein export or ER/Golgi-independent protein secretion) from eukaryotic cells was discovered more than 10 years ago and yet the molecular mechanism and the molecular identity of machinery components that mediate this process remain elusive. This problem appears to be even more complex as several lines of evidence indicate that various kinds of mechanistically distinct nonclassical export routes may exist. In most cases these secretory mechanisms are gated in a tightly controlled fashion. This review aims to provide a comprehensive overview of our current knowledge as a basis for the development of new experimental strategies designed to unravel the molecular machineries mediating ER/Golgi-independent protein secretion. Beyond solving a fundamental problem in current cell biology, the molecular analysis of these processes is of major biomedical importance as these export routes are taken by proteins such as angiogenic growth factors, inflammatory cytokines, components of the extracellular matrix which regulate cell differentiation, proliferation and apoptosis, viral proteins, and parasite surface proteins potentially involved in host infection.

  4. Transferable Coarse-Grained Potential for De Novo Protein Folding and Design

    PubMed Central

    Coluzza, Ivan

    2014-01-01

    Protein folding and design are major biophysical problems, the solution of which would lead to important applications especially in medicine. Here we provide evidence of how a novel parametrization of the Caterpillar model may be used for both quantitative protein design and folding. With computer simulations it is shown that, for a large set of real protein structures, the model produces designed sequences with similar physical properties to the corresponding natural occurring sequences. The designed sequences require further experimental testing. For an independent set of proteins, previously used as benchmark, the correct folded structure of both the designed and the natural sequences is also demonstrated. The equilibrium folding properties are characterized by free energy calculations. The resulting free energy profiles not only are consistent among natural and designed proteins, but also show a remarkable precision when the folded structures are compared to the experimentally determined ones. Ultimately, the updated Caterpillar model is unique in the combination of its fundamental three features: its simplicity, its ability to produce natural foldable designed sequences, and its structure prediction precision. It is also remarkable that low frustration sequences can be obtained with such a simple and universal design procedure, and that the folding of natural proteins shows funnelled free energy landscapes without the need of any potentials based on the native structure. PMID:25436908

  5. Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors.

    PubMed

    Phillips, J J; Javadi, Y; Millership, C; Main, E R G

    2012-03-01

    Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.

  6. Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures

    NASA Astrophysics Data System (ADS)

    Lammert, Heiko; Noel, Jeffrey K.; Haglund, Ellinor; Schug, Alexander; Onuchic, José N.

    2015-12-01

    The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein's functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMR structures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism.

  7. Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin

    NASA Astrophysics Data System (ADS)

    Homouz, D.; Stagg, L.; Wittungstafshede, P.; Cheung, M.

    2009-01-01

    Protein dynamics in cells may be different from that in dilute solutions in vitro since the environment in cells is highly concentrated with other macromolecules. This volume exclusion due to macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, here we have investigated the folding energy landscape of an alpha/beta protein, apoflavodoxin, in the presence of inert macromolecular crowding agents using in silico and in vitro approaches. By coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fraction of crowding agents (phi_c) as well as of crowding agent geometry (sphere or spherocylinder) at high phi_c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we have identified in silico crowding conditions that best enhance protein stability and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. The test-tube experiments confirmed that apoflavodoxin's time-resolved folding path is modulated by crowding agent geometry. We propose that macromolecular crowding effects may be a tool for manipulation of protein folding and function in living cells.

  8. De Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints.

    PubMed

    Smock, Robert G; Yadid, Itamar; Dym, Orly; Clarke, Jane; Tawfik, Dan S

    2016-01-28

    Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of β-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence. PMID:26806127

  9. A new zinc binding fold underlines the versatility of zinc binding modules in protein evolution.

    PubMed

    Sharpe, Belinda K; Matthews, Jacqueline M; Kwan, Ann H Y; Newton, Anthea; Gell, David A; Crossley, Merlin; Mackay, Joel P

    2002-05-01

    Many different zinc binding modules have been identified. Their abundance and variety suggests that the formation of zinc binding folds might be relatively common. We have determined the structure of CH1(1), a 27-residue peptide derived from the first cysteine/histidine-rich region (CH1) of CREB binding protein (CBP). This peptide forms a highly ordered zinc-dependent fold that is distinct from known folds. The structure differs from a subsequently determined structure of a larger region from the CH3 region of CBP, and the CH1(1) fold probably represents a nonphysiologically active form. Despite this, the fold is thermostable and tolerant to both multiple alanine mutations and changes in the zinc-ligand spacing. Our data support the idea that zinc binding domains may arise frequently. Additionally, such structures may prove useful as scaffolds for protein design, given their stability and robustness.

  10. De Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints.

    PubMed

    Smock, Robert G; Yadid, Itamar; Dym, Orly; Clarke, Jane; Tawfik, Dan S

    2016-01-28

    Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of β-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence.

  11. De Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints

    PubMed Central

    Smock, Robert G.; Yadid, Itamar; Dym, Orly; Clarke, Jane; Tawfik, Dan S.

    2016-01-01

    Summary Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of β-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence. PMID:26806127

  12. Complex Pathways in Folding of Protein G Explored by Simulation and Experiment

    PubMed Central

    Lapidus, Lisa J.; Acharya, Srabasti; Schwantes, Christian R.; Wu, Ling; Shukla, Diwakar; King, Michael; DeCamp, Stephen J.; Pande, Vijay S.

    2014-01-01

    The B1 domain of protein G has been a classic model system of folding for decades, the subject of numerous experimental and computational studies. Most of the experimental work has focused on whether the protein folds via an intermediate, but the evidence is mostly limited to relatively slow kinetic observations with a few structural probes. In this work we observe folding on the submillisecond timescale with microfluidic mixers using a variety of probes including tryptophan fluorescence, circular dichroism, and photochemical oxidation. We find that each probe yields different kinetics and compare these observations with a Markov State Model constructed from large-scale molecular dynamics simulations and find a complex network of states that yield different kinetics for different observables. We conclude that there are many folding pathways before the final folding step and that these paths do not have large free energy barriers. PMID:25140430

  13. Acceleration of protein folding by four orders of magnitude through a single amino acid substitution

    PubMed Central

    Roderer, Daniel J. A.; Schärer, Martin A.; Rubini, Marina; Glockshuber, Rudi

    2015-01-01

    Cis prolyl peptide bonds are conserved structural elements in numerous protein families, although their formation is energetically unfavorable, intrinsically slow and often rate-limiting for folding. Here we investigate the reasons underlying the conservation of the cis proline that is diagnostic for the fold of thioredoxin-like thiol-disulfide oxidoreductases. We show that replacement of the conserved cis proline in thioredoxin by alanine can accelerate spontaneous folding to the native, thermodynamically most stable state by more than four orders of magnitude. However, the resulting trans alanine bond leads to small structural rearrangements around the active site that impair the function of thioredoxin as catalyst of electron transfer reactions by more than 100-fold. Our data provide evidence for the absence of a strong evolutionary pressure to achieve intrinsically fast folding rates, which is most likely a consequence of proline isomerases and molecular chaperones that guarantee high in vivo folding rates and yields. PMID:26121966

  14. Phosphatidylethanolamine binding protein 4 (PEBP4) is a secreted protein and has multiple functions.

    PubMed

    He, Huan; Liu, Dan; Lin, Hui; Jiang, Shanshan; Ying, Ying; Chun, Shao; Deng, Haiteng; Zaia, Joseph; Wen, Rong; Luo, Zhijun

    2016-07-01

    Phosphatidylethanolamine binding proteins (PEBP) represent a superfamily of proteins that are conserved from bacteria to humans. In mammals, four members have been identified, PEBP1-4. To determine the functional differences among PEBP1-4 and the underlying mechanism for their actions, we performed a sequence alignment and found that PEBP4 contains a signal peptide and potential glycosylation sites, whereas PEBP1-3 are intracellular proteins. To test if PEBP4 is secreted, we made constructs with Myc epitope at the amino (N) terminus or carboxyl (C) terminus to mask the signal sequence or keep it free, respectively. Our data revealed that both mouse and human PEBP4 were secreted when the epitope was tagged at their C-terminus. To our surprise, secretion was dependent upon the C-terminal conserved domain in addition to the N-terminal signal sequence. When the epitope was placed to the N-terminus, the recombinant protein failed to secrete and instead, was retained in the cytoplasm. Mass spectrometry detected asparagine (N)-glycosylation on the secreted PEBP4. Although overexpression of N-terminal tagged PEBP4 resulted in an inhibition of ERK activation by EGF, that with a C-terminal epitope tag did not have such an effect. Likewise, transfection of PEBP4 shRNA did not appear to affect ERK activation, suggesting that PEBP4 does not participate in the regulation of this pathway. In contrast, PEBP4 siRNA suppressed phosphorylation of Act at S473. Therefore, our results suggest that PEBP4 is a multifunctional protein and can be secreted. It will be important to investigate the mechanism by which PEBP4 is secreted and regulates cellular events.

  15. How Adequate are One- and Two-Dimensional Free Energy Landscapes for Protein Folding Dynamics?

    PubMed Central

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

    2009-01-01

    The molecular dynamics trajectories of protein folding or unfolding, generated with the coarse-grained united-residue force field for the B domain of staphylococcal protein A, were analyzed by principal component analysis (PCA). The folding or unfolding process was examined by using free-energy landscapes (FELs) in PC space. By introducing a novel multidimensional FEL, it was shown that the low-dimensional FELs are not always sufficient for the description of folding or unfolding processes. Similarities between the topographies of FELs along low- and high-indexed principal components were observed. PMID:19658975

  16. Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants

    PubMed Central

    Hueck, Christoph J.

    1998-01-01

    Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli

  17. Single-molecule observation of protein folding in symmetric GroEL-(GroES)2 complexes.

    PubMed

    Takei, Yodai; Iizuka, Ryo; Ueno, Taro; Funatsu, Takashi

    2012-11-30

    The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding. PMID:23048033

  18. Balance Between Folding and Degradation for Hsp90-Dependent Client Proteins: A Key Role for CHIP

    PubMed Central

    Kundrat, Lenka; Regan, Lynne

    2011-01-01

    Cells must regulate the synthesis and degradation of their proteins to maintain a balance that is appropriate for their specific growth conditions. Here we present the results of an investigation of the balance between protein folding and degradation for mammalian chaperone Hsp90-dependent client proteins. The central players are the molecular chaperones Hsp70 and Hsp90, the co-chaperone HOP, and ubiquitin ligase, CHIP. Hsp70 and Hsp90 bind to HOP thus forming a ternary folding complex whereas the binding of CHIP to the chaperones has previously been shown to lead to ubiquitination and ultimately to degradation of the client proteins as well as the chaperones. To understand the folding/degradation balance in more detail, we characterized the stoichiometries of the CHIP-Hsp70 and CHIP-Hsp90 complexes and measured the corresponding dissociation constants to be ~ 1 µM and ~ 4.5 µM respectively. We quantified the rate of ubiquitination of various substrates by CHIP in vitro. We further determined that the folding and degradation machineries cannot coexist in one complex. Lastly, we measured the in vivo concentrations of Hsp70, Hsp90, HOP, and CHIP under normal conditions and when client proteins are being degraded due to inhibition of the folding pathway. These in vivo measurements along with the in vitro data allowed us to calculate the approximate cellular concentrations of the folding and degradation complexes under both conditions and formulate a quantitative model for the balance between protein folding and degradation as well as an explanation for the shift to client protein degradation when the folding pathway is inhibited. PMID:20704274

  19. Integral and differential form of the protein folding problem

    NASA Astrophysics Data System (ADS)

    Tramontano, Anna

    2004-07-01

    The availability of the complete genomic sequences of many species, including human, has raised enormous expectations in medicine, pharmacology, ecology, biotechnology and forensic sciences. However, knowledge is only a first step toward understanding, and we are only at the early stage of a scientific process that might lead us to satisfy all the expectations raised by the genomic projects. In this review I will discuss the present status of computational methods that attempt to infer the unique three-dimensional structure of proteins from their amino acid sequences. Although this problem has been defined as the “holy grail” of biology, it represents only one of the many hurdles in our path towards the understanding of life at a molecular level.

  20. Btc22 chaperone is required for secretion and stability of the type III secreted protein Bsp22 in Bordetella bronchiseptica.

    PubMed

    Kurushima, Jun; Kuwae, Asaomi; Abe, Akio

    2012-06-01

    The type III secretion system (T3SS) is a sophisticated protein secretion machinery that delivers bacterial virulence proteins into host cells. A needle-tip protein, Bsp22 , is one of the secreted substrates of the T3SS and plays an essential role in the full function of the T3SS in Bordetella bronchiseptica. In this study, we found that BB1618 functions as a chaperone for Bsp22 . The deletion of BB1618 resulted in a dramatic impairment of Bsp22 secretion into the culture supernatants and Bsp22 stability in the bacterial cytosol. In contrast, the secretion of other type III secreted proteins was not affected by the BB1618 mutation. Furthermore, the BB1618 mutant strain could not induce cytotoxicity and displayed the same phenotypes as the Bsp22 mutant strain. An immunoprecipitation assay demonstrated that BB1618 interacts with Bsp22 , but not with BopB and BopD . Thus, we identified BB1618 as a specific type III chaperone for Bsp22 . Therefore, we propose that BB1618 be renamed Btc22 for the Bordetella type III chaperone for Bsp22 .

  1. Analyzing protein folding cooperativity by differential scanning calorimetry and NMR spectroscopy.

    PubMed

    Farber, Patrick; Darmawan, Hariyanto; Sprules, Tara; Mittermaier, Anthony

    2010-05-01

    Some marginally stable proteins undergo microsecond time scale folding reactions that involve significant populations of partly ordered forms, making it difficult to discern individual steps in their folding pathways. It has been suggested that many of these proteins fold non-cooperatively, with no significant barriers to separate the energy landscape into distinct thermodynamic states. Here we present an approach for studying the cooperativity of rapid protein folding with a combination of differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) relaxation dispersion experiments, and an analysis of the temperature dependence of amide (1)H and (15)N chemical shifts. We applied this method to the PBX homeodomain (PBX-HD), which folds on the microsecond time scale and produces a broad DSC thermogram with an elevated and steeply sloping native-state heat capacity baseline, making it a candidate for barrierless folding. However, by globally fitting the NMR thermal melt and DSC data, and by comparing these results to those obtained from the NMR relaxation dispersion experiments, we show that the native form of the protein undergoes two-state exchange with a small population of the thermally denatured form, well below the melting temperature. This result directly demonstrates the coexistence of distinct folded and unfolded forms and firmly establishes that folding of PBX-HD is cooperative. Further, we see evidence of large-scale structural and dynamical changes within the native state by NMR, which helps to explain the broad and shallow DSC profile. This study illustrates the potential of combining calorimetry with NMR dynamics experiments to dissect mechanisms of protein folding.

  2. Nuclear Magnetic Resonance Characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa.

    PubMed

    Chaudhury, Sukanya; Nordhues, Bryce A; Kaur, Kawaljit; Zhang, Na; De Guzman, Roberto N

    2015-11-01

    Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS), to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologues are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residue 16 to 41 and from residue 55 to 76. The helices of PcrG are partially formed, have similar backbone dynamics, and are flexible. NMR titrations show that the entire length of PcrG residues from position 9 to 76 is involved in binding to PcrV. PcrG adds to the growing list of partially folded or unstructured proteins with important roles in type III secretion.

  3. Microscopic theory of protein folding rates. I. Fine structure of the free energy profile and folding routes from a variational approach

    NASA Astrophysics Data System (ADS)

    Portman, John J.; Takada, Shoji; Wolynes, Peter G.

    2001-03-01

    A microscopic theory of the free energy barriers and folding routes for minimally frustrated proteins is presented, greatly expanding on the presentation of the variational approach outlined previously [J. J. Portman, S. Takada, and P. G. Wolynes, Phys. Rev. Lett. 81, 5237 (1998)]. We choose the λ-repressor protein as an illustrative example and focus on how the polymer chain statistics influence free energy profiles and partially ordered ensembles of structures. In particular, we investigate the role of chain stiffness on the free energy profile and folding routes. We evaluate the applicability of simpler approximations in which the conformations of the protein molecule along the folding route are restricted to have residues that are either entirely folded or unfolded in contiguous stretches. We find that the folding routes obtained from only one contiguous folded region corresponds to a chain with a much greater persistence length than appropriate for natural protein chains, while the folding route obtained from two contiguous folded regions is able to capture the relatively folded regions calculated within the variational approach. The free energy profiles obtained from the contiguous sequence approximations have larger barriers than the more microscopic variational theory which is understood as a consequence of partial ordering.

  4. Are there folding pathways in the functional stages of intrinsically disordered proteins?

    NASA Astrophysics Data System (ADS)

    Ilieva, N.; Liu, J.; Marinova, R.; Petkov, P.; Litov, L.; He, J.; Niemi, A. J.

    2016-10-01

    We proceed from the description of protein folding by means of molecular dynamics (MD) simulations with all-atom force fields, with folding pathways interpreted in terms of soliton structures, to identify possible systematic dynamical patterns of self-organisation that govern protein folding process. We perform in silico investigations of the conformational transformations of three different proteins - MYC protein (an α-helical protein), amylin and indolicidin (IDPs with different length and binding dynamics). We discuss the emergence of soliton-mediated secondary motifs, in the case of IDPs - in the context of their functional activity. We hypothesize that soliton-like quasi-ordered conformations appear as an important intermediate stage in this process.

  5. Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

    SciTech Connect

    Klinefelter, G.R.; Hamilton, D.W.

    1985-11-01

    We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-(35S)methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-(35S)methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB(3H)4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis.

  6. Modulation of the maladaptive stress response to manage diseases of protein folding.

    PubMed

    Roth, Daniela Martino; Hutt, Darren M; Tong, Jiansong; Bouchecareilh, Marion; Wang, Ning; Seeley, Theo; Dekkers, Johanna F; Beekman, Jeffrey M; Garza, Dan; Drew, Lawrence; Masliah, Eliezer; Morimoto, Richard I; Balch, William E

    2014-11-01

    Diseases of protein folding arise because of the inability of an altered peptide sequence to properly engage protein homeostasis components that direct protein folding and function. To identify global principles of misfolding disease pathology we examined the impact of the local folding environment in alpha-1-antitrypsin deficiency (AATD), Niemann-Pick type C1 disease (NPC1), Alzheimer's disease (AD), and cystic fibrosis (CF). Using distinct models, including patient-derived cell lines and primary epithelium, mouse brain tissue, and Caenorhabditis elegans, we found that chronic expression of misfolded proteins not only triggers the sustained activation of the heat shock response (HSR) pathway, but that this sustained activation is maladaptive. In diseased cells, maladaptation alters protein structure-function relationships, impacts protein folding in the cytosol, and further exacerbates the disease state. We show that down-regulation of this maladaptive stress response (MSR), through silencing of HSF1, the master regulator of the HSR, restores cellular protein folding and improves the disease phenotype. We propose that restoration of a more physiological proteostatic environment will strongly impact the management and progression of loss-of-function and gain-of-toxic-function phenotypes common in human disease. PMID:25406061

  7. Modulation of the Maladaptive Stress Response to Manage Diseases of Protein Folding

    PubMed Central

    Roth, Daniela Martino; Hutt, Darren M.; Tong, Jiansong; Bouchecareilh, Marion; Wang, Ning; Seeley, Theo; Dekkers, Johanna F.; Beekman, Jeffrey M.; Garza, Dan; Drew, Lawrence; Masliah, Eliezer; Morimoto, Richard I.; Balch, William E.

    2014-01-01

    Diseases of protein folding arise because of the inability of an altered peptide sequence to properly engage protein homeostasis components that direct protein folding and function. To identify global principles of misfolding disease pathology we examined the impact of the local folding environment in alpha-1-antitrypsin deficiency (AATD), Niemann-Pick type C1 disease (NPC1), Alzheimer's disease (AD), and cystic fibrosis (CF). Using distinct models, including patient-derived cell lines and primary epithelium, mouse brain tissue, and Caenorhabditis elegans, we found that chronic expression of misfolded proteins not only triggers the sustained activation of the heat shock response (HSR) pathway, but that this sustained activation is maladaptive. In diseased cells, maladaptation alters protein structure–function relationships, impacts protein folding in the cytosol, and further exacerbates the disease state. We show that down-regulation of this maladaptive stress response (MSR), through silencing of HSF1, the master regulator of the HSR, restores cellular protein folding and improves the disease phenotype. We propose that restoration of a more physiological proteostatic environment will strongly impact the management and progression of loss-of-function and gain-of-toxic-function phenotypes common in human disease. PMID:25406061

  8. Ab initio protein folding simulations using atomic burials as informational intermediates between sequence and structure.

    PubMed

    van der Linden, Marx Gomes; Ferreira, Diogo César; de Oliveira, Leandro Cristante; Onuchic, José N; de Araújo, Antônio F Pereira

    2014-07-01

    The three-dimensional structure of proteins is determined by their linear amino acid sequences but decipherment of the underlying protein folding code has remained elusive. Recent studies have suggested that burials, as expressed by atomic distances to the molecular center, are sufficiently informative for structural determination while potentially obtainable from sequences. Here we provide direct evidence for this distinctive role of burials in the folding code, demonstrating that burial propensities estimated from local sequence can indeed be used to fold globular proteins in ab initio simulations. We have used a statistical scheme based on a Hidden Markov Model (HMM) to classify all heavy atoms of a protein into a small number of burial atomic types depending on sequence context. Molecular dynamics simulations were then performed with a potential that forces all atoms of each type towards their predicted burial level, while simple geometric constraints were imposed on covalent structure and hydrogen bond formation. The correct folded conformation was obtained and distinguished in simulations that started from extended chains for a selection of structures comprising all three folding classes and high burial prediction quality. These results demonstrate that atomic burials can act as informational intermediates between sequence and structure, providing a new conceptual framework for improving structural prediction and understanding the fundamentals of protein folding.

  9. Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures

    SciTech Connect

    Lammert, Heiko; Noel, Jeffrey K.; Haglund, Ellinor; Onuchic, José N.; Schug, Alexander

    2015-12-28

    The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein’s functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMR structures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism.

  10. Folding and signaling share the same pathway in a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter D.

    2002-03-01

    The photoreceptor photoactive yellow protein (PYP) was used as a model system to study receptor activation and protein folding. Refolding was studied by stopped-flow absorbance spectroscopy for PYP with either a trans or a cis chromophore. Chromophore trans to cis isomerization, the mechanism of light detection by PYP, greatly affects the protein folding process. When the cis chromophore is present, the unfolded state refolding proceeds through the putative signaling state of PYP as an on-pathway intermediate. In addition, moderate denaturant concentrations result in the specific unfolding of the signaling state of PYP. Thus, the signaling state is common to the pathways of folding and signaling. This provides a novel avenue for the study of protein folding. We demonstrate how this approach can be used to establish whether a folding intermediate is on-pathway or off-pathway. The results also reveal transient partial unfolding as a molecular mechanism for signaling. The signaling intermediate of PYP exhibits properties characteristic of a molten globule, providing a challenge for the current paradigm for the relay of signals along a signal transduction chain by highly specific interactions between fully folded proteins.

  11. Folding Proteins at 500 ns/hour with Work Queue

    PubMed Central

    Abdul-Wahid, Badi’; Yu, Li; Rajan, Dinesh; Feng, Haoyun; Darve, Eric; Thain, Douglas; Izaguirre, Jesús A.

    2014-01-01

    Molecular modeling is a field that traditionally has large computational costs. Until recently, most simulation techniques relied on long trajectories, which inherently have poor scalability. A new class of methods is proposed that requires only a large number of short calculations, and for which minimal communication between computer nodes is required. We considered one of the more accurate variants called Accelerated Weighted Ensemble Dynamics (AWE) and for which distributed computing can be made efficient. We implemented AWE using the Work Queue framework for task management and applied it to an all atom protein model (Fip35 WW domain). We can run with excellent scalability by simultaneously utilizing heterogeneous resources from multiple computing platforms such as clouds (Amazon EC2, Microsoft Azure), dedicated clusters, grids, on multiple architectures (CPU/GPU, 32/64bit), and in a dynamic environment in which processes are regularly added or removed from the pool. This has allowed us to achieve an aggregate sampling rate of over 500 ns/hour. As a comparison, a single process typically achieves 0.1 ns/hour. PMID:25540799

  12. Connecting thermal and mechanical protein (un)folding landscapes

    NASA Astrophysics Data System (ADS)

    Sun, Li; Noel, Jeffrey; Sulkowska, Joanna; Levine, Herbert; Onuchic, José

    2015-03-01

    Molecular dynamics simulations supplement single-molecule pulling experiments by providing the possibility of examining the full free energy landscape using many coordinates. Here, we use an all-atom structure-based model to study the force and temperature dependence of the unfolding of the protein filamin by applying force at both termini. The unfolding time-force relation τ(F) indicates that the unfolding behavior can be characterized into three regimes: barrier-limited low- and intermediate-force regimes, and a barrierless high-force regime. Slope changes of τ(F) separate the three regimes. We show that the behavior of τ(F) can be understood from a two-dimensional free energy landscape projected onto the extension X and the fraction of native contacts Q. In the low-force regime, the unfolding rate is roughly force-independent due to the small (even negative) separation in X between the native ensemble and transition state ensemble (TSE). In the intermediate-force regime, force sufficiently separates the TSE from the native ensemble such that τ(F) roughly follows an exponential relation. The TSE becomes increasingly structured with force. The high-force regime is characterized by barrierless unfolding, approaching a time limit of around 10 μs.

  13. A Model in which Hsp90 Targets Protein Folding Clefts: rationale for a New Approach to Neuroprotective Treatment of Protein Folding Diseases

    PubMed Central

    Pratt, William B.; Morishima, Yoshihiro; Gestwicki, Jason E.; Lieberman, Andrew P.; Osawa, Yoichi

    2015-01-01

    In an EBM Minireview published in 2010, we proposed that the Hsp90/Hsp70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation (1). The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand binding clefts (2). The model provides a framework for thinking about the development of neuroprotective therapies for protein folding diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD) and the polyglutamine expansion disorders, such as Huntington’s disease (HD) and spinal and bulbar muscular atrophy (SBMA). Major aberrant proteins that misfold and accumulate in these diseases are ‘client’ proteins of the abundant and ubiquitous stress chaperone Hsp90 (3). These Hsp90 client proteins include tau (AD), α-synuclein (PD), huntingtin (HD) and the expanded glutamine androgen receptor (polyQ AR) (SBMA). In this minireview we update our model in which Hsp90 acts on protein folding clefts and show how it forms a rational basis for developing drugs that promote the targeted elimination of these aberrant proteins. PMID:24990484

  14. Autonomously Folding Protein Fragments Reveal Differences in the Energy Landscapes of Homologous RNases H

    PubMed Central

    Rosen, Laura E.; Marqusee, Susan

    2015-01-01

    An important approach to understanding how a protein sequence encodes its energy landscape is to compare proteins with different sequences that fold to the same general native structure. In this work, we compare E. coli and T. thermophilus homologs of the protein RNase H. Using protein fragments, we create equilibrium mimics of two different potential partially-folded intermediates (Icore and Icore+1) hypothesized to be present on the energy landscapes of these two proteins. We observe that both T. thermophilus RNase H (ttRNH) fragments are folded and have distinct stabilities, indicating that both regions are capable of autonomous folding and that both intermediates are present as local minima on the ttRNH energy landscape. In contrast, the two E. coli RNase H (ecRNH) fragments have very similar stabilities, suggesting that the presence of additional residues in the Icore+1 fragment does not affect the folding or structure as compared to Icore. NMR experiments provide additional evidence that only the Icore intermediate is populated by ecRNH. This is one of the biggest differences that has been observed between the energy landscapes of these two proteins. Additionally, we used a FRET experiment in the background of full-length ttRNH to specifically monitor the formation of the Icore+1 intermediate. We determine that the ttRNH Icore+1 intermediate is likely the intermediate populated prior to the rate-limiting barrier to global folding, in contrast to E. coli RNase H for which Icore is the folding intermediate. This result provides new insight into the nature of the rate-limiting barrier for the folding of RNase H. PMID:25803034

  15. Role of Autocleavage in the Function of a Type III Secretion Specificity Switch Protein in Salmonella enterica Serovar Typhimurium

    PubMed Central

    Monjarás Feria, Julia V.; Lefebre, Matthew D.; Stierhof, York-Dieter

    2015-01-01

    ABSTRACT Type III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the “switch protein,” a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of the Salmonella enterica serovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage event per se does not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function. PMID:26463164

  16. Protein fold recognition using HMM-HMM alignment and dynamic programming.

    PubMed

    Lyons, James; Paliwal, Kuldip K; Dehzangi, Abdollah; Heffernan, Rhys; Tsunoda, Tatsuhiko; Sharma, Alok

    2016-03-21

    Detecting three dimensional structures of protein sequences is a challenging task in biological sciences. For this purpose, protein fold recognition has been utilized as an intermediate step which helps in classifying a novel protein sequence into one of its folds. The process of protein fold recognition encompasses feature extraction of protein sequences and feature identification through suitable classifiers. Several feature extractors are developed to retrieve useful information from protein sequences. These features are generally extracted by constituting protein's sequential, physicochemical and evolutionary properties. The performance in terms of recognition accuracy has also been gradually improved over the last decade. However, it is yet to reach a well reasonable and accepted level. In this work, we first applied HMM-HMM alignment of protein sequence from HHblits to extract profile HMM (PHMM) matrix. Then we computed the distance between respective PHMM matrices using kernalized dynamic programming. We have recorded significant improvement in fold recognition over the state-of-the-art feature extractors. The improvement of recognition accuracy is in the range of 2.7-11.6% when experimented on three benchmark datasets from Structural Classification of Proteins. PMID:26801876

  17. Statistical Mechanical Model for pH-Induced Protein Folding: Application to Apomyoglobin.

    PubMed

    Mizukami, Takuya; Sakuma, Yosuke; Maki, Kosuke

    2016-09-01

    Despite the major role of pH in protein folding and stability, a quantitative understanding of the pH-induced protein folding mechanism remains elusive. Two conventional models, the Monod-Wyman-Changeux and Linderstrøm-Lang smeared charge models, respectively, have been used to analyze the formation/disruption of specific native structures and fluctuating non-native states. However, there are only a few models that can represent the overall kinetic events of folding/unfolding independent of the properties of relevant molecular species, which has hampered the efforts to systematically analyze pH-induced folding. Here, we constructed a statistical mechanical model that incorporates the protonation mechanism of conventional models along with a combined manual search and least-squares fitting procedure, which was used to investigate the folding of horse apomyoglobin over a wide pH range (2.2-6.7), with a time window ranging from ∼40 μs to ∼100 s, using continuous-/stopped-flow fluorescence at 8 °C. Quantitative analysis assuming a five-state sequential scheme indicated that (1) pH-induced folding/unfolding is represented by both specific binding and Coulombic interactions; (2) kinetic folding/unfolding intermediates share kinetic mechanisms with the equilibrium intermediate, indicating their equivalence; and (3) native-like properties are acquired successively during folding by intermediates and in transition states. This model could also be applied to a variety of association/dissociation processes. PMID:27491483

  18. Two-state protein-like folding of a homopolymer chain

    NASA Astrophysics Data System (ADS)

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

    Many small proteins fold via a first-order 'all-or-none' transition directly from an expanded coil to a compact native state. Here we study an analogous direct freezing transition from an expanded coil to a compact crystallite for a simple flexible homopolymer. Wang-Landau sampling is used to construct the 1D density of states for square-well chains of length 128. Analysis within both the micro-canonical and canonical ensembles shows that, for a chain with sufficiently short-range interactions, the usual polymer collapse transition is preempted by a direct freezing or 'folding' transition. A 2D free-energy landscape, built via subsequent multi-canonical sampling, reveals a dominant folding pathway over a single free-energy barrier. This barrier separates a high entropy ensemble of unfolded states from a low entropy set of crystallite states and the transition proceeds via the formation of a transition-state folding nucleus. Despite the non-unique homopolymer ground state, the thermodynamics of this direct freezing transition are identical to the thermodynamics of two-state protein folding. The model chain satisfies the van't Hoff calorimetric criterion for two-state folding and an Arrhenius analysis of the folding/unfolding free energy barrier yields a Chevron plot characteristic of small proteins.

  19. The cytosolic chaperone α-crystallin B rescues folding and compartmentalization of misfolded multispan transmembrane proteins.

    PubMed

    D'Agostino, Massimo; Lemma, Valentina; Chesi, Giancarlo; Stornaiuolo, Mariano; Cannata Serio, Magda; D'Ambrosio, Chiara; Scaloni, Andrea; Polishchuk, Roman; Bonatti, Stefano

    2013-09-15

    The α-crystallin B chain (CRYAB or HspB5) is a cytosolic chaperone belonging to the small heat shock protein family, which is known to help in the folding of cytosolic proteins. Here we show that CRYAB binds the mutant form of at least two multispan transmembrane proteins (TMPs), exerting an anti-aggregation activity. It rescues the folding of mutant Frizzled4, which is responsible for a rare autosomal dominant form of familial exudative vitreoretinopathy (Fz4-FEVR), and the mutant ATP7B Cu transporter (ATP7B-H1069Q) associated with a common form of Wilson's disease. In the case of Fz4-FEVR, CRYAB prevents the formation of inter-chain disulfide bridges between the lumenal ectodomains of the aggregated mutant chains, which enables correct folding and promotes appropriate compartmentalization on the plasma membrane. ATP7B-H1069Q, with help from CRYAB, folds into the proper conformation, moves to the Golgi complex, and responds to copper overload in the same manner as wild-type ATP7B. These findings strongly suggest that CRYAB plays a pivotal role, previously undetected, in the folding of multispan TMPs and, from the cytosol, is able to orchestrate folding events that take place in the lumen of the ER. Our results contribute to the explanation of the complex scenario behind multispan TMP folding; additionally, they serve to expose interesting avenues for novel therapeutic approaches. PMID:23843626

  20. Effects of N-glycan precursor length diversity on quality control of protein folding and on protein glycosylation

    PubMed Central

    Samuelson, John; Robbins, Phillips W.

    2014-01-01

    Asparagine-linked glycans (N-glycans) of medically important protists have much to tell us about the evolution of N-glycosylation and of N-glycan-dependent quality control (N-glycan QC) of protein folding in the endoplasmic reticulum. While host N-glycans are built upon a dolichol-pyrophosphate-linked precursor with 14 sugars (Glc3Man9GlcNAc2), protist N-glycan precursors vary from Glc3Man9GlcNAc2 (Acanthamoeba) to Man9GlcNAc2 (Trypanosoma) to Glc3Man5GlcNAc2 (Toxoplasma) to Man5GlcNAc2 (Entamoeba, Trichomonas, and Eimeria) to GlcNAc2 (Plasmodium and Giardia) to zero (Theileria). As related organisms have differing N-glycan lengths (e.g. Toxoplasma, Eimeria, Plasmodium, and Theileria), the present N-glycan variation is based upon secondary loss of Alg genes, which encode enzymes that add sugars to the N-glycan precursor. An N-glycan precursor with Man5GlcNAc2 is necessary but not sufficient for N-glycan QC, which is predicted by the presence of the UDP-glucose:glucosyltransferase (UGGT) plus calreticulin and/or calnexin. As many parasites lack glucose in their N-glycan precursor, UGGT product may be identified by inhibition of glucosidase II. The presence of an armless calnexin in Toxoplasma suggests secondary loss of N-glycan QC from coccidia. Positive selection for N-glycan sites occurs in secreted proteins of organisms with NG-QC and is based upon an increased likelihood of threonine but not serine in the second position versus asparagine. In contrast, there appears to be selection against N-glycan length in Plasmodium and N-glycan site density in Toxoplasma. Finally, there is suggestive evidence for N-glycan-dependent ERAD in Trichomonas, which glycosylates and degrades the exogenous reporter mutant carboxypeptidase Y (CPY*). PMID:25475176

  1. Effects of N-glycan precursor length diversity on quality control of protein folding and on protein glycosylation.

    PubMed

    Samuelson, John; Robbins, Phillips W

    2015-05-01

    Asparagine-linked glycans (N-glycans) of medically important protists have much to tell us about the evolution of N-glycosylation and of N-glycan-dependent quality control (N-glycan QC) of protein folding in the endoplasmic reticulum. While host N-glycans are built upon a dolichol-pyrophosphate-linked precursor with 14 sugars (Glc3Man9GlcNAc2), protist N-glycan precursors vary from Glc3Man9GlcNAc2 (Acanthamoeba) to Man9GlcNAc2 (Trypanosoma) to Glc3Man5GlcNAc2 (Toxoplasma) to Man5GlcNAc2 (Entamoeba, Trichomonas, and Eimeria) to GlcNAc2 (Plasmodium and Giardia) to zero (Theileria). As related organisms have differing N-glycan lengths (e.g. Toxoplasma, Eimeria, Plasmodium, and Theileria), the present N-glycan variation is based upon secondary loss of Alg genes, which encode enzymes that add sugars to the N-glycan precursor. An N-glycan precursor with Man5GlcNAc2 is necessary but not sufficient for N-glycan QC, which is predicted by the presence of the UDP-glucose:glucosyltransferase (UGGT) plus calreticulin and/or calnexin. As many parasites lack glucose in their N-glycan precursor, UGGT product may be identified by inhibition of glucosidase II. The presence of an armless calnexin in Toxoplasma suggests secondary loss of N-glycan QC from coccidia. Positive selection for N-glycan sites occurs in secreted proteins of organisms with N-glycan QC and is based upon an increased likelihood of threonine but not serine in the +2 position versus asparagine. In contrast, there appears to be selection against N-glycan length in Plasmodium and N-glycan site density in Toxoplasma. Finally, there is suggestive evidence for N-glycan-dependent ERAD in Trichomonas, which glycosylates and degrades the exogenous reporter mutant carboxypeptidase Y (CPY*).

  2. Application of physical organic chemistry to engineered mutants of proteins: Hammond postulate behavior in the transition state of protein folding.

    PubMed Central

    Matouschek, A; Fersht, A R

    1993-01-01

    Transition states in protein folding may be analyzed by linear free-energy relationships (LFERs) analogous to the Brønsted equation for changes in reactivity with changes in structure. There is an additional source of LFERs in protein folding: the perturbation of the equilibrium and rate constants by denaturants. These LFERs give a measure of the position of the transition state along the reaction coordinate. The transition state for folding/unfolding of barnase has been analyzed by both types of LFERs: changing the structure by protein engineering and perturbation by denaturants. The combination has allowed the direct monitoring of Hammond postulate behavior of the transition state on the reaction pathway. Movement of the transition state has been found and analyzed to give further details of the order of events in protein folding. PMID:8356089

  3. Fluorescent In Situ Folding Control for Rapid Optimization of Cell-Free Membrane Protein Synthesis

    PubMed Central

    Müller-Lucks, Annika; Bock, Sinja; Wu, Binghua; Beitz, Eric

    2012-01-01

    Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP) indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD), proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality. PMID:22848743

  4. Recognition and delivery of effector proteins into eukaryotic cells by bacterial secretion systems.

    PubMed

    Cambronne, Eric D; Roy, Craig R

    2006-08-01

    The direct transport of virulence proteins from bacterium to host has emerged as a common strategy employed by Gram-negative pathogens to establish infections. Specialized secretion systems function to facilitate this process. The delivery of 'effector' proteins by these secretion systems is currently confined to two functionally similar but mechanistically distinct pathways, termed type III and type IV secretion. The type III secretion pathway is ancestrally related to the multiprotein complexes that assemble flagella, whereas the type IV mechanism probably emerged from the protein complexes that support conjugal transfer of DNA. Although both pathways serve to transport proteins from the bacterium to host, the recognition of the effector protein substrates and the secretion information contained in these proteins appear highly distinct. Here, we review the mechanisms involved in the selection of substrates by each of these transport systems and secretion signal information required for substrate transport. PMID:16734660

  5. The Role of Electrostatic Interactions in Folding of β-Proteins

    PubMed Central

    Davis, Caitlin M.; Dyer, R. Brian

    2016-01-01

    Atomic-level molecular dynamic simulations are capable of fully folding structurally diverse proteins; however, they are limited in their ability to accurately represent electrostatic interactions. Here we have experimentally tested the role of charged residues on stability and folding kinetics of one of the most widely simulated β-proteins, the WW domain. The folding of wild type Pin1 WW domain, which has two positively charged residues in the first turn, was compared to the fast folding mutant FiP35 Pin1, which introduces a negative charge into the first turn. A combination of FTIR spectroscopy and laser-induced temperature-jump coupled with infrared spectroscopy was used to probe changes in the amide I region. The relaxation dynamics of the peptide backbone, β-sheets and β-turns, and negatively charged aspartic acid side chain of FiP35 were measured independently by probing the corresponding bands assigned in the amide I region. Folding is initiated in the turns and the β-sheets form last. While the global folding mechanism is in good agreement with simulation predictions, we observe changes in the protonation state of aspartic acid during folding that have not been captured by simulation methods. The protonation state of aspartic acid is coupled to protein folding; the apparent pKa of aspartic acid in the folded protein is 6.4. The dynamics of the aspartic acid follow the dynamics of the intermediate phase, supporting assignment of this phase to formation of the first hairpin. These results demonstrate the importance of electrostatic interactions in turn stability and formation of extended β-sheet structures. PMID:26750867

  6. Schistosoma mansoni secretes a chemokine binding protein with antiinflammatory activity.

    PubMed

    Smith, Philip; Fallon, Rosie E; Mangan, Niamh E; Walsh, Caitriona M; Saraiva, Margarida; Sayers, Jon R; McKenzie, Andrew N J; Alcami, Antonio; Fallon, Padraic G

    2005-11-21

    The coevolution of humans and infectious agents has exerted selective pressure on the immune system to control potentially lethal infections. Correspondingly, pathogens have evolved with various strategies to modulate and circumvent the host's innate and adaptive immune response. Schistosoma species are helminth parasites with genes that have been selected to modulate the host to tolerate chronic worm infections, often for decades, without overt morbidity. The modulation of immunity by schistosomes has been shown to prevent a range of immune-mediated diseases, including allergies and autoimmunity. Individual immune-modulating schistosome molecules have, therefore, therapeutic potential as selective manipulators of the immune system to prevent unrelated diseases. Here we show that S. mansoni eggs secrete a protein into host tissues that binds certain chemokines and inhibits their interaction with host chemokine receptors and their biological activity. The purified recombinant S. mansoni chemokine binding protein (smCKBP) suppressed inflammation in several disease models. smCKBP is unrelated to host proteins and is the first described chemokine binding protein encoded by a pathogenic human parasite and may have potential as an antiinflammatory agent.

  7. Precursory signatures of protein folding/unfolding: From time series correlation analysis to atomistic mechanisms

    SciTech Connect

    Hsu, P. J.; Lai, S. K.; Cheong, S. A.

    2014-05-28

    Folded conformations of proteins in thermodynamically stable states have long lifetimes. Before it folds into a stable conformation, or after unfolding from a stable conformation, the protein will generally stray from one random conformation to another leading thus to rapid fluctuations. Brief structural changes therefore occur before folding and unfolding events. These short-lived movements are easily overlooked in studies of folding/unfolding for they represent momentary excursions of the protein to explore conformations in the neighborhood of the stable conformation. The present study looks for precursory signatures of protein folding/unfolding within these rapid fluctuations through a combination of three techniques: (1) ultrafast shape recognition, (2) time series segmentation, and (3) time series correlation analysis. The first procedure measures the differences between statistical distance distributions of atoms in different conformations by calculating shape similarity indices from molecular dynamics simulation trajectories. The second procedure is used to discover the times at which the protein makes transitions from one conformation to another. Finally, we employ the third technique to exploit spatial fingerprints of the stable conformations; this procedure is to map out the sequences of changes preceding the actual folding and unfolding events, since strongly correlated atoms in different conformations are different due to bond and steric constraints. The aforementioned high-frequency fluctuations are therefore characterized by distinct correlational and structural changes that are associated with rate-limiting precursors that translate into brief segments. Guided by these technical procedures, we choose a model system, a fragment of the protein transthyretin, for identifying in this system not only the precursory signatures of transitions associated with α helix and β hairpin, but also the important role played by weaker correlations in such protein

  8. Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast

    PubMed Central

    Xu, Ping; Robinson, Anne Skaja

    2009-01-01

    Heterologous protein expression can easily overwhelm a cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. In the current model of the unfolded protein response, the chaperone BiP modulates the activation of the UPR due to its interactions with the signaling protein Ire1p and newly synthesized proteins. In this research, 4−4−20 scFv variants were generated by rational design to alter BiP binding to newly synthesized scFv proteins or via directed evolution aimed at improved secretion. Interestingly, the predicted BiP binding ability did not correlate significantly with the unfolded protein response. However, pulse-chase analysis of scFv fate revealed that mutants with a decreased ER residence time were more highly secreted, indicating that improved protein folding was more likely the cause for improved secretion. In fact, decreased secretion correlated with increased binding by BiP, as determined by co-immune precipitation studies. This suggests that the algorithm is not useful for in vivo prediction of variants, and that in vivo screens are more effective for finding variants with improved properties. PMID:19415776

  9. Universality and diversity of folding mechanics for three-helix bundle proteins

    PubMed Central

    Yang, Jae Shick; Wallin, Stefan; Shakhnovich, Eugene I.

    2008-01-01

    In this study we evaluate, at full atomic detail, the folding processes of two small helical proteins, the B domain of protein A and the Villin headpiece. Folding kinetics are studied by performing a large number of ab initio Monte Carlo folding simulations using a single transferable all-atom potential. Using these trajectories, we examine the relaxation behavior, secondary structure formation, and transition-state ensembles (TSEs) of the two proteins and compare our results with experimental data and previous computational studies. To obtain a detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Moreover, rigorous pfold analysis is used to obtain representative samples of the TSEs and a good quantitative agreement between experimental and simulated Φ values is obtained for protein A. Φ values for Villin also are obtained and left as predictions to be tested by future experiments. Our analysis shows that the two-helix hairpin is a common partially stable structural motif that gets formed before entering the TSE in the studied proteins. These results together with our earlier study of Engrailed Homeodomain and recent experimental studies provide a comprehensive, atomic-level picture of folding mechanics of three-helix bundle proteins. PMID:18195374

  10. The Histone Database: a comprehensive resource for histones and histone fold-containing proteins.

    PubMed

    Mariño-Ramírez, Leonardo; Hsu, Benjamin; Baxevanis, Andreas D; Landsman, David

    2006-03-01

    The Histone Database is a curated and searchable collection of full-length sequences and structures of histones and nonhistone proteins containing histone-like folds, compiled from major public databases. Several new histone fold-containing proteins have been identified, including the huntingtin-interacting protein HYPM. Additionally, based on the recent crystal structure of the Son of Sevenless protein, an interpretation of the sequence analysis of the histone fold domain is presented. The database contains an updated collection of multiple sequence alignments for the four core histones (H2A, H2B, H3, and H4) and the linker histones (H1/H5) from a total of 975 organisms. The database also contains information on the human histone gene complement and provides links to three-dimensional structures of histone and histone fold-containing proteins. The Histone Database is a comprehensive bioinformatics resource for the study of structure and function of histones and histone fold-containing proteins. The database is available at http://research.nhgri.nih.gov/histones/.

  11. Intermediates in the folding equilibrium of repeat proteins from the TPR family.

    PubMed

    González-Charro, Vicente; Rey, Antonio

    2014-09-01

    In recent decades, advances in computational methods and experimental biophysical techniques have improved our understanding of protein folding. Although some of these advances have been remarkable, the structural variability of globular proteins usually encountered makes it difficult to extract general features of their folding processes. To overcome this difficulty, experimental and computational studies of the folding of repeat (or modular) proteins are of interest. Because their native structures can be described as linear arrays of the same, repeated, supersecondary structure unit, it is possible to seek a possibly independent behavior of the different modules without taking into account the intrinsic stability associated with different secondary structure motifs. In this work we have used a Monte Carlo-based simulation to study the folding equilibrium of four repeat proteins belonging to the tetratricopeptide repeat family. Our studies provide new insights into their energy profiles, enabling investigation about the existence of intermediate states and their relative stabilities. We have also performed structural analyses to describe the structure of these intermediates, going through the vast number of conformations obtained from the simulations. In this way, we have tried to identify the regions of each protein in which the modular structure yields a different behavior and, more specifically, regions of the proteins that can stay folded when the rest of the chain has been thermally denatured.

  12. Role of the chaperonin cofactor Hsp10 in protein folding and sorting in yeast mitochondria

    PubMed Central

    1994-01-01

    Protein folding in mitochondria is mediated by the chaperonin Hsp60, the homologue of E. coli GroEL. Mitochondria also contain a homologue of the cochaperonin GroES, called Hsp10, which is a functional regulator of the chaperonin. To define the in vivo role of the co- chaperonin, we have used the genetic and biochemical potential of the yeast S. cerevisiae. The HSP10 gene was cloned and sequenced and temperature-sensitive lethal hsp10 mutants were generated. Our results identify Hsp10 as an essential component of the mitochondrial protein folding apparatus, participating in various aspects of Hsp60 function. Hsp10 is required for the folding and assembly of proteins imported into the matrix compartment, and is involved in the sorting of certain proteins, such as the Rieske Fe/S protein, passing through the matrix en route to the intermembrane space. The folding of the precursor of cytosolic dihydrofolate reductase (DHFR), imported into mitochondria as a fusion protein, is apparently independent of Hsp10 function consistent with observations made for the chaperonin-mediated folding of DHFR in vitro. The temperature-sensitive mutations in Hsp10 map to a domain (residues 25-40) that corresponds to a previously identified mobile loop region of bacterial GroES and result in a reduced binding affinity of hsp10 for the chaperonin at the non-permissive temperature. PMID:7913473

  13. Beta-Barrel Scaffold of Fluorescent Proteins: Folding, Stability and Role in Chromophore Formation

    PubMed Central

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K.

    2013-01-01

    This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal helix. The chromophore originates from the polypeptide chain and its properties are influenced by the surrounding protein matrix of the β-barrel. On the other hand, it appears that a chromophore tightens the β-barrel scaffold and plays a crucial role in its stability. Furthermore, the presence of a mature chromophore causes hysteresis of protein unfolding and refolding. We survey studies measuring protein unfolding and refolding using traditional methods as well as new approaches, such as mechanical unfolding and reassembly of truncated fluorescent proteins. We also analyze models of fluorescent protein unfolding and refolding obtained through different approaches, and compare the results of protein folding in vitro to co-translational folding of a newly synthesized polypeptide chain. PMID:23351712

  14. Natural constraints, folding, motion, and structural stability in transmembrane helical proteins

    NASA Astrophysics Data System (ADS)

    Harrington, Susan E.; Ben-Tal, Nir

    Transmembrane (TM) helical proteins are of fundamental importance in many diverse biological processes. To understand these proteins functionally, it is necessary to characterize the forces that stabilize them. What are these forces (both within the protein itself and between the protein and membrane) and how do they give rise to the multiple conformational states and complex activity of TM helical proteins? How do they act in concert to fold TM helical proteins, create their low-energy stable states, and guide their motion? These central questions have led to the description of critical natural constraints and partial answers, which we will review. We will then describe how these constraints can be tracked through homologs and proteins of similar folds in order to better understand how amino acid sequence can specify structure and guide motion. Our emphasis throughout will be on structural features of TM helix bundles themselves, but we will also sketch the membrane-related aspects of these questions.

  15. CASP10-BCL::Fold efficiently samples topologies of large proteins.

    PubMed

    Heinze, Sten; Putnam, Daniel K; Fischer, Axel W; Kohlmann, Tim; Weiner, Brian E; Meiler, Jens

    2015-03-01

    During CASP10 in summer 2012, we tested BCL::Fold for prediction of free modeling (FM) and template-based modeling (TBM) targets. BCL::Fold assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs) omitting more flexible loop regions early on. This approach enables the sampling of conformational space for larger proteins with more complex topologies. In preparation of CASP11, we analyzed the quality of CASP10 models throughout the prediction pipeline to understand BCL::Fold's ability to sample the native topology, identify native-like models by scoring and/or clustering approaches, and our ability to add loop regions and side chains to initial SSE-only models. The standout observation is that BCL::Fold sampled topologies with a GDT_TS score > 33% for 12 of 18 and with a topology score > 0.8 for 11 of 18 test cases de novo. Despite the sampling success of BCL::Fold, significant challenges still exist in clustering and loop generation stages of the pipeline. The clustering approach employed for model selection often failed to identify the most native-like assembly of SSEs for further refinement and submission. It was also observed that for some β-strand proteins model refinement failed as β-strands were not properly aligned to form hydrogen bonds removing otherwise accurate models from the pool. Further, BCL::Fold samples frequently non-natural topologies that require loop regions to pass through the center of the protein.

  16. Folding behavior of ribosomal protein S6 studied by modified Go¯ -like model

    NASA Astrophysics Data System (ADS)

    Wu, L.; Zhang, J.; Wang, J.; Li, W. F.; Wang, W.

    2007-03-01

    Recent experimental and theoretical studies suggest that, although topology is the determinant factor in protein folding, especially for small single-domain proteins, energetic factors also play an important role in the folding process. The ribosomal protein S6 has been subjected to intensive studies. A radical change of the transition state in its circular permutants has been observed, which is believed to be caused by a biased distribution of contact energies. Since the simplistic topology-only Gō -like model is not able to reproduce such an observation, we modify the model by introducing variable contact energies between residues based on their physicochemical properties. The modified Gō -like model can successfully reproduce the Φ -value distributions, folding nucleus, and folding pathways of both the wild-type and circular permutants of S6. Furthermore, by comparing the results of the modified and the simplistic models, we find that the hydrophobic effect constructs the major force that balances the loop entropies. This may indicate that nature maintains the folding cooperativity of this protein by carefully arranging the location of hydrophobic residues in the sequence. Our study reveals a strategy or mechanism used by nature to get out of the dilemma when the native structure, possibly required by biological function, conflicts with folding cooperativity. Finally, the possible relationship between such a design of nature and amyloidosis is also discussed.

  17. Introducing the Levinthal's Protein Folding Paradox and Its Solution

    ERIC Educational Resources Information Center

    Martínez, Leandro

    2014-01-01

    The protein folding (Levinthal's) paradox states that it would not be possible in a physically meaningful time to a protein to reach the native (functional) conformation by a random search of the enormously large number of possible structures. This paradox has been solved: it was shown that small biases toward the native conformation result…

  18. Protein folding in hydrophobic-polar lattice model: a flexible ant-colony optimization approach.

    PubMed

    Hu, Xiao-Min; Zhang, Jun; Xiao, Jing; Li, Yun

    2008-01-01

    This paper proposes a flexible ant colony (FAC) algorithm for solving protein folding problems based on the hydrophobic-polar square lattice model. Collaborations of novel pheromone and heuristic strategies in the proposed algorithm make it more effective in predicting structures of proteins compared with other state-of-the-art algorithms. PMID:18537736

  19. Participation of Low Molecular Weight Electron Carriers in Oxidative Protein Folding

    PubMed Central

    Margittai, Éva; Csala, Miklós; Mandl, József; Bánhegyi, Gábor

    2009-01-01

    Oxidative protein folding is mediated by a proteinaceous electron relay system, in which the concerted action of protein disulfide isomerase and Ero1 delivers the electrons from thiol groups to the final acceptor. Oxygen appears to be the final oxidant in aerobic living organisms, although the existence of alternative electron acceptors, e.g. fumarate or nitrate, cannot be excluded. Whilst the protein components of the system are well-known, less attention has been turned to the role of low molecular weight electron carriers in the process. The function of ascorbate, tocopherol and vitamin K has been raised recently. In vitro and in vivo evidence suggests that these redox-active compounds can contribute to the functioning of oxidative folding. This review focuses on the participation of small molecular weight redox compounds in oxidative protein folding. PMID:19399252

  20. New insights into structural determinants of prion protein folding and stability.

    PubMed

    Benetti, Federico; Legname, Giuseppe

    2015-01-01

    Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

  1. Simulation study of the role of the ribosomal exit tunnel on protein folding

    NASA Astrophysics Data System (ADS)

    Chen, Changjun; Wang, Ercheng; Liu, Pengyu; Xiao, Yi

    2013-02-01

    To investigate the role of the ribosomal exit tunnel on protein folding, we simulate the initial-stage folding behavior of the protein villin headpiece subdomain HP35 (PDB id: 1yrf) with and without prefolding in the exit tunnel by using an all-atom model and find that prefolding in the exit tunnel could effectively help the protein form native secondary structures. Furthermore, our results show that, after releasing from the exit tunnel, the prefolded chains may have a tendency to form more native contacts than those only in free space and this reduces the conformational space of sampling. Our results may provide an alternative way to explain the fast folding mechanism of proteins in vivo.

  2. Direct observation of transition paths during the folding of proteins and nucleic acids.

    PubMed

    Neupane, Krishna; Foster, Daniel A N; Dee, Derek R; Yu, Hao; Wang, Feng; Woodside, Michael T

    2016-04-01

    Transition paths, the fleeting trajectories through the transition states that dominate the dynamics of biomolecular folding reactions, encapsulate the critical information about how structure forms. Owing to their brief duration, however, they have not previously been observed directly. We measured transition paths for both nucleic acid and protein folding, using optical tweezers to observe the microscopic diffusive motion of single molecules traversing energy barriers. The average transit times and the shapes of the transit-time distributions agreed well with theoretical expectations for motion over the one-dimensional energy landscapes reconstructed for the same molecules, validating the physical theory of folding reactions. These measurements provide a first look at the critical microscopic events that occur during folding, opening exciting new avenues for investigating folding phenomena. PMID:27124461

  3. Regulation of Protein Secretion Through Controlled Aggregation in the Endoplasmic Reticulum

    NASA Astrophysics Data System (ADS)

    Rivera, Victor M.; Wang, Xiurong; Wardwell, Scott; Courage, Nancy L.; Volchuk, Allen; Keenan, Terence; Holt, Dennis A.; Gilman, Michael; Orci, Lelio; Cerasoli, Frank; Rothman, James E.; Clackson, Tim

    2000-02-01

    A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

  4. Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum.

    PubMed

    Rivera, V M; Wang, X; Wardwell, S; Courage, N L; Volchuk, A; Keenan, T; Holt, D A; Gilman, M; Orci, L; Cerasoli, F; Rothman, J E; Clackson, T

    2000-02-01

    A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery. PMID:10657290

  5. Folding behaviors of lattice model proteins with three kinds of contact potentials

    NASA Astrophysics Data System (ADS)

    Qin, Meng; Wang, Jun; Tang, Yi; Wang, Wei

    2003-06-01

    The interaction potentials between the amino acids are very important in the study of protein folding and design. In this work, the folding behaviors of lattice model protein chains are studied using three kinds of contact potentials between the beads. For these three cases, a number of sequences are designed using the Z-score method, and then their folding behaviors are obtained via Monte Carlo simulations for different sizes of the chains. It is found that the proper weakening of hydrophobicity may speed up the folding and the elimination of the mixing interaction terms may deteriorate the foldability. The different features of the foldability are discussed by comparing the characteristics of the energy landscapes of these model chains. The formations of various contacts are also analyzed, which provide us with some microscopic information on the model systems and interaction potentials.

  6. Configuration-dependent diffusion can shift the kinetic transition state and barrier height of protein folding

    PubMed Central

    Chahine, Jorge; Oliveira, Ronaldo J.; Leite, Vitor B. P.; Wang, Jin

    2007-01-01

    We show that diffusion can play an important role in protein-folding kinetics. We explicitly calculate the diffusion coefficient of protein folding in a lattice model. We found that diffusion typically is configuration- or reaction coordinate-dependent. The diffusion coefficient is found to be decreasing with respect to the progression of folding toward the native state, which is caused by the collapse to a compact state constraining the configurational space for exploration. The configuration- or position-dependent diffusion coefficient has a significant contribution to the kinetics in addition to the thermodynamic free-energy barrier. It effectively changes (increases in this case) the kinetic barrier height as well as the position of the corresponding transition state and therefore modifies the folding kinetic rates as well as the kinetic routes. The resulting folding time, by considering both kinetic diffusion and the thermodynamic folding free-energy profile, thus is slower than the estimation from the thermodynamic free-energy barrier with constant diffusion but is consistent with the results from kinetic simulations. The configuration- or coordinate-dependent diffusion is especially important with respect to fast folding, when there is a small or no free-energy barrier and kinetics is controlled by diffusion. Including the configurational dependence will challenge the transition state theory of protein folding. The classical transition state theory will have to be modified to be consistent. The more detailed folding mechanistic studies involving phi value analysis based on the classical transition state theory also will have to be modified quantitatively. PMID:17804812

  7. Mutation of the protein-O-mannosyltransferase enhances secretion of the human urokinase-type plasminogen activator in Hansenula polymorpha.

    PubMed

    Agaphonov, Michael O; Sokolov, Sviatoslav S; Romanova, Nina V; Sohn, Jung-Hoon; Kim, So-Young; Kalebina, Tatyana S; Choi, Eui-Sung; Ter-Avanesyan, Michael D

    2005-10-15

    Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein-O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal. PMID:16200504

  8. Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain

    PubMed Central

    Gruber, Tobias; Balbach, Jochen

    2015-01-01

    The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state. PMID:26368922

  9. Förster resonance energy transfer as a probe of membrane protein folding

    PubMed Central

    Kang, Guipeun; López-Peña, Ignacio; Oklejas, Vanessa; Gary, Cyril S.; Cao, Weihan; Kim, Judy E.

    2011-01-01

    The folding reaction of a β-barrel membrane protein, Outer Membrane Protein A (OmpA), is probed with Förster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, 1,5-IAEDANS, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding. PMID:21925139

  10. MOLECULAR GENETIC AND BIOCHEMICAL APPROACHES FOR DEFINING LIPID-DEPENDENT MEMBRANE PROTEIN FOLDING

    PubMed Central

    Dowhan, William; Bogdanov, Mikhail

    2011-01-01

    We provide an overview of lipid-dependent polytopic membrane protein folding and topogenesis. Lipid dependence of this process was determined by employing Escherichia coli cells in which specific lipids can be eliminated, substituted, tightly titrated or controlled temporally during membrane protein synthesis and assembly. The secondary transport protein lactose permease (LacY) was used to establish general principles underlying the molecular basis of lipid-dependent effects on protein domain folding, protein transmembrane domain (TM) orientation, and function. These principles were then extended to several other secondary transport proteins of E. coli. The methods used to follow proper conformational organization of protein domains and the topological organization of protein TMs in whole cells and membranes are described. The proper folding of an extramembrane domain of LacY that is crucial for energy dependent uphill transport function depends on specific lipids acting as non-protein molecular chaperones. Correct TM topogenesis is dependent on charge interactions between the cytoplasmic surface of membrane proteins and a proper balance of the membrane surface net charge defined by the lipid head groups. Short-range interactions between the nascent protein chain and the translocon are necessary but not sufficient for establishment of final topology. After release from the translocon short-range interactions between lipid head groups and the nascent protein chain, partitioning of protein hydrophobic domains into the membrane bilayer, and long–range interactions within the protein thermodynamically drive final membrane protein organization. Given the diversity of membrane lipid compositions throughout nature, it is tempting to speculate that during the course of evolution the physical and chemical properties of proteins and lipids have co-evolved in the context of the lipid environment of membrane systems in which both are mutually depend on each other for

  11. Induction of a secreted protein by the myxoid liposarcoma oncogene

    PubMed Central

    Kuroda, Masahiko; Wang, XiaoZhong; Sok, John; Yin, Yin; Chung, Peter; Giannotti, JoAnn W.; Jacobs, Kenneth A.; Fitz, Lori J.; Murtha-Riel, Patricia; Turner, Katherine J.; Ron, David

    1999-01-01

    The TLS-CHOP oncoprotein, found in the majority of human myxoid liposarcomas, consists of a fusion between the transcription factor CHOP/GADD153 and the N terminus of an RNA-binding protein TLS/FUS. Clinical correlation and in vitro transformation assays indicate that the N terminus of TLS plays an important role in oncogenesis by TLS-CHOP. Until now, however, the only activity attributed to the oncoprotein is that of inhibiting the binding of transcription factors of the C/EBP class to certain adipogenic target genes, a function that TLS-CHOP shares with the nononcogenic CHOP protein. Here we report the isolation of a gene, DOL54, that is activated in primary fibroblasts by the expression of TLS-CHOP. DOL54 is expressed in the neoplastic component of human myxoid liposarcomas and increases the tumorigenicity of cells injected in nude mice. Activation of DOL54 requires an intact DNA-binding and dimerization domain in TLS-CHOP, a suitable cellular dimerization partner, and depends on the TLS N terminus. Normal adipocytic differentiation is associated with an early and transient expression of DOL54, and the gene encodes a secreted protein that is tightly associated with the cell surface or extracellular matrix. TLS-CHOP thus leads to the unscheduled expression of a gene that is normally associated with adipocytic differentiation. PMID:10220412

  12. Unconventional protein secretion in plants: a critical assessment.

    PubMed

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

    2016-01-01

    Unconventional protein secretion (UPS) is a collective term for mechanisms by which cytosolic proteins that lack a signal peptide ("leaderless secretory proteins" (LSPs)) can gain access to the cell exterior. Numerous examples of UPS have been well documented in animal and yeast cells. In contrast, our understanding of the mechanism(s) and function of UPS in plants is very limited. This review evaluates the available literature on this subject. The apparent large numbers of LSPs in the plant secretome suggest that UPS also occurs in plants but is not a proof. Although the direct transport of LSPs across the plant plasma membrane (PM) has not yet been described, it is possible that as in other eukaryotes, exosomes may be released from plant cells through fusion of multivesicular bodies (MVBs) with the PM. In this way, LSPs, but also small RNAs (sRNAs), that are passively taken up from the cytosol into the intraluminal vesicles of MVBs, could reach the apoplast. Another possible mechanism is the recently discovered exocyst-positive organelle (EXPO), a double-membrane-bound compartment, distinct from autophagosomes, which appears to sequester LSPs.

  13. Efficient Numerical Reconstruction of Protein Folding Kinetics with Partial Path Sampling and Pathlike Variables

    NASA Astrophysics Data System (ADS)

    Juraszek, J.; Saladino, G.; van Erp, T. S.; Gervasio, F. L.

    2013-03-01

    Numerically predicting rate constants of protein folding and other relevant biological events is still a significant challenge. We show that the combination of partial path transition interface sampling with the optimal interfaces and free-energy profiles provided by path collective variables makes the rate calculation for practical biological applications feasible and efficient. This methodology can reproduce the experimental rate constant of Trp-cage miniprotein folding with the same level of accuracy as transition path sampling at a fraction of the cost.

  14. Assessing the effect of dynamics on the closed-loop protein-folding hypothesis.

    PubMed

    Chintapalli, Sree V; Illingworth, Christopher J R; Upton, Graham J G; Sacquin-Mora, Sophie; Reeves, Philip J; Mohammedali, Hani S; Reynolds, Christopher A

    2014-02-01

    The closed-loop (loop-n-lock) hypothesis of protein folding suggests that loops of about 25 residues, closed through interactions between the loop ends (locks), play an important role in protein structure. Coarse-grain elastic network simulations, and examination of loop lengths in a diverse set of proteins, each supports a bias towards loops of close to 25 residues in length between residues of high stability. Previous studies have established a correlation between total contact distance (TCD), a metric of sequence distances between contacting residues (cf. contact order), and the log-folding rate of a protein. In a set of 43 proteins, we identify an improved correlation (r(2) = 0.76), when the metric is restricted to residues contacting the locks, compared to the equivalent result when all residues are considered (r(2) = 0.65). This provides qualified support for the hypothesis, albeit with an increased emphasis upon the importance of a much larger set of residues surrounding the locks. Evidence of a similar-sized protein core/extended nucleus (with significant overlap) was obtained from TCD calculations in which residues were successively eliminated according to their hydrophobicity and connectivity, and from molecular dynamics simulations. Our results suggest that while folding is determined by a subset of residues that can be predicted by application of the closed-loop hypothesis, the original hypothesis is too simplistic; efficient protein folding is dependent on a considerably larger subset of residues than those involved in lock formation.

  15. Nucleic acid chaperons: a theory of an RNA-assisted protein folding

    PubMed Central

    Biro, Jan C

    2005-01-01

    Background Proteins are assumed to contain all the information necessary for unambiguous folding (Anfinsen's principle). However, ab initio structure prediction is often not successful because the amino acid sequence itself is not sufficient to guide between endless folding possibilities. It seems to be a logical to try to find the "missing" information in nucleic acids, in the redundant codon base. Results mRNA energy dot plots and protein residue contact maps were found to be rather similar. The structure of mRNA is also conserved if the protein structure is conserved, even if the sequence similarity is low. These observations led me to suppose that some similarity might exist between nucleic acid and protein folding. I found that amino acid pairs, which are co-located in the protein structure, are preferentially coded by complementary codons. This codon complementarity is not perfect; it is suboptimal where the 1st and 3rd codon residues are complementary to each other in reverse orientation, while the 2nd codon letters may be, but are not necessarily, complementary. Conclusion Partial complementary coding of co-locating amino acids in protein structures suggests that mRNA assists in protein folding and functions not only as a template but even as a chaperon during translation. This function explains the role of wobble bases and answers the mystery of why we have a redundant codon base. PMID:16137324

  16. Ostreopexin: a hemopexin fold protein from the oyster mushroom, Pleurotus ostreatus.

    PubMed

    Ota, Katja; Mikelj, Miha; Papler, Tadeja; Leonardi, Adrijana; Križaj, Igor; Maček, Peter

    2013-08-01

    Proteins with hemopexin repeats are widespread in viruses, prokaryotes and eukaryotes. We report here for the first time the existence of a protein in fungi with the four-bladed β-propeller fold that is typical for hemopexin-like proteins. This protein was isolated from the edible basidiomycetous fungus Pleurotus ostreatus and is named ostreopexin. It binds to Ni(2+)-NTA-agarose, and is structurally and functionally very similar to PA2 albumins isolated from legume seeds and the hemopexin fold protein from rice. Like these plant proteins, ostreopexin shows reversible binding to hemin with moderate affinity, but does not bind to polyamines. We suggest that ostreopexin participates in intracellular management of metal (II or III)-chelates. PMID:23567905

  17. Exploring the folding pathway of green fluorescent protein through disulfide engineering.

    PubMed

    Pitman, Derek J; Banerjee, Shounak; Macari, Stephen J; Castaldi, Christopher A; Crone, Donna E; Bystroff, Christopher

    2015-03-01

    We have introduced two disulfide crosslinks into the loop regions on opposite ends of the beta barrel in superfolder green fluorescent protein (GFP) in order to better understand the nature of its folding pathway. When the disulfide on the side opposite the N/C-termini is formed, folding is 2× faster, unfolding is 2000× slower, and the protein is stabilized by 16 kJ/mol. But when the disulfide bond on the side of the termini is formed we see little change in the kinetics and stability. The stabilization upon combining the two crosslinks is approximately additive. When the kinetic effects are broken down into multiple phases, we observe Hammond behavior in the upward shift of the kinetic m-value of unfolding. We use these results in conjunction with structural analysis to assign folding intermediates to two parallel folding pathways. The data are consistent with a view that the two fastest transition states of folding are "barrel closing" steps. The slower of the two phases passes through an intermediate with the barrel opening occurring between strands 7 and 8, while the faster phase opens between 9 and 4. We conclude that disulfide crosslink-induced perturbations in kinetics are useful for mapping the protein folding pathway.

  18. Protein-like folding and free energy landscape of a homopolymer chain

    NASA Astrophysics Data System (ADS)

    Taylor, Mark; Paul, Wolfgang; Binder, Kurt

    2011-03-01

    Many small proteins fold via a first-order ``all-or-none'' transition directly from an expanded coil to a compact native state. We have recently reported an analogous direct coil-to-crystallite transition for a flexible homopolymer. Wang-Landau sampling was used to construct the 1D density of states for square-well chains up to length 256 and a microcanonical analysis shows that for short-range interactions the usual polymer collapse transition is preempted by a direct freezing transition. A 2D configurational probability landscape, built via multi-canonical sampling, reveals a dominant folding pathway and an inherent configurational barrier to folding. Despite the non-unique homopolymer ground state, the thermodynamics of this direct freezing transition are identical to those of two-state protein folding. Homopolymer folding proceeds over a free energy barrier via a transition state folding nucleus, displays a protein-like Chevron plot, and satisfies the van't Hoff two-state criterion. Funding: NSF DMR-0804370, DFG SFB-625/A3.

  19. Protein-like folding and free energy landscape of a homopolymer chain

    NASA Astrophysics Data System (ADS)

    Taylor, Mark; Paul, Wolfgang; Binder, Kurt

    2011-04-01

    Many small proteins fold via a first-order "all-or-none" transition directly from an expanded coil to a compact native state. We have recently reported an analogous direct coil-to-crystallite transition for a flexible homopolymer [1]. Wang-Landau sampling was used to construct the 1D density of states for square-well chains up to length 256 and a microcanonical analysis shows that for short-range interactions the usual polymer collapse transition is preempted by a direct freezing transition. A 2D configurational probability landscape, built via multi-canonical sampling, reveals a dominant folding pathway and an inherent configurational barrier to folding. Despite the non-unique homopolymer ground state, the thermodynamics of this direct freezing transition are identical to those of two-state protein folding. Homopolymer folding proceeds over a free energy barrier via a transition state folding nucleus, displays a protein-like Chevron plot, and satisfies the van't Hoff two-state criterion.[4pt] [1] Phys. Rev. E 79, 050801(R) (2009); J. Chem. Phys. 131, 114907 (2009).

  20. MICROFLUIDIC MIXERS FOR THE INVESTIGATION OF PROTEIN FOLDING USING SYNCHROTRON RADIATION CIRCULAR DICHROISM SPECTROSCOPY

    SciTech Connect

    Kane, A; Hertzog, D; Baumgartel, P; Lengefeld, J; Horsley, D; Schuler, B; Bakajin, O

    2006-03-20

    The purpose of this study is to design, fabricate and optimize microfluidic mixers to investigate the kinetics of protein secondary structure formation with Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The mixers are designed to rapidly initiate protein folding reaction through the dilution of denaturant. The devices are fabricated out of fused silica, so that they are transparent in the UV. We present characterization of mixing in the fabricated devices, as well as the initial SRCD data on proteins inside the mixers.

  1. The Unfolded Protein Response in Retinal Vascular Diseases: Implications and Therapeutic Potential Beyond Protein Folding

    PubMed Central

    Zhang, Sarah X.; Ma, Jacey H.; Bhatta, Maulasri; Fliesler, Steven J.; Wang, Joshua J.

    2015-01-01

    Angiogenesis is a complex, step-wise process of new vessel formation that is involved in both normal embryonic development as well as postnatal pathological processes, such as cancer, cardiovascular disease, and diabetes. Aberrant blood vessel growth, also known as neovascularization, in the retina and the choroid is a major cause of vision loss in severe eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and central and branch retinal vein occlusion. Yet, retinal neovascularization is causally and dynamically associated with vasodegeneration, ischemia, and vascular remodeling in retinal tissues. Understanding the mechanisms of retinal neovascularization is an urgent unmet need for developing new treatments for these devastating diseases. Accumulating evidence suggests a vital role for the unfolded protein response (UPR) in regulation of angiogenesis, in part through coordinating the secretion of pro-angiogenic growth factors, such as VEGF, and modulating endothelial cell survival and activity. Herein, we summarize current research in the context of endoplasmic reticulum (ER) stress and UPR signaling in retinal angiogenesis and vascular remodeling, highlighting potential implications of targeting these stress response pathways in the prevention and treatment of retinal vascular diseases that result in visual deficits and blindness. PMID:25529848

  2. Effect of glycosylation on protein folding: A close look at thermodynamic stabilization

    PubMed Central

    Shental-Bechor, Dalit; Levy, Yaakov

    2008-01-01

    Glycosylation is one of the most common posttranslational modifications to occur in protein biosynthesis, yet its effect on the thermodynamics and kinetics of proteins is poorly understood. A minimalist model based on the native protein topology, in which each amino acid and sugar ring was represented by a single bead, was used to study the effect of glycosylation on protein folding. We studied in silico the folding of 63 engineered SH3 domain variants that had been glycosylated with different numbers of conjugated polysaccharide chains at different sites on the protein's surface. Thermal stabilization of the protein by the polysaccharide chains was observed in proportion to the number of attached chains. Consistent with recent experimental data, the degree of thermal stabilization depended on the position of the glycosylation sites, but only very weakly on the size of the glycans. A thermodynamic analysis showed that the origin of the enhanced protein stabilization by glycosylation is destabilization of the unfolded state rather than stabilization of the folded state. The higher free energy of the unfolded state is enthalpic in origin because the bulky polysaccharide chains force the unfolded ensemble to adopt more extended conformations by prohibiting formation of a residual structure. The thermodynamic stabilization induced by glycosylation is coupled with kinetic stabilization. The effects introduced by the glycans on the biophysical properties of proteins are likely to be relevant to other protein polymeric conjugate systems that regularly occur in the cell as posttranslational modifications or for biotechnological purposes. PMID:18550810

  3. Catalysis of protein folding by chaperones accelerates evolutionary dynamics in adapting cell populations.

    PubMed

    Cetinbaş, Murat; Shakhnovich, Eugene I

    2013-01-01

    Although molecular chaperones are essential components of protein homeostatic machinery, their mechanism of action and impact on adaptation and evolutionary dynamics remain controversial. Here we developed a physics-based ab initio multi-scale model of a living cell for population dynamics simulations to elucidate the effect of chaperones on adaptive evolution. The 6-loci genomes of model cells encode model proteins, whose folding and interactions in cellular milieu can be evaluated exactly from their genome sequences. A genotype-phenotype relationship that is based on a simple yet non-trivially postulated protein-protein interaction (PPI) network determines the cell division rate. Model proteins can exist in native and molten globule states and participate in functional and all possible promiscuous non-functional PPIs. We find that an active chaperone mechanism, whereby chaperones directly catalyze protein folding, has a significant impact on the cellular fitness and the rate of evolutionary dynamics, while passive chaperones, which just maintain misfolded proteins in soluble complexes have a negligible effect on the fitness. We find that by partially releasing the constraint on protein stability, active chaperones promote a deeper exploration of sequence space to strengthen functional PPIs, and diminish the non-functional PPIs. A key experimentally testable prediction emerging from our analysis is that down-regulation of chaperones that catalyze protein folding significantly slows down the adaptation dynamics. PMID:24244114

  4. Iron-sulfur protein folds, iron-sulfur chemistry, and evolution.

    PubMed

    Meyer, Jacques

    2008-02-01

    An inventory of unique local protein folds around Fe-S clusters has been derived from the analysis of protein structure databases. Nearly 50 such folds have been identified, and over 90% of them harbor low-potential [2Fe-2S](2+,+) or [4Fe-4S](2+,+) clusters. In contrast, high-potential Fe-S clusters, notwithstanding their structural diversity, occur in only three different protein folds. These observations suggest that the extant population of Fe-S protein folds has to a large extent been shaped in the reducing iron- and sulfur-rich environment that is believed to have predominated on this planet until approximately two billion years ago. High-potential active sites are then surmised to be rarer because they emerged later, in a more oxidizing biosphere, in conditions where iron and sulfide had become poorly available, Fe-S clusters were less stable, and in addition faced competition from heme iron and copper active sites. Among the low-potential Fe-S active sites, protein folds hosting [4Fe-4S](2+,+) clusters outnumber those with [2Fe-2S](2+,+) ones by a factor of 3 at least. This is in keeping with the higher chemical stability and versatility of the tetranuclear clusters, compared with the binuclear ones. It is therefore suggested that, at least while novel Fe-S sites are evolving within proteins, the intrinsic chemical stability of the inorganic moiety may be more important than the stabilizing effect of the polypeptide chain. The discovery rate of novel Fe-S-containing protein folds underwent a sharp increase around 1995, and has remained stable to this day. The current trend suggests that the mapping of the Fe-S fold space is not near completion, in agreement with predictions made for protein folds in general. Altogether, the data collected and analyzed here suggest that the extant structural landscape of Fe-S proteins has been shaped to a large extent by primeval geochemical conditions on one hand, and iron-sulfur chemistry on the other.

  5. Gentamicin inhibits HSP70-assisted protein folding by interfering with substrate recognition.

    PubMed

    Yamamoto, Soh; Nakano, Shunsuke; Owari, Kensuke; Fuziwara, Kazuhiko; Ogawa, Nobuaki; Otaka, Michiro; Tamaki, Kumiko; Watanabe, Sumio; Komatsuda, Atsushi; Wakui, Hideki; Sawada, Ken-Ichi; Kubota, Hiroshi; Itoh, Hideaki

    2010-02-19

    We previously reported that gentamicin (GM) specifically binds to heat-shock protein with subunit molecular masses of 70 kDa (HSP70). In the present study, we have investigated the effects of GM binding on HSP70-assisted protein folding in vitro. The C-terminal, and not the N-terminal of HSP70 was found to bind to GM. GM significantly suppressed refolding of firefly luciferase in the presence of HSP70 and HSP40, although the ATPase activity of HSP70 was unaffected by GM. A surface plasmon resonance analysis revealed that GM specifically interferes with the binding of HSP70 to a model peptide that mimics the exposed hydrophobic surface of the folding intermediates. These results indicated that GM inhibits the chaperone activity of HSP70 and may suppress protein folding via inhibition of HSP70 in vivo.

  6. Ancylostoma ceylanicum excretory-secretory protein 2 adopts a netrin-like fold and defines a novel family of nematode proteins.

    PubMed

    Kucera, Kaury; Harrison, Lisa M; Cappello, Michael; Modis, Yorgo

    2011-04-22

    Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory-secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75 Å resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinant AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2's netrin-like fold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.

  7. Outer Membrane Protein Folding and Topology from a Computational Transfer Free Energy Scale.

    PubMed

    Lin, Meishan; Gessmann, Dennis; Naveed, Hammad; Liang, Jie

    2016-03-01

    Knowledge of the transfer free energy of amino acids from aqueous solution to a lipid bilayer is essential for understanding membrane protein folding and for predicting membrane protein structure. Here we report a computational approach that can calculate the folding free energy of the transmembrane region of outer membrane β-barrel proteins (OMPs) by combining an empirical energy function with a reduced discrete state space model. We quantitatively analyzed the transfer free energies of 20 amino acid residues at the center of the lipid bilayer of OmpLA. Our results are in excellent agreement with the experimentally derived hydrophobicity scales. We further exhaustively calculated the transfer free energies of 20 amino acids at all positions in the TM region of OmpLA. We found that the asymmetry of the Gram-negative bacterial outer membrane as well as the TM residues of an OMP determine its functional fold in vivo. Our results suggest that the folding process of an OMP is driven by the lipid-facing residues in its hydrophobic