WorldWideScience

Sample records for assisted protein folding

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

    Directory of Open Access Journals (Sweden)

    Biro Jan C

    2005-09-01

    Full Text Available Summary 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.

  2. Exploring the mechanisms used by promiscuous chaperones to assist protein folding in the cell

    Science.gov (United States)

    Jewett, Andrew I.

    There are two popular theories to explain how molecular chaperones boost the yield of folded protein in the cell: According to the Anfinsen cage model, (ACM) chaperonins protect denatured proteins from aggregation. A competing theory, the iterative annealing model (IAM) claims that ATP regulated chaperone binding and release accelerates folding by freeing proteins from long-lived kinetic traps. We present experimental and kinetic evidence to argue that the IAM is not a complete picture of how the GroEL/ES chaperonin works. Surprisingly some substrate proteins experience folding rate enhancements without undergoing multiple rounds of ATP-induced binding and release from the chaperonin. An explanation of this data requires going beyond the ACM and IAM models. Our work uses molecular dynamics simulations to investigate the folding of a highly frustrated protein within a chaperonin cavity. The chaperonin interior is modeled by a sphere with variable degree of attraction to the protein inside. We demonstrate that this cavity, similar to the weakly hydrophobic interior of the GroEL cavity upon complexion with ATP and GroES, is sufficient to accelerate the folding of a frustrated protein by more than an order of magnitude. Our simulations uncover a novel form of the IAM in which the substrate exhibits spontaneous binding and release from the wall of the chaperonin cage. This mimics the behavior observed in the standard IAM, with the difference that thermal fluctuations, rather than ATP, allow the substrate to unbind from the chaperone. An growing number of smaller cageless chaperones have been discovered that can assist protein folding without the consumption of ATP, including artificial "minichaperones" (fragments of larger chaperones). It is tempting to speculate that the same thermally-driven IAM mechanism could play a role with these chaperones as well. We performed additional simulations of protein folding outside the sphere. We find that in order to accelerate

  3. Discovery of Proteomic Code with mRNA Assisted Protein Folding

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    Jan C. Biro

    2008-12-01

    Full Text Available The 3x redundancy of the Genetic Code is usually explained as a necessity to increase the mutation-resistance of the genetic information. However recent bioinformatical observations indicate that the redundant Genetic Code contains more biological information than previously known and which is additional to the 64/20 definition of amino acids. It might define the physico-chemical and structural properties of amino acids, the codon boundaries, the amino acid co-locations (interactions in the coded proteins and the free folding energy of mRNAs. This additional information, which seems to be necessary to determine the 3D structure of coding nucleic acids as well as the coded proteins, is known as the Proteomic Code and mRNA Assisted Protein Folding.

  4. Folding of newly translated membrane protein CCR5 is assisted by the chaperonin GroEL-GroES

    Science.gov (United States)

    Chi, Haixia; Wang, Xiaoqiang; Li, Jiqiang; Ren, Hao; Huang, Fang

    2015-11-01

    The in vitro folding of newly translated human CC chemokine receptor type 5 (CCR5), which belongs to the physiologically important family of G protein-coupled receptors (GPCRs), has been studied in a cell-free system supplemented with the surfactant Brij-35. The freshly synthesized CCR5 can spontaneously fold into its biologically active state but only slowly and inefficiently. However, on addition of the GroEL-GroES molecular chaperone system, the folding of the nascent CCR5 was significantly enhanced, as was the structural stability and functional expression of the soluble form of CCR5. The chaperonin GroEL was partially effective on its own, but for maximum efficiency both the GroEL and its GroES lid were necessary. These results are direct evidence for chaperone-assisted membrane protein folding and therefore demonstrate that GroEL-GroES may be implicated in the folding of membrane proteins.

  5. Assisted protein folding at low temperature: evolutionary adaptation of the Antarctic fish chaperonin CCT and its client proteins

    Science.gov (United States)

    Cuellar, Jorge; Yébenes, Hugo; Parker, Sandra K.; Carranza, Gerardo; Serna, Marina; Valpuesta, José María; Zabala, Juan Carlos; Detrich, H. William

    2014-01-01

    ABSTRACT Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs) have co-evolved molecular adaptations that facilitate CCT–CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT–CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lönnberg) (habitat/body T = −1.9 to +2°C), and of the cow (body T = 37°C). We examined the temperature dependence of the binding of denatured CPs (β-actin, β-tubulin) by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between −4°C and 20°C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2°C. The ATPase activity of apo-CCT from G. gibberifrons at 4°C was ∼2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20°C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits. PMID:24659247

  6. Fast protein folding kinetics

    Science.gov (United States)

    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

  7. Disassembly of yeast 80S ribosomes into subunits is a concerted action of ribosome-assisted folding of denatured protein.

    Science.gov (United States)

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-22

    It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded. In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg(+2) ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg(+2) ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing. For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. A Self-Assisting Protein Folding Model for Teaching Structural Molecular Biology.

    Science.gov (United States)

    Davenport, Jodi; Pique, Michael; Getzoff, Elizabeth; Huntoon, Jon; Gardner, Adam; Olson, Arthur

    2017-04-04

    Structural molecular biology is now becoming part of high school science curriculum thus posing a challenge for teachers who need to convey three-dimensional (3D) structures with conventional text and pictures. In many cases even interactive computer graphics does not go far enough to address these challenges. We have developed a flexible model of the polypeptide backbone using 3D printing technology. With this model we have produced a polypeptide assembly kit to create an idealized model of the Triosephosphate isomerase mutase enzyme (TIM), which forms a structure known as TIM barrel. This kit has been used in a laboratory practical where students perform a step-by-step investigation into the nature of protein folding, starting with the handedness of amino acids to the formation of secondary and tertiary structure. Based on the classroom evidence we collected, we conclude that these models are valuable and inexpensive resource for teaching structural molecular biology. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Evolutionary optimization of protein folding.

    Directory of Open Access Journals (Sweden)

    Cédric Debès

    Full Text Available 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.

  10. Teaching computers to fold proteins

    DEFF Research Database (Denmark)

    Winther, Ole; Krogh, Anders Stærmose

    2004-01-01

    A new general algorithm for optimization of potential functions for protein folding is introduced. It is based upon gradient optimization of the thermodynamic stability of native folds of a training set of proteins with known structure. The iterative update rule contains two thermodynamic average...

  11. Protein folding and wring resonances

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren

    1997-01-01

    The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested...... that protein folding takes place when the amplitude of a wring excitation becomes so large that it is energetically favorable to bend the protein backbone. The condition under which such structural transformations can occur is found, and it is shown that both cold and hot denaturation (the unfolding...... of proteins) are natural consequences of the suggested wring mode model. Native (folded) proteins are found to possess an intrinsic standing wring mode....

  12. Protein folding by motion planning.

    Science.gov (United States)

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

    2005-11-09

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

  13. Protein folding by motion planning

    Science.gov (United States)

    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.

  14. Use of Protein Folding Reagents

    Science.gov (United States)

    Wingfield, Paul T.

    2016-01-01

    The reagents and methods for purification of the most commonly used denaturants guanidine hydrochloride (guanidine-HCl) and urea are described. Other protein denaturants and reagents used to fold proteins are briefly mentioned. Sulfhydryl reagents (reducing agents) and “oxido-shuffling” (or oxidative regeneration) systems are also described. PMID:18429069

  15. Understanding Protein Non-Folding

    Science.gov (United States)

    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

  16. Parser for protein folding units.

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    Holm, L; Sander, C

    1994-07-01

    General patterns of protein structural organization have emerged from studies of hundreds of structures elucidated by X-ray crystallography and nuclear magnetic resonance. Structural units are commonly identified by visual inspection of molecular models using qualitative criteria. Here, we propose an algorithm for identification of structural units by objective, quantitative criteria based on atomic interactions. The underlying physical concept is maximal interactions within each unit and minimal interaction between units (domains). In a simple harmonic approximation, interdomain dynamics is determined by the strength of the interface and the distribution of masses. The most likely domain decomposition involves units with the most correlated motion, or largest interdomain fluctuation time. The decomposition of a convoluted 3-D structure is complicated by the possibility that the chain can cross over several times between units. Grouping the residues by solving an eigenvalue problem for the contact matrix reduces the problem to a one-dimensional search for all reasonable trial bisections. Recursive bisection yields a tree of putative folding units. Simple physical criteria are used to identify units that could exist by themselves. The units so defined closely correspond to crystallographers' notion of structural domains. The results are useful for the analysis of folding principles, for modular protein design and for protein engineering.

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

    Science.gov (United States)

    Pappu, Rohit V.; Nussinov, Ruth

    2009-03-01

    multi-scale dynamical problem when one considers the synergies between protein expression, spontaneous folding, chaperonin-assisted folding, protein targeting, the kinetics of post-translational modifications, protein degradation, and of course the drive to avoid aggregation. Further, there is growing recognition that cells not only tolerate but select for proteins that are intrinsically disordered. These proteins are essential for many crucial activities, and yet their inability to fold in isolation makes them prone to proteolytic processing and aggregation. In the series of papers that make up this special focus on protein folding in physical biology, leading researchers provide insights into diverse cross-sections of problems in protein folding. Barrick provides a concise review of what we have learned from the study of two-state folders and draws attention to how several unanswered questions are being approached using studies on large repeat proteins. Dissecting the contribution of hydration-mediated interactions to driving forces for protein folding and assembly has been extremely challenging. There is renewed interest in using hydrostatic pressure as a tool to access folding intermediates and decipher the role of partially hydrated states in folding, misfolding, and aggregation. Silva and Foguel review many of the nuances that have been uncovered by perturbing hydrostatic pressure as a thermodynamic parameter. As noted above, protein folding in vivo is expected to be considerably more complex than the folding of two-state proteins in dilute solutions. Lucent et al review the state-of-the-art in the development of quantitative theories to explain chaperonin-assisted folding in vivo. Additionally, they highlight unanswered questions pertaining to the processing of unfolded/misfolded proteins by the chaperone machinery. Zhuang et al present results that focus on the effects of surface tethering on transition state ensembles and folding mechanisms of a model two

  18. The concept of RNA-assisted protein folding: representation of amino acid kinetics at the tRNA level.

    Science.gov (United States)

    Biro, Jan C; Biro, Josephine M K

    2013-01-21

    Transfer RNA molecules (tRNA) have the thermodynamic potential to interact with each other to form dimers (hybridization energy, dG=-33.2±6.7 kcal/mole, compared to the folding energy of the cloverleaf configuration, dG=-25±4.4 kcal/mole, p<0.0001). The dG values have a strong negative correlation with the frequency of co-locations and substitutions of associated amino acids. We suggest that tRNA interactions participate in determining amino acid interactions (co-locations) and consequently the 3D structures of peptides. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Reduced alphabet for protein folding prediction.

    Science.gov (United States)

    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. © 2015 Wiley Periodicals, Inc.

  20. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: A novel strategy exploiting disulfide assisted protein folding

    DEFF Research Database (Denmark)

    Ferré, Henrik; Ruffet, E.; Blicher, T.

    2003-01-01

    disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta(2...

  1. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

    DEFF Research Database (Denmark)

    Ferré, Henrik; Ruffet, Emmanuel; Blicher, Thomas

    2003-01-01

    with correct disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta...

  2. Protein Folding and Self-Organized Criticality

    Science.gov (United States)

    Bajracharya, Arun; Murray, Joelle

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

  3. Protein folding on a chip

    CERN Multimedia

    2004-01-01

    "Scientists at the U.S. Department of Energy's Brookhaven National Laboratory are proposing to use a super- computer originally developed to simulate elementary particles in high- energy physics to help determine the structures and functions of proteins, including, for example, the 30,000 or so proteins encoded by the human genome" (1 page)

  4. Towards a systematic classification of protein folds

    DEFF Research Database (Denmark)

    Lindgård, Per-Anker; Bohr, Henrik

    1997-01-01

    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...

  5. The folding of disulfide-rich proteins.

    Science.gov (United States)

    Craik, David J

    2011-01-01

    The articles in this forum issue describe various aspects of the folding of disulfide-rich proteins. They include review articles using proteins such as bovine pancreatic trypsin inhibitor as models to highlight the range of folding pathways seen in disulfide-rich proteins, along with a detailed analysis of the methods used to study them. Following two comprehensive reviews on the methods and applications of protein folding, three original articles in this issue focus on two specific classes of disulfide-rich proteins that have applications in drug design and development, namely cyclotides and conotoxins. Cyclotides are head-to-tail cyclic and disulfide-rich proteins from plants and function as a defense against insect attack. Conotoxins are the disulfide-rich components of the venom of marine cone snails that is used to capture prey. These research articles report on factors that modulate protein folding pathways in these molecules and determine the outcomes of protein folding, that is, yield and heterogeneity of products. Finally, the issue concludes with a comprehensive review on a different type of disulfide bond, namely those that have a functional rather than structural role in proteins, with a particular focus on allosteric disulfide bonds that modify protein function.

  6. Coherent topological phenomena in protein folding

    DEFF Research Database (Denmark)

    Bohr, Henrik; Brunak, Søren; Bohr, Jakob

    1997-01-01

    A theory is presented for coherent topological phenomena in protein dynamics with implications for protein folding and stability. We discuss the relationship to the writhing number used in knot diagrams of DNA. The winding state defines a long-range order along the backbone of a protein with long...

  7. Protein folding and tRNA biology.

    Science.gov (United States)

    Marín, Mónica; Fernández-Calero, Tamara; Ehrlich, Ricardo

    2017-10-01

    Polypeptides can fold into tertiary structures while they are synthesized by the ribosome. In addition to the amino acid sequence, protein folding is determined by several factors within the cell. Among others, the folding pathway of a nascent polypeptide can be affected by transient interactions with other proteins, ligands, or the ribosome, as well as by the translocation through membrane pores. Particularly, the translation machinery and the population of tRNA under different physiological or adaptive responses can dramatically affect protein folding. This review summarizes the scientific evidence describing the role of translation kinetics and tRNA populations on protein folding and addresses current efforts to better understand tRNA biology. It is organized into three main parts, which are focused on: (i) protein folding in the cellular context; (ii) tRNA biology and the complexity of the tRNA population; and (iii) available methods and technical challenges in the characterization of tRNA pools. In this manner, this work illustrates the ways by which functional properties of proteins may be modulated by cellular tRNA populations.

  8. Cotranslational folding of deeply knotted proteins

    CERN Document Server

    Chwastyk, Mateusz

    2015-01-01

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

  9. Melody discrimination and protein fold classification

    Directory of Open Access Journals (Sweden)

    Robert P. Bywater

    2016-10-01

    Full Text Available One of the greatest challenges in theoretical biophysics and bioinformatics is the identification of protein folds from sequence data. This can be regarded as a pattern recognition problem. In this paper we report the use of a melody generation software where the inputs are derived from calculations of evolutionary information, secondary structure, flexibility, hydropathy and solvent accessibility from multiple sequence alignment data. The melodies so generated are derived from the sequence, and by inference, of the fold, in ways that give each fold a sound representation that may facilitate analysis, recognition, or comparison with other sequences.

  10. Effects of knots on protein folding properties.

    Directory of Open Access Journals (Sweden)

    Miguel A Soler

    Full Text Available 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.

  11. Analysis of protein folds using protein contact networks

    Indian Academy of Sciences (India)

    Proteins are important biomolecules, which perform diverse structural and functional roles in living systems. Starting from a linear chain of amino acids, proteins fold to different secondary structures, which then fold through short- and long-range interactions to give rise to the final three-dimensional shapes useful to carry out ...

  12. Glycoprotein folding and quality-control mechanisms in protein-folding diseases

    Directory of Open Access Journals (Sweden)

    Sean P. Ferris

    2014-03-01

    Full Text Available Biosynthesis of proteins – from translation to folding to export – encompasses a complex set of events that are exquisitely regulated and scrutinized to ensure the functional quality of the end products. Cells have evolved to capitalize on multiple post-translational modifications in addition to primary structure to indicate the folding status of nascent polypeptides to the chaperones and other proteins that assist in their folding and export. These modifications can also, in the case of irreversibly misfolded candidates, signal the need for dislocation and degradation. The current Review focuses on the glycoprotein quality-control (GQC system that utilizes protein N-glycosylation and N-glycan trimming to direct nascent glycopolypeptides through the folding, export and dislocation pathways in the endoplasmic reticulum (ER. A diverse set of pathological conditions rooted in defective as well as over-vigilant ER quality-control systems have been identified, underlining its importance in human health and disease. We describe the GQC pathways and highlight disease and animal models that have been instrumental in clarifying our current understanding of these processes.

  13. Protein Folding: Search for Basic Physical Models

    Directory of Open Access Journals (Sweden)

    Ivan Y. Torshin

    2003-01-01

    Full Text Available How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context of in vivo protein folding (which has been studied only for a few proteins, the roles of the fundamental physical forces in the in vitro folding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces. Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

  14. Protein folding and the organization of the protein topology universe

    DEFF Research Database (Denmark)

    Lindorff-Larsen,, Kresten; Røgen, Peter; Paci, Emanuele

    2005-01-01

    residues and, in addition, that the topology of the transition state is closer to that of the native state than to that of any other fold in the protein universe. Here, we review the evidence for these conclusions and suggest a molecular mechanism that rationalizes these findings by presenting a view...... of protein folds that is based on the topological features of the polypeptide backbone, rather than the conventional view that depends on the arrangement of different types of secondary-structure elements. By linking the folding process to the organization of the protein structure universe, we propose...

  15. Analysis of protein folds using protein contact networks

    Indian Academy of Sciences (India)

    - range non-local interactions among these secondary structural elements lead the protein to fold into a functionally active, native, three-dimensional, tertiary struc- ture. Often more than one tertiary subunits interact among themselves to form.

  16. Folding Membrane Proteins by Deep Transfer Learning

    KAUST Repository

    Wang, Sheng

    2017-08-29

    Computational elucidation of membrane protein (MP) structures is challenging partially due to lack of sufficient solved structures for homology modeling. Here, we describe a high-throughput deep transfer learning method that first predicts MP contacts by learning from non-MPs and then predicts 3D structure models using the predicted contacts as distance restraints. Tested on 510 non-redundant MPs, our method has contact prediction accuracy at least 0.18 better than existing methods, predicts correct folds for 218 MPs, and generates 3D models with root-mean-square deviation (RMSD) less than 4 and 5 Å for 57 and 108 MPs, respectively. A rigorous blind test in the continuous automated model evaluation project shows that our method predicted high-resolution 3D models for two recent test MPs of 210 residues with RMSD ∼2 Å. We estimated that our method could predict correct folds for 1,345–1,871 reviewed human multi-pass MPs including a few hundred new folds, which shall facilitate the discovery of drugs targeting at MPs.

  17. Improving decoy databases for protein folding algorithms

    KAUST Repository

    Lindsey, Aaron

    2014-01-01

    Copyright © 2014 ACM. Predicting protein structures and simulating protein folding are two of the most important problems in computational biology today. Simulation methods rely on a scoring function to distinguish the native structure (the most energetically stable) from non-native structures. Decoy databases are collections of non-native structures used to test and verify these functions. We present a method to evaluate and improve the quality of decoy databases by adding novel structures and removing redundant structures. We test our approach on 17 different decoy databases of varying size and type and show significant improvement across a variety of metrics. We also test our improved databases on a popular modern scoring function and show that they contain a greater number of native-like structures than the original databases, thereby producing a more rigorous database for testing scoring functions.

  18. Hydrophobic-hydrophilic forces in protein folding.

    Science.gov (United States)

    Durell, Stewart R; Ben-Naim, Arieh

    2017-08-01

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

  19. Algorithm discovery by protein folding game players.

    Science.gov (United States)

    Khatib, Firas; Cooper, Seth; Tyka, Michael D; Xu, Kefan; Makedon, Ilya; Popovic, Zoran; Baker, David; Players, Foldit

    2011-11-22

    Foldit is a multiplayer online game in which players collaborate and compete to create accurate protein structure models. For specific hard problems, Foldit player solutions can in some cases outperform state-of-the-art computational methods. However, very little is known about how collaborative gameplay produces these results and whether Foldit player strategies can be formalized and structured so that they can be used by computers. To determine whether high performing player strategies could be collectively codified, we augmented the Foldit gameplay mechanics with tools for players to encode their folding strategies as "recipes" and to share their recipes with other players, who are able to further modify and redistribute them. Here we describe the rapid social evolution of player-developed folding algorithms that took place in the year following the introduction of these tools. Players developed over 5,400 different recipes, both by creating new algorithms and by modifying and recombining successful recipes developed by other players. The most successful recipes rapidly spread through the Foldit player population, and two of the recipes became particularly dominant. Examination of the algorithms encoded in these two recipes revealed a striking similarity to an unpublished algorithm developed by scientists over the same period. Benchmark calculations show that the new algorithm independently discovered by scientists and by Foldit players outperforms previously published methods. Thus, online scientific game frameworks have the potential not only to solve hard scientific problems, but also to discover and formalize effective new strategies and algorithms.

  20. Co- and post-translational protein folding in the ER

    DEFF Research Database (Denmark)

    Ellgaard, Lars; McCaul, Nicholas; Chatsisvili, Anna

    2016-01-01

    The biophysical rules that govern folding of small, single-domain proteins in dilute solutions are now quite well understood. The mechanisms underlying co-translational folding of multidomain and membrane-spanning proteins in complex cellular environments are often less clear. The endoplasmic...... to monitor the progression of folding as it occurs inside living cells, while at the same time probing the intricate relationship between protein modifications during folding....

  1. Improving Protein Fold Recognition by Extracting Fold-specific Features from Predicted Residue-residue Contacts.

    Science.gov (United States)

    Zhu, Jianwei; Zhang, Haicang; Li, Shuai Cheng; Wang, Chao; Kong, Lupeng; Sun, Shiwei; Zheng, Wei-Mou; Bu, Dongbo

    2017-08-16

    Accurate recognition of protein fold types is a key step for template-based prediction of protein structures. The existing approaches to fold recognition mainly exploit the features derived from alignments of query protein against templates. These approaches have been shown to be successful for fold recognition at family level, but usually failed at superfamily/fold levels. To overcome this limitation, one of the key points is to explore more structurally-informative features of proteins. Although residue-residue contacts carry abundant structural information, how to thoroughly exploit these information for fold recognition still remains a challenge. In this study, we present an approach (called DeepFR) to improve fold recognition at superfamily/fold levels. The basic idea of our approach is to extract fold-specific features from predicted residue-residue contacts of proteins using deep convolutional neural network (DCNN) technique. Based on these fold-specific features, we calculated similarity between query protein and templates, and then assigned query protein with fold type of the most similar template. DCNN has showed excellent performance in image feature extraction and image recognition; the rational underlying the application of DCNN for fold recognition is that contact likelihood maps are essentially analogy to images, as they both display compositional hierarchy. Experimental results on the LINDAHL dataset suggest that even using the extracted fold-specific features alone, our approach achieved success rate comparable to the state-of-the-art approaches. When further combining these features with traditional alignment-related features, the success rate of our approach increased to 92.3%, 82.5%, and 78.8% at family, superfamily, and fold levels, respectively, which is about 18% higher than the state-of-the-art approach at fold level, 6% higher at superfamily level, and 1% higher at family level. An independent assessment on SCOP_TEST dataset showed

  2. Prediction of protein folding rates from simplified secondary structure alphabet.

    Science.gov (United States)

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

    2015-10-21

    Protein folding is a very complicated and highly cooperative dynamic process. However, the folding kinetics is likely to depend more on a few key structural features. Here we find that secondary structures can determine folding rates of only large, multi-state folding proteins and fails to predict those for small, two-state proteins. The importance of secondary structures for protein folding is ordered as: extended β strand > α helix > bend > turn > undefined secondary structure>310 helix > isolated β strand > π helix. Only the first three secondary structures, extended β strand, α helix and bend, can achieve a good correlation with folding rates. This suggests that the rate-limiting step of protein folding would depend upon the formation of regular secondary structures and the buckling of chain. The reduced secondary structure alphabet provides a simplified description for the machine learning applications in protein design. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Green fluorescent protein as a reporter of prion protein folding

    Directory of Open Access Journals (Sweden)

    Dalton Kevin

    2006-08-01

    Full Text Available Abstract Background The amino terminal half of the cellular prion protein PrPc is implicated in both the binding of copper ions and the conformational changes that lead to disease but has no defined structure. However, as some structure is likely to exist we have investigated the use of an established protein refolding technology, fusion to green fluorescence protein (GFP, as a method to examine the refolding of the amino terminal domain of mouse prion protein. Results Fusion proteins of PrPc and GFP were expressed at high level in E.coli and could be purified to near homogeneity as insoluble inclusion bodies. Following denaturation, proteins were diluted into a refolding buffer whereupon GFP fluorescence recovered with time. Using several truncations of PrPc the rate of refolding was shown to depend on the prion sequence expressed. In a variation of the format, direct observation in E.coli, mutations introduced randomly in the PrPc protein sequence that affected folding could be selected directly by recovery of GFP fluorescence. Conclusion Use of GFP as a measure of refolding of PrPc fusion proteins in vitro and in vivo proved informative. Refolding in vitro suggested a local structure within the amino terminal domain while direct selection via fluorescence showed that as little as one amino acid change could significantly alter folding. These assay formats, not previously used to study PrP folding, may be generally useful for investigating PrPc structure and PrPc-ligand interaction.

  4. Stabilization of partially folded states in protein folding/misfolding transitions by hydrostatic pressure

    Directory of Open Access Journals (Sweden)

    S.T. Ferreira

    2005-08-01

    Full Text Available In the last few years, hydrostatic pressure has been extensively used in the study of both protein folding and misfolding/aggregation. Compared to other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, which allow the stabilization of partially folded intermediate states that are usually not significantly populated under more drastic conditions (e.g., in the presence of chemical denaturants or at high temperatures. Much of the recent research in the field of protein folding has focused on the characterization of folding intermediates since these species appear to be involved in a variety of disease-causing protein misfolding and aggregation events. The exact mechanisms of these biologicalphenomena, however, are still poorly understood. Here, we review recent examples of the use of hydrostatic pressure as a tool to obtain insight into the forces and energetics governing the productive folding or the misfolding and aggregation of proteins.

  5. Chloroplast Chaperonin: An Intricate Protein Folding Machine for Photosynthesis

    Directory of Open Access Journals (Sweden)

    Qian Zhao

    2018-01-01

    Full Text Available Group I chaperonins are large cylindrical-shaped nano-machines that function as a central hub in the protein quality control system in the bacterial cytosol, mitochondria and chloroplasts. In chloroplasts, proteins newly synthesized by chloroplast ribosomes, unfolded by diverse stresses, or translocated from the cytosol run the risk of aberrant folding and aggregation. The chloroplast chaperonin system assists these proteins in folding into their native states. A widely known protein folded by chloroplast chaperonin is the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, an enzyme responsible for the fixation of inorganic CO2 into organic carbohydrates during photosynthesis. Chloroplast chaperonin was initially identified as a Rubisco-binding protein. All photosynthetic eucaryotes genomes encode multiple chaperonin genes which can be divided into α and β subtypes. Unlike the homo-oligomeric chaperonins from bacteria and mitochondria, chloroplast chaperonins are more complex and exists as intricate hetero-oligomers containing both subtypes. The Group I chaperonin requires proper interaction with a detachable lid-like co-chaperonin in the presence of ATP and Mg2+ for substrate encapsulation and conformational transition. Besides the typical Cpn10-like co-chaperonin, a unique co-chaperonin consisting of two tandem Cpn10-like domains joined head-to-tail exists in chloroplasts. Since chloroplasts were proposed as sensors to various environmental stresses, this diversified chloroplast chaperonin system has the potential to adapt to complex conditions by accommodating specific substrates or through regulation at both the transcriptional and post-translational levels. In this review, we discuss recent progress on the unique structure and function of the chloroplast chaperonin system based on model organisms Chlamydomonas reinhardtii and Arabidopsis thaliana. Knowledge of the chloroplast chaperonin system may ultimately lead

  6. Protein-Folding Landscapes in Multi-Chain Systems

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    CERN Document Server

    Chwastyk, Mateusz

    2015-01-01

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

  8. Folding of proteins with a flavodoxin-like architecture.

    Science.gov (United States)

    Houwman, Joseline A; van Mierlo, Carlo P M

    2017-10-01

    The flavodoxin-like fold is a protein architecture that can be traced back to the universal ancestor of the three kingdoms of life. Many proteins share this α-β parallel topology and hence it is highly relevant to illuminate how they fold. Here, we review experiments and simulations concerning the folding of flavodoxins and CheY-like proteins, which share the flavodoxin-like fold. These polypeptides tend to temporarily misfold during unassisted folding to their functionally active forms. This susceptibility to frustration is caused by the more rapid formation of an α-helix compared to a β-sheet, particularly when a parallel β-sheet is involved. As a result, flavodoxin-like proteins form intermediates that are off-pathway to native protein and several of these species are molten globules (MGs). Experiments suggest that the off-pathway species are of helical nature and that flavodoxin-like proteins have a nonconserved transition state that determines the rate of productive folding. Folding of flavodoxin from Azotobacter vinelandii has been investigated extensively, enabling a schematic construction of its folding energy landscape. It is the only flavodoxin-like protein of which cotranslational folding has been probed. New insights that emphasize differences between in vivo and in vitro folding energy landscapes are emerging: the ribosome modulates MG formation in nascent apoflavodoxin and forces this polypeptide toward the native state. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  9. Folding of a large protein at high structural resolution.

    Science.gov (United States)

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

    2013-11-19

    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 (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.

  10. Effects of confinement on protein folding and protein stability

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Arviv, Oshrit; Levy, Yaakov

    2012-12-01

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

  12. Using motion planning to map protein folding landscapes and analyze folding kinetics of known native structures.

    Science.gov (United States)

    Amato, Nancy M; Dill, Ken A; Song, Guang

    2003-01-01

    We investigate a novel approach for studying the kinetics of protein folding. Our framework has evolved from robotics motion planning techniques called probabilistic roadmap methods (PRMs) that have been applied in many diverse fields with great success. In our previous work, we presented our PRM-based technique and obtained encouraging results studying protein folding pathways for several small proteins. In this paper, we describe how our motion planning framework can be used to study protein folding kinetics. In particular, we present a refined version of our PRM-based framework and describe how it can be used to produce potential energy landscapes, free energy landscapes, and many folding pathways all from a single roadmap which is computed in a few hours on a desktop PC. Results are presented for 14 proteins. Our ability to produce large sets of unrelated folding pathways may potentially provide crucial insight into some aspects of folding kinetics, such as proteins that exhibit both two-state and three-state kinetics that are not captured by other theoretical techniques.

  13. Co- and Post-Translational Protein Folding in the ER

    NARCIS (Netherlands)

    Ellgaard, Lars; McCaul, Nicholas|info:eu-repo/dai/nl/375286659; Chatsisvili, Anna|info:eu-repo/dai/nl/371747058; Braakman, Ineke|info:eu-repo/dai/nl/073923737

    The biophysical rules that govern folding of small, single-domain proteins in dilute solutions are now quite well understood. The mechanisms underlying co-translational folding of multidomain and membrane-spanning proteins in complex cellular environments are often less clear. The endoplasmic

  14. Fluorescence of Alexa Fluor dye tracks protein folding

    NARCIS (Netherlands)

    Lindhoud, S.; Westphal, A.H.; Borst, J.W.; Visser, A.J.W.G.; Mierlo, van C.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

  15. Identification of intermediate species in protein-folding by ...

    Indian Academy of Sciences (India)

    Identification of intermediate species in protein-folding by quantitative analysis of amplitudes in time-domain fluorescence spectroscopy ... This differentiation of a two-state from a multi-state structural transition is non-trivial when studied by the several steady-state spectroscopic methods that are popular in protein-folding ...

  16. Structure formation during translocon-unassisted co-translational membrane protein folding.

    Science.gov (United States)

    Harris, Nicola J; Reading, Eamonn; Ataka, Kenichi; Grzegorzewski, Lucjan; Charalambous, Kalypso; Liu, Xia; Schlesinger, Ramona; Heberle, Joachim; Booth, Paula J

    2017-08-14

    Correctly folded membrane proteins underlie a plethora of cellular processes, but little is known about how they fold. Knowledge of folding mechanisms centres on reversible folding of chemically denatured membrane proteins. However, this cannot replicate the unidirectional elongation of the protein chain during co-translational folding in the cell, where insertion is assisted by translocase apparatus. We show that a lipid membrane (devoid of translocase components) is sufficient for successful co-translational folding of two bacterial α-helical membrane proteins, DsbB and GlpG. Folding is spontaneous, thermodynamically driven, and the yield depends on lipid composition. Time-resolving structure formation during co-translational folding revealed different secondary and tertiary structure folding pathways for GlpG and DsbB that correlated with membrane interfacial and biological transmembrane amino acid hydrophobicity scales. Attempts to refold DsbB and GlpG from chemically denatured states into lipid membranes resulted in extensive aggregation. Co-translational insertion and folding is thus spontaneous and minimises aggregation whilst maximising correct folding.

  17. Microwave-enhanced folding and denaturation of globular proteins

    DEFF Research Database (Denmark)

    Bohr, Henrik; Bohr, Jakob

    2000-01-01

    It is shown that microwave irradiation can affect the kinetics of the folding process of some globular proteins, especially beta-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...

  18. Computational Solutions to the Protein Folding Problem,

    Science.gov (United States)

    1994-05-19

    the chemical construction of proteins. The use of knowledge such as the Ramachandran plot ( Lehninger 1970) which specifies the possible values for...Note that similar information has been developed for proteins using the Ramachandran plot ( Lehninger 1970). B. The Conformation of Heptane Heptane is an...Boston. 60 Lehninger , A.L. (1970), Biochemistqy: The Molecular Basis of Cell Structure and Func- tion, Worth Publishers, New York. Maranas, C.D. and

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

    Science.gov (United States)

    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.

  20. Fluorescence of Alexa fluor dye tracks protein folding.

    Science.gov (United States)

    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.

  1. Fluorescence of Alexa fluor dye tracks protein folding.

    Directory of Open Access Journals (Sweden)

    Simon Lindhoud

    Full Text Available 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.

  2. Chemical methods for producing disulfide bonds in peptides and proteins to study folding regulation.

    Science.gov (United States)

    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. Copyright © 2014 John Wiley & Sons, Inc.

  3. Using motion planning to study protein folding pathways.

    Science.gov (United States)

    Amato, Nancy M; Song, Guang

    2002-01-01

    We present a framework for studying protein folding pathways and potential landscapes which is based on techniques recently developed in the robotics motion planning community. Our focus in this work is to study the protein folding mechanism assuming we know the native fold. That is, instead of performing fold prediction, we aim to study issues related to the folding process, such as the formation of secondary and tertiary structure, and the dependence of the folding pathway on the initial denatured conformation. Our work uses probabilistic roadmap (PRM) motion planning techniques which have proven successful for problems involving high-dimensional configuration spaces. A strength of these methods is their efficiency in rapidly covering the planning space without becoming trapped in local minima. We have applied our PRM technique to several small proteins (~60 residues) and validated the pathways computed by comparing the secondary structure formation order on our paths to known hydrogen exchange experimental results. An advantage of the PRM framework over other simulation methods is that it enables one to easily and efficiently compute folding pathways from any denatured starting state to the (known) native fold. This aspect makes our approach ideal for studying global properties of the protein's potential landscape, most of which are difficult to simulate and study with other methods. For example, in the proteins we study, the folding pathways starting from different denatured states sometimes share common portions when they are close to the native fold, and moreover, the formation order of the secondary structure appears largely independent of the starting denatured conformation. Another feature of our technique is that the distribution of the sampled conformations is correlated with the formation of secondary structure and, in particular, appears to differentiate situations in which secondary structure clearly forms first and those in which the tertiary

  4. A PDZ domain recapitulates a unifying mechanism for protein folding

    NARCIS (Netherlands)

    Gianni, S.; Geierhaas, C.D.; Calosci, N.; Jemth, P.; Vuister, G.W.; Travaglini-Allocatelli, C.; Vendruscolo, M.; Brunori, M.

    2007-01-01

    A unifying view has been recently proposed according to which the classical diffusion-collision and nucleation-condensation models may represent two extreme manifestations of an underlying common mechanism for the folding of small globular proteins. We report here the characterization of the folding

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

    Science.gov (United States)

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

    2013-01-01

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

  6. Mechanical Modeling and Computer Simulation of Protein Folding

    Science.gov (United States)

    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…

  7. Protein Collapse is Encoded in the Folded State Architecture

    CERN Document Server

    Samanta, Himadri S; Hinczewski, Michael; Hori, Naoto; Chakrabarti, Shaon; Thirumalai, D

    2016-01-01

    Natural protein sequences that self-assemble to form globular structures are compact with high packing densities in the folded states. It is known that proteins unfold upon addition of denaturants, adopting random coil structures. The dependence of the radii of gyration on protein size in the folded and unfolded states obeys the same scaling laws as synthetic polymers. Thus, one might surmise that the mechanism of collapse in proteins and polymers ought to be similar. However, because the number of amino acids in single domain proteins is not significantly greater than about two hundred, it has not been resolved if the unfolded states of proteins are compact under conditions that favor the folded states - a problem at the heart of how proteins fold. By adopting a theory used to derive polymer-scaling laws, we find that the propensity for the unfolded state of a protein to be compact is universal and is encoded in the contact map of the folded state. Remarkably, analysis of over 2000 proteins shows that protei...

  8. Lymphotactin: How a protein can adopt two folds

    Science.gov (United States)

    Camilloni, Carlo; Sutto, Ludovico

    2009-12-01

    Metamorphic proteins such as lymphotactin are a notable exception of the empirical principle that structured natural proteins possess a unique three-dimensional structure. In particular, the human chemokine lymphotactin protein exists in two distinct conformations (one monomeric and one dimeric) under physiological conditions. In this work, we use a Cα Go¯ model to show how this very peculiar behavior can be reproduced. From the study of the thermodynamics and of the kinetics, we characterize the interconversion mechanism. In particular, this takes place through the docking of the two chains living in a third monomeric, partially unfolded, state which shows a residual structure involving a set of local contacts common to the two native conformations. The main feature of two fold proteins appears to be the sharing of a common set of local contacts between the two distinct folds as confirmed by the study of two designed two fold proteins. Metamorphic proteins may be more common than expected.

  9. Solitons and protein folding: An In Silico experiment

    Energy Technology Data Exchange (ETDEWEB)

    Ilieva, N., E-mail: nevena.ilieva@parallel.bas.bg [Institute of Information and Communication Technologies, Bulgarian Aacademy of Sciences, Sofia (Bulgaria); Dai, J., E-mail: daijing491@gmail.com [School of Physics, Beijing Institute of Technology, Beijing (China); Sieradzan, A., E-mail: adams86@wp.pl [Faculty of Chemistry, University of Gdańsk, Gdańsk (Poland); Niemi, A., E-mail: Antti.Niemi@physics.uu.se [Department of Physics and Astronomy, Uppsala University, Uppsala (Sweden); LMPT–CNRS, Université de Tours, Tours (France)

    2015-10-28

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

  10. Folding of Aggregated Proteins to Functionally Active Form

    Science.gov (United States)

    2006-06-01

    Advances in the production of human therapeutic proteins in yeasts and filamentous fungi . Nat Biotechnol 2004, 22:1409-1414. 4. Wildt S, Gerngross TU: The...Folding aggregated proteins into functionally active forms Wieslaw SwietnickiThe successful expression and purification of proteins in an active form...number of proteins are being identified as potential drug targets but are difficult to obtain in a form suitable for structural or biochemical studies

  11. Folding of small proteins using constrained molecular dynamics.

    Science.gov (United States)

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

    2011-06-16

    The focus of this paper is to examine whether conformational search using constrained molecular dynamics (MD) method is more enhanced and enriched toward "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 time scale 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 co-workers 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. © 2011 American Chemical Society

  12. Topological aspects of DNA function and protein folding.

    Science.gov (United States)

    Stasiak, Andrzej; Bates, Andrew D; Buck, Dorothy E; Harris, Sarah A; Sumners, De Witt

    2013-04-01

    The Topological Aspects of DNA Function and Protein Folding international meeting provided an interdisciplinary forum for biological scientists, physicists and mathematicians to discuss recent developments in the application of topology to the study of DNA and protein structure. It had 111 invited participants, 48 talks and 21 posters. The present article discusses the importance of topology and introduces the articles from the meeting's speakers.

  13. Protein folding and misfolding shining light by infrared spectroscopy

    CERN Document Server

    Fabian, Heinz

    2012-01-01

    Infrared spectroscopy is a new and innovative technology to study protein folding/misfolding events in the broad arsenal of techniques conventionally used in this field. The progress in understanding protein folding and misfolding is primarily due to the development of biophysical methods which permit to probe conformational changes with high kinetic and structural resolution. The most commonly used approaches rely on rapid mixing methods to initiate the folding event via a sudden change in solvent conditions. Traditionally, techniques such as fluorescence, circular dichroism or visible absorption are applied to probe the process. In contrast to these techniques, infrared spectroscopy came into play only very recently, and the progress made in this field up to date which now permits to probe folding events over the time scale from picoseconds to minutes has not yet been discussed in a book. The aim of this book is to provide an overview of the developments as seen by some of the main contributors to the field...

  14. Folding landscapes of ankyrin repeat proteins: experiments meet theory.

    Science.gov (United States)

    Barrick, Doug; Ferreiro, Diego U; Komives, Elizabeth A

    2008-02-01

    Nearly 6% of eukaryotic protein sequences contain ankyrin repeat (AR) domains, which consist of several repeats and often function in binding. AR proteins show highly cooperative folding despite a lack of long-range contacts. Both theory and experiment converge to explain that formation of the interface between elements is more favorable than formation of any individual repeat unit. IkappaBalpha and Notch both undergo partial folding upon binding perhaps influencing the binding free energy. The simple architecture, combined with identification of consensus residues that are important for stability, has enabled systematic perturbation of the energy landscape by single point mutations that affect stability or by addition of consensus repeats. The folding energy landscapes appear highly plastic, with small perturbations re-routing folding pathways.

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

    CERN Document Server

    Zhang, Yuanzhao; 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 ({\\alpha}-helix, \\b{eta}-hairpin, {\\alpha}-helix bundle, and {\\alpha}/\\b{eta}-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 in influenced by protein size and the flexibility of the native hydrophobic core. For \\b{eta}-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 \\b{eta}-turn region. This systematic look at reverse sequence duality sheds new light on t...

  16. Fluctuations within folded proteins: implications for thermodynamic and allosteric regulation.

    Science.gov (United States)

    DuBay, Kateri H; Bowman, Gregory R; Geissler, Phillip L

    2015-04-21

    Folded protein structures are both stable and dynamic. Historically, our clearest window into these structures came from X-ray crystallography, which generally provided a static image of each protein's singular "folded state", highlighting its stability. Deviations away from that crystallographic structure were difficult to quantify, and as a result, their potential functional consequences were often neglected. However, several dynamical and statistical studies now highlight the structural variability that is present within the protein's folded state. Here we review mounting evidence of the importance of these structural rearrangements; both experiment and computation indicate that folded proteins undergo substantial fluctuations that can greatly influence their function. Crucially, recent studies have shown that structural elements of proteins, especially their side-chain degrees of freedom, fluctuate in ways that generate significant conformational heterogeneity. The entropy associated with these motions contributes to the folded structure's thermodynamic stability. In addition, since these fluctuations can shift in response to perturbations such as ligand binding, they may play an important role in the protein's capacity to respond to environmental cues. In one compelling example, the entropy associated with side-chain fluctuations contributes significantly to regulating the binding of calmodulin to a set of peptide ligands. The neglect of fluctuations within proteins' native states was often justified by the dense packing within folded proteins, which has inspired comparisons with crystalline solids. Many liquids, however, can achieve similarly dense packing yet fluidity is maintained through correlated molecular motions. Indeed, the studies we discuss favor comparison of folded proteins not with solids but instead with dense liquids, where the internal side chain fluidity is facilitated by collective motions that are correlated over long distances. These

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

    Science.gov (United States)

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

    2012-01-27

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

  18. Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

    Science.gov (United States)

    Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

    2016-01-12

    We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.

  19. Aggregation of natively folded proteins: a theoretical approach

    Energy Technology Data Exchange (ETDEWEB)

    Trovato, Antonio [CNISM, Unita di Padova, Via Marzolo 8, 35131 Padova (Italy); Maritan, Amos [CNISM, Unita di Padova, Via Marzolo 8, 35131 Padova (Italy); Seno, Flavio [CNISM, Unita di Padova, Via Marzolo 8, 35131 Padova (Italy)

    2007-07-18

    The reliable identification of {beta}-aggregating stretches in protein sequences is essential for the development of therapeutic agents for Alzheimer's and Parkinson's diseases, as well as other pathological conditions associated with protein deposition. While the list of aggregation related diseases is growing, it has also been shown that many proteins that are normally well behaved can be induced to aggregate in vitro. This fact suggests the existence of a unified framework that could explain both folding and aggregation. By assuming this universal behaviour, we have recently introduced an algorithm (PASTA: prediction of amyloid structure aggregation), which is based on a sequence-specific energy function derived from the propensity of two residue types to be found paired in neighbouring strands within {beta}-sheets in globular proteins. The algorithm is able to predict the most aggregation-prone portions of several proteins initially unfolded, in excellent agreement with experimental results. Here, we apply the method to a set of proteins which are known to aggregate, but which are natively folded. The quality of the prediction is again very high, corroborating the hypothesis that the amyloid structure is stabilized by the same physico-chemical determinants as those operating in folded proteins.

  20. Kinetics and thermodynamics of the thermal inactivation and chaperone assisted folding of zebrafish dihydrofolate reductase.

    Science.gov (United States)

    Thapliyal, Charu; Jain, Neha; Rashid, Naira; Chaudhuri Chattopadhyay, Pratima

    2017-11-11

    The maintenance of thermal stability is a major issue in protein engineering as many proteins tend to form inactive aggregates at higher temperatures. Zebrafish DHFR, an essential protein for the survival of cells, shows irreversible thermal unfolding transition. The protein exhibits complete unfolding and loss of activity at 50 °C as monitored by UV-Visible, fluorescence and far UV-CD spectroscopy. The heat induced inactivation of zDHFR follows first-order kinetics and Arrhenius law. The variation in the value of inactivation rate constant, k with increasing temperatures depicts faster inactivation at elevated temperatures. We have attempted to study the chaperoning ability of a shorter variant of GroEL (minichaperone) and compared it with that of conventional GroEL-GroES chaperone system. Both the chaperone system prevented the aggregation and assisted in refolding of zDHFR. The rate of thermal inactivation was significantly retarded in the presence of chaperones which indicate that it enhances the thermal stability of the enzyme. As minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Identification of intermediate species in protein-folding by ...

    Indian Academy of Sciences (India)

    TECS

    Identification of intermediate species in protein-folding by quantitative analysis of amplitudes in time-domain fluorescence spectroscopy. ANOOP M SAXENA,. 1. G KRISHNAMOORTHY,. 1,. * JAYANT B UDGAONKAR. 2 and. N PERIASAMY. 1. *. 1. Department of Chemical Sciences, Tata Institute of Fundamental Research, ...

  2. Cotranslational protein folding reveals the selective use of ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 93; Issue 3. Cotranslational protein folding reveals the selective use of synonymous codons along the coding sequence of a low expression gene. Suvendra Kumar Ray Vishwa Jyoti Baruah Siddhartha Sankar Satapathy Rajat Banerjee. Research Commentary Volume 93 Issue ...

  3. Probing folding free energy landscape of small proteins through ...

    Indian Academy of Sciences (India)

    Unknown

    folding. A protein could be thought of as a heteropolymer, consisting of mainly hydro- phobic and hydrophilic amino acid residues. The water repellent hydrophobic residues are the ones that drive the initial collapse. Many theoretical studies have been carried out. ¶Dedicated to Professor C N R Rao on his 70th birthday.

  4. A 9-state hidden Markov model using protein secondary structure information for protein fold recognition.

    Science.gov (United States)

    Lee, Sun Young; Lee, Jong Yun; Jung, Kwang Su; Ryu, Keun Ho

    2009-06-01

    In protein fold recognition, the main disadvantage of hidden Markov models (HMMs) is the employment of large-scale model architectures which require large data sets and high computational resources for training. Also, HMMs must consider sequential information about secondary structures of proteins, to improve prediction performance and reduce model parameters. Therefore, we propose a novel method for protein fold recognition based on a hidden Markov model, called a 9-state HMM. The method can (i) reduce the number of states using secondary structure information about proteins for each fold and (ii) recognize protein folds more accurately than other HMMs.

  5. Is Poisson-Boltzmann theory insufficient for protein folding simulations?

    Science.gov (United States)

    Lwin, Thu Zar; Zhou, Ruhong; Luo, Ray

    2006-01-21

    The Poisson-Boltzmann theory has been widely used in the studies of energetics and conformations of biological macromolecules. Recently, introduction of the efficient generalized Born approximation has greatly extended its applicability to areas such as protein folding simulations where highly efficient computation is crucial. However, limitations have been found in the folding simulations of a well-studied beta hairpin with several generalized Born implementations and different force fields. These studies have raised the question whether the underlining Poisson-Boltzmann theory, on which the generalized Born model is calibrated, is adequate in the treatment of polar interactions for the challenging protein folding simulations. To address the question whether the Poisson-Boltzmann theory in the current formalism might be insufficient, we directly tested our efficient numerical Poisson-Boltzmann implementation in the beta-hairpin folding simulation. Good agreement between simulation and experiment was found for the beta-hairpin equilibrium structures when the numerical Poisson-Boltzmann solvent and a recently improved generalized Born solvent were used. In addition simulated thermodynamic properties also agree well with experiment in both solvents. Finally, an overall agreement on the beta-hairpin folding mechanism was found between the current and previous studies. Thus, our simulations indicate that previously observed limitations are most likely due to imperfect calibration in previous generalized Born models but not due to the limitation of the Poisson-Boltzmann theory.

  6. Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway.

    Directory of Open Access Journals (Sweden)

    Alistair G Irvine

    Full Text Available In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding. However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10(-5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding - differential affinity, rapid ligand exchange and conformational flexibility.

  7. Visualization of protein folding funnels in lattice models.

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yaman Arkun

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

  9. Fold Recognition Using Sequence Fingerprints of Protein Local Substructures

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Enhanced sampling and applications in protein folding in explicit solvent

    Science.gov (United States)

    Zhang, Cheng; Ma, Jianpeng

    2010-01-01

    We report a single-copy tempering method for simulating large complex systems. In a generalized ensemble, the method uses runtime estimate of the thermal average energy computed from a novel integral identity to guide a continuous temperature-space random walk. We first validated the method in a two-dimensional Ising model and a Lennard-Jones liquid system. It was then applied to folding of three small proteins, trpzip2, trp-cage, and villin headpiece in explicit solvent. Within 0.5∼1 microsecond, all three systems were reversibly folded into atomic accuracy: the alpha carbon root mean square deviations of the best folded conformations from the native states were 0.2, 0.4, and 0.4 Å, for trpzip2, trp-cage, and villin headpiece, respectively. PMID:20590175

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

    Science.gov (United States)

    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/

  12. Theoretical and computational studies in protein folding, design, and function

    Science.gov (United States)

    Morrissey, Michael Patrick

    2000-10-01

    In this work, simplified statistical models are used to understand an array of processes related to protein folding and design. In Part I, lattice models are utilized to test several theories about the statistical properties of protein-like systems. In Part II, sequence analysis and all-atom simulations are used to advance a novel theory for the behavior of a particular protein. Part I is divided into five chapters. In Chapter 2, a method of sequence design for model proteins, based on statistical mechanical first-principles, is developed. The cumulant design method uses a mean-field approximation to expand the free energy of a sequence in temperature. The method successfully designs sequences which fold to a target lattice structure at a specific temperature, a feat which was not possible using previous design methods. The next three chapters are computational studies of the double mutant cycle, which has been used experimentally to predict intra-protein interactions. Complete structure prediction is demonstrated for a model system using exhaustive, and also sub-exhaustive, double mutants. Nonadditivity of enthalpy, rather than of free energy, is proposed and demonstrated to be a superior marker for inter-residue contact. Next, a new double mutant protocol, called exchange mutation, is introduced. Although simple statistical arguments predict exchange mutation to be a more accurate contact predictor than standard mutant cycles, this hypothesis was not upheld in lattice simulations. Reasons for this inconsistency will be discussed. Finally, a multi-chain folding algorithm is introduced. Known as LINKS, this algorithm was developed to test a method of structure prediction which utilizes chain-break mutants. While structure prediction was not successful, LINKS should nevertheless be a useful tool for the study of protein-protein and protein-ligand interactions. The last chapter of Part I utilizes the lattice to explore the differences between standard folding, from

  13. Monitoring protein folding through high pressure NMR spectroscopy.

    Science.gov (United States)

    Roche, Julien; Royer, Catherine A; Roumestand, Christian

    2017-11-01

    High-pressure is a well-known perturbation method used to destabilize globular proteins. It is perfectly reversible, which is essential for a proper thermodynamic characterization of a protein equilibrium. In contrast to other perturbation methods such as heat or chemical denaturant that destabilize protein structures uniformly, pressure exerts local effects on regions or domains of a protein containing internal cavities. When combined with NMR spectroscopy, hydrostatic pressure offers the possibility to monitor at a residue level the structural transitions occurring upon unfolding and to determine the kinetic properties of the process. High-pressure NMR experiments can now be routinely performed, owing to the recent development of commercially available high-pressure sample cells. This review summarizes recent advances and some future directions of high-pressure NMR techniques for the characterization at atomic resolution of the energy landscape of protein folding. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Acceleration through passive destabilization: protein folding in a weak hydrophobic environment

    Science.gov (United States)

    Jewett, Andrew; Baumketner, Andrij; Shea, Joan-Emma

    2004-03-01

    The GroEL chaperonin is a biomolecule which assists the folding of an extremely diverse range of proteins in Eubacteria. Some proteins undergo many rounds of ATP-regulated binding and dissociation from GroEL/ES before folding. It has been proposed that transient stress from ATP-regulated binding and release from GroEL/ES frees frustrated proteins from misfolded conformations. However recent evidence suggests that chaperonin-accelerated protein folding can take place entirely within a mutated GroEL+ES cavity that is unable to open and release the protein. Using molecular dynamics, we demonstrate that static confinement within a weakly hydrophobic (attractive) cavity (similar to the interior of the cavity formed by the GroEL+ES complex) is sufficient to significantly accelerate the folding of a highly frustrated protein-like heteropolymer. Our frustrated molecule benifits kinetically from a static hydrophobic environment that destabilizes misfolded conformations. This may shed light on the mechanisms used by other chaperones which do not depend on ATP.

  15. A folding pathway-dependent score to recognize membrane proteins.

    Directory of Open Access Journals (Sweden)

    Hamid Hadi-Alijanvand

    Full Text Available While various approaches exist to study protein localization, it is still a challenge to predict where proteins localize. Here, we consider a mechanistic viewpoint for membrane localization. Taking into account the steps for the folding pathway of α-helical membrane proteins and relating biophysical parameters to each of these steps, we create a score capable of predicting the propensity for membrane localization and call it FP(3mem. This score is driven from the principal component analysis (PCA of the biophysical parameters related to membrane localization. FP(3mem allows us to rationalize the colocalization of a number of channel proteins with the Cav1.2 channel by their fewer propensities for membrane localization.

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

    Science.gov (United States)

    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

  17. Multiple folding pathways for heterologously expressed human prion protein.

    Science.gov (United States)

    Jackson, G S; Hill, A F; Joseph, C; Hosszu, L; Power, A; Waltho, J P; Clarke, A R; Collinge, J

    1999-04-12

    Human PrP (residues 91-231) expressed in Escherichia coli can adopt several conformations in solution depending on pH, redox conditions and denaturant concentration. Oxidised PrP at neutral pH, with the disulphide bond intact, is a soluble monomer which contains 47% alpha-helix and corresponds to PrPC. Denaturation studies show that this structure has a relatively small, solvent-excluded core and unfolds to an unstructured state in a single, co-operative transition with a DeltaG for folding of -5.6 kcal mol-1. The unfolding behaviour is sensitive to pH and at 4.0 or below the molecule unfolds via a stable folding intermediate. This equilibrium intermediate has a reduced helical content and aggregates over several hours. When the disulphide bond is reduced the protein adopts different conformations depending upon pH. At neutral pH or above, the reduced protein has an alpha-helical fold, which is identical to that observed for the oxidised protein. At pH 4 or below, the conformation rearranges to a fold that contains a high proportion of beta-sheet structure. In the reduced state the alpha- and beta-forms are slowly inter-convertible whereas when oxidised the protein can only adopt an alpha-conformation in free solution. The data we present here shows that the human prion protein can exist in multiple conformations some of which are known to be capable of forming fibrils. The precise conformation that human PrP adopts and the pathways for unfolding are dependent upon solvent conditions. The conditions we examined are within the range that a protein may encounter in sub-cellular compartments and may have implications for the mechanism of conversion of PrPC to PrPSc in vivo. Since the conversion of PrPC to PrPSc is accompanied by a switch in secondary structure from alpha to beta, this system provides a useful model for studying major structural rearrangements in the prion protein.

  18. Benchmarking consensus model quality assessment for protein fold recognition

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    McGuffin Liam J

    2007-09-01

    Full Text Available Abstract Background Selecting the highest quality 3D model of a protein structure from a number of alternatives remains an important challenge in the field of structural bioinformatics. Many Model Quality Assessment Programs (MQAPs have been developed which adopt various strategies in order to tackle this problem, ranging from the so called "true" MQAPs capable of producing a single energy score based on a single model, to methods which rely on structural comparisons of multiple models or additional information from meta-servers. However, it is clear that no current method can separate the highest accuracy models from the lowest consistently. In this paper, a number of the top performing MQAP methods are benchmarked in the context of the potential value that they add to protein fold recognition. Two novel methods are also described: ModSSEA, which based on the alignment of predicted secondary structure elements and ModFOLD which combines several true MQAP methods using an artificial neural network. Results The ModSSEA method is found to be an effective model quality assessment program for ranking multiple models from many servers, however further accuracy can be gained by using the consensus approach of ModFOLD. The ModFOLD method is shown to significantly outperform the true MQAPs tested and is competitive with methods which make use of clustering or additional information from multiple servers. Several of the true MQAPs are also shown to add value to most individual fold recognition servers by improving model selection, when applied as a post filter in order to re-rank models. Conclusion MQAPs should be benchmarked appropriately for the practical context in which they are intended to be used. Clustering based methods are the top performing MQAPs where many models are available from many servers; however, they often do not add value to individual fold recognition servers when limited models are available. Conversely, the true MQAP methods

  19. Entropic formulation for the protein folding process: Hydrophobic stability correlates with folding rates

    Science.gov (United States)

    Dal Molin, J. P.; Caliri, A.

    2018-01-01

    Here we focus on the conformational search for the native structure when it is ruled by the hydrophobic effect and steric specificities coming from amino acids. Our main tool of investigation is a 3D lattice model provided by a ten-letter alphabet, the stereochemical model. This minimalist model was conceived for Monte Carlo (MC) simulations when one keeps in mind the kinetic behavior of protein-like chains in solution. We have three central goals here. The first one is to characterize the folding time (τ) by two distinct sampling methods, so we present two sets of 103 MC simulations for a fast protein-like sequence. The resulting sets of characteristic folding times, τ and τq were obtained by the application of the standard Metropolis algorithm (MA), as well as by an enhanced algorithm (Mq A). The finding for τq shows two things: (i) the chain-solvent hydrophobic interactions {hk } plus a set of inter-residues steric constraints {ci,j } are able to emulate the conformational search for the native structure. For each one of the 103MC performed simulations, the target is always found within a finite time window; (ii) the ratio τq / τ ≅ 1 / 10 suggests that the effect of local thermal fluctuations, encompassed by the Tsallis weight, provides to the chain an innate efficiency to escape from energetic and steric traps. We performed additional MC simulations with variations of our design rule to attest this first result, both algorithms the MA and the Mq A were applied to a restricted set of targets, a physical insight is provided. Our second finding was obtained by a set of 600 independent MC simulations, only performed with the Mq A applied to an extended set of 200 representative targets, our native structures. The results show how structural patterns should modulate τq, which cover four orders of magnitude; this finding is our second goal. The third, and last result, was obtained with a special kind of simulation performed with the purpose to explore a

  20. Calculation of the free energy and cooperativity of protein folding.

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    Alex Kentsis

    2007-05-01

    Full Text Available Calculation of the free energy of protein folding and delineation of its pre-organization are of foremost importance for understanding, predicting and designing biological macromolecules. Here, we introduce an energy smoothing variant of parallel tempering replica exchange Monte Carlo (REMS that allows for efficient configurational sampling of flexible solutes under the conditions of molecular hydration. Its usage to calculate the thermal stability of a model globular protein, Trp cage TC5b, achieves excellent agreement with experimental measurements. We find that the stability of TC5b is attained through the coupled formation of local and non-local interactions. Remarkably, many of these structures persist at high temperature, concomitant with the origin of native-like configurations and mesostates in an otherwise macroscopically disordered unfolded state. Graph manifold learning reveals that the conversion of these mesostates to the native state is structurally heterogeneous, and that the cooperativity of their formation is encoded largely by the unfolded state ensemble. In all, these studies establish the extent of thermodynamic and structural pre-organization of folding of this model globular protein, and achieve the calculation of macromolecular stability ab initio, as required for ab initio structure prediction, genome annotation, and drug design.

  1. Mapping the Geometric Evolution of Protein Folding Motor.

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    Gaurav Jerath

    Full Text Available 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.

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

    Science.gov (United States)

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

    2008-12-02

    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.

  3. Is tensegrity a unifying concept of protein folds?

    Science.gov (United States)

    Zanotti, Giuseppe; Guerra, Concettina

    2003-01-16

    We suggest that the three-dimensional architecture of globular proteins can be described in terms of tensegrets, i.e. structural elements that are held together through attractive and repulsive forces. Hard elements of tensegrets are represented by secondary structure elements, i.e. alpha-helices and beta-strands, while the role of elastic elements is played by attractive and repulsive atomic forces. Characteristics of tensegrets is that they can auto-assemble and that they respond to changes of tension in some part of the entire object through a deformation in another part, thus partially preserving their structure, despite their deformation. This latter property well explains both the folding process and the behavior of globular proteins under mild denaturing conditions, as revealed by the molten globule state.

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

    Science.gov (United States)

    Dyson, H. Jane

    2011-01-01

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

  5. Species-specific protein sequence and fold optimizations

    Directory of Open Access Journals (Sweden)

    Michalickova Katerina

    2002-12-01

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

  6. Using robotics to fold proteins and dock ligands.

    Science.gov (United States)

    Brutlag, Douglas; Apaydin, Serkan; Guestrin, Carlos; Hsu, David; Varma, Chris; Singh, Amit; Latombe, Jean-Claude

    2002-01-01

    The problems of protein folding and ligand docking have been explored largely using molecular dynamics or Monte Carlo methods. These methods are very compute intensive because they often explore a much wider range of energies, conformations and time than necessary. In addition, Monte Carlo methods often get trapped in local minima. We initially showed that robotic motion planning permitted one to determine the energy of binding and dissociation of ligands from protein binding sites (Singh et al., 1999). The robotic motion planning method maps complicated three-dimensional conformational states into a much simpler, but higher dimensional space in which conformational rearrangements can be represented as linear paths. The dimensionality of the conformation space is of the same order as the number of degrees of conformational freedom in three-dimensional space. We were able to determine the relative energy of association and dissociation of a ligand to a protein by calculating the energetics of interaction for a few thousand conformational states in the vicinity of the protein and choosing the best path from the roadmap. More recently, we have applied roadmap planning to the problem of protein folding (Apaydin et al., 2002a). We represented multiple conformations of a protein as nodes in a compact graph with the edges representing the probability of moving between neighboring states. Instead of using Monte Carlo simulation to simulate thousands of possible paths through various conformational states, we were able to use Markov methods to calculate the steady state occupancy of each conformation, needing to calculate the energy of each conformation only once. We referred to this Markov method of representing multiple conformations and transitions as stochastic roadmap simulation or SRS. We demonstrated that the distribution of conformational states calculated with exhaustive Monte Carlo simulations asymptotically approached the Markov steady state if the same Boltzman

  7. Statistical analyses of protein folding rates from the view of quantum transition.

    Science.gov (United States)

    Lv, Jun; Luo, LiaoFu

    2014-12-01

    Understanding protein folding rate is the primary key to unlock the fundamental physics underlying protein structure and its folding mechanism. Especially, the temperature dependence of the folding rate remains unsolved in the literature. Starting from the assumption that protein folding is an event of quantum transition between molecular conformations, we calculated the folding rate for all two-state proteins in a database and studied their temperature dependencies. The non-Arrhenius temperature relation for 16 proteins, whose experimental data had previously been available, was successfully interpreted by comparing the Arrhenius plot with the first-principle calculation. A statistical formula for the prediction of two-state protein folding rate was proposed based on quantum folding theory. The statistical comparisons of the folding rates for 65 two-state proteins were carried out, and the theoretical vs. experimental correlation coefficient was 0.73. Moreover, the maximum and the minimum folding rates given by the theory were consistent with the experimental results.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  9. Chaotic Multiquenching Annealing Applied to the Protein Folding Problem

    Directory of Open Access Journals (Sweden)

    Juan Frausto-Solis

    2014-01-01

    Full Text Available The Chaotic Multiquenching Annealing algorithm (CMQA is proposed. CMQA is a new algorithm, which is applied to protein folding problem (PFP. This algorithm is divided into three phases: (i multiquenching phase (MQP, (ii annealing phase (AP, and (iii dynamical equilibrium phase (DEP. MQP enforces several stages of quick quenching processes that include chaotic functions. The chaotic functions can increase the exploration potential of solutions space of PFP. AP phase implements a simulated annealing algorithm (SA with an exponential cooling function. MQP and AP are delimited by different ranges of temperatures; MQP is applied for a range of temperatures which goes from extremely high values to very high values; AP searches for solutions in a range of temperatures from high values to extremely low values. DEP phase finds the equilibrium in a dynamic way by applying least squares method. CMQA is tested with several instances of PFP.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  11. Electrostatics, structure prediction, and the energy landscapes for protein folding and binding.

    Science.gov (United States)

    Tsai, Min-Yeh; Zheng, Weihua; Balamurugan, D; Schafer, Nicholas P; Kim, Bobby L; Cheung, Margaret S; Wolynes, Peter G

    2016-01-01

    While being long in range and therefore weakly specific, electrostatic interactions are able to modulate the stability and folding landscapes of some proteins. The relevance of electrostatic forces for steering the docking of proteins to each other is widely acknowledged, however, the role of electrostatics in establishing specifically funneled landscapes and their relevance for protein structure prediction are still not clear. By introducing Debye-Hückel potentials that mimic long-range electrostatic forces into the Associative memory, Water mediated, Structure, and Energy Model (AWSEM), a transferable protein model capable of predicting tertiary structures, we assess the effects of electrostatics on the landscapes of thirteen monomeric proteins and four dimers. For the monomers, we find that adding electrostatic interactions does not improve structure prediction. Simulations of ribosomal protein S6 show, however, that folding stability depends monotonically on electrostatic strength. The trend in predicted melting temperatures of the S6 variants agrees with experimental observations. Electrostatic effects can play a range of roles in binding. The binding of the protein complex KIX-pKID is largely assisted by electrostatic interactions, which provide direct charge-charge stabilization of the native state and contribute to the funneling of the binding landscape. In contrast, for several other proteins, including the DNA-binding protein FIS, electrostatics causes frustration in the DNA-binding region, which favors its binding with DNA but not with its protein partner. This study highlights the importance of long-range electrostatics in functional responses to problems where proteins interact with their charged partners, such as DNA, RNA, as well as membranes. © 2015 The Protein Society.

  12. Analyzing the Folding and Binding Steps of an Intrinsically Disordered Protein by Protein Engineering.

    Science.gov (United States)

    Bonetti, Daniela; Troilo, Francesca; Toto, Angelo; Brunori, Maurizio; Longhi, Sonia; Gianni, Stefano

    2017-07-25

    Intrinsically disordered proteins (IDPs) are functionally active despite lacking a well-defined three-dimensional structure. Such proteins often undergo a disorder-to-order transition, or induced folding, when binding to their specific physiological partner. Because of cooperativity, the folding and binding steps typically appear as a single event, and therefore, induced folding is extremely difficult to characterize experimentally. In this perspective, the interaction between the disordered C-terminal domain of the measles virus nucleoprotein NTAIL and the folded X domain of the viral phosphoprotein (XD) is particularly interesting because the inherent complexity of the observed kinetics allows characterization of the binding and folding steps individually. Here we present a detailed structural description of the folding and binding events occurring in the recognition between NTAIL and XD. This result was achieved by measuring the effect of single-amino acid substitutions in NTAIL on the reaction mechanism. Analysis of the experimental data allowed us (i) to identify the key residues involved in the initial recognition between the two molecules and (ii) to depict the general features of the folding pathway of NTAIL. Furthermore, an analysis of the changes in stability obtained for the whole set of variants highlights how the sequence of this IDP has not been selected during evolution to fold efficiently. This feature might be a consequence of the weakly funneled nature of the energy landscape of IDPs in their unbound state and represents a plausible explanation of their highly dynamic nature even in the bound state, typically defined as "fuzziness".

  13. A simple quantitative model of macromolecular crowding effects on protein folding: Application to the murine prion protein(121-231)

    Science.gov (United States)

    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.

  14. Universal distribution of protein evolution rates as a consequence of protein folding physics.

    Science.gov (United States)

    Lobkovsky, Alexander E; Wolf, Yuri I; Koonin, Eugene V

    2010-02-16

    The hypothesis that folding robustness is the primary determinant of the evolution rate of proteins is explored using a coarse-grained off-lattice model. The simplicity of the model allows rapid computation of the folding probability of a sequence to any folded conformation. For each robust folder, the network of sequences that share its native structure is identified. The fitness of a sequence is postulated to be a simple function of the number of misfolded molecules that have to be produced to reach a characteristic protein abundance. After fixation probabilities of mutants are computed under a simple population dynamics model, a Markov chain on the fold network is constructed, and the fold-averaged evolution rate is computed. The distribution of the logarithm of the evolution rates across distinct networks exhibits a peak with a long tail on the low rate side and resembles the universal empirical distribution of the evolutionary rates more closely than either distribution resembles the log-normal distribution. The results suggest that the universal distribution of the evolutionary rates of protein-coding genes is a direct consequence of the basic physics of protein folding.

  15. Protein-disulphide isomerase and prolyl isomerase act differently and independently as catalysts of protein folding.

    Science.gov (United States)

    Lang, K; Schmid, F X

    1988-02-04

    Two enzymes are now known that catalyse slow steps in protein folding. Peptidyl-prolyl cis-trans isomerase catalyses the cis-trans isomerization of Xaa-Pro peptide bonds in oligopeptides and during the refolding of several proteins. The other enzyme, protein-disulphide isomerase, accelerates the reactivation of reduced proteins, presumably by catalysis of thiol-disulphide exchange reactions. Recent evidence indicates that the beta-subunit of prolyl 4-hydroxylase, an enzyme involved in collagen biosynthesis, is identical with disulphide isomerase. On the basis of this important finding, it was suggested that disulphide isomerase accelerates protein folding, not by 'reshuffling' incorrect disulphide bonds, but in the same way as prolyl isomerase by catalysing proline isomerization which is known to be important for the folding of collagen and other proteins. Here we show that the catalytic activities of these two enzymes are different. Disulphide isomerase accelerates the reformation of native disulphide bonds during protein reoxidation. We find no evidence that this enzyme can catalyse the isomerization of proline peptide bonds, a reaction efficiently accelerated by prolyl isomerase. When both enzymes are present simultaneously during protein folding, they act independently of one another.

  16. Protein folding and protein metallocluster studies using synchrotron small angler X-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

    Science.gov (United States)

    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.

  18. Engineering protein folding and translocation improves heterologous protein secretion in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tang, Hongting; Bao, Xiaoming; Shen, Yu; Song, Meihui; Wang, Shenghuan; Wang, Chengqiang; Hou, Jin

    2015-09-01

    Saccharomyces cerevisiae is widely used as a producer of heterologous proteins of medical and industrial interest. Numerous efforts have been made to overcome bottlenecks in protein expression and secretion. However, the effect of engineering protein translocation to heterologous protein secretion has not been studied extensively in S. cerevisiae. In this work, we confirmed that heterologous protein expression in S. cerevisiae induced the unfolded protein response (UPR). To enhance protein folding capacity, the endoplasmic reticulum (ER) chaperone protein BiP and the disulfide isomerase Pdi1p were each over-expressed, and the secretion of three heterologous proteins, β-glucosidase, endoglucanase, and α-amylase, was improved. The impact of engineering key translocation components was also studied. The over-expression of co-translational translocation components Srp14p and Srp54p enhanced the secretion of three heterologous proteins (β-glucosidase, endoglucanase, and α-amylase), but over-expressing the cytosolic chaperone Ssa1p (involved in post-translational translocation) only enhanced the secretion of β-glucosidase. By engineering both co-translational translocation and protein folding, we obtained strains with β-glucosidase, endoglucanase, and α-amylase activities increased by 72%, 60%, and 103% compared to the controls. Our results show that protein translocation may be a limiting factor for heterologous protein production. © 2015 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    Andreeva, Antonina; Murzin, Alexey G

    2010-10-01

    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.

  20. Predicting protein folding pathways at the mesoscopic level based on native interactions between secondary structure elements

    Directory of Open Access Journals (Sweden)

    Sze Sing-Hoi

    2008-07-01

    Full Text Available Abstract Background Since experimental determination of protein folding pathways remains difficult, computational techniques are often used to simulate protein folding. Most current techniques to predict protein folding pathways are computationally intensive and are suitable only for small proteins. Results By assuming that the native structure of a protein is known and representing each intermediate conformation as a collection of fully folded structures in which each of them contains a set of interacting secondary structure elements, we show that it is possible to significantly reduce the conformation space while still being able to predict the most energetically favorable folding pathway of large proteins with hundreds of residues at the mesoscopic level, including the pig muscle phosphoglycerate kinase with 416 residues. The model is detailed enough to distinguish between different folding pathways of structurally very similar proteins, including the streptococcal protein G and the peptostreptococcal protein L. The model is also able to recognize the differences between the folding pathways of protein G and its two structurally similar variants NuG1 and NuG2, which are even harder to distinguish. We show that this strategy can produce accurate predictions on many other proteins with experimentally determined intermediate folding states. Conclusion Our technique is efficient enough to predict folding pathways for both large and small proteins at the mesoscopic level. Such a strategy is often the only feasible choice for large proteins. A software program implementing this strategy (SSFold is available at http://faculty.cs.tamu.edu/shsze/ssfold.

  1. N-Glycan-dependent protein folding and endoplasmic reticulum retention regulate GPI-anchor processing.

    Science.gov (United States)

    Liu, Yi-Shi; Guo, Xin-Yu; Hirata, Tetsuya; Rong, Yao; Motooka, Daisuke; Kitajima, Toshihiko; Murakami, Yoshiko; Gao, Xiao-Dong; Nakamura, Shota; Kinoshita, Taroh; Fujita, Morihisa

    2017-12-18

    Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in the endoplasmic reticulum (ER). Soon after GPI is attached, an acyl chain on the GPI inositol is removed by post-GPI attachment to proteins 1 (PGAP1), a GPI-inositol deacylase. This is crucial for switching GPI-anchored proteins (GPI-APs) from protein folding to transport states. We performed haploid genetic screens to identify factors regulating GPI-inositol deacylation, identifying seven genes. In particular, calnexin cycle impairment caused inefficient GPI-inositol deacylation. Calnexin was specifically associated with GPI-APs, dependent on N-glycan and GPI moieties, and assisted efficient GPI-inositol deacylation by PGAP1. Under chronic ER stress caused by misfolded GPI-APs, inositol-acylated GPI-APs were exposed on the cell surface. These results indicated that N-glycans participate in quality control and temporal ER retention of GPI-APs, ensuring their correct folding and GPI processing before exiting from the ER. Once the system is disrupted by ER stress, unprocessed GPI-APs become exposed on the cell surface. © 2018 Liu et al.

  2. The family feud: do proteins with similar structures fold via the same pathway?

    Science.gov (United States)

    Zarrine-Afsar, Arash; Larson, Stefan M; Davidson, Alan R

    2005-02-01

    Theoretical and experimental studies of protein folding have suggested that the topology of the native state may be the most important factor determining the folding pathway of a protein, independent of its specific amino acid sequence. To test this concept, many experimental studies have been carried out with the aim of comparing the folding pathways of proteins that possess similar tertiary structures, but divergent sequences. Many of these studies focus on quantitative comparisons of folding transition state structures, as determined by Phi(f) value analysis of folding kinetic data. In some of these studies, folding transition state structures are found to be highly conserved, whereas in others they are not. We conclude that folds displaying more conserved transition state structures may have the most restricted number of possible folding pathways and that folds displaying low transition state structural conservation possess many potential pathways for reaching the native state.

  3. Protein folding: Over half a century lasting quest. Comment on "There and back again: Two views on the protein folding puzzle" by Alexei V. Finkelstein et al.

    Science.gov (United States)

    Krokhotin, Andrey; Dokholyan, Nikolay V.

    2017-07-01

    Most proteins fold into unique three-dimensional (3D) structures that determine their biological functions, such as catalytic activity or macromolecular binding. Misfolded proteins can pose a threat through aberrant interactions with other proteins leading to a number of diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [1,2]. What does determine 3D structure of proteins? The first clue to this question came more than fifty years ago when Anfinsen demonstrated that unfolded proteins can spontaneously fold to their native 3D structures [3,4]. Anfinsen's experiments lead to the conclusion that proteins fold to unique native structure corresponding to the stable and kinetically accessible free energy minimum, and protein native structure is solely determined by its amino acid sequence. The question of how exactly proteins find their free energy minimum proved to be a difficult problem. One of the puzzles, initially pointed out by Levinthal, was an inconsistency between observed protein folding times and theoretical estimates. A self-avoiding polymer model of a globular protein of 100-residues length on a cubic lattice can sample at least 1047 states. Based on the assumption that conformational sampling occurs at the highest vibrational mode of proteins (∼picoseconds), predicted folding time by searching among all the possible conformations leads to ∼1027 years (much larger than the age of the universe) [5]. In contrast, observed protein folding time range from microseconds to minutes. Due to tremendous theoretical progress in protein folding field that has been achieved in past decades, the source of this inconsistency is currently understood that is thoroughly described in the review by Finkelstein et al. [6].

  4. In-Situ Observation of Membrane Protein Folding during Cell-Free Expression.

    Science.gov (United States)

    Baumann, Axel; Kerruth, Silke; 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.

  5. In-Situ Observation of Membrane Protein Folding during Cell-Free Expression.

    Directory of Open Access Journals (Sweden)

    Axel Baumann

    Full Text Available 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.

  6. Defective folding and rapid degradation of mutant proteins is a common disease mechanism in genetic disorders

    DEFF Research Database (Denmark)

    Gregersen, N; Bross, P; Jørgensen, M M

    2000-01-01

    Many disease-causing point mutations do not seriously compromise synthesis of the affected polypeptide but rather exert their effects by impairing subsequent protein folding or stability of the folded protein. This often results in rapid degradation of the affected protein. The concepts...

  7. Defective folding and rapid degradation of mutant proteins is a common disease mechanism in genetic disorders

    DEFF Research Database (Denmark)

    Gregersen, Niels; Bross, Peter; Jørgensen, Malene Munk

    2000-01-01

    Many disease-causing point mutations do not seriously compromise synthesis of the affected polypeptides but rather exert their effects by impairing subsequent protein folding or stability of the folded protein. This often results in rapid degradation of the affected protein. The concepts...

  8. Mathematics, Thermodynamics, and Modeling to Address Ten Common Misconceptions about Protein Structure, Folding, and Stability

    Science.gov (United States)

    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…

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

    Science.gov (United States)

    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.

  10. An expanded view of the protein folding landscape of PDZ domains

    DEFF Research Database (Denmark)

    Hultqvist, Greta; Pedersen, Søren W; Chi, Celestine N.

    2012-01-01

    Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting for the ......Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting...

  11. A novel reaction of peroxiredoxin 4 towards substrates in oxidative protein folding.

    Directory of Open Access Journals (Sweden)

    Li Zhu

    Full Text Available Peroxiredoxin 4 (Prx4 is the only endoplasmic reticulum localized peroxiredoxin. It functions not only to eliminate peroxide but also to promote oxidative protein folding via oxidizing protein disulfide isomerase (PDI. In Prx4-mediated oxidative protein folding we discovered a new reaction that the sulfenic acid form of Prx4 can directly react with thiols in folding substrates, resulting in non-native disulfide cross-linking and aggregation. We also found that PDI can inhibit this reaction by exerting its reductase and chaperone activities. This discovery discloses an off-pathway reaction in the Prx4-mediated oxidative protein folding and the quality control role of PDI.

  12. Sequence and structural analysis of two designed proteins with 88% identity adopting different folds.

    Science.gov (United States)

    Saravanan, K Mani; Balasubramanian, Harihar; Nallusamy, Saranya; Samuel, Selvaraj

    2010-12-01

    Protein folding is a natural phenomenon by which a sequence of amino acids folds into a unique functional three-dimensional structure. Although the sequence code that governs folding remains a mystery, one can identify key inter-residue contacts responsible for a given topology. In nature, there are many pairs of proteins of a given length that share little or no sequence identity. Similarly, there are many proteins that share a common topology but lack significant evidence of homology. In order to tackle this problem, protein engineering studies have been used to determine the minimal number of amino acid residues that codes for a particular fold. In recent years, the coupling of theoretical models and experiments in the study of protein folding has resulted in providing some fruitful clues. He et al. have designed two proteins with 88% sequence identity, which adopt different folds and functions. In this work, we have systematically analysed these two proteins by performing pentapeptide search, secondary structure predictions, variation in inter-residue interactions and residue-residue pair preferences, surrounding hydrophobicity computations, conformational switching and energy computations. We conclude that the local secondary structural preference of the two designed proteins at the Nand C-terminal ends to adopt either coil or strand conformation may be a crucial factor in adopting the different folds. Early on during the process of folding, both proteins may choose different energetically favourable pathways to attain the different folds.

  13. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

    Directory of Open Access Journals (Sweden)

    Lobstein Julie

    2012-05-01

    Full Text Available Abstract Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using

  14. Fast mapping of global protein folding states by multivariate NMR: a GPS for proteins

    DEFF Research Database (Denmark)

    Malmendal, Anders; Underhaug, Jarl; Otzen, Daniel E

    2010-01-01

    that provides such an overview. GPS NMR exploits the unique ability of NMR to simultaneously record signals from individual hydrogen atoms in complex macromolecular systems and of multivariate analysis to describe spectral variations from these by a few variables for establishment of, and positioning in......, protein-folding state maps. The method is fast, sensitive, and robust, and it works without isotope-labelling. The unique capabilities of GPS NMR to identify different folding states and to compare different unfolding processes are demonstrated by mapping of the equilibrium folding space of bovine alpha......-lactalbumin in the presence of the anionic surfactant sodium dodecyl sulfate, SDS, and compare these with other surfactants, acid, denaturants and heat....

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

    DEFF Research Database (Denmark)

    Lyngsø, C.; Kjaerulff, S.; Muller, S.

    2010-01-01

    -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......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...

  16. Extracting features from protein sequences to improve deep extreme learning machine for protein fold recognition.

    Science.gov (United States)

    Ibrahim, Wisam; Abadeh, Mohammad Saniee

    2017-05-21

    Protein fold recognition is an important problem in bioinformatics to predict three-dimensional structure of a protein. One of the most challenging tasks in protein fold recognition problem is the extraction of efficient features from the amino-acid sequences to obtain better classifiers. In this paper, we have proposed six descriptors to extract features from protein sequences. These descriptors are applied in the first stage of a three-stage framework PCA-DELM-LDA to extract feature vectors from the amino-acid sequences. Principal Component Analysis PCA has been implemented to reduce the number of extracted features. The extracted feature vectors have been used with original features to improve the performance of the Deep Extreme Learning Machine DELM in the second stage. Four new features have been extracted from the second stage and used in the third stage by Linear Discriminant Analysis LDA to classify the instances into 27 folds. The proposed framework is implemented on the independent and combined feature sets in SCOP datasets. The experimental results show that extracted feature vectors in the first stage could improve the performance of DELM in extracting new useful features in second stage. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Theory and simulation of explicit solvent effects on protein folding in vitro and in vivo

    Science.gov (United States)

    England, Jeremy L.

    The aim of this work is to develop theoretical tools for understanding what happens to water that is confined in amphipathic cavities, and for testing the consequences of this understanding for protein folding in vitro and in vivo. We begin in the first chapter with a brief review of the theoretical and simulation literature on the hydrophobic effect and the aqueous solvation of charged species that also puts forward a simple theoretical framework within which various solvation phenomena reported in past studies may be unified. Subsequently, in the second chapter we also review past computational and theoretical work on the specific question of how chaperonin complexes assist the folding of their substrates. With the context set, we turn in Chapter 3 to the case of an open system with water trapped between hydrophobic plates that experiences a uniform electric field normal to and between the plates. Classic bulk theory of electrostriction in polarizable fluids tells us that the electric field should cause an increase in local water density as it rises, yet some simulations have suggested the opposite. We present a mean-field Potts model we have developed to explain this discrepancy, and show how such a simple, coarse-grained lattice description can capture the fundamental consequences of the fact that external electric fields can frustrate the hydrogen bond network in confined water. Chapter 4 continues to pursue the issue of solvent evacuation between hydrophobic plates, but focuses on the impact of chemical denaturants on hydrophobic effects using molecular dynamics simulations of hydrophobic dewetting. We find that while urea and guanidinium have similar qualitative effects at the bulk level, they seem to differ in the microscopic mechanism by which they denature proteins, although both inhibit the onset of dewetting. Lastly, Chapters 5 and 6 examine the potential importance of solvent-mediated forces to protein folding in vivo. Chapter 5 develops a Landau

  18. A Particle Swarm Optimization-Based Approach with Local Search for Predicting Protein Folding.

    Science.gov (United States)

    Yang, Cheng-Hong; Lin, Yu-Shiun; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2017-10-01

    The hydrophobic-polar (HP) model is commonly used for predicting protein folding structures and hydrophobic interactions. This study developed a particle swarm optimization (PSO)-based algorithm combined with local search algorithms; specifically, the high exploration PSO (HEPSO) algorithm (which can execute global search processes) was combined with three local search algorithms (hill-climbing algorithm, greedy algorithm, and Tabu table), yielding the proposed HE-L-PSO algorithm. By using 20 known protein structures, we evaluated the performance of the HE-L-PSO algorithm in predicting protein folding in the HP model. The proposed HE-L-PSO algorithm exhibited favorable performance in predicting both short and long amino acid sequences with high reproducibility and stability, compared with seven reported algorithms. The HE-L-PSO algorithm yielded optimal solutions for all predicted protein folding structures. All HE-L-PSO-predicted protein folding structures possessed a hydrophobic core that is similar to normal protein folding.

  19. A Soluble, Folded Protein without Charged Amino Acid Residues

    DEFF Research Database (Denmark)

    Højgaard, Casper; Kofoed, Christian; Espersen, Roall

    2016-01-01

    side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find......, this chargeless protein is produced reasonably well in :Escherichia coli, retains its stable three-dimensional structure, and is still capable of strong cellulose binding. To further deprive this protein of charges, we removed the N-terminal charge by acetylation and studied the protein at pH 2, where the C...

  20. NMR structures of two designed proteins with high sequence identity but different fold and function.

    Science.gov (United States)

    He, Yanan; Chen, Yihong; Alexander, Patrick; Bryan, Philip N; Orban, John

    2008-09-23

    How protein sequence codes for 3D structure remains a fundamental question in biology. One approach to understanding the folding code is to design a pair of proteins with maximal sequence identity but retaining different folds. Therefore, the nonidentities must be responsible for determining which fold topology prevails and constitute a fold-specific folding code. We recently designed two proteins, G(A)88 and G(B)88, with 88% sequence identity but different folds and functions [Alexander et al. (2007) Proc Natl Acad Sci USA 104:11963-11968]. Here, we describe the detailed 3D structures of these proteins determined in solution by NMR spectroscopy. Despite a large number of mutations taking the sequence identity level from 16 to 88%, G(A)88 and G(B)88 maintain their distinct wild-type 3-alpha and alpha/beta folds, respectively. To our knowledge, the 3D-structure determination of two monomeric proteins with such high sequence identity but different fold topology is unprecedented. The geometries of the seven nonidentical residues (of 56 total) provide insights into the structural basis for switching between 3-alpha and alpha/beta conformations. Further mutation of a subset of these nonidentities, guided by the G(A)88 and G(B)88 structures, leads to proteins with even higher levels of sequence identity (95%) and different folds. Thus, conformational switching to an alternative monomeric fold of comparable stability can be effected with just a handful of mutations in a small protein. This result has implications for understanding not only the folding code but also the evolution of new folds.

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

    Science.gov (United States)

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

    2014-01-01

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

  2. Genetic Algorithms and Their Application to the Protein Folding Problem

    Science.gov (United States)

    1993-12-01

    Example of a Mutation ............................................................................. 16 Figure 4. A Three Amino Acid ProteinA ...promotion of evolutionary computation. The proceedings of these conferences provide a wealth of information perWt g to both theory and application. The...given its primary structure. HH Figure 4. A Three Amino Acid ProteinA 28 3.1 Background Proteins are an integral part of everyday life. The function of

  3. A fusion tag to fold on: the S-layer protein SgsE confers improved folding kinetics to translationally fused enhanced green fluorescent protein.

    Science.gov (United States)

    Ristl, Robin; Kainz, Birgit; Stadlmayr, Gerhard; Schuster, Heinrich; Pum, Dietmar; Messner, Paul; Obinger, Christian; Schaffer, Christina

    2012-09-01

    Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

  4. The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Moye Wang

    2016-04-01

    Full Text Available As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5–10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design.

  5. The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations.

    Science.gov (United States)

    Wang, Moye; Hu, Jie; Zhang, Zhuqing

    2016-04-26

    As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD) simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD) simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5-10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design.

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

    Science.gov (United States)

    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.

  7. Electrostatically accelerated encounter and folding for facile recognition of intrinsically disordered proteins.

    Directory of Open Access Journals (Sweden)

    Debabani Ganguly

    Full Text Available Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012 demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via "electrostatic steering" and at the same time promote "folding-competent" encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association

  8. There and back again: Two views on the protein folding puzzle

    Science.gov (United States)

    Finkelstein, Alexei V.; Badretdin, Azat J.; Galzitskaya, Oxana V.; Ivankov, Dmitry N.; Bogatyreva, Natalya S.; Garbuzynskiy, Sergiy O.

    2017-07-01

    The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of single-domain globular proteins range from microseconds to hours: the difference (10-11 orders of magnitude) is the same as that between the life span of a mosquito and the age of the universe. This review describes physical theories of rates of overcoming the free-energy barrier separating the natively folded (N) and unfolded (U) states of protein chains in both directions: ;U-to-N; and ;N-to-U;. In the theory of protein folding rates a special role is played by the point of thermodynamic (and kinetic) equilibrium between the native and unfolded state of the chain; here, the theory obtains the simplest form. Paradoxically, a theoretical estimate of the folding time is easier to get from consideration of protein unfolding (the ;N-to-U; transition) rather than folding, because it is easier to outline a good unfolding pathway of any structure than a good folding pathway that leads to the stable fold, which is yet unknown to the folding protein chain. And since the rates of direct and reverse reactions are equal at the equilibrium point (as follows from the physical ;detailed balance; principle), the estimated folding time can be derived from the estimated unfolding time. Theoretical analysis of the ;N-to-U; transition outlines the range of protein folding rates in a good agreement with experiment. Theoretical analysis of folding (the ;U-to-N; transition), performed at the level of formation and assembly of protein secondary structures, outlines the upper limit of protein folding times (i.e., of the time of search for the most stable fold). Both theories come to essentially the same results; this is not a surprise, because they describe overcoming one and the same free-energy barrier, although the way to the top of this barrier from the side of the unfolded state is very different from the way from the

  9. M1 RNA is important for the in-cell solubility of its cognate C5 protein: Implications for RNA-mediated protein folding.

    Science.gov (United States)

    Son, Ahyun; Choi, Seong Il; Han, Gyoonhee; Seong, Baik L

    2015-01-01

    It is one of the fundamental questions in biology how proteins efficiently fold into their native conformations despite off-pathway events such as misfolding and aggregation in living cells. Although molecular chaperones have been known to assist the de novo folding of certain types of proteins, the role of a binding partner (or a ligand) in the folding and in-cell solubility of its interacting protein still remains poorly defined. RNase P is responsible for the maturation of tRNAs as adaptor molecules of amino acids in ribosomal protein synthesis. The RNase P from Escherichia coli, composed of M1 RNA and C5 protein, is a prototypical ribozyme in which the RNA subunit contains the catalytic activity. Using E. coli RNase P, we demonstrate that M1 RNA plays a pivotal role in the in-cell solubility of C5 protein both in vitro and in vivo. Mutations in either the C5 protein or M1 RNA that affect their interactions significantly abolished the folding of C5 protein. Moreover, we find that M1 RNA provides quality insurance of interacting C5 protein, either by promoting the degradation of C5 mutants in the presence of functional proteolytic machinery, or by abolishing their solubility if the machinery is non-functional. Our results describe a crucial role of M1 RNA in the folding, in-cell solubility, and, consequently, the proteostasis of the client C5 protein, giving new insight into the biological role of RNAs as chaperones and mediators that ensure the quality of interacting proteins.

  10. Repeat protein architectures predicted by a continuum representation of fold space

    OpenAIRE

    Hausrath, Andrew C.; Goriely, Alain

    2006-01-01

    It is an open question whether nature has utilized all possible protein folds. For a simple protein architecture, the helical repeats, we report a method to address this question based on a mapping between the set of repetitive curves and a space of parameters specifying the curve. The exploration of the parameter space for a particular architecture enables a systematic exploration of the fold space for that protein architecture. In a planar subspace of the parameter space of helical repeats ...

  11. Evidence for the principle of minimal frustration in the evolution of protein folding landscapes.

    Science.gov (United States)

    Tzul, Franco O; Vasilchuk, Daniel; Makhatadze, George I

    2017-02-28

    Theoretical and experimental studies have firmly established that protein folding can be described by a funneled energy landscape. This funneled energy landscape is the result of foldable protein sequences evolving following the principle of minimal frustration, which allows proteins to rapidly fold to their native biologically functional conformations. For a protein family with a given functional fold, the principle of minimal frustration suggests that, independent of sequence, all proteins within this family should fold with similar rates. However, depending on the optimal living temperature of the organism, proteins also need to modulate their thermodynamic stability. Consequently, the difference in thermodynamic stability should be primarily caused by differences in the unfolding rates. To test this hypothesis experimentally, we performed comprehensive thermodynamic and kinetic analyses of 15 different proteins from the thioredoxin family. Eight of these thioredoxins were extant proteins from psychrophilic, mesophilic, or thermophilic organisms. The other seven protein sequences were obtained using ancestral sequence reconstruction and can be dated back over 4 billion years. We found that all studied proteins fold with very similar rates but unfold with rates that differ up to three orders of magnitude. The unfolding rates correlate well with the thermodynamic stability of the proteins. Moreover, proteins that unfold slower are more resistant to proteolysis. These results provide direct experimental support to the principle of minimal frustration hypothesis.

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

    Science.gov (United States)

    Corrales, Marc; Cuscó, Pol; Usmanova, Dinara R; 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.

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

    Directory of Open Access Journals (Sweden)

    Marc Corrales

    Full Text Available 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.

  14. ER Dysfunction and Protein Folding Stress in ALS

    Science.gov (United States)

    Matus, Soledad; Valenzuela, Vicente; Medinas, Danilo B.; Hetz, Claudio

    2013-01-01

    Amyotrophic lateral sclerosis (ALS) is the most frequent paralytic disease in adults. Most ALS cases are considered sporadic with no clear genetic component. The disruption of protein homeostasis due to chronic stress responses at the endoplasmic reticulum (ER) and the accumulation of abnormal protein inclusions are extensively described in ALS mouse models and patient-derived tissue. Recent studies using pharmacological and genetic manipulation of the unfolded protein response (UPR), an adaptive reaction against ER stress, have demonstrated a complex involvement of the pathway in experimental models of ALS. In addition, quantitative changes in ER stress-responsive chaperones in body fluids have been proposed as possible biomarkers to monitor the disease progression. Here we review most recent advances attributing a causal role of ER stress in ALS. PMID:24324498

  15. Analysis of the kinetics of folding of proteins and peptides using circular dichroism

    Science.gov (United States)

    Greenfield, Norma J.

    2009-01-01

    Circular dichroism (CD) is a useful spectroscopic technique for studying the secondary structure, folding and binding properties of proteins. This protocol covers how to use the intrinsic circular dichroic properties of proteins to follow their folding and unfolding as a function of time. Included will be methods of obtaining data and how to analyze the folding and unfolding data to determine the rate constants and the order of the folding/unfolding reactions. The protocol focuses on the use of CD to follow folding when it is relatively slow, on the order of minutes to days. The methods for analyzing the data, however, can also be applied to data collected with a CD machine equipped with stopped-flow accessories in the millisecond to second range and folding analyzed by other spectroscopic methods including changes in absorption or fluorescence spectra as a function of time. PMID:17406548

  16. Cleavage Mapping the Topology of Protein Folding Intermediates

    Science.gov (United States)

    1995-03-13

    Connecticut, 06520 3Instituto de Qufmica y Fisicoquimica Biolögicas, Facultad de Farmacia y Bioqufmica (UBA-CONICET), Buenos Aires, Argentina 4...calculate the moles of carbonyl per mole of protein using molar extinction coefficients of 22 000 at 370 nm and 9460 at 276 nm for hydrazones ( Levine ...that observed in other metal-catalyzed oxidation systems ( Levine et al., 1994; Ue et al., 1992; Kulmacz et al., 1994; Soundar & Colman, 1993). These

  17. Extent of hydrogen-bond protection in folded proteins: a constraint on packing architectures.

    OpenAIRE

    Fernández, Ariel; Berry, R. Stephen

    2002-01-01

    Progressive structuring and ultimately exclusion of water by hydrophobes surrounding backbone hydrogen bonds turn the latter into guiding factors of protein folding. Here we demonstrate that an arrangement of five hydrophobes yields an optimal hydrogen-bond stabilization. This motif is shown to be nearly ubiquitous in native folds.

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

    Science.gov (United States)

    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.

  19. SCOWLP update: 3D classification of protein-protein, -peptide, -saccharide and -nucleic acid interactions, and structure-based binding inferences across folds

    Directory of Open Access Journals (Sweden)

    Schreiber Sven

    2011-10-01

    Full Text Available Abstract Background Protein interactions are essential for coordinating cellular functions. Proteomic studies have already elucidated a huge amount of protein-protein interactions that require detailed functional analysis. Understanding the structural basis of each individual interaction through their structural determination is necessary, yet an unfeasible task. Therefore, computational tools able to predict protein binding regions and recognition modes are required to rationalize putative molecular functions for proteins. With this aim, we previously created SCOWLP, a structural classification of protein binding regions at protein family level, based on the information obtained from high-resolution 3D protein-protein and protein-peptide complexes. Description We present here a new version of SCOWLP that has been enhanced by the inclusion of protein-nucleic acid and protein-saccharide interactions. SCOWLP takes interfacial solvent into account for a detailed characterization of protein interactions. In addition, the binding regions obtained per protein family have been enriched by the inclusion of predicted binding regions, which have been inferred from structurally related proteins across all existing folds. These inferences might become very useful to suggest novel recognition regions and compare structurally similar interfaces from different families. Conclusions The updated SCOWLP has new functionalities that allow both, detection and comparison of protein regions recognizing different types of ligands, which include other proteins, peptides, nucleic acids and saccharides, within a solvated environment. Currently, SCOWLP allows the analysis of predicted protein binding regions based on structure-based inferences across fold space. These predictions may have a unique potential in assisting protein docking, in providing insights into protein interaction networks, and in guiding rational engineering of protein ligands. The newly designed

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

    Science.gov (United States)

    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

  1. Why and how does native topology dictate the folding speed of a protein?

    Science.gov (United States)

    Rustad, Mark; Ghosh, Kingshuk

    2012-11-28

    Since the pioneering work of Plaxco, Simons, and Baker, it is now well known that the rates of protein folding strongly correlate with the average sequence separation (absolute contact order (ACO)) of native contacts. In spite of multitude of papers, our understanding to the basis of the relation between folding speed and ACO is still lacking. We model the transition state as a gaussian polymer chain decorated with weak springs between native contacts while the unfolded state is modeled as a gaussian chain only. Using these hamiltonians, our perturbative calculation explicitly shows folding speed and ACO are linearly related when only the first order term in the series is considered. However, to the second order, we notice the existence of two new topological metrics, termed COC(1) and COC(2) (COC stands for contact order correction). These additional correction terms are needed to properly account for the entropy loss due to overlapping (nested or linked) loops that are not well described by simple addition of entropies in ACO. COC(1) and COC(2) are related to fluctuations and correlations among different sequence separations. The new metric combining ACO, COC(1), and COC(2) improves folding speed dependence on native topology when applied to three different databases: (i) two-state proteins with only α∕β and β proteins, (ii) two-state proteins (α∕β, β and purely helical proteins all combined), and (iii) master set (multi-state and two-state) folding proteins. Furthermore, the first principle calculation provides us direct physical insights to the meaning of the fit parameters. The coefficient of ACO, for example, is related to the average strength of the contacts, while the constant term is related to the protein folding speed limit. With the new scaling law, our estimate of the folding speed limit is in close agreement with the widely accepted value of 1 μs observed in proteins and RNA. Analyzing an exhaustive set (7367) of monomeric proteins from

  2. Effective inter-residue contact definitions for accurate protein fold recognition

    Directory of Open Access Journals (Sweden)

    Yuan Chao

    2012-11-01

    Full Text Available Abstract Background Effective encoding of residue contact information is crucial for protein structure prediction since it has a unique role to capture long-range residue interactions compared to other commonly used scoring terms. The residue contact information can be incorporated in structure prediction in several different ways: It can be incorporated as statistical potentials or it can be also used as constraints in ab initio structure prediction. To seek the most effective definition of residue contacts for template-based protein structure prediction, we evaluated 45 different contact definitions, varying bases of contacts and distance cutoffs, in terms of their ability to identify proteins of the same fold. Results We found that overall the residue contact pattern can distinguish protein folds best when contacts are defined for residue pairs whose Cβ atoms are at 7.0 Å or closer to each other. Lower fold recognition accuracy was observed when inaccurate threading alignments were used to identify common residue contacts between protein pairs. In the case of threading, alignment accuracy strongly influences the fraction of common contacts identified among proteins of the same fold, which eventually affects the fold recognition accuracy. The largest deterioration of the fold recognition was observed for β-class proteins when the threading methods were used because the average alignment accuracy was worst for this fold class. When results of fold recognition were examined for individual proteins, we found that the effective contact definition depends on the fold of the proteins. A larger distance cutoff is often advantageous for capturing spatial arrangement of the secondary structures which are not physically in contact. For capturing contacts between neighboring β strands, considering the distance between Cα atoms is better than the Cβ−based distance because the side-chain of interacting residues on β strands sometimes point to

  3. Thermodynamics of model prions and its implications for the problem of prion protein folding.

    Science.gov (United States)

    Harrison, P M; Chan, H S; Prusiner, S B; Cohen, F E

    1999-02-19

    Prion disease is caused by the propagation of a particle containing PrPSc, a misfolded form of the normal cellular prion protein (PrPC). PrPC can re-fold to form PrPSc with loss of alpha-helical structure and formation of extensive beta-sheet structure. Here, we model this prion folding problem with a simple, low-resolution lattice model of protein folding. If model proteins are allowed to re-fold upon dimerization, a minor proportion of them (up to approximately 17%) encrypts an alternative native state as a homodimer. The structures in this homodimeric native state re-arrange so that they are very different in conformation from the monomeric native state. We find that model proteins that are relatively less stable as monomers are more susceptible to the formation of alternative native states as homodimers. These results suggest that less-stable proteins have a greater need for a well-designed energy landscape for protein folding to overcome an increased chance of encrypting substantially different native conformations stabilized by multimeric interactions. This conceptual framework for aberrant folding should be relevant in Alzheimer's disease and other disorders associated with protein aggregation. Copyright 1999 Academic Press.

  4. Measuring "unmeasurable" folding kinetics of proteins by single-molecule force spectroscopy.

    Science.gov (United States)

    Jollymore, Ashlee; Li, Hongbin

    2010-09-24

    Folding and unfolding are fundamental biological processes in cell and are important for the biological functions of proteins. Characterizing the folding and unfolding kinetics of proteins is important for understanding the energetic landscape leading to the active native conformations of these molecules. However, the thermal or chemical-induced unfolding of many proteins is irreversible in vitro, precluding characterization of the folding kinetics of such proteins, just as it is impossible to "un-boil" an egg. Irreversible unfolding often manifests as irreversible aggregation of unfolded polypeptide chains, which typically occurs between denatured protein molecules in response to the exposure of hydrophobic residues to solvent. An example of such a protein where thermal denaturation results in irreversible aggregation is the β-1,4 endoxylanase from Bacillus circulans (BCX). Here, we report the use of single-molecule atomic force microscopy to directly measure the folding kinetics of BCX in vitro. By mechanically unfolding BCX, we essentially allowed only one unfolded molecule to exist in solution at a given time, effectively eliminating the possibility for aggregation. We found that BCX can readily refold back to the native state, allowing us to measure its folding kinetics for the first time. Our results demonstrate that single-molecule force-spectroscopy-based methods can adequately tackle the challenge of "un-boiling eggs", providing a general methodology to characterize the folding kinetics of many proteins that suffer from irreversible denaturation and thus cannot be characterized using traditional equilibrium methodologies. Copyright © 2010 Elsevier Ltd. All rights reserved.

  5. Global analysis of protein folding using massively parallel design, synthesis, and testing.

    Science.gov (United States)

    Rocklin, Gabriel J; Chidyausiku, Tamuka M; Goreshnik, Inna; Ford, Alex; Houliston, Scott; Lemak, Alexander; Carter, Lauren; Ravichandran, Rashmi; Mulligan, Vikram K; Chevalier, Aaron; Arrowsmith, Cheryl H; Baker, David

    2017-07-14

    Proteins fold into unique native structures stabilized by thousands of weak interactions that collectively overcome the entropic cost of folding. Although these forces are "encoded" in the thousands of known protein structures, "decoding" them is challenging because of the complexity of natural proteins that have evolved for function, not stability. We combined computational protein design, next-generation gene synthesis, and a high-throughput protease susceptibility assay to measure folding and stability for more than 15,000 de novo designed miniproteins, 1000 natural proteins, 10,000 point mutants, and 30,000 negative control sequences. This analysis identified more than 2500 stable designed proteins in four basic folds-a number sufficient to enable us to systematically examine how sequence determines folding and stability in uncharted protein space. Iteration between design and experiment increased the design success rate from 6% to 47%, produced stable proteins unlike those found in nature for topologies where design was initially unsuccessful, and revealed subtle contributions to stability as designs became increasingly optimized. Our approach achieves the long-standing goal of a tight feedback cycle between computation and experiment and has the potential to transform computational protein design into a data-driven science. Copyright © 2017, American Association for the Advancement of Science.

  6. Integral and differential form of the protein folding problem

    Science.gov (United States)

    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.

  7. The energy landscape, folding pathways and the kinetics of a knotted protein.

    Directory of Open Access Journals (Sweden)

    Michael C Prentiss

    2010-07-01

    Full Text Available The folding pathway and rate coefficients of the folding of a knotted protein are calculated for a potential energy function with minimal energetic frustration. A kinetic transition network is constructed using the discrete path sampling approach, and the resulting potential energy surface is visualized by constructing disconnectivity graphs. Owing to topological constraints, the low-lying portion of the landscape consists of three distinct regions, corresponding to the native knotted state and to configurations where either the N or C terminus is not yet folded into the knot. The fastest folding pathways from denatured states exhibit early formation of the N terminus portion of the knot and a rate-determining step where the C terminus is incorporated. The low-lying minima with the N terminus knotted and the C terminus free therefore constitute an off-pathway intermediate for this model. The insertion of both the N and C termini into the knot occurs late in the folding process, creating large energy barriers that are the rate limiting steps in the folding process. When compared to other protein folding proteins of a similar length, this system folds over six orders of magnitude more slowly.

  8. The energy landscape, folding pathways and the kinetics of a knotted protein.

    Science.gov (United States)

    Prentiss, Michael C; Wales, David J; Wolynes, Peter G

    2010-07-01

    The folding pathway and rate coefficients of the folding of a knotted protein are calculated for a potential energy function with minimal energetic frustration. A kinetic transition network is constructed using the discrete path sampling approach, and the resulting potential energy surface is visualized by constructing disconnectivity graphs. Owing to topological constraints, the low-lying portion of the landscape consists of three distinct regions, corresponding to the native knotted state and to configurations where either the N or C terminus is not yet folded into the knot. The fastest folding pathways from denatured states exhibit early formation of the N terminus portion of the knot and a rate-determining step where the C terminus is incorporated. The low-lying minima with the N terminus knotted and the C terminus free therefore constitute an off-pathway intermediate for this model. The insertion of both the N and C termini into the knot occurs late in the folding process, creating large energy barriers that are the rate limiting steps in the folding process. When compared to other protein folding proteins of a similar length, this system folds over six orders of magnitude more slowly.

  9. An update of the DEF database of protein fold class predictions

    DEFF Research Database (Denmark)

    Reczko, Martin; Karras, Dimitris; Bohr, Henrik

    1997-01-01

    An update is given on the Database of Expected Fold classes (DEF) that contains a collection of fold-class predictions made from protein sequences and a mail server that provides new predictions for new sequences. To any given sequence one of 49 fold-classes is chosen to classify the structure...... related to the sequence with high accuracy. The updated predictions system is developed using data from the new version of the 3D-ALI database of aligned protein structures and thus is giving more reliable and more detailed predictions than the previous DEF system....

  10. Classification of protein fold classes by knot theory and prediction of folds by neural networks: A combined theoretical and experimental approach

    DEFF Research Database (Denmark)

    Ramnarayan, K.; Bohr, Henrik; Jalkanen, Karl J.

    2008-01-01

    classifications, we utilize standard neural network methods for predicting protein fold classes from amino acid sequences. We also make an analysis of the redundancy of the structural classifications in relation to function and ligand binding. Finally we advocate the use of combining the measurement of the VA......We present different means of classifying protein structure. One is made rigorous by mathematical knot invariants that coincide reasonably well with ordinary graphical fold classification and another classification is by packing analysis. Furthermore when constructing our mathematical fold......, VCD, Raman, ROA, EA and ECD spectra with the primary sequence as a way to improve both the accuracy and reliability of fold class prediction schemes....

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

    Science.gov (United States)

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

    2012-10-01

    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.

  12. Correlation between drug release kinetics from proteineous matrices and protein folding: elasticity and compressibility study.

    Science.gov (United States)

    Katzhendler, I; Priev, A; Friedman1, M

    2000-07-03

    Naproxen sodium (NS) release mechanism from proteineous matrices based on egg albumin (EA) and bovine serum albumin (BSA) was investigated by several physico-chemical methods. The gel strength, modulus of elasticity and erosion properties of the matrices were studied and correlated with drug release kinetics. The results revealed that NS release rate from EA and BSA matrices was markedly different, indicating the significant role of protein nature and conformation on matrix behavior. Unexpectedly it was found that incorporation of NS to EA matrix increased gel strength and modulus of elasticity and decreased matrix erosion. This effect was dependent on NS concentration in the matrix. In contrast to EA, BSA behaved as a non-gelling matrix and was unable to retard drug release because of its high solubility. The influence of NS on protein folding and compressibility in protein solutions was studied using densitometric and ultrasonic techniques. Adiabatic compressibility measurements revealed that NS caused unfolding of EA, an effect which led to a decrease in EA intrinsic compressibility and the exposure of atomic side groups buried in protein interior. Unfolding of EA led to an increase of modulus of elasticity in solution (measured by ultrasonic velocimetry technique) which is in correlation with the modulus of elasticity measurements of gelled tablets (measured by Instron). In concentrated EA solutions, the results showed a large increase in EA compressibility and ultrasonic absorption in the presence of NS indicating a strong aggregation of the denatured state of EA. Regarding BSA, the results suggested that NS affected the packing of the protein interior, transforming it to a molten globule intermediate state, an effect that led to an increase in BSA compressibility. At high BSA concentrations, aggregation of the molten globule state was observed as indicated by an increase of BSA compressibility and ultrasonic absorption values.

  13. Improvement of Structure-Based Potentials for Protein Folding by Native and Nonnative Hydrogen Bonds

    Science.gov (United States)

    Enciso, Marta; Rey, Antonio

    2011-01-01

    Pure Gō models (where every native interaction equally stabilizes the folded state) have widely proved their convenience in the computational investigation of protein folding. However, a chemistry-based description of the real interactions also provides a desirable tune in the analysis of the folding process, and thus some hybrid Gō potentials that combine both aspects have been proposed. Among all the noncovalent interactions that contribute to protein folding, hydrogen bonds are the only ones with a partial covalent character. This feature makes them directional and, thus, more difficult to model as part of the coarse-grained descriptions that are typically employed in Gō models. Thanks to a simplified but rigorous representation of backbone hydrogen bonds that we have recently proposed, we present in this article a combined potential (Gō + backbone hydrogen bond) to study the thermodynamics of protein folding in the frame of very simple simulation models. We show that the explicit inclusion of hydrogen bonds leads to a systematic improvement in the description of protein folding. We discuss a representative set of examples (from two-state folders to downhill proteins, with different types of native structures) that reveal a relevant agreement with experimental data. PMID:21943429

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

    Energy Technology Data Exchange (ETDEWEB)

    Lammert, Heiko; Noel, Jeffrey K.; Haglund, Ellinor; Onuchic, José N., E-mail: jonuchic@rice.edu [Center for Theoretical Biological Physics and Department of Physics, Rice University, Houston, Texas 77005 (United States); Schug, Alexander [Steinbuch Centre for Computing, Karlsruhe Institute of Technology, Karlsruhe (Germany)

    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.

  15. Making connections between ultrafast protein folding kinetics and molecular dynamics simulations

    Science.gov (United States)

    Cellmer, Troy; Buscaglia, Marco; Henry, Eric R.; Hofrichter, James; Eaton, William A.

    2011-01-01

    Determining the rate of forming the truly folded conformation of ultrafast folding proteins is an important issue for both experiments and simulations. The double-norleucine mutant of the 35-residue villin subdomain is the focus of recent computer simulations with atomistic molecular dynamics because it is currently the fastest folding protein. The folding kinetics of this protein have been measured in laser temperature-jump experiments using tryptophan fluorescence as a probe of overall folding. The conclusion from the simulations, however, is that the rate determined by fluorescence is significantly larger than the rate of overall folding. We have therefore employed an independent experimental method to determine the folding rate. The decay of the tryptophan triplet-state in photoselection experiments was used to monitor the change in the unfolded population for a sequence of the villin subdomain with one amino acid difference from that of the laser temperature-jump experiments, but with almost identical equilibrium properties. Folding times obtained in a two-state analysis of the results from the two methods at denaturant concentrations varying from 1.5–6.0 M guanidinium chloride are in excellent agreement, with an average difference of only 20%. Polynomial extrapolation of all the data to zero denaturant yields a folding time of 220 (+100,-70) ns at 283 K, suggesting that under these conditions the barrier between folded and unfolded states has effectively disappeared—the so-called “downhill scenario.” PMID:21441105

  16. Hidden protein folding pathways in free-energy landscapes uncovered by network analysis.

    Science.gov (United States)

    Yin, Yanping; Maisuradze, Gia G; Liwo, Adam; Scheraga, Harold A

    2012-04-10

    A network analysis is used to uncover hidden folding pathways in free-energy landscapes usually defined in terms of such arbitrary order parameters as root-mean-square deviation from the native structure, radius of gyration, etc. The analysis has been applied to molecular dynamics (MD) trajectories of the B-domain of staphylococcal protein A, generated with the coarse-grained united-residue (UNRES) force field in a broad range of temperatures (270K ≤ T ≤ 325K). Thousands of folding pathways have been identified at each temperature. Out of these many folding pathways, several most probable ones were selected for investigation of the conformational transitions during protein folding. Unlike other conformational space network (CSN) methods, a node in the CSN variant implemented in this work is defined according to the nativelikeness class of the structure, which defines the similarity of segments of the compared structures in terms of secondary-structure, contact-pattern, and local geometry, as well as the overall geometric similarity of the conformation under consideration to that of the reference (experimental) structure. Our previous findings, regarding the folding model and conformations found at the folding-transition temperature for protein A (Maisuradze et al., J. Am. Chem. Soc. 132, 9444, 2010), were confirmed by the conformational space network analysis. In the methodology and in the analysis of the results, the shortest path identified by using the shortest-path algorithm corresponds to the most probable folding pathway in the conformational space network.

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

    Science.gov (United States)

    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

  18. PyFolding: Open-Source Graphing, Simulation, and Analysis of the Biophysical Properties of Proteins.

    Science.gov (United States)

    Lowe, Alan R; Perez-Riba, Albert; Itzhaki, Laura S; Main, Ewan R G

    2018-02-06

    For many years, curve-fitting software has been heavily utilized to fit simple models to various types of biophysical data. Although such software packages are easy to use for simple functions, they are often expensive and present substantial impediments to applying more complex models or for the analysis of large data sets. One field that is reliant on such data analysis is the thermodynamics and kinetics of protein folding. Over the past decade, increasingly sophisticated analytical models have been generated, but without simple tools to enable routine analysis. Consequently, users have needed to generate their own tools or otherwise find willing collaborators. Here we present PyFolding, a free, open-source, and extensible Python framework for graphing, analysis, and simulation of the biophysical properties of proteins. To demonstrate the utility of PyFolding, we have used it to analyze and model experimental protein folding and thermodynamic data. Examples include: 1) multiphase kinetic folding fitted to linked equations, 2) global fitting of multiple data sets, and 3) analysis of repeat protein thermodynamics with Ising model variants. Moreover, we demonstrate how PyFolding is easily extensible to novel functionality beyond applications in protein folding via the addition of new models. Example scripts to perform these and other operations are supplied with the software, and we encourage users to contribute notebooks and models to create a community resource. Finally, we show that PyFolding can be used in conjunction with Jupyter notebooks as an easy way to share methods and analysis for publication and among research teams. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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. © 2014 The Protein Society.

  20. Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis

    Directory of Open Access Journals (Sweden)

    Schlegel Brigitte

    2004-03-01

    Full Text Available Abstract Background High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination. Results 88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D 1H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains were quickly identified as being well folded and suitable for structural analysis. Conclusion The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D 1H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics.

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

    Directory of Open Access Journals (Sweden)

    József Mandl

    2009-03-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  3. The ribosome can prevent aggregation of partially folded protein intermediates: studies using the Escherichia coli ribosome.

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    Bani Kumar Pathak

    Full Text Available BACKGROUND: Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone 'foldases' that are distinct from chaperone' holdases' that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains. RESULTS: We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein. CONCLUSION: The ribosome can behave like a 'holdase' chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.

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

    Directory of Open Access Journals (Sweden)

    Murat Cetinbaş

    Full Text Available 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.

  5. Protein structure prediction using a docking-based hierarchical folding scheme.

    Science.gov (United States)

    Kifer, Ilona; Nussinov, Ruth; Wolfson, Haim J

    2011-06-01

    The pathways by which proteins fold into their specific native structure are still an unsolved mystery. Currently, many methods for protein structure prediction are available, and most of them tackle the problem by relying on the vast amounts of data collected from known protein structures. These methods are often not concerned with the route the protein follows to reach its final fold. This work is based on the premise that proteins fold in a hierarchical manner. We present FOBIA, an automated method for predicting a protein structure. FOBIA consists of two main stages: the first finds matches between parts of the target sequence and independently folding structural units using profile-profile comparison. The second assembles these units into a 3D structure by searching and ranking their possible orientations toward each other using a docking-based approach. We have previously reported an application of an initial version of this strategy to homology based targets. Since then we have considerably enhanced our method's abilities to allow it to address the more difficult template-based target category. This allows us to now apply FOBIA to the template-based targets of CASP8 and to show that it is both very efficient and promising. Our method can provide an alternative for template-based structure prediction, and in particular, the docking-basedranking technique presented here can be incorporated into any profile-profile comparison based method. Copyright © 2011 Wiley-Liss, Inc.

  6. An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.

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    Marcin Michalik

    Full Text Available An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

  7. Folding and unfolding of a non-fluorescent mutant of green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Wielgus-Kutrowska, Beata [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Narczyk, Marta [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Buszko, Anna [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Bzowska, Agnieszka [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Clark, Patricia L [Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556-5670 (United States)

    2007-07-18

    Green fluorescent protein (GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and it remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; i.e., the principles of microscopic reversibility do not apply. We have generated the mutant S65T/G67A-GFP, which is unable to efficiently form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of guanidinium hydrochloride (GdnHCl). The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of 1,1'-bis(4-anilino-5-naphthalenesulphonic acid) (bis-ANS). Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements that S65T/G67A-GFP folding is largely reversible. During equilibrium folding, the first step is the formation of a molten globule, prone to aggregation.

  8. Sampling-based exploration of folded state of a protein under kinematic and geometric constraints

    KAUST Repository

    Yao, Peggy

    2011-10-04

    Flexibility is critical for a folded protein to bind to other molecules (ligands) and achieve its functions. The conformational selection theory suggests that a folded protein deforms continuously and its ligand selects the most favorable conformations to bind to. Therefore, one of the best options to study protein-ligand binding is to sample conformations broadly distributed over the protein-folded state. This article presents a new sampler, called kino-geometric sampler (KGS). This sampler encodes dominant energy terms implicitly by simple kinematic and geometric constraints. Two key technical contributions of KGS are (1) a robotics-inspired Jacobian-based method to simultaneously deform a large number of interdependent kinematic cycles without any significant break-up of the closure constraints, and (2) a diffusive strategy to generate conformation distributions that diffuse quickly throughout the protein folded state. Experiments on four very different test proteins demonstrate that KGS can efficiently compute distributions containing conformations close to target (e.g., functional) conformations. These targets are not given to KGS, hence are not used to bias the sampling process. In particular, for a lysine-binding protein, KGS was able to sample conformations in both the intermediate and functional states without the ligand, while previous work using molecular dynamics simulation had required the ligand to be taken into account in the potential function. Overall, KGS demonstrates that kino-geometric constraints characterize the folded subset of a protein conformation space and that this subset is small enough to be approximated by a relatively small distribution of conformations. © 2011 Wiley Periodicals, Inc.

  9. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding.

    Science.gov (United States)

    Tottey, Steve; Waldron, Kevin J; Firbank, Susan J; Reale, Brian; Bessant, Conrad; Sato, Katsuko; Cheek, Timothy R; Gray, Joe; Banfield, Mark J; Dennison, Christopher; Robinson, Nigel J

    2008-10-23

    Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).

  10. BCL::MP-Fold: membrane protein structure prediction guided by EPR restraints

    CERN Document Server

    Fischer, Axel Walter; Woetzel, Nils; Karakas, Mert; Weiner, Brian; Meiler, Jens

    2015-01-01

    For many membrane proteins the determination of their topology remains a challenge for methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Electron paramagnetic resonance (EPR) spectroscopy has evolved as an alternative technique to study structure and dynamics of membrane proteins. The present study demonstrates the feasibility of membrane protein topology determination using limited EPR distance and accessibility measurements. The BCL::MP-Fold (BioChemical Library membrane protein fold) algorithm assembles secondary structure elements (SSEs) in the membrane using a Monte Carlo Metropolis (MCM) approach. Sampled models are evaluated using knowledge-based potential functions and agreement with the EPR data and a knowledge-based energy function. Twenty-nine membrane proteins of up to 696 residues are used to test the algorithm. The RMSD100 value of the most accurate model is better than 8{\\AA} for twenty-seven, better than 6{\\AA} for twenty-two and better than 4{\\AA} for fifte...

  11. Structural insights into the evolution of a non-biological protein: importance of surface residues in protein fold optimization.

    Directory of Open Access Journals (Sweden)

    Matthew D Smith

    Full Text Available Phylogenetic profiling of amino acid substitution patterns in proteins has led many to conclude that most structural information is carried by interior core residues that are solvent inaccessible. This conclusion is based on the observation that buried residues generally tolerate only conserved sequence changes, while surface residues allow more diverse chemical substitutions. This notion is now changing as it has become apparent that both core and surface residues play important roles in protein folding and stability. Unfortunately, the ability to identify specific mutations that will lead to enhanced stability remains a challenging problem. Here we discuss two mutations that emerged from an in vitro selection experiment designed to improve the folding stability of a non-biological ATP binding protein. These mutations alter two solvent accessible residues, and dramatically enhance the expression, solubility, thermal stability, and ligand binding affinity of the protein. The significance of both mutations was investigated individually and together, and the X-ray crystal structures of the parent sequence and double mutant protein were solved to a resolution limit of 2.8 and 1.65 A, respectively. Comparative structural analysis of the evolved protein to proteins found in nature reveals that our non-biological protein evolved certain structural features shared by many thermophilic proteins. This experimental result suggests that protein fold optimization by in vitro selection offers a viable approach to generating stable variants of many naturally occurring proteins whose structures and functions are otherwise difficult to study.

  12. Comparing Fast Pressure Jump and Temperature Jump Protein Folding Experiments and Simulations.

    Science.gov (United States)

    Wirth, Anna Jean; Liu, Yanxin; Prigozhin, Maxim B; Schulten, Klaus; Gruebele, Martin

    2015-06-10

    The unimolecular folding reaction of small proteins is now amenable to a very direct mechanistic comparison between experiment and simulation. We present such a comparison of microsecond pressure and temperature jump refolding kinetics of the engineered WW domain FiP35, a model system for β-sheet folding. Both perturbations produce experimentally a faster and a slower kinetic phase, and the "slow" microsecond phase is activated. The fast phase shows differences between perturbation methods and is closer to the downhill limit by temperature jump, but closer to the transiently populated intermediate limit by pressure jump. These observations make more demands on simulations of the folding process than just a rough comparison of time scales. To complement experiments, we carried out several pressure jump and temperature jump all-atom molecular dynamics trajectories in explicit solvent, where FiP35 folded in five of the six simulations. We analyzed our pressure jump simulations by kinetic modeling and found that the pressure jump experiments and MD simulations are most consistent with a 4-state kinetic mechanism. Together, our experimental and computational data highlight FiP35's position at the boundary where activated intermediates and downhill folding meet, and we show that this model protein is an excellent candidate for further pressure jump molecular dynamics studies to compare experiment and modeling at the folding mechanism level.

  13. Exploring the correlation between the folding rates of proteins and the entanglement of their native states

    Science.gov (United States)

    Baiesi, Marco; Orlandini, Enzo; Seno, Flavio; Trovato, Antonio

    2017-12-01

    The folding of a protein towards its native state is a rather complicated process. However, there is empirical evidence that the folding time correlates with the contact order, a simple measure of the spatial organization of the native state of the protein. Contact order is related to the average length of the main chain loops formed by amino acids that are in contact. Here we argue that folding kinetics can also be influenced by the entanglement that loops may undergo within the overall three-dimensional protein structure. In order to explore such a possibility, we introduce a novel descriptor, which we call ‘maximum intrachain contact entanglement’. Specifically, we measure the maximum Gaussian entanglement between any looped portion of a protein and any other non-overlapping subchain of the same protein, which is easily computed by discretized line integrals on the coordinates of the Cα atoms. By analyzing experimental data sets of two-state and multi-state folders, we show that the new index is also a good predictor of the folding rate. Moreover, being only partially correlated with previous methods, it can be integrated with them to yield more accurate predictions.

  14. Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT

    Directory of Open Access Journals (Sweden)

    Gregory R. Kowald

    2016-01-01

    Full Text Available Earthworms express, as most animals, metallothioneins (MTs—small, cysteine-rich proteins that bind d10 metal ions (Zn(II, Cd(II, or Cu(I in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II and Zn(II. Crucially, whilst a single Cd7wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II, expressions in the presence of Zn(II yielded mixtures. The average affinities of wMT-2 determined for either Cd(II or Zn(II are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by 1H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo.

  15. CATHEDRAL: a fast and effective algorithm to predict folds and domain boundaries from multidomain protein structures.

    Directory of Open Access Journals (Sweden)

    Oliver C Redfern

    2007-11-01

    Full Text Available We present CATHEDRAL, an iterative protocol for determining the location of previously observed protein folds in novel multidomain protein structures. CATHEDRAL builds on the features of a fast secondary-structure-based method (using graph theory to locate known folds within a multidomain context and a residue-based, double-dynamic programming algorithm, which is used to align members of the target fold groups against the query protein structure to identify the closest relative and assign domain boundaries. To increase the fidelity of the assignments, a support vector machine is used to provide an optimal scoring scheme. Once a domain is verified, it is excised, and the search protocol is repeated in an iterative fashion until all recognisable domains have been identified. We have performed an initial benchmark of CATHEDRAL against other publicly available structure comparison methods using a consensus dataset of domains derived from the CATH and SCOP domain classifications. CATHEDRAL shows superior performance in fold recognition and alignment accuracy when compared with many equivalent methods. If a novel multidomain structure contains a known fold, CATHEDRAL will locate it in 90% of cases, with <1% false positives. For nearly 80% of assigned domains in a manually validated test set, the boundaries were correctly delineated within a tolerance of ten residues. For the remaining cases, previously classified domains were very remotely related to the query chain so that embellishments to the core of the fold caused significant differences in domain sizes and manual refinement of the boundaries was necessary. To put this performance in context, a well-established sequence method based on hidden Markov models was only able to detect 65% of domains, with 33% of the subsequent boundaries assigned within ten residues. Since, on average, 50% of newly determined protein structures contain more than one domain unit, and typically 90% or more of these

  16. Molecular chaperone assisted expression systems: obtaining pure soluble and active recombinant proteins for structural and therapeutic purposes

    CSIR Research Space (South Africa)

    Makhoba, XH

    2015-09-01

    Full Text Available during recombinant proteins production in E. coli. Molecular chaperones are proteins that are known to assist the newly synthesized proteins to complete their folding stages. This system has improved various proteins that are difficult to produce in E...

  17. Engineering of protein folding and secretion-strategies to overcome bottlenecks for efficient production of recombinant proteins.

    Science.gov (United States)

    Delic, Marizela; Göngrich, Rebecca; Mattanovich, Diethard; Gasser, Brigitte

    2014-07-20

    Recombinant protein production has developed into a huge market with enormous positive implications for human health and for the future direction of a biobased economy. Limitations in the economic and technical feasibility of production processes are often related to bottlenecks of in vivo protein folding. Based on cell biological knowledge, some major bottlenecks have been overcome by the overexpression of molecular chaperones and other folding related proteins, or by the deletion of deleterious pathways that may lead to misfolding, mistargeting, or degradation. While important success could be achieved by this strategy, the list of reported unsuccessful cases is disappointingly long and obviously dependent on the recombinant protein to be produced. Singular engineering of protein folding steps may not lead to desired results if the pathway suffers from several limitations. In particular, the connection between folding quality control and proteolytic degradation needs further attention. Based on recent understanding that multiple steps in the folding and secretion pathways limit productivity, synergistic combinations of the cell engineering approaches mentioned earlier need to be explored. In addition, systems biology-based whole cell analysis that also takes energy and redox metabolism into consideration will broaden the knowledge base for future rational engineering strategies.

  18. Atomic force microscopy and force spectroscopy on the assessment of protein folding and functionality.

    Science.gov (United States)

    Carvalho, Filomena A; Martins, Ivo C; Santos, Nuno C

    2013-03-01

    Atomic force microscopy (AFM) applied to biological systems can, besides generating high-quality and well-resolved images, be employed to study protein folding via AFM-based force spectroscopy. This approach allowed remarkable advances in the measurement of inter- and intramolecular interaction forces with piconewton resolution. The detection of specific interaction forces between molecules based on the AFM sensitivity and the manipulation of individual molecules greatly advanced the understanding of intra-protein and protein-ligand interactions. Apart from the academic interest in the resolution of basic scientific questions, this technique has also key importance on the clarification of several biological questions of immediate biomedical relevance. Force spectroscopy is an especially appropriate technique for "mechanical proteins" that can provide crucial information on single protein molecules and/or domains. Importantly, it also has the potential of combining in a single experiment spatial and kinetic measurements. Here, the main principles of this methodology are described, after which the ability to measure interactions at the single-molecule level is discussed, in the context of relevant protein-folding examples. We intend to demonstrate the potential of AFM-based force spectroscopy in the study of protein folding, especially since this technique is able to circumvent some of the difficulties typically encountered in classical thermal/chemical denaturation studies. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. The double life of the ribosome: When its protein folding activity supports prion propagation.

    Science.gov (United States)

    Voisset, Cécile; Blondel, Marc; Jones, Gary W; Friocourt, Gaëlle; Stahl, Guillaume; Chédin, Stéphane; Béringue, Vincent; Gillet, Reynald

    2017-03-04

    It is no longer necessary to demonstrate that ribosome is the central machinery of protein synthesis. But it is less known that it is also key player of the protein folding process through another conserved function: the protein folding activity of the ribosome (PFAR). This ribozyme activity, discovered more than 2 decades ago, depends upon the domain V of the large rRNA within the large subunit of the ribosome. Surprisingly, we discovered that anti-prion compounds are also potent PFAR inhibitors, highlighting an unexpected link between PFAR and prion propagation. In this review, we discuss the ancestral origin of PFAR in the light of the ancient RNA world hypothesis. We also consider how this ribosomal activity fits into the landscape of cellular protein chaperones involved in the appearance and propagation of prions and other amyloids in mammals. Finally, we examine how drugs targeting the protein folding activity of the ribosome could be active against mammalian prion and other protein aggregation-based diseases, making PFAR a promising therapeutic target for various human protein misfolding diseases.

  20. Antibody Epitope Analysis to Investigate Folded Structure, Allosteric Conformation, and Evolutionary Lineage of Proteins.

    Science.gov (United States)

    Wong, Sienna; Jin, J-P

    2017-01-01

    Study of folded structure of proteins provides insights into their biological functions, conformational dynamics and molecular evolution. Current methods of elucidating folded structure of proteins are laborious, low-throughput, and constrained by various limitations. Arising from these methods is the need for a sensitive, quantitative, rapid and high-throughput method not only analysing the folded structure of proteins, but also to monitor dynamic changes under physiological or experimental conditions. In this focused review, we outline the foundation and limitations of current protein structure-determination methods prior to discussing the advantages of an emerging antibody epitope analysis for applications in structural, conformational and evolutionary studies of proteins. We discuss the application of this method using representative examples in monitoring allosteric conformation of regulatory proteins and the determination of the evolutionary lineage of related proteins and protein isoforms. The versatility of the method described herein is validated by the ability to modulate a variety of assay parameters to meet the needs of the user in order to monitor protein conformation. Furthermore, the assay has been used to clarify the lineage of troponin isoforms beyond what has been depicted by sequence homology alone, demonstrating the nonlinear evolutionary relationship between primary structure and tertiary structure of proteins. The antibody epitope analysis method is a highly adaptable technique of protein conformation elucidation, which can be easily applied without the need for specialized equipment or technical expertise. When applied in a systematic and strategic manner, this method has the potential to reveal novel and biomedically meaningful information for structure-function relationship and evolutionary lineage of proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  1. Folded Proteins Occur Frequently in Libraries of Random Amino Acid Sequences

    Science.gov (United States)

    Davidson, Alan R.; Sauer, Robert T.

    1994-03-01

    A library of synthetic genes encoding 80- to 100-residue proteins composed mainly of random combinations of glutamine (Q), leucine (L), and arginine (R) has been expressed in Escherichia coli. These genes also encode an epitope tag and six carboxyl-terminal histidines. Screening of this library by immunoblotting showed that 5% of these QLR proteins are expressed at readily detectable levels. Three well-expressed QLR proteins were purified and characterized. Each of these proteins has significant α-helical content, is largely resistant to degradation by Pronase, and has a distinct oligomeric structure. In addition, one protein unfolds in a highly cooperative manner. These properties of the QLR proteins demonstrate that they possess folded structures with some native-like properties. The QLR proteins differ from most natural proteins, however, in being remarkably resistant to denaturant-induced and thermal-induced unfolding and in being relatively insoluble in the absence of denaturants.

  2. Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory

    Science.gov (United States)

    Satomura, Atsushi; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

    Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROLimp was more stable than mROLWT. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity. PMID:25970342

  3. Native contact density and nonnative hydrophobic effects in the folding of bacterial immunity proteins.

    Directory of Open Access Journals (Sweden)

    Tao Chen

    2015-05-01

    Full Text Available The bacterial colicin-immunity proteins Im7 and Im9 fold by different mechanisms. Experimentally, at pH 7.0 and 10°C, Im7 folds in a three-state manner via an intermediate but Im9 folding is two-state-like. Accordingly, Im7 exhibits a chevron rollover, whereas the chevron arm for Im9 folding is linear. Here we address the biophysical basis of their different behaviors by using native-centric models with and without additional transferrable, sequence-dependent energies. The Im7 chevron rollover is not captured by either a pure native-centric model or a model augmented by nonnative hydrophobic interactions with a uniform strength irrespective of residue type. By contrast, a more realistic nonnative interaction scheme that accounts for the difference in hydrophobicity among residues leads simultaneously to a chevron rollover for Im7 and an essentially linear folding chevron arm for Im9. Hydrophobic residues identified by published experiments to be involved in nonnative interactions during Im7 folding are found to participate in the strongest nonnative contacts in this model. Thus our observations support the experimental perspective that the Im7 folding intermediate is largely underpinned by nonnative interactions involving large hydrophobics. Our simulation suggests further that nonnative effects in Im7 are facilitated by a lower local native contact density relative to that of Im9. In a one-dimensional diffusion picture of Im7 folding with a coordinate- and stability-dependent diffusion coefficient, a significant chevron rollover is consistent with a diffusion coefficient that depends strongly on native stability at the conformational position of the folding intermediate.

  4. EM-Fold: De novo atomic-detail protein structure determination from medium resolution density maps

    Science.gov (United States)

    Lindert, Steffen; Alexander, Nathan; Wötzel, Nils; Karakaş, Mert; Stewart, Phoebe L.; Meiler, Jens

    2012-01-01

    Electron density maps of membrane proteins or large macromolecular complexes are frequently only determined at medium resolution between 4 Å and 10 Å, either by cryo-electron microscopy (cryoEM) or X-ray crystallography. In these density maps the general arrangement of secondary structure elements is revealed while their directionality and connectivity remain elusive. We demonstrate that the topology of proteins with up to 250 amino acids can be determined from such density maps when combined with a computational protein folding protocol. Furthermore, we accurately reconstruct atomic detail in loop regions and amino acid side chains not visible in the experimental data. The EM-Fold algorithm assembles the secondary structure elements de novo before atomic detail is added using Rosetta. In a benchmark of 27 proteins the protocol consistently and reproducibly achieves models with RMSD values smaller than 3 Å. PMID:22405005

  5. Soft Computing Techniques for the Protein Folding Problem on High Performance Computing Architectures.

    Science.gov (United States)

    Llanes, Antonio; Muñoz, Andrés; Bueno-Crespo, Andrés; García-Valverde, Teresa; Sánchez, Antonia; Arcas-Túnez, Francisco; Pérez-Sánchez, Horacio; Cecilia, José M

    2016-01-01

    The protein-folding problem has been extensively studied during the last fifty years. The understanding of the dynamics of global shape of a protein and the influence on its biological function can help us to discover new and more effective drugs to deal with diseases of pharmacological relevance. Different computational approaches have been developed by different researchers in order to foresee the threedimensional arrangement of atoms of proteins from their sequences. However, the computational complexity of this problem makes mandatory the search for new models, novel algorithmic strategies and hardware platforms that provide solutions in a reasonable time frame. We present in this revision work the past and last tendencies regarding protein folding simulations from both perspectives; hardware and software. Of particular interest to us are both the use of inexact solutions to this computationally hard problem as well as which hardware platforms have been used for running this kind of Soft Computing techniques.

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

    Science.gov (United States)

    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. © 2015 Wiley Periodicals, Inc.

  7. The ribosome and its role in protein folding: looking through a magnifying glass.

    Science.gov (United States)

    Javed, Abid; Christodoulou, John; Cabrita, Lisa D; Orlova, Elena V

    2017-06-01

    Protein folding, a process that underpins cellular activity, begins co-translationally on the ribosome. During translation, a newly synthesized polypeptide chain enters the ribosomal exit tunnel and actively interacts with the ribosome elements - the r-proteins and rRNA that line the tunnel - prior to emerging into the cellular milieu. While understanding of the structure and function of the ribosome has advanced significantly, little is known about the process of folding of the emerging nascent chain (NC). Advances in cryo-electron microscopy are enabling visualization of NCs within the exit tunnel, allowing early glimpses of the interplay between the NC and the ribosome. Once it has emerged from the exit tunnel into the cytosol, the NC (still attached to its parent ribosome) can acquire a range of conformations, which can be characterized by NMR spectroscopy. Using experimental restraints within molecular-dynamics simulations, the ensemble of NC structures can be described. In order to delineate the process of co-translational protein folding, a hybrid structural biology approach is foreseeable, potentially offering a complete atomic description of protein folding as it occurs on the ribosome.

  8. Structural determinants of Neosartorya fischeri antifungal protein (NFAP) for folding, stability and antifungal activity

    OpenAIRE

    Galg?czy, L?szl?; Borics, Attila; Vir?gh, M?t?; Ficze, Hargita; V?radi, Gy?rgyi; Kele, Zolt?n; Marx, Florentine

    2017-01-01

    The recent global challenges to prevent and treat fungal infections strongly demand for the development of new antifungal strategies. The structurally very similar cysteine-rich antifungal proteins from ascomycetes provide a feasible basis for designing new antifungal molecules. The main structural elements responsible for folding, stability and antifungal activity are not fully understood, although this is an essential prerequisite for rational protein design. In this study, we used the Neos...

  9. Application of Time-Resolved Tryptophan Phosphorescence Spectroscopy to Protein Folding Studies.

    Science.gov (United States)

    Subramaniam, Vinod

    This thesis presents studies of the protein folding problem, one of the most significant questions in contemporary biophysics. Sensitive biophysical techniques, including room temperature tryptophan phosphorescence, which reports on the local environment of the residue, and the lability of proteins to denaturation, a global parameter, were used to assess the validity of the traditional assumption that the biologically active state of a protein is the 'native' state, and to determine whether the pathways of folding in vitro lead to the folded state achieved in vivo. Phosphorescence techniques have also been extended to study, for the first time, emission from tryptophan residues engineered into specific positions as reporters of protein structure. During in vitro refolding of E. coli alkaline phosphatase and bovine 13-lactoglobulin, significant differences were found between the refolded proteins and the native conformations, which have no apparent effect on the biological functions. Slow conformational transitions, termed 'annealing,' that occur long after the return of enzyme activity of alkaline phosphatase are manifested in the retarded recovery of phosphorescence intensity, lifetime, and protein lability. While 'annealing' is not observed for beta -lactoglobulin, both phosphorescence and lability experiments reveal changes in the structure of the refolded protein, even though its biological activity, retinol binding, is fully recovered. This result suggests that the pathways of folding in vitro need not lead to the structure formed in vivo. We have used phosphorescence techniques to study the refolding of ribonuclease T1, which exhibits slow kinetics characteristic of proline isomerization. Furthermore, the ability to extract structural information from phosphorescent tryptophan probes engineered into selected regions represents an important advance in studying protein structure; we have reported the first such results from a mutant staphylococcal nuclease. The

  10. High-pressure NMR techniques for the study of protein dynamics, folding and aggregation

    Science.gov (United States)

    Nguyen, Luan M.; Roche, Julien

    2017-04-01

    High-pressure is a well-known perturbation method used to destabilize globular proteins and dissociate protein complexes or aggregates. The heterogeneity of the response to pressure offers a unique opportunity to dissect the thermodynamic contributions to protein stability. In addition, pressure perturbation is generally reversible, which is essential for a proper thermodynamic characterization of a protein equilibrium. When combined with NMR spectroscopy, hydrostatic pressure offers the possibility of monitoring at an atomic resolution the structural transitions occurring upon unfolding and determining the kinetic properties of the process. The recent development of commercially available high-pressure sample cells greatly increased the potential applications for high-pressure NMR experiments that can now be routinely performed. This review summarizes the recent applications and future directions of high-pressure NMR techniques for the characterization of protein conformational fluctuations, protein folding and the stability of protein complexes and aggregates.

  11. A simple lattice model that captures protein folding, aggregation and amyloid formation.

    Directory of Open Access Journals (Sweden)

    Sanne Abeln

    Full Text Available The ability of many proteins to convert from their functional soluble state to amyloid fibrils can be attributed to inter-molecular beta strand formation. Such amyloid formation is associated with neurodegenerative disorders like Alzheimer's and Parkinson's. Molecular modelling can play a key role in providing insight into the factors that make proteins prone to fibril formation. However, fully atomistic models are computationally too expensive to capture the length and time scales associated with fibril formation. As the ability to form fibrils is the rule rather than the exception, much insight can be gained from the study of coarse-grained models that capture the key generic features associated with amyloid formation. Here we present a simple lattice model that can capture both protein folding and beta strand formation. Unlike standard lattice models, this model explicitly incorporates the formation of hydrogen bonds and the directionality of side chains. The simplicity of our model makes it computationally feasible to investigate the interplay between folding, amorphous aggregation and fibril formation, and maintains the capability of classic lattice models to simulate protein folding with high specificity. In our model, the folded proteins contain structures that resemble naturally occurring beta-sheets, with alternating polar and hydrophobic amino acids. Moreover, fibrils with intermolecular cross-beta strand conformations can be formed spontaneously out of multiple short hydrophobic peptide sequences. Both the formation of hydrogen bonds in folded structures and in fibrils is strongly dependent on the amino acid sequence, indicating that hydrogen-bonding interactions alone are not strong enough to initiate the formation of beta sheets. This result agrees with experimental observations that beta sheet and amyloid formation is strongly sequence dependent, with hydrophobic sequences being more prone to form such structures. Our model should

  12. A simple lattice model that captures protein folding, aggregation and amyloid formation.

    Science.gov (United States)

    Abeln, Sanne; Vendruscolo, Michele; Dobson, Christopher M; Frenkel, Daan

    2014-01-01

    The ability of many proteins to convert from their functional soluble state to amyloid fibrils can be attributed to inter-molecular beta strand formation. Such amyloid formation is associated with neurodegenerative disorders like Alzheimer's and Parkinson's. Molecular modelling can play a key role in providing insight into the factors that make proteins prone to fibril formation. However, fully atomistic models are computationally too expensive to capture the length and time scales associated with fibril formation. As the ability to form fibrils is the rule rather than the exception, much insight can be gained from the study of coarse-grained models that capture the key generic features associated with amyloid formation. Here we present a simple lattice model that can capture both protein folding and beta strand formation. Unlike standard lattice models, this model explicitly incorporates the formation of hydrogen bonds and the directionality of side chains. The simplicity of our model makes it computationally feasible to investigate the interplay between folding, amorphous aggregation and fibril formation, and maintains the capability of classic lattice models to simulate protein folding with high specificity. In our model, the folded proteins contain structures that resemble naturally occurring beta-sheets, with alternating polar and hydrophobic amino acids. Moreover, fibrils with intermolecular cross-beta strand conformations can be formed spontaneously out of multiple short hydrophobic peptide sequences. Both the formation of hydrogen bonds in folded structures and in fibrils is strongly dependent on the amino acid sequence, indicating that hydrogen-bonding interactions alone are not strong enough to initiate the formation of beta sheets. This result agrees with experimental observations that beta sheet and amyloid formation is strongly sequence dependent, with hydrophobic sequences being more prone to form such structures. Our model should open the way to a

  13. The IntFOLD server: an integrated web resource for protein fold recognition, 3D model quality assessment, intrinsic disorder prediction, domain prediction and ligand binding site prediction.

    Science.gov (United States)

    Roche, Daniel B; Buenavista, Maria T; Tetchner, Stuart J; McGuffin, Liam J

    2011-07-01

    The IntFOLD server is a novel independent server that integrates several cutting edge methods for the prediction of structure and function from sequence. Our guiding principles behind the server development were as follows: (i) to provide a simple unified resource that makes our prediction software accessible to all and (ii) to produce integrated output for predictions that can be easily interpreted. The output for predictions is presented as a simple table that summarizes all results graphically via plots and annotated 3D models. The raw machine readable data files for each set of predictions are also provided for developers, which comply with the Critical Assessment of Methods for Protein Structure Prediction (CASP) data standards. The server comprises an integrated suite of five novel methods: nFOLD4, for tertiary structure prediction; ModFOLD 3.0, for model quality assessment; DISOclust 2.0, for disorder prediction; DomFOLD 2.0 for domain prediction; and FunFOLD 1.0, for ligand binding site prediction. Predictions from the IntFOLD server were found to be competitive in several categories in the recent CASP9 experiment. The IntFOLD server is available at the following web site: http://www.reading.ac.uk/bioinf/IntFOLD/.

  14. Microsecond simulations of the folding/unfolding thermodynamics of the Trp-cage mini protein

    Science.gov (United States)

    Day, Ryan; Paschek, Dietmar; Garcia, Angel E.

    2012-01-01

    We study the unbiased folding/unfolding thermodynamics of the Trp-cage miniprotein using detailed molecular dynamics simulations of an all-atom model of the protein in explicit solvent, using the Amberff99SB force field. Replica-exchange molecular dynamics (REMD) simulations are used to sample the protein ensembles over a broad range of temperatures covering the folded and unfolded states, and at two densities. The obtained ensembles are shown to reach equilibrium in the 1 μs per replica timescale. The total simulation time employed in the calculations exceeds 100 μs. Ensemble averages of the fraction folded, pressure, and energy differences between the folded and unfolded states as a function of temperature are used to model the free energy of the folding transition, ΔG(P,T), over the whole region of temperature and pressures sampled in the simulations. The ΔG(P,T) diagram describes an ellipse over the range of temperatures and pressures sampled, predicting that the system can undergo pressure induced unfolding and cold denaturation at low temperatures and high pressures, and unfolding at low pressures and high temperatures. The calculated free energy function exhibits remarkably good agreement with the experimental folding transition temperature (Tf = 321 K), free energy and specific heat changes. However, changes in enthalpy and entropy are significantly different than the experimental values. We speculate that these differences may be due to the simplicity of the semi-empirical force field used in the simulations and that more elaborate force fields may be required to describe appropriately the thermodynamics of proteins. PMID:20408169

  15. A fully automatic evolutionary classification of protein folds: Dali Domain Dictionary version 3.

    Science.gov (United States)

    Dietmann, S; Park, J; Notredame, C; Heger, A; Lappe, M; Holm, L

    2001-01-01

    The Dali Domain Dictionary (http://www.ebi.ac.uk/dali/domain) is a numerical taxonomy of all known structures in the Protein Data Bank (PDB). The taxonomy is derived fully automatically from measurements of structural, functional and sequence similarities. Here, we report the extension of the classification to match the traditional four hierarchical levels corresponding to: (i) supersecondary structural motifs (attractors in fold space), (ii) the topology of globular domains (fold types), (iii) remote homologues (functional families) and (iv) homologues with sequence identity above 25% (sequence families). The computational definitions of attractors and functional families are new. In September 2000, the Dali classification contained 10 531 PDB entries comprising 17 101 chains, which were partitioned into five attractor regions, 1375 fold types, 2582 functional families and 3724 domain sequence families. Sequence families were further associated with 99 582 unique homologous sequences in the HSSP database, which increases the number of effectively known structures several-fold. The resulting database contains the description of protein domain architecture, the definition of structural neighbours around each known structure, the definition of structurally conserved cores and a comprehensive library of explicit multiple alignments of distantly related protein families.

  16. Long range correlations and folding angle with applications to α-helical proteins

    Energy Technology Data Exchange (ETDEWEB)

    Krokhotin, Andrey, E-mail: Andrei.Krokhotine@cern.ch [Department of Physics and Astronomy, Uppsala University, P.O. Box 803, S-75108, Uppsala (Sweden); Nicolis, Stam, E-mail: Stam.Nicolis@lmpt.univ-tours.fr [Laboratoire de Mathematiques et Physique Theorique CNRS UMR 6083, Fédération Denis Poisson, Université de Tours, Parc de Grandmont, F37200 Tours (France); Niemi, Antti J., E-mail: Antti.Niemi@physics.uu.se [Department of Physics and Astronomy, Uppsala University, P.O. Box 803, S-75108, Uppsala (Sweden); Laboratoire de Mathematiques et Physique Theorique CNRS UMR 6083, Fédération Denis Poisson, Université de Tours, Parc de Grandmont, F37200 Tours (France); Department of Physics, Beijing Institute of Technology, Haidian District, Beijing 100081 (China)

    2014-03-07

    The conformational complexity of chain-like macromolecules such as proteins and other linear polymers is much larger than that of point-like atoms and molecules. Unlike particles, chains can bend, twist, and even become knotted. Thus chains might also display a much richer phase structure. Unfortunately, it is not very easy to characterize the phase of a long chain. Essentially, the only known attribute is the radius of gyration. The way how it changes when the degree of polymerization becomes different, and how it evolves when the ambient temperature and solvent properties change, is commonly used to disclose the phase. But in any finite length chain there are corrections to scaling that complicate the detailed analysis of the phase structure. Here we introduce a quantity that we call the folding angle to identify and scrutinize the phase structure, as a complement to the radius of gyration. We argue for a mean-field level relationship between the folding angle and the scaling exponent in the radius of gyration. We then estimate the value of the folding angle in the case of crystallographic α-helical protein structures in the Protein Data Bank. We also show how the experimental value of the folding angle can be obtained computationally, using a semiclassical Born-Oppenheimer description of α-helical chiral chains.

  17. Mapping the Protein Fold Universe Using the CamTube Force Field in Molecular Dynamics Simulations.

    Science.gov (United States)

    Kukic, Predrag; Kannan, Arvind; Dijkstra, Maurits J J; Abeln, Sanne; Camilloni, Carlo; Vendruscolo, Michele

    2015-10-01

    It has been recently shown that the coarse-graining of the structures of polypeptide chains as self-avoiding tubes can provide an effective representation of the conformational space of proteins. In order to fully exploit the opportunities offered by such a 'tube model' approach, we present here a strategy to combine it with molecular dynamics simulations. This strategy is based on the incorporation of the 'CamTube' force field into the Gromacs molecular dynamics package. By considering the case of a 60-residue polyvaline chain, we show that CamTube molecular dynamics simulations can comprehensively explore the conformational space of proteins. We obtain this result by a 20 μs metadynamics simulation of the polyvaline chain that recapitulates the currently known protein fold universe. We further show that, if residue-specific interaction potentials are added to the CamTube force field, it is possible to fold a protein into a topology close to that of its native state. These results illustrate how the CamTube force field can be used to explore efficiently the universe of protein folds with good accuracy and very limited computational cost.

  18. Topology-based potentials and the study of the competition between protein folding and aggregation.

    Science.gov (United States)

    Prieto, Lidia; Rey, Antonio

    2009-03-21

    Topology-based or Go-type potentials have been shown to be very useful in the understanding of the relations between the structure of the native state of a protein and some of its folding characteristics. A different question is whether they can also make such a contribution when the aggregation process of misfolded or partially folded structures is under study. In this work, in spite of the obvious trend of these simulation models toward the native state, we show that there are some aspects about aggregation that can be addressed by topology-based potentials: the role of the thermodynamic characteristics of the transition on preventing the aggregation process, or the larger propensity of highly symmetric protein structures to form domain swapped dimers. In a second part of this work, we use the possibilities of computer simulation as a design of numerical experiments to analyze the fundamental role of intermediate states in the aggregation process of globular proteins.

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

    DEFF Research Database (Denmark)

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.

    2016-01-01

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

  20. A multi-directional rapidly exploring random graph (mRRG) for protein folding

    KAUST Repository

    Nath, Shuvra Kanti

    2012-01-01

    Modeling large-scale protein motions, such as those involved in folding and binding interactions, is crucial to better understanding not only how proteins move and interact with other molecules but also how proteins misfold, thus causing many devastating diseases. Robotic motion planning algorithms, such as Rapidly Exploring Random Trees (RRTs), have been successful in simulating protein folding pathways. Here, we propose a new multi-directional Rapidly Exploring Random Graph (mRRG) specifically tailored for proteins. Unlike traditional RRGs which only expand a parent conformation in a single direction, our strategy expands the parent conformation in multiple directions to generate new samples. Resulting samples are connected to the parent conformation and its nearest neighbors. By leveraging multiple directions, mRRG can model the protein motion landscape with reduced computational time compared to several other robotics-based methods for small to moderate-sized proteins. Our results on several proteins agree with experimental hydrogen out-exchange, pulse-labeling, and F-value analysis. We also show that mRRG covers the conformation space better as compared to the other computation methods. Copyright © 2012 ACM.

  1. ProtASR: An Evolutionary Framework for Ancestral Protein Reconstruction with Selection on Folding Stability.

    Science.gov (United States)

    Arenas, Miguel; Weber, Claudia C; Liberles, David A; Bastolla, Ugo

    2017-11-01

    The computational reconstruction of ancestral proteins provides information on past biological events and has practical implications for biomedicine and biotechnology. Currently available tools for ancestral sequence reconstruction (ASR) are often based on empirical amino acid substitution models that assume that all sites evolve at the same rate and under the same process. However, this assumption is frequently violated because protein evolution is highly heterogeneous due to different selective constraints among sites. Here, we present ProtASR, a new evolutionary framework to infer ancestral protein sequences accounting for selection on protein stability. First, ProtASR generates site-specific substitution matrices through the structurally constrained mean-field (MF) substitution model, which considers both unfolding and misfolding stability. We previously showed that MF models outperform empirical amino acid substitution models, as well as other structurally constrained substitution models, both in terms of likelihood and correctly inferring amino acid distributions across sites. In the second step, ProtASR adapts a well-established maximum-likelihood (ML) ASR procedure to infer ancestral proteins under MF models. A known bias of ML ASR methods is that they tend to overestimate the stability of ancestral proteins by underestimating the frequency of deleterious mutations. We compared ProtASR under MF to two empirical substitution models (JTT and CAT), reconstructing the ancestral sequences of simulated proteins. ProtASR yields reconstructed proteins with less biased stabilities, which are significantly closer to those of the simulated proteins. Analysis of extant protein families suggests that folding stability evolves through time across protein families, potentially reflecting neutral fluctuation. Some families exhibit a more constant protein folding stability, while others are more variable. ProtASR is freely available from https

  2. Different secondary structure elements as scaffolds for protein folding transition states of two homologous four-helix bundles

    DEFF Research Database (Denmark)

    Teilum, Kaare; Thormann, Thorsten; Caterer, Nigel R.

    2005-01-01

    Comparison of the folding processes for homologue proteins can provide valuable information about details in the interactions leading to the formation of the folding transition state. Here the folding kinetics of 18 variants of yACBP and 3 variants of bACBP have been studied by -value analysis...

  3. Rapid measurement of residual dipolar couplings for fast fold elucidation of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Rasia, Rodolfo M. [Jean-Pierre Ebel CNRS/CEA/UJF, Institut de Biologie Structurale (France); Lescop, Ewen [CNRS, Institut de Chimie des Substances Naturelles (France); Palatnik, Javier F. [Universidad Nacional de Rosario, Instituto de Biologia Molecular y Celular de Rosario, Facultad de Ciencias Bioquimicas y Farmaceuticas (Argentina); Boisbouvier, Jerome, E-mail: jerome.boisbouvier@ibs.fr; Brutscher, Bernhard, E-mail: Bernhard.brutscher@ibs.fr [Jean-Pierre Ebel CNRS/CEA/UJF, Institut de Biologie Structurale (France)

    2011-11-15

    It has been demonstrated that protein folds can be determined using appropriate computational protocols with NMR chemical shifts as the sole source of experimental restraints. While such approaches are very promising they still suffer from low convergence resulting in long computation times to achieve accurate results. Here we present a suite of time- and sensitivity optimized NMR experiments for rapid measurement of up to six RDCs per residue. Including such an RDC data set, measured in less than 24 h on a single aligned protein sample, greatly improves convergence of the Rosetta-NMR protocol, allowing for overnight fold calculation of small proteins. We demonstrate the performance of our fast fold calculation approach for ubiquitin as a test case, and for two RNA-binding domains of the plant protein HYL1. Structure calculations based on simulated RDC data highlight the importance of an accurate and precise set of several complementary RDCs as additional input restraints for high-quality de novo structure determination.

  4. Structure-based phylogeny as a diagnostic for functional characterization of proteins with a cupin fold.

    Science.gov (United States)

    Agarwal, Garima; Rajavel, Malligarjunan; Gopal, Balasubramanian; Srinivasan, Narayanaswamy

    2009-05-29

    The members of cupin superfamily exhibit large variations in their sequences, functions, organization of domains, quaternary associations and the nature of bound metal ion, despite having a conserved beta-barrel structural scaffold. Here, an attempt has been made to understand structure-function relationships among the members of this diverse superfamily and identify the principles governing functional diversity. The cupin superfamily also contains proteins for which the structures are available through world-wide structural genomics initiatives but characterized as "hypothetical". We have explored the feasibility of obtaining clues to functions of such proteins by means of comparative analysis with cupins of known structure and function. A 3-D structure-based phylogenetic approach was undertaken. Interestingly, a dendrogram generated solely on the basis of structural dissimilarity measure at the level of domain folds was found to cluster functionally similar members. This clustering also reflects an independent evolution of the two domains in bicupins. Close examination of structural superposition of members across various functional clusters reveals structural variations in regions that not only form the active site pocket but are also involved in interaction with another domain in the same polypeptide or in the oligomer. Structure-based phylogeny of cupins can influence identification of functions of proteins of yet unknown function with cupin fold. This approach can be extended to other proteins with a common fold that show high evolutionary divergence. This approach is expected to have an influence on the function annotation in structural genomics initiatives.

  5. Structure-based phylogeny as a diagnostic for functional characterization of proteins with a cupin fold.

    Directory of Open Access Journals (Sweden)

    Garima Agarwal

    2009-05-01

    Full Text Available The members of cupin superfamily exhibit large variations in their sequences, functions, organization of domains, quaternary associations and the nature of bound metal ion, despite having a conserved beta-barrel structural scaffold. Here, an attempt has been made to understand structure-function relationships among the members of this diverse superfamily and identify the principles governing functional diversity. The cupin superfamily also contains proteins for which the structures are available through world-wide structural genomics initiatives but characterized as "hypothetical". We have explored the feasibility of obtaining clues to functions of such proteins by means of comparative analysis with cupins of known structure and function.A 3-D structure-based phylogenetic approach was undertaken. Interestingly, a dendrogram generated solely on the basis of structural dissimilarity measure at the level of domain folds was found to cluster functionally similar members. This clustering also reflects an independent evolution of the two domains in bicupins. Close examination of structural superposition of members across various functional clusters reveals structural variations in regions that not only form the active site pocket but are also involved in interaction with another domain in the same polypeptide or in the oligomer.Structure-based phylogeny of cupins can influence identification of functions of proteins of yet unknown function with cupin fold. This approach can be extended to other proteins with a common fold that show high evolutionary divergence. This approach is expected to have an influence on the function annotation in structural genomics initiatives.

  6. Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

    Science.gov (United States)

    Malhotra, Pooja; Jethva, Prashant N; Udgaonkar, Jayant B

    2017-08-08

    To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

  7. IntFOLD: an integrated server for modelling protein structures and functions from amino acid sequences.

    Science.gov (United States)

    McGuffin, Liam J; Atkins, Jennifer D; Salehe, Bajuna R; Shuid, Ahmad N; Roche, Daniel B

    2015-07-01

    IntFOLD is an independent web server that integrates our leading methods for structure and function prediction. The server provides a simple unified interface that aims to make complex protein modelling data more accessible to life scientists. The server web interface is designed to be intuitive and integrates a complex set of quantitative data, so that 3D modelling results can be viewed on a single page and interpreted by non-expert modellers at a glance. The only required input to the server is an amino acid sequence for the target protein. Here we describe major performance and user interface updates to the server, which comprises an integrated pipeline of methods for: tertiary structure prediction, global and local 3D model quality assessment, disorder prediction, structural domain prediction, function prediction and modelling of protein-ligand interactions. The server has been independently validated during numerous CASP (Critical Assessment of Techniques for Protein Structure Prediction) experiments, as well as being continuously evaluated by the CAMEO (Continuous Automated Model Evaluation) project. The IntFOLD server is available at: http://www.reading.ac.uk/bioinf/IntFOLD/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Studies of protein structure in solution and protein folding using synchrotron small-angle x-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Lingling [Stanford Univ., CA (United States)

    1996-04-01

    Synchrotron small angle x-ray scattering (SAXS) has been applied to the structural study of several biological systems, including the nitrogenase complex, the heat shock cognate protein (hsc70), and lysozyme folding. The structural information revealed from the SAXS experiments is complementary to information obtained by other physical and biochemical methods, and adds to our knowledge and understanding of these systems.

  9. The free energy landscape analysis of protein (FIP35) folding dynamics.

    Science.gov (United States)

    Krivov, Sergei V

    2011-10-27

    A fundamental problem in the analysis of protein folding and other complex reactions is the determination of the reaction free energy landscape. The current experimental techniques lack the necessary spatial and temporal resolution to construct such landscapes. The properties of the landscapes can be probed only indirectly. Simulation, assuming that it reproduces the experimental dynamics, can provide the necessary spatial and temporal resolution. It is, arguably, the only way for direct rigorous construction of the quantitatively accurate free energy landscapes. Here, such landscape is constructed from the equilibrium folding simulation of FIP35 protein reported by Shaw et al. Science 2010, 330, 341-346. For the dynamics to be accurately described as diffusion on the free energy landscape, the choice of reaction coordinates is crucial. The reaction coordinate used here is such that the dynamics projected on it is diffusive, so the description is consistent and accurate. The obtained landscape suggests an alternative interpretation of the simulation, markedly different from that of Shaw et al. In particular, FIP35 is not an incipient downhill folder, it folds via a populated on-pathway intermediate separated by high free energy barriers; the high free energy barriers rather than landscape roughness are a major determinant of the rates for conformational transitions; the preexponential factor of folding kinetics 1/k(0) ∼ 10 ns rather than 1 μs.

  10. Folding helical proteins in explicit solvent using dihedral-biased tempering.

    Science.gov (United States)

    Zhang, Cheng; Ma, Jianpeng

    2012-05-22

    Using a single-trajectory-based tempering method with a high-temperature dihedral bias, we repeatedly folded four helical proteins [α(3)D (PDB ID: 2A3D, 73 residues), α(3)W (1LQ7, 67 residues), Fap1-NR(α) (2KUB, 81 residues) and S-836 (2JUA, 102 residues)] and some of the mutants in explicit solvent within several microseconds. The lowest root-mean-square deviations of backbone atoms from the experimentally determined structures were 1.9, 1.4, 1.0, and 2.1 Å, respectively. Cluster analyses of folding trajectories showed the native conformation usually occupied the most populated cluster. The simulation protocol can be applied to large-scale simulations of other helical proteins on commonly accessible computing platforms.

  11. Folding induced supramolecular assembly into pH-responsive nanorods with a protein repellent shell.

    Science.gov (United States)

    Otter, R; Klinker, K; Spitzer, D; Schinnerer, M; Barz, M; Besenius, P

    2018-01-04

    We report the synthesis of ABA' triblock peptide-polysarcosine-peptide conjugates featuring two complementary phenylalanine-histidine pentapeptide strands A/A'. These sequences encode for antiparallel β-sheet formation into folded conjugates, which promote the self-assembly into polysarcosine-shielded core-shell nanorods. These do not cause aggregation of serum proteins in human blood plasma underlining an enhanced stability.

  12. Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions

    OpenAIRE

    Cieplak, Andrzej Stanis?aw

    2017-01-01

    Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of foldin...

  13. Amino acid alphabet reduction preserves fold information contained in contact interactions in proteins.

    Science.gov (United States)

    Solis, Armando D

    2015-12-01

    To reduce complexity, understand generalized rules of protein folding, and facilitate de novo protein design, the 20-letter amino acid alphabet is commonly reduced to a smaller alphabet by clustering amino acids based on some measure of similarity. In this work, we seek the optimal alphabet that preserves as much of the structural information found in long-range (contact) interactions among amino acids in natively-folded proteins. We employ the Information Maximization Device, based on information theory, to partition the amino acids into well-defined clusters. Numbering from 2 to 19 groups, these optimal clusters of amino acids, while generated automatically, embody well-known properties of amino acids such as hydrophobicity/polarity, charge, size, and aromaticity, and are demonstrated to maintain the discriminative power of long-range interactions with minimal loss of mutual information. Our measurements suggest that reduced alphabets (of less than 10) are able to capture virtually all of the information residing in native contacts and may be sufficient for fold recognition, as demonstrated by extensive threading tests. In an expansive survey of the literature, we observe that alphabets derived from various approaches-including those derived from physicochemical intuition, local structure considerations, and sequence alignments of remote homologs-fare consistently well in preserving contact interaction information, highlighting a convergence in the various factors thought to be relevant to the folding code. Moreover, we find that alphabets commonly used in experimental protein design are nearly optimal and are largely coherent with observations that have arisen in this work. © 2015 Wiley Periodicals, Inc.

  14. Right- and left-handed three-helix proteins. I. Experimental and simulation analysis of differences in folding and structure.

    Science.gov (United States)

    Glyakina, Anna V; Pereyaslavets, Leonid B; Galzitskaya, Oxana V

    2013-09-01

    Despite the large number of publications on three-helix protein folding, there is no study devoted to the influence of handedness on the rate of three-helix protein folding. From the experimental studies, we make a conclusion that the left-handed three-helix proteins fold faster than the right-handed ones. What may explain this difference? An important question arising in this paper is whether the modeling of protein folding can catch the difference between the protein folding rates of proteins with similar structures but with different folding mechanisms. To answer this question, the folding of eight three-helix proteins (four right-handed and four left-handed), which are similar in size, was modeled using the Monte Carlo and dynamic programming methods. The studies allowed us to determine the orders of folding of the secondary-structure elements in these domains and amino acid residues which are important for the folding. The obtained data are in good correlation with each other and with the experimental data. Structural analysis of these proteins demonstrated that the left-handed domains have a lesser number of contacts per residue and a smaller radius of cross section than the right-handed domains. This may be one of the explanations of the observed fact. The same tendency is observed for the large dataset consisting of 332 three-helix proteins (238 right- and 94 left-handed). From our analysis, we found that the left-handed three-helix proteins have some less-dense packing that should result in faster folding for some proteins as compared to the case of right-handed proteins. Copyright © 2013 Wiley Periodicals, Inc.

  15. Highly anomalous energetics of protein cold denaturation linked to folding-unfolding kinetics.

    Science.gov (United States)

    Romero-Romero, M Luisa; Inglés-Prieto, Alvaro; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2011-01-01

    Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a "mirror image" of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions.

  16. Systematic analysis of short internal indels and their impact on protein folding

    Directory of Open Access Journals (Sweden)

    Guo Jun-tao

    2010-08-01

    Full Text Available Abstract Background Protein sequence insertions/deletions (indels can be introduced during evolution or through alternative splicing (AS. Alternative splicing is an important biological phenomenon and is considered as the major means of expanding structural and functional diversity in eukaryotes. Knowledge of the structural changes due to indels is critical to our understanding of the evolution of protein structure and function. In addition, it can help us probe the evolution of alternative splicing and the diversity of functional isoforms. However, little is known about the effects of indels, in particular the ones involving core secondary structures, on the folding of protein structures. The long term goal of our study is to accurately predict the protein AS isoform structures. As a first step towards this goal, we performed a systematic analysis on the structural changes caused by short internal indels through mining highly homologous proteins in Protein Data Bank (PDB. Results We compiled a non-redundant dataset of short internal indels (2-40 amino acids from highly homologous protein pairs and analyzed the sequence and structural features of the indels. We found that about one third of indel residues are in disordered state and majority of the residues are exposed to solvent, suggesting that these indels are generally located on the surface of proteins. Though naturally occurring indels are fewer than engineered ones in the dataset, there are no statistically significant differences in terms of amino acid frequencies and secondary structure types between the "Natural" indels and "All" indels in the dataset. Structural comparisons show that all the protein pairs with short internal indels in the dataset preserve the structural folds and about 85% of protein pairs have global RMSDs (root mean square deviations of 2Å or less, suggesting that protein structures tend to be conserved and can tolerate short insertions and deletions. A few pairs

  17. Protein folding: Defining a standard set of experimental conditions and a preliminary kinetic data set of two-state proteins

    DEFF Research Database (Denmark)

    Maxwell, Karen L.; Wildes, D.; Zarrine-Afsar, A.

    2005-01-01

    rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized...... constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a consensus set of experimental conditions (25°C at pH 7.0, 50 m......M buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our...

  18. Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

    Directory of Open Access Journals (Sweden)

    Christen Y. L. Yuen

    2013-10-01

    Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

  19. Targeting the OB-Folds of Replication Protein A with Small Molecules

    Directory of Open Access Journals (Sweden)

    Victor J. Anciano Granadillo

    2010-01-01

    Full Text Available Replication protein A (RPA is the main eukaryotic single-strand (ss DNA-binding protein involved in DNA replication and repair. We have identified and developed two classes of small molecule inhibitors (SMIs that show in vitro inhibition of the RPA-DNA interaction. We present further characterization of these SMIs with respect to their target binding, mechanism of action, and specificity. Both reversible and irreversible modes of inhibition are observed for the different classes of SMIs with one class found to specifically interact with DNA-binding domains A and B (DBD-A/B of RPA. In comparison with other oligonucleotide/oligosaccharide binding-fold (OB-fold containing ssDNA-binding proteins, one class of SMIs displayed specificity for the RPA protein. Together these data demonstrate that the specific targeting of a protein-DNA interaction can be exploited towards interrogating the cellular activity of RPA as well as increasing the efficacy of DNA-damaging chemotherapeutics used in cancer treatment.

  20. MFIB: a repository of protein complexes with mutual folding induced by binding.

    Science.gov (United States)

    Fichó, Erzsébet; Reményi, István; Simon, István; Mészáros, Bálint

    2017-11-15

    It is commonplace that intrinsically disordered proteins (IDPs) are involved in crucial interactions in the living cell. However, the study of protein complexes formed exclusively by IDPs is hindered by the lack of data and such analyses remain sporadic. Systematic studies benefited other types of protein-protein interactions paving a way from basic science to therapeutics; yet these efforts require reliable datasets that are currently lacking for synergistically folding complexes of IDPs. Here we present the Mutual Folding Induced by Binding (MFIB) database, the first systematic collection of complexes formed exclusively by IDPs. MFIB contains an order of magnitude more data than any dataset used in corresponding studies and offers a wide coverage of known IDP complexes in terms of flexibility, oligomeric composition and protein function from all domains of life. The included complexes are grouped using a hierarchical classification and are complemented with structural and functional annotations. MFIB is backed by a firm development team and infrastructure, and together with possible future community collaboration it will provide the cornerstone for structural and functional studies of IDP complexes. MFIB is freely accessible at http://mfib.enzim.ttk.mta.hu/. The MFIB application is hosted by Apache web server and was implemented in PHP. To enrich querying features and to enhance backend performance a MySQL database was also created. simon.istvan@ttk.mta.hu, meszaros.balint@ttk.mta.hu. Supplementary data are available at Bioinformatics online.

  1. Aromatic thiol-mediated cleavage of N-O bonds enables chemical ubiquitylation of folded proteins.

    Science.gov (United States)

    Weller, Caroline E; Dhall, Abhinav; Ding, Feizhi; Linares, Edlaine; Whedon, Samuel D; Senger, Nicholas A; Tyson, Elizabeth L; Bagert, John D; Li, Xiaosong; Augusto, Ohara; Chatterjee, Champak

    2016-09-29

    Access to protein substrates homogenously modified by ubiquitin (Ub) is critical for biophysical and biochemical investigations aimed at deconvoluting the myriad biological roles for Ub. Current chemical strategies for protein ubiquitylation, however, employ temporary ligation auxiliaries that are removed under harsh denaturing conditions and have limited applicability. We report an unprecedented aromatic thiol-mediated N-O bond cleavage and its application towards native chemical ubiquitylation with the ligation auxiliary 2-aminooxyethanethiol. Our interrogation of the reaction mechanism suggests a disulfide radical anion as the active species capable of cleaving the N-O bond. The successful semisynthesis of full-length histone H2B modified by the small ubiquitin-like modifier-3 (SUMO-3) protein further demonstrates the generalizability and compatibility of our strategy with folded proteins.

  2. Adaptive local learning in sampling based motion planning for protein folding.

    Science.gov (United States)

    Ekenna, Chinwe; Thomas, Shawna; Amato, Nancy M

    2016-08-01

    Simulating protein folding motions is an important problem in computational biology. Motion planning algorithms, such as Probabilistic Roadmap Methods, have been successful in modeling the folding landscape. Probabilistic Roadmap Methods and variants contain several phases (i.e., sampling, connection, and path extraction). Most of the time is spent in the connection phase and selecting which variant to employ is a difficult task. Global machine learning has been applied to the connection phase but is inefficient in situations with varying topology, such as those typical of folding landscapes. We develop a local learning algorithm that exploits the past performance of methods within the neighborhood of the current connection attempts as a basis for learning. It is sensitive not only to different types of landscapes but also to differing regions in the landscape itself, removing the need to explicitly partition the landscape. We perform experiments on 23 proteins of varying secondary structure makeup with 52-114 residues. We compare the success rate when using our methods and other methods. We demonstrate a clear need for learning (i.e., only learning methods were able to validate against all available experimental data) and show that local learning is superior to global learning producing, in many cases, significantly higher quality results than the other methods. We present an algorithm that uses local learning to select appropriate connection methods in the context of roadmap construction for protein folding. Our method removes the burden of deciding which method to use, leverages the strengths of the individual input methods, and it is extendable to include other future connection methods.

  3. A replica exchange Monte Carlo algorithm for protein folding in the HP model

    Directory of Open Access Journals (Sweden)

    Shmygelska Alena

    2007-09-01

    Full Text Available Abstract Background The ab initio protein folding problem consists of predicting protein tertiary structure from a given amino acid sequence by minimizing an energy function; it is one of the most important and challenging problems in biochemistry, molecular biology and biophysics. The ab initio protein folding problem is computationally challenging and has been shown to be NP MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaat0uy0HwzTfgDPnwy1egaryqtHrhAL1wy0L2yHvdaiqaacqWFneVtcqqGqbauaaa@3961@-hard even when conformations are restricted to a lattice. In this work, we implement and evaluate the replica exchange Monte Carlo (REMC method, which has already been applied very successfully to more complex protein models and other optimization problems with complex energy landscapes, in combination with the highly effective pull move neighbourhood in two widely studied Hydrophobic Polar (HP lattice models. Results We demonstrate that REMC is highly effective for solving instances of the square (2D and cubic (3D HP protein folding problem. When using the pull move neighbourhood, REMC outperforms current state-of-the-art algorithms for most benchmark instances. Additionally, we show that this new algorithm provides a larger ensemble of ground-state structures than the existing state-of-the-art methods. Furthermore, it scales well with sequence length, and it finds significantly better conformations on long biological sequences and sequences with a provably unique ground-state structure, which is believed to be a characteristic of real proteins. We also present evidence that our REMC algorithm can fold sequences which exhibit significant interaction between termini in the hydrophobic core relatively easily. Conclusion We demonstrate that REMC utilizing the pull move

  4. Protein folding optimization based on 3D off-lattice model via an improved artificial bee colony algorithm.

    Science.gov (United States)

    Li, Bai; Lin, Mu; Liu, Qiao; Li, Ya; Zhou, Changjun

    2015-10-01

    Protein folding is a fundamental topic in molecular biology. Conventional experimental techniques for protein structure identification or protein folding recognition require strict laboratory requirements and heavy operating burdens, which have largely limited their applications. Alternatively, computer-aided techniques have been developed to optimize protein structures or to predict the protein folding process. In this paper, we utilize a 3D off-lattice model to describe the original protein folding scheme as a simplified energy-optimal numerical problem, where all types of amino acid residues are binarized into hydrophobic and hydrophilic ones. We apply a balance-evolution artificial bee colony (BE-ABC) algorithm as the minimization solver, which is featured by the adaptive adjustment of search intensity to cater for the varying needs during the entire optimization process. In this work, we establish a benchmark case set with 13 real protein sequences from the Protein Data Bank database and evaluate the convergence performance of BE-ABC algorithm through strict comparisons with several state-of-the-art ABC variants in short-term numerical experiments. Besides that, our obtained best-so-far protein structures are compared to the ones in comprehensive previous literature. This study also provides preliminary insights into how artificial intelligence techniques can be applied to reveal the dynamics of protein folding. Graphical Abstract Protein folding optimization using 3D off-lattice model and advanced optimization techniques.

  5. The Folding process of Human Profilin-1, a novel protein associated with familial amyotrophic lateral sclerosis.

    Science.gov (United States)

    Del Poggetto, Edoardo; Chiti, Fabrizio; Bemporad, Francesco

    2015-07-31

    Human profilin-1 is a novel protein associated with a recently discovered form of familial amyotrophic lateral sclerosis. This urges the characterization of possible conformational states, different from the fully folded state, potentially able to initiate self-assembly. Under native conditions, profilin-1 is monomeric and possesses a well-defined secondary and tertiary structure. When incubated at low pH or with high urea concentrations, profilin-1 remains monomeric but populates unfolded states exhibiting larger hydrodynamic radius and disordered structure, as assessed by dynamic light scattering, far-UV circular dichroism and intrinsic fluorescence. Refolding from the urea-unfolded state was studied at equilibrium and in real-time using a stopped-flow apparatus. The results obtained with intrinsic fluorescence and circular dichroism indicate a single phase without significant changes of the corresponding signals before the major refolding transition. However, such a transition is preceded by a burst phase with an observed increase of ANS fluorescence, which indicates the conversion into a transiently populated collapsed state possessing solvent-exposed hydrophobic clusters. Kinetic analysis reveals that such state has a conformational stability comparable to that of the fully unfolded state. To our knowledge, profilin-1 is the first example of an amyloid-related protein where folding occurs in the absence of thermodynamically stable partially folded states.

  6. Optimized parameter selection reveals trends in Markov state models for protein folding.

    Science.gov (United States)

    Husic, Brooke E; McGibbon, Robert T; Sultan, Mohammad M; Pande, Vijay S

    2016-11-21

    As molecular dynamics simulations access increasingly longer time scales, complementary advances in the analysis of biomolecular time-series data are necessary. Markov state models offer a powerful framework for this analysis by describing a system's states and the transitions between them. A recently established variational theorem for Markov state models now enables modelers to systematically determine the best way to describe a system's dynamics. In the context of the variational theorem, we analyze ultra-long folding simulations for a canonical set of twelve proteins [K. Lindorff-Larsen et al., Science 334, 517 (2011)] by creating and evaluating many types of Markov state models. We present a set of guidelines for constructing Markov state models of protein folding; namely, we recommend the use of cross-validation and a kinetically motivated dimensionality reduction step for improved descriptions of folding dynamics. We also warn that precise kinetics predictions rely on the features chosen to describe the system and pose the description of kinetic uncertainty across ensembles of models as an open issue.

  7. Shedding Light on Protein Folding, Structural and Functional Dynamics by Single Molecule Studies

    Directory of Open Access Journals (Sweden)

    Krutika Bavishi

    2014-11-01

    Full Text Available The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions.

  8. Fluorescence quenching of buried Trp residues by acrylamide does not require penetration of the protein fold.

    Science.gov (United States)

    Strambini, Giovanni B; Gonnelli, Margherita

    2010-01-21

    The accessibility of acrylamide to buried Trp residues in proteins, as attested by dynamic quenching of their fluorescence emission, is often interpreted in terms of migration of the quencher (Q) through the globular fold. The quencher penetration mechanism, however, has long been debated because, on one hand, solutes the size of acrylamide are not expected to diffuse within the protein matrix on the nanosecond time scale of fluorescence and, on the other hand, alternative reactions pathways where Q remains in the solvent cannot be ruled out. To test the Q penetration hypothesis, we compared the quenching rates of acrylamide analogs of increasing molecular size (acrylonitrile, acrylamide, and bis-acrylamide) on the buried Trp residues of RNaseT1 and parvalbumin. The results show that the largest molecule, bis-acrylamide, is also the most efficient quencher and that in general the quenching rate is not correlated to quencher size, as expected for a penetration mechanism. Whereas these results rule out significant internal Q migration in the times of fluorescence, it is also demonstrated that up to a depth of burial of 3 A, through-space interactions with acrylamide in the solvent satisfactorily account for the small rate constants reported for these proteins. More generally, this analysis emphasizes that reduced dynamic quenching of protein fluorescence by acrylamide rather than reporting on the structural rigidity of the globular fold reflects the distance of closest approach between the internal chromophore and Q in the aqueous phase.

  9. Improving protein fold recognition and structural class prediction accuracies using physicochemical properties of amino acids.

    Science.gov (United States)

    Raicar, Gaurav; Saini, Harsh; Dehzangi, Abdollah; Lal, Sunil; Sharma, Alok

    2016-08-07

    Predicting the three-dimensional (3-D) structure of a protein is an important task in the field of bioinformatics and biological sciences. However, directly predicting the 3-D structure from the primary structure is hard to achieve. Therefore, predicting the fold or structural class of a protein sequence is generally used as an intermediate step in determining the protein's 3-D structure. For protein fold recognition (PFR) and structural class prediction (SCP), two steps are required - feature extraction step and classification step. Feature extraction techniques generally utilize syntactical-based information, evolutionary-based information and physicochemical-based information to extract features. In this study, we explore the importance of utilizing the physicochemical properties of amino acids for improving PFR and SCP accuracies. For this, we propose a Forward Consecutive Search (FCS) scheme which aims to strategically select physicochemical attributes that will supplement the existing feature extraction techniques for PFR and SCP. An exhaustive search is conducted on all the existing 544 physicochemical attributes using the proposed FCS scheme and a subset of physicochemical attributes is identified. Features extracted from these selected attributes are then combined with existing syntactical-based and evolutionary-based features, to show an improvement in the recognition and prediction performance on benchmark datasets. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Linking computation and experiments to study the role of charge-charge interactions in protein folding and stability

    Science.gov (United States)

    Makhatadze, George I.

    2017-02-01

    Over the past two decades there has been an increase in appreciation for the role of surface charge-charge interactions in protein folding and stability. The perception shifted from the belief that charge-charge interactions are not important for protein folding and stability to the near quantitative understanding of how these interactions shape the folding energy landscape. This led to the ability of computational approaches to rationally redesign surface charge-charge interactions to modulate thermodynamic properties of proteins. Here we summarize our progress in understanding the role of charge-charge interactions for protein stability using examples drawn from my own laboratory and touch upon unanswered questions.

  11. Potyvirus virion structure shows conserved protein fold and RNA binding site in ssRNA viruses.

    Science.gov (United States)

    Zamora, Miguel; Méndez-López, Eduardo; Agirrezabala, Xabier; Cuesta, Rebeca; Lavín, José L; Sánchez-Pina, M Amelia; Aranda, Miguel A; Valle, Mikel

    2017-09-01

    Potyviruses constitute the second largest genus of plant viruses and cause important economic losses in a large variety of crops; however, the atomic structure of their particles remains unknown. Infective potyvirus virions are long flexuous filaments where coat protein (CP) subunits assemble in helical mode bound to a monopartite positive-sense single-stranded RNA [(+)ssRNA] genome. We present the cryo-electron microscopy (cryoEM) structure of the potyvirus watermelon mosaic virus at a resolution of 4.0 Å. The atomic model shows a conserved fold for the CPs of flexible filamentous plant viruses, including a universally conserved RNA binding pocket, which is a potential target for antiviral compounds. This conserved fold of the CP is widely distributed in eukaryotic viruses and is also shared by nucleoproteins of enveloped viruses with segmented (-)ssRNA (negative-sense ssRNA) genomes, including influenza viruses.

  12. Circadian rhythms. A protein fold switch joins the circadian oscillator to clock output in cyanobacteria.

    Science.gov (United States)

    Chang, Yong-Gang; Cohen, Susan E; Phong, Connie; Myers, William K; Kim, Yong-Ick; Tseng, Roger; Lin, Jenny; Zhang, Li; Boyd, Joseph S; Lee, Yvonne; Kang, Shannon; Lee, David; Li, Sheng; Britt, R David; Rust, Michael J; Golden, Susan S; LiWang, Andy

    2015-07-17

    Organisms are adapted to the relentless cycles of day and night, because they evolved timekeeping systems called circadian clocks, which regulate biological activities with ~24-hour rhythms. The clock of cyanobacteria is driven by a three-protein oscillator composed of KaiA, KaiB, and KaiC, which together generate a circadian rhythm of KaiC phosphorylation. We show that KaiB flips between two distinct three-dimensional folds, and its rare transition to an active state provides a time delay that is required to match the timing of the oscillator to that of Earth's rotation. Once KaiB switches folds, it binds phosphorylated KaiC and captures KaiA, which initiates a phase transition of the circadian cycle, and it regulates components of the clock-output pathway, which provides the link that joins the timekeeping and signaling functions of the oscillator. Copyright © 2015, American Association for the Advancement of Science.

  13. Silent substitutions predictably alter translation elongation rates and protein folding efficiencies.

    Science.gov (United States)

    Spencer, Paige S; Siller, Efraín; Anderson, John F; Barral, José M

    2012-09-21

    Genetic code redundancy allows most amino acids to be encoded by multiple codons that are non-randomly distributed along coding sequences. An accepted theory explaining the biological significance of such non-uniform codon selection is that codons are translated at different speeds. Thus, varying codon placement along a message may confer variable rates of polypeptide emergence from the ribosome, which may influence the capacity to fold toward the native state. Previous studies report conflicting results regarding whether certain codons correlate with particular structural or folding properties of the encoded protein. This is partly due to different criteria traditionally utilized for predicting translation speeds of codons, including their usage frequencies and the concentration of tRNA species capable of decoding them, which do not always correlate. Here, we developed a metric to predict organism-specific relative translation rates of codons based on the availability of tRNA decoding mechanisms: Watson-Crick, non-Watson-Crick or both types of interactions. We determine translation rates of messages by pulse-chase analyses in living Escherichia coli cells and show that sequence engineering based on these concepts predictably modulates translation rates in a manner that is superior to codon usage frequency, which occur during the elongation phase, and significantly impacts folding of the encoded polypeptide. Finally, we demonstrate that sequence harmonization based on expression host tRNA pools, designed to mimic ribosome movement of the original organism, can significantly increase the folding of the encoded polypeptide. These results illuminate how genetic code degeneracy may function to specify properties beyond amino acid encoding, including folding. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Effect of hydrogen bond networks on the nucleation mechanism of protein folding.

    Science.gov (United States)

    Djikaev, Y S; Ruckenstein, Eli

    2009-12-01

    We have recently developed a kinetic model for the nucleation mechanism of protein folding (NMPF) in terms of ternary nucleation by using the first passage time analysis. A protein was considered as a random heteropolymer consisting of hydrophobic, hydrophilic (some of which are negatively or positively ionizable), and neutral beads. The main idea of the NMPF model consisted of averaging the dihedral potential in which a selected residue is involved over all possible configurations of all neighboring residues along the protein chain. The combination of the average dihedral, effective pairwise (due to Lennard-Jones-type and electrostatic interactions), and confining (due to the polymer connectivity constraint) potentials gives rise to an overall potential around the cluster that, as a function of the distance from the cluster center, has a double-well shape. This allows one to evaluate the protein folding time. In the original NMPF model hydrogen bonding was not taken into account explicitly. To improve the NMPF model and make it more realistic, in this paper we modify our (previously developed) probabilistic hydrogen bond model and combine it with the former. Thus, a contribution due to the disruption of hydrogen bond networks around the interacting particles (cluster of native residues and residue in the protein unfolded part) appears in the overall potential field around a cluster. The modified model is applied to the folding of the same model proteins that were examined in the original model: a short protein consisting of 124 residues (roughly mimicking bovine pancreatic ribonuclease) and a long one consisting of 2500 residues (as a representative of large proteins with superlong polypeptide chains), at pH=8.3 , 7.3, and 6.3. The hydrogen bond contribution now plays a dominant role in the total potential field around the cluster (except for very short distances thereto where the repulsive energy tends to infinity). It is by an order of magnitude stronger for

  15. Shedding light on protein folding, structural and functional dynamics by single molecule studies

    DEFF Research Database (Denmark)

    Bavishi, Krutika; Hatzakis, Nikos

    2014-01-01

    The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean...... property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out...... in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions...

  16. The folding trajectory of RNase H is dominated by its topology and not local stability: a protein engineering study of variants that fold via two-state and three-state mechanisms.

    Science.gov (United States)

    Connell, Katelyn B; Miller, Erik J; Marqusee, Susan

    2009-08-14

    Proteins can sample a variety of partially folded conformations during the transition between the unfolded and native states. Some proteins never significantly populate these high-energy states and fold by an apparently two-state process. However, many proteins populate detectable, partially folded forms during the folding process. The role of such intermediates is a matter of considerable debate. A single amino acid change can convert Escherichia coli ribonuclease H from a three-state folder that populates a kinetic intermediate to one that folds in an apparent two-state fashion. We have compared the folding trajectories of the three-state RNase H and the two-state RNase H, proteins with the same native-state topology but altered regional stability, using a protein engineering approach. Our data suggest that both versions of RNase H fold through a similar trajectory with similar high-energy conformations. Mutations in the core and the periphery of the protein affect similar aspects of folding for both variants, suggesting a common trajectory with folding of the core region followed by the folding of the periphery. Our results suggest that formation of specific partially folded conformations may be a general feature of protein folding that can promote, rather than hinder, efficient folding.

  17. Crystallization of small proteins assisted by green fluorescent protein.

    Science.gov (United States)

    Suzuki, Nobuhiro; Hiraki, Masahiko; Yamada, Yusuke; Matsugaki, Naohiro; Igarashi, Noriyuki; Kato, Ryuichi; Dikic, Ivan; Drew, David; Iwata, So; Wakatsuki, Soichi; Kawasaki, Masato

    2010-10-01

    The generation of crystal lattice contacts by proteinaceous tags fused to target proteins is an attractive approach to aid in the crystallization of otherwise intractable proteins. Here, the use of green fluorescent protein (GFP) fusions for this purpose is demonstrated, using ubiquitin and the ubiquitin-binding motif (UBM) of Y-family polymerase ι as examples. The structure of the GFP-ubiquitin fusion protein revealed that the crystal lattice was formed by GFP moieties. Ubiquitin was accommodated in the lattice through interactions with the peripheral loops of GFP. However, in the GFP-UBM fusion crystal UBM formed extensive interactions with GFP and these interactions, together with UBM dimerization, mediated the crystal packing. Interestingly, the tyrosine residues that are involved in mediating crystal contacts in both GFP-ubiquitin and GFP-UBM crystals are arranged in a belt on the surface of the β-barrel structure of GFP. Therefore, it is likely that GFP can assist in the crystallization of small proteins and of protein domains in general.

  18. Unraveling protein folding mechanism by analyzing the hierarchy of models with increasing level of detail.

    Science.gov (United States)

    Hayashi, Tomohiko; Yasuda, Satoshi; Škrbić, Tatjana; Giacometti, Achille; Kinoshita, Masahiro

    2017-09-28

    Taking protein G with 56 residues for a case study, we investigate the mechanism of protein folding. In addition to its native structure possessing α-helix and β-sheet contents of 27% and 39%, respectively, we construct a number of misfolded decoys with a wide variety of α-helix and β-sheet contents. We then consider a hierarchy of 8 different models with increasing level of detail in terms of the number of entropic and energetic physical factors incorporated. The polyatomic structure is always taken into account, but the side chains are removed in half of the models. The solvent is formed by either neutral hard spheres or water molecules. Protein intramolecular hydrogen bonds (H-bonds) and protein-solvent H-bonds (the latter is present only in water) are accounted for or not, depending on the model considered. We then apply a physics-based free-energy function (FEF) corresponding to each model and investigate which structures are most stabilized. This special approach taken on a step-by-step basis enables us to clarify the role of each physical factor in contributing to the structural stability and separately elucidate its effect. Depending on the model employed, significantly different structures such as very compact configurations with no secondary structures and configurations of associated α-helices are optimally stabilized. The native structure can be identified as that with lowest FEF only when the most detailed model is employed. This result is significant for at least the two reasons: The most detailed model considered here is able to capture the fundamental aspects of protein folding notwithstanding its simplicity; and it is shown that the native structure is stabilized by a complex interplay of minimal multiple factors that must be all included in the description. In the absence of even a single of these factors, the protein is likely to be driven towards a different, more stable state.

  19. Unraveling protein folding mechanism by analyzing the hierarchy of models with increasing level of detail

    Science.gov (United States)

    Hayashi, Tomohiko; Yasuda, Satoshi; Škrbić, Tatjana; Giacometti, Achille; Kinoshita, Masahiro

    2017-09-01

    Taking protein G with 56 residues for a case study, we investigate the mechanism of protein folding. In addition to its native structure possessing α-helix and β-sheet contents of 27% and 39%, respectively, we construct a number of misfolded decoys with a wide variety of α-helix and β-sheet contents. We then consider a hierarchy of 8 different models with increasing level of detail in terms of the number of entropic and energetic physical factors incorporated. The polyatomic structure is always taken into account, but the side chains are removed in half of the models. The solvent is formed by either neutral hard spheres or water molecules. Protein intramolecular hydrogen bonds (H-bonds) and protein-solvent H-bonds (the latter is present only in water) are accounted for or not, depending on the model considered. We then apply a physics-based free-energy function (FEF) corresponding to each model and investigate which structures are most stabilized. This special approach taken on a step-by-step basis enables us to clarify the role of each physical factor in contributing to the structural stability and separately elucidate its effect. Depending on the model employed, significantly different structures such as very compact configurations with no secondary structures and configurations of associated α-helices are optimally stabilized. The native structure can be identified as that with lowest FEF only when the most detailed model is employed. This result is significant for at least the two reasons: The most detailed model considered here is able to capture the fundamental aspects of protein folding notwithstanding its simplicity; and it is shown that the native structure is stabilized by a complex interplay of minimal multiple factors that must be all included in the description. In the absence of even a single of these factors, the protein is likely to be driven towards a different, more stable state.

  20. The Protein Kingdom Extended: Ordered and Intrinsically Disordered Proteins, Their Folding, Supramolecular Complex Formation, and Aggregation

    Science.gov (United States)

    Turoverov, Konstantin K.; Kuznetsova, Irina M.; Uversky, Vladimir N.

    2010-01-01

    The native state of a protein is usually associated with a compact globular conformation possessing a rigid and highly ordered structure. At the turn of the last century certain studies arose which concluded that many proteins cannot, in principle, form a rigid globular structure in an aqueous environment, but they are still able to fulfill their specific functions — i.e., they are native. The existence of the disordered regions allows these proteins to interact with their numerous binding partners. Such interactions are often accompanied by the formation of complexes that possess a more ordered structure than the original components. The functional diversity of these proteins, combined with the variability of signals related to the various intra-and intercellular processes handled by these proteins and their capability to produce multi-variant and multi-directional responses allow them to form a unique regulatory net in a cell. The abundance of disordered proteins inside the cell is precisely controlled at the synthesis and clearance levels as well as via interaction with specific binding partners and posttranslational modifications. Another recently recognized biologically active state of proteins is the functional amyloid. The formation of such functional amyloids is tightly controlled and therefore differs from the uncontrolled formation of pathogenic amyloids which are associated with the pathogenesis of several conformational diseases, the development of which is likely to be determined by the failures of the cellular regulatory systems rather than by the formation of the proteinaceous deposits and/or by the protofibril toxicity. PMID:20097220

  1. Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism

    Science.gov (United States)

    Greenfield, Norma J.

    2009-01-01

    Circular dichroism (CD) is an excellent tool for examining the interactions and stability of proteins. This protocol covers methods to obtain and analyze circular dichroism spectra to measure changes in the folding of proteins as a function of denaturants, osmolytes or ligands. Applications include determination of the free energy of folding of a protein, the effects of mutations on protein stability and the estimation of binding constants for the interactions of proteins with other proteins, DNA or ligands, such as substrates or inhibitors. The experiments take 2-5 h. PMID:17406529

  2. RMI, a new OB-fold complex essential for Bloom syndrome protein to maintain genome stability.

    Science.gov (United States)

    Xu, Dongyi; Guo, Rong; Sobeck, Alexandra; Bachrati, Csanad Z; Yang, Jay; Enomoto, Takemi; Brown, Grant W; Hoatlin, Maureen E; Hickson, Ian D; Wang, Weidong

    2008-10-15

    BLM, the helicase mutated in Bloom syndrome, associates with topoisomerase 3alpha, RMI1 (RecQ-mediated genome instability), and RPA, to form a complex essential for the maintenance of genome stability. Here we report a novel component of the BLM complex, RMI2, which interacts with RMI1 through two oligonucleotide-binding (OB)-fold domains similar to those in RPA. The resulting complex, named RMI, differs from RPA in that it lacks obvious DNA-binding activity. Nevertheless, RMI stimulates the dissolution of a homologous recombination intermediate in vitro and is essential for the stability, localization, and function of the BLM complex in vivo. Notably, inactivation of RMI2 in chicken DT40 cells results in an increased level of sister chromatid exchange (SCE)--the hallmark feature of Bloom syndrome cells. Epistasis analysis revealed that RMI2 and BLM suppress SCE within the same pathway. A point mutation in the OB domain of RMI2 disrupts the association between BLM and the rest of the complex, and abrogates the ability of RMI2 to suppress elevated SCE. Our data suggest that multi-OB-fold complexes mediate two modes of BLM action: via RPA-mediated protein-DNA interaction, and via RMI-mediated protein-protein interactions.

  3. Determining Protein Folding Pathway and Associated Energetics through Partitioned Integrated-Tempering-Sampling Simulation.

    Science.gov (United States)

    Shao, Qiang; Shi, Jiye; Zhu, Weiliang

    2017-03-14

    Replica exchange molecular dynamics (REMD) and integrated-tempering-sampling (ITS) are two representative enhanced sampling methods which utilize parallel and integrated tempering approaches, respectively. In this work, a partitioned integrated-tempering-sampling (P-ITS) method is proposed which takes advantage of the benefits of both parallel and integrated tempering approaches. Using P-ITS, the folding pathways of a series of proteins with diverse native structures are explored on multidimensional free-energy landscapes, and the associated thermodynamics are evaluated. In comparison to the original form of ITS, P-ITS improves the sampling efficiency and measures the folding/unfolding thermodynamic quantities more consistently with experimental data. In comparison to REMD, P-ITS significantly reduces the requirement of computational resources and meanwhile achieves similar simulation results. The observed structural characterizations of transition and intermediate states of the proteins under study are in good agreement with previous experimental and simulation studies on the same proteins and homologues. Therefore, the P-ITS method has great potential in simulating the structural dynamics of complex biomolecular systems.

  4. Folding 19 proteins to their native state and stability of large proteins from a coarse-grained model.

    Science.gov (United States)

    Kapoor, Abhijeet; Travesset, Alex

    2014-03-01

    We develop an intermediate resolution model, where the backbone is modeled with atomic resolution but the side chain with a single bead, by extending our previous model (Proteins (2013) DOI: 10.1002/prot.24269) to properly include proline, preproline residues and backbone rigidity. Starting from random configurations, the model properly folds 19 proteins (including a mutant 2A3D sequence) into native states containing β sheet, α helix, and mixed α/β. As a further test, the stability of H-RAS (a 169 residue protein, critical in many signaling pathways) is investigated: The protein is stable, with excellent agreement with experimental B-factors. Despite that proteins containing only α helices fold to their native state at lower backbone rigidity, and other limitations, which we discuss thoroughly, the model provides a reliable description of the dynamics as compared with all atom simulations, but does not constrain secondary structures as it is typically the case in more coarse-grained models. Further implications are described. Copyright © 2013 Wiley Periodicals, Inc.

  5. TMFoldWeb: a web server for predicting transmembrane protein fold class.

    Science.gov (United States)

    Kozma, Dániel; Tusnády, Gábor E

    2015-09-17

    Here we present TMFoldWeb, the web server implementation of TMFoldRec, a transmembrane protein fold recognition algorithm. TMFoldRec uses statistical potentials and utilizes topology filtering and a gapless threading algorithm. It ranks template structures and selects the most likely candidates and estimates the reliability of the obtained lowest energy model. The statistical potential was developed in a maximum likelihood framework on a representative set of the PDBTM database. According to the benchmark test the performance of TMFoldRec is about 77 % in correctly predicting fold class for a given transmembrane protein sequence. An intuitive web interface has been developed for the recently published TMFoldRec algorithm. The query sequence goes through a pipeline of topology prediction and a systematic sequence to structure alignment (threading). Resulting templates are ordered by energy and reliability values and are colored according to their significance level. Besides the graphical interface, a programmatic access is available as well, via a direct interface for developers or for submitting genome-wide data sets. The TMFoldWeb web server is unique and currently the only web server that is able to predict the fold class of transmembrane proteins while assigning reliability scores for the prediction. This method is prepared for genome-wide analysis with its easy-to-use interface, informative result page and programmatic access. Considering the info-communication evolution in the last few years, the developed web server, as well as the molecule viewer, is responsive and fully compatible with the prevalent tablets and mobile devices.

  6. Template-assisted fabrication of protein nanocapsules

    Science.gov (United States)

    Dougherty, Shelley A.; Liang, Jianyu; Kowalik, Timothy F.

    2009-02-01

    Bionanomaterials have recently begun to spark a great amount of interest and could potentially revolutionize biomedical research. Nanoparticles, nanocapsules, and nanotubular structures are becoming attractive options in drug and gene delivery. The size of the delivery vehicles greatly impacts cellular uptake and makes it highly desirable to precisely control the diameter and length of nanocarriers to make uniform nanoparticles at low cost. Carbon nanotubes have shown great potential within the field of drug and gene delivery. However, their insolubility and cytotoxicity could severely delay FDA approval. A desirable alternative would be to fabricate nanostructures from biomaterials such as proteins, peptides, or liposomes, which are already FDA approved. In this article we demonstrate the preparation of protein nanocapsules with both ends sealed using a template-assisted alternate immersion method combined with controlled cleaving. Glucose oxidase nanocapsules with controllable diameter, wall thickness, and length were fabricated and characterized with SEM and TEM. The biochemical activity of glucose oxidase in the form of nanocapsules after processing was confirmed using UV spectrometry. Our future work will explore proteins suitable for drug encapsulation and cellular uptake and will focus on optimizing the cleaving process to gain precise control over the length of the nanocapsules.

  7. Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding.

    Science.gov (United States)

    Chen, Daming; Xiang, Qiwang; Nicholas, John

    2017-11-15

    Viral interleukin-6 (vIL-6) encoded by human herpesvirus 8 (HHV-8) is believed to contribute via mitogenic, survival, and angiogenic activities to HHV-8-associated Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease through autocrine or paracrine mechanisms during latency or productive replication. There is direct evidence that vIL-6 promotes latently infected PEL cell viability and proliferation and also viral productive replication in PEL and endothelial cells. These activities are mediated largely through endoplasmic reticulum (ER)-localized vIL-6, which can induce signal transduction via the gp130 signaling receptor, activating mitogen-activated protein kinase and signal transducer and activator of transcription signaling, and interactions of vIL-6 with the ER membrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). The latter functional axis involves suppression of proapoptotic lysosomal protein cathepsin D by promotion of the ER-associated degradation of ER-transiting, preproteolytically processed procathepsin D. Other interactions of VKORC1v2 and activities of vIL-6 via the receptor have not been reported. We show here that both vIL-6 and VKORC1v2 interact with calnexin cycle proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), which catalyzes monoglucosylation of N-glycans, and oppositely acting glucosidase II (GlucII), and that vIL-6 can promote protein folding. This activity was found to require VKORC1v2 and UGGT1, to involve vIL-6 associations with VKORC1v2, UGGT1, and GlucII, and to operate in the context of productively infected cells. These findings document new VKORC1v2-associated interactions and activities of vIL-6, revealing novel mechanisms of vIL-6 function within the ER compartment. IMPORTANCE HHV-8 vIL-6 prosurvival (latent) and proreplication functions are mediated from the ER compartment through both gp130 receptor-mediated signal transduction and interaction of v

  8. A structural basis for cellular uptake of GST-fold proteins.

    Directory of Open Access Journals (Sweden)

    Melanie J Morris

    Full Text Available It has recently emerged that glutathione transferase enzymes (GSTs and other structurally related molecules can be translocated from the external medium into many different cell types. In this study we aim to explore in detail, the structural features that govern cell translocation and by dissecting the human GST enzyme GSTM2-2 we quantatively demonstrate that the α-helical C-terminal domain (GST-C is responsible for this property. Attempts to further examine the constituent helices within GST-C resulted in a reduction in cell translocation efficiency, indicating that the intrinsic GST-C domain structure is necessary for maximal cell translocation capacity. In particular, it was noted that the α-6 helix of GST-C plays a stabilising role in the fold of this domain. By destabilising the conformation of GST-C, an increase in cell translocation efficiency of up to ∼2-fold was observed. The structural stability profiles of these protein constructs have been investigated by circular dichroism and differential scanning fluorimetry measurements and found to impact upon their cell translocation efficiency. These experiments suggest that the globular, helical domain in the 'GST-fold' structural motif plays a role in influencing cellular uptake, and that changes that affect the conformational stability of GST-C can significantly influence cell translocation efficiency.

  9. Modeling the effect of codon translation rates on co-translational protein folding mechanisms of arbitrary complexity.

    Science.gov (United States)

    Caniparoli, Luca; O'Brien, Edward P

    2015-04-14

    In a cell, the folding of a protein molecule into tertiary structure can begin while it is synthesized by the ribosome. The rate at which individual amino acids are incorporated into the elongating nascent chain has been shown to affect the likelihood that proteins will populate their folded state, indicating that co-translational protein folding is a far from equilibrium process. Developing a theoretical framework to accurately describe this process is, therefore, crucial for advancing our understanding of how proteins acquire their functional conformation in living cells. Current state-of-the-art computational approaches, such as molecular dynamics simulations, are very demanding in terms of the required computer resources, making the simulation of co-translational protein folding difficult. Here, we overcome this limitation by introducing an efficient approach that predicts the effects that variable codon translation rates have on co-translational folding pathways. Our approach is based on Markov chains. By using as an input a relatively small number of molecular dynamics simulations, it allows for the computation of the probability that a nascent protein is in any state as a function of the translation rate of individual codons along a mRNA's open reading frame. Due to its computational efficiency and favorable scalability with the complexity of the folding mechanism, this approach could enable proteome-wide computational studies of the influence of translation dynamics on co-translational folding.

  10. Mutational Analysis of the Binding-Induced Folding Reaction of the Mixed-Lineage Leukemia Protein to the KIX Domain.

    Science.gov (United States)

    Toto, Angelo; Gianni, Stefano

    2016-07-19

    Intrinsically disordered proteins represent a large class of proteins that lack a well-defined three-dimensional structure in isolation but can undergo a disorder to order transition upon binding to their physiological ligands. Understanding the mechanism by which these proteins fold upon binding represents a challenge. Here we present a detailed mutational study of the kinetics of the binding reaction between the transactivation domain of the mixed-lineage leukemia protein, an intrinsically disordered protein, and the KIX domain, performed under different experimental conditions. The experimental data allow us to infer the mechanism of folding upon binding and to pinpoint the key interactions present in the transition state. Furthermore, we identify a peculiar malleability of the observed mechanism upon changes in reaction conditions. This finding, which is in opposition to the robustness typically observed in the folding of globular proteins, is discussed in the context of previous work on intrinsically disordered proteins.

  11. Folding and association of an extremely stable dimeric protein from Sulfolobus islandicus.

    Science.gov (United States)

    Zeeb, Markus; Lipps, Georg; Lilie, Hauke; Balbach, Jochen

    2004-02-06

    ORF56 is a plasmid-encoded protein from Sulfolobus islandicus, which probably controls the copy number of the pRN1 plasmid by binding to its own promotor. The protein showed an extremely high stability in denaturant, heat, and pH-induced unfolding transitions, which can be well described by a two-state reaction between native dimers and unfolded monomers. The homodimeric character of native ORF56 was confirmed by analytical ultracentrifugation. Far-UV circular dichroism and fluorescence spectroscopy gave superimposable denaturant-induced unfolding transitions and the midpoints of both heat as well as denaturant-induced unfolding depend on the protein concentration supporting the two-state model. This model was confirmed by GdmSCN-induced unfolding monitored by heteronuclear 2D NMR spectroscopy. Chemical denaturation was accomplished by GdmCl and GdmSCN, revealing a Gibbs free energy of stabilization of -85.1 kJ/mol at 25 degrees C. Thermal unfolding was possible only above 1 M GdmCl, which shifted the melting temperature (t(m)) below the boiling point of water. Linear extrapolation of t(m) to 0 M GdmCl yielded a t(m) of 107.5 degrees C (5 microM monomer concentration). Additionally, ORF56 remains natively structured over a remarkable pH range from pH 2 to pH 12. Folding kinetics were followed by far-UV CD and fluorescence after either stopped-flow or manual mixing. All kinetic traces showed only a single phase and the two probes revealed coincident folding rates (k(f), k(u)), indicating the absence of intermediates. Apparent first-order refolding rates depend linearly on the protein concentration, whereas the unfolding rates do not. Both lnk(f) and lnk(u) depend linearly on the GdmCl concentration. Together, folding and association of homodimeric ORF56 are concurrent events. In the absence of denaturant ORF56 refolds fast (7.0 x 10(7)M(-1)s(-1)) and unfolds extremely slowly (5.7 year(-1)). Therefore, high stability is coupled to a slow unfolding rate, which is

  12. Thermodynamic Stabilization of the Folded Domain of Prion Protein Inhibits Prion Infection in Vivo

    Directory of Open Access Journals (Sweden)

    Qingzhong Kong

    2013-07-01

    Full Text Available Prion diseases, or transmissible spongiform encephalopathies (TSEs, are associated with the conformational conversion of the cellular prion protein, PrPC, into a protease-resistant form, PrPSc. Here, we show that mutation-induced thermodynamic stabilization of the folded, α-helical domain of PrPC has a dramatic inhibitory effect on the conformational conversion of prion protein in vitro, as well as on the propagation of TSE disease in vivo. Transgenic mice expressing a human prion protein variant with increased thermodynamic stability were found to be much more resistant to infection with the TSE agent than those expressing wild-type human prion protein, in both the primary passage and three subsequent subpassages. These findings not only provide a line of evidence in support of the protein-only model of TSEs but also yield insight into the molecular nature of the PrPC→PrPSc conformational transition, and they suggest an approach to the treatment of prion diseases.

  13. Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions.

    Science.gov (United States)

    Cieplak, Andrzej Stanisław

    2017-01-01

    Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of folding and misfolding is limited for want of a physicochemical theory of protein secondary and tertiary structure. Here we demonstrate that electronic configuration and hyperconjugation of the peptide amide bonds ought to be taken into account to advance such a theory. To capture the effect of polarization of peptide linkages on conformational and H-bonding propensity of the polypeptide backbone, we introduce a function of shielding tensors of the Cα atoms. Carrying no information about side chain-side chain interactions, this function nonetheless identifies basic features of the secondary and tertiary structure, establishes sequence correlates of the metamorphic and pH-driven equilibria, relates binding affinities and folding rate constants to secondary structure preferences, and manifests common patterns of backbone density distribution in amyloidogenic regions of Alzheimer's amyloid β and tau, Parkinson's α-synuclein and prions. Based on those findings, a split-intein like mechanism of molecular recognition is proposed to underlie dimerization of Aβ, tau, αS and PrPC, and divergent pathways for subsequent association of dimers are outlined; a related mechanism is proposed to underlie formation of PrPSc fibrils. The model does account for: (i) structural features of paranuclei, off-pathway oligomers, non-fibrillar aggregates and fibrils; (ii) effects of incubation conditions, point mutations, isoform lengths, small-molecule assembly modulators and chirality of solid

  14. Proteomic screening of glucose-responsive and glucose non-reponsive MIN-6 beta cells reveals differential expression of protein involved in protein folding, secretion and oxidative stress

    DEFF Research Database (Denmark)

    Dowling, P.; O´Driscoll, L.; O´Sullivan, F.

    2006-01-01

    .8%). From the differentially expressed proteins identified in this study, groups of proteins associated with the endoplasmic reticulum (ER) and proteins involved in oxidative stress were found to be significantly decreased in the high-passage (H passage) cells. These proteins included endoplasmic reticulum......-6 cells to successfully fold, modify or secrete proteins and counteract the problems associated with oxidative stress...

  15. Structure optimisation by thermal cycling for the hydrophobic-polar lattice model of protein folding

    Science.gov (United States)

    Günther, Florian; Möbius, Arnulf; Schreiber, Michael

    2017-03-01

    The function of a protein depends strongly on its spatial structure. Therefore the transition from an unfolded stage to the functional fold is one of the most important problems in computational molecular biology. Since the corresponding free energy landscapes exhibit huge numbers of local minima, the search for the lowest-energy configurations is very demanding. Because of that, efficient heuristic algorithms are of high value. In the present work, we investigate whether and how the thermal cycling (TC) approach can be applied to the hydrophobic-polar (HP) lattice model of protein folding. Evaluating the efficiency of TC for a set of two- and three-dimensional examples, we compare the performance of this strategy with that of multi-start local search (MSLS) procedures and that of simulated annealing (SA). For this aim, we incorporated several simple but rather efficient modifications into the standard procedures: in particular, a strong improvement was achieved by also allowing energy conserving state modifications. Furthermore, the consideration of ensembles instead of single samples was found to greatly improve the efficiency of TC. In the framework of different benchmarks, for all considered HP sequences, we found TC to be far superior to SA, and to be faster than Wang-Landau sampling.

  16. Transition path times reveal memory effects and anomalous diffusion in the dynamics of protein folding

    Science.gov (United States)

    Satija, Rohit; Das, Atanu; Makarov, Dmitrii E.

    2017-10-01

    Recent single-molecule experiments probed transition paths of biomolecular folding and, in particular, measured the time biomolecules spend while crossing their free energy barriers. A surprising finding from these studies is that the transition barriers crossed by transition paths, as inferred from experimentally observed transition path times, are often lower than the independently determined free energy barriers. Here we explore memory effects leading to anomalous diffusion as a possible origin of this discrepancy. Our analysis of several molecular dynamics trajectories shows that the dynamics of common reaction coordinates used to describe protein folding is subdiffusive, at least at sufficiently short times. We capture this effect using a one-dimensional fractional Brownian motion (FBM) model, in which the system undergoes a subdiffusive process in the presence of a potential of mean force, and show that this model yields much broader distributions of transition path times with stretched exponential long-time tails. Without any adjustable parameters, these distributions agree well with the transition path times computed directly from protein trajectories. We further discuss how the FBM model can be tested experimentally.

  17. Protein folding in mode space: a collective coordinate approach to structure prediction.

    Science.gov (United States)

    Abseher, Roger; Nilges, Michael

    2002-11-15

    Does the dynamics of a protein encode its structure? Many studies have addressed the inverse of this question-how a three-dimensional structure determines its dynamics-using molecular dynamics simulation, normal mode analysis, and similar methods. Recently we have developed a molecular dynamics (MD) simulation method to impose dynamic properties on ensembles of MD trajectories in the form of restraints on structural diversity in the directions of the principal components of motion of the molecule. In the current work, we investigate if these restraints in combination with a standard MD force field are sufficient to generate native structure in disordered structural ensembles. We present simulations from a series of increasingly disordered structural ensembles obtained by thermal unfolding or randomization of the coordinates of the native structure of two src-homology 3 (SH3) domains. Native structure formation is observed under the sole action of the diversity restraint and the MD force field. We investigate the importance of accuracy of the description of native dynamics. Protein folding is a highly cooperative process. The dynamic restraints may enforce long-range cooperativity and thus speed up the folding from unstructured states. Applications of the restraints to structure refinement and structure prediction are possible. Copyright 2002 Wiley-Liss, Inc.

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

    CERN Document Server

    Simmons, Walter

    2013-01-01

    Processes that proceed reliably from a variety of initial conditions to a unique final form, regardless of moderately changing conditions, are of obvious importance in biophysics. Protein folding is a case in point. We show that the action principle can be applied directly to study the stability of biological processes. The action principle in classical physics starts with the first variation of the action and leads immediately to the equations of motion. The second variation of the action leads in a natural way to powerful theorems that provide quantitative treatment of stability and focusing and also explain how some very complex processes can behave as though some seemingly important forces drop out. We first apply these ideas to the non-equilibrium states involved in two-state folding. We treat torsional waves and use the action principle to talk about critical points in the dynamics. For some proteins the theory resembles TST. We reach several quantitative and qualitative conclusions. Besides giving an e...

  19. Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

    Science.gov (United States)

    Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

    2016-06-01

    The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

  20. Kinks, loops, and protein folding, with protein A as an example

    Energy Technology Data Exchange (ETDEWEB)

    Krokhotin, Andrey, E-mail: Andrei.Krokhotine@cern.ch [Department of Physics and Astronomy and Science for Life Laboratory, Uppsala University, P.O. Box 803, S-75108 Uppsala (Sweden); Liwo, Adam, E-mail: adam@chem.univ.gda.pl [Faculty of Chemistry, University of Gdansk, ul. Sobieskiego 18, 80-952 Gdansk (Poland); Maisuradze, Gia G., E-mail: gm56@cornell.edu; Scheraga, Harold A., E-mail: has5@cornell.edu [Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301 (United States); Niemi, Antti J., E-mail: Antti.Niemi@physics.uu.se [Department of Physics and Astronomy and Science for Life Laboratory, Uppsala University, P.O. Box 803, S-75108 Uppsala (Sweden); Laboratoire de Mathematiques et Physique Theorique CNRS UMR 6083, Fédération Denis Poisson, Université de Tours, Parc de Grandmont, F37200 Tours, France and Department of Physics, Beijing Institute of Technology, Haidian District, Beijing 100081 (China)

    2014-01-14

    The dynamics and energetics of formation of loops in the 46-residue N-terminal fragment of the B-domain of staphylococcal protein A has been studied. Numerical simulations have been performed using coarse-grained molecular dynamics with the united-residue (UNRES) force field. The results have been analyzed in terms of a kink (heteroclinic standing wave solution) of a generalized discrete nonlinear Schrödinger (DNLS) equation. In the case of proteins, the DNLS equation arises from a C{sup α}-trace-based energy function. Three individual kink profiles were identified in the experimental three-α-helix structure of protein A, in the range of the Glu16-Asn29, Leu20-Asn29, and Gln33-Asn44 residues, respectively; these correspond to two loops in the native structure. UNRES simulations were started from the full right-handed α-helix to obtain a clear picture of kink formation, which would otherwise be blurred by helix formation. All three kinks emerged during coarse-grained simulations. It was found that the formation of each is accompanied by a local free energy increase; this is expressed as the change of UNRES energy which has the physical sense of the potential of mean force of a polypeptide chain. The increase is about 7 kcal/mol. This value can thus be considered as the free energy barrier to kink formation in full α-helical segments of polypeptide chains. During the simulations, the kinks emerge, disappear, propagate, and annihilate each other many times. It was found that the formation of a kink is initiated by an abrupt change in the orientation of a pair of consecutive side chains in the loop region. This resembles the formation of a Bloch wall along a spin chain, where the C{sup α} backbone corresponds to the chain, and the amino acid side chains are interpreted as the spin variables. This observation suggests that nearest-neighbor side chain–side chain interactions are responsible for initiation of loop formation. It was also found that the individual

  1. Single-molecule kinetics under force: probing protein folding and enzymatic activity with optical tweezers

    Science.gov (United States)

    Wong, Wesley

    2010-03-01

    Weak non-covalent bonds between and within single molecules govern many aspects of biological structure and function (e.g. DNA base-paring, receptor-ligand binding, protein folding, etc.) In living systems, these interactions are often subject to mechanical forces, which can greatly alter their kinetics and activity. My group develops and applies novel single-molecule manipulation techniques to explore and quantify these force-dependent kinetics. Using optical tweezers, we have quantified the force-dependent unfolding and refolding kinetics of different proteins, including the cytoskeletal protein spectrin in collaboration with E. Evans's group [1], and the A2 domain of the von Willebrand factor blood clotting protein in collaboration with T. Springer's group [2]. Furthermore, we have studied the kinetics of the ADAMTS13 enzyme acting on a single A2 domain, and have shown that physiolgical forces in the circulation can act as a cofactor for enzymatic cleavage, regulating hemostatic activity [2]. References: 1. E. Evans, K. Halvorsen, K. Kinoshita, and W.P. Wong, Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer (2009). 2. X. Zhang, K. Halvorsen, C.-Z. Zhang, W.P. Wong, and T.A. Springer, Science 324 (5932), 1330-1334 (2009).

  2. Web-based computational chemistry education with CHARMMing II: Coarse-grained protein folding.

    Science.gov (United States)

    Pickard, Frank C; Miller, Benjamin T; Schalk, Vinushka; Lerner, Michael G; Woodcock, H Lee; Brooks, Bernard R

    2014-07-01

    A lesson utilizing a coarse-grained (CG) Gō-like model has been implemented into the CHARMM INterface and Graphics (CHARMMing) web portal (www.charmming.org) to the Chemistry at HARvard Macromolecular Mechanics (CHARMM) molecular simulation package. While widely used to model various biophysical processes, such as protein folding and aggregation, CG models can also serve as an educational tool because they can provide qualitative descriptions of complex biophysical phenomena for a relatively cheap computational cost. As a proof of concept, this lesson demonstrates the construction of a CG model of a small globular protein, its simulation via Langevin dynamics, and the analysis of the resulting data. This lesson makes connections between modern molecular simulation techniques and topics commonly presented in an advanced undergraduate lecture on physical chemistry. It culminates in a straightforward analysis of a short dynamics trajectory of a small fast folding globular protein; we briefly describe the thermodynamic properties that can be calculated from this analysis. The assumptions inherent in the model and the data analysis are laid out in a clear, concise manner, and the techniques used are consistent with those employed by specialists in the field of CG modeling. One of the major tasks in building the Gō-like model is determining the relative strength of the nonbonded interactions between coarse-grained sites. New functionality has been added to CHARMMing to facilitate this process. The implementation of these features into CHARMMing helps automate many of the tedious aspects of constructing a CG Gō model. The CG model builder and its accompanying lesson should be a valuable tool to chemistry students, teachers, and modelers in the field.

  3. ModFOLD6: an accurate web server for the global and local quality estimation of 3D protein models.

    Science.gov (United States)

    Maghrabi, Ali H A; McGuffin, Liam J

    2017-04-29

    Methods that reliably estimate the likely similarity between the predicted and native structures of proteins have become essential for driving the acceptance and adoption of three-dimensional protein models by life scientists. ModFOLD6 is the latest version of our leading resource for Estimates of Model Accuracy (EMA), which uses a pioneering hybrid quasi-single model approach. The ModFOLD6 server integrates scores from three pure-single model methods and three quasi-single model methods using a neural network to estimate local quality scores. Additionally, the server provides three options for producing global score estimates, depending on the requirements of the user: (i) ModFOLD6_rank, which is optimized for ranking/selection, (ii) ModFOLD6_cor, which is optimized for correlations of predicted and observed scores and (iii) ModFOLD6 global for balanced performance. The ModFOLD6 methods rank among the top few for EMA, according to independent blind testing by the CASP12 assessors. The ModFOLD6 server is also continuously automatically evaluated as part of the CAMEO project, where significant performance gains have been observed compared to our previous server and other publicly available servers. The ModFOLD6 server is freely available at: http://www.reading.ac.uk/bioinf/ModFOLD/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. RECOVERY ACT - Thylakoid Assembly and Folded Protein Transport by the Tat Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Dabney-Smith, Carole [Miami Univ., Oxford, OH (United States)

    2016-07-18

    Assembly of functional photosystems complete with necessary intrinsic (membrane-bound) and extrinsic proteins requires the function of at least 3 protein transport pathways in thylakoid membranes. Our research focuses on one of those pathways, a unique and essential protein transport pathway found in the chloroplasts of plants, bacteria, and some archaebacteria, the Twin arginine translocation (Tat) system. The chloroplast Tat (cpTat) system is thought to be responsible for the proper location of ~50% of thylakoid lumen proteins, several of which are necessary for proper photosystem assembly, maintenance, and function. Specifically, cpTat systems are unique because they transport fully folded and assembled proteins across ion tight membranes using only three membrane components, Tha4, Hcf106, and cpTatC, and the protonmotive force generated by photosynthesis. Despite the importance of the cpTat system in plants, the mechanism of transport of a folded precursor is not well known. Our long-term goal is to investigate the role protein transport systems have on organelle biogenesis, particularly the assembly of membrane protein complexes in thylakoids of chloroplasts. The objective of this proposal is to correlate structural changes in the membrane-bound cpTat component, Tha4, to the mechanism of translocation of folded-precursor substrates across the membrane bilayer by using a cysteine accessibility and crosslinking approach. Our central hypothesis is that the precursor passes through a proteinaceous pore of assembled Tha4 protomers that have undergone a conformational or topological change in response to transport. This research is predicated upon the observations that Tha4 exists in molar excess in the membrane relative to the other cpTat components; its regulated assembly to the precursor-bound receptor; and our data showing oligomerization of Tha4 into very large complexes in response to transport. Our rationale for these studies is that understanding cp

  5. The structure of HasB reveals a new class of TonB protein fold.

    Directory of Open Access Journals (Sweden)

    Gisele Cardoso de Amorim

    Full Text Available TonB is a key protein in active transport of essential nutrients like vitamin B12 and metal sources through the outer membrane transporters of Gram-negative bacteria. This inner membrane protein spans the periplasm, contacts the outer membrane receptor by its periplasmic domain and transduces energy from the cytoplasmic membrane pmf to the receptor allowing nutrient internalization. Whereas generally a single TonB protein allows the acquisition of several nutrients through their cognate receptor, in some species one particular TonB is dedicated to a specific system. Despite a considerable amount of data available, the molecular mechanism of TonB-dependent active transport is still poorly understood. In this work, we present a structural study of a TonB-like protein, HasB dedicated to the HasR receptor. HasR acquires heme either free or via an extracellular heme transporter, the hemophore HasA. Heme is used as an iron source by bacteria. We have solved the structure of the HasB periplasmic domain of Serratia marcescens and describe its interaction with a critical region of HasR. Some important differences are observed between HasB and TonB structures. The HasB fold reveals a new structural class of TonB-like proteins. Furthermore, we have identified the structural features that explain the functional specificity of HasB. These results give a new insight into the molecular mechanism of nutrient active transport through the bacterial outer membrane and present the first detailed structural study of a specific TonB-like protein and its interaction with the receptor.

  6. Evaluating the effects of cutoffs and treatment of long-range electrostatics in protein folding simulations.

    Directory of Open Access Journals (Sweden)

    Stefano Piana

    Full Text Available The use of molecular dynamics simulations to provide atomic-level descriptions of biological processes tends to be computationally demanding, and a number of approximations are thus commonly employed to improve computational efficiency. In the past, the effect of these approximations on macromolecular structure and stability has been evaluated mostly through quantitative studies of small-molecule systems or qualitative observations of short-timescale simulations of biological macromolecules. Here we present a quantitative evaluation of two commonly employed approximations, using a test system that has been the subject of a number of previous protein folding studies--the villin headpiece. In particular, we examined the effect of (i the use of a cutoff-based force-shifting technique rather than an Ewald summation for the treatment of electrostatic interactions, and (ii the length of the cutoff used to determine how many pairwise interactions are included in the calculation of both electrostatic and van der Waals forces. Our results show that the free energy of folding is relatively insensitive to the choice of cutoff beyond 9 Å, and to whether an Ewald method is used to account for long-range electrostatic interactions. In contrast, we find that the structural properties of the unfolded state depend more strongly on the two approximations examined here.

  7. ASTRO-FOLD 2.0: an Enhanced Framework for Protein Structure Prediction.

    Science.gov (United States)

    Subramani, A; Wei, Y; Floudas, C A

    2012-05-01

    The three-dimensional (3-D) structure prediction of proteins, given their amino acid sequence, is addressed using the first principles-based approach ASTRO-FOLD 2.0. The key features presented are: (1) Secondary structure prediction using a novel optimization-based consensus approach, (2) β-sheet topology prediction using mixed-integer linear optimization (MILP), (3) Residue-to-residue contact prediction using a high-resolution distance-dependent force field and MILP formulation, (4) Tight dihedral angle and distance bound generation for loop residues using dihedral angle clustering and non-linear optimization (NLP), (5) 3-D structure prediction using deterministic global optimization, stochastic conformational space annealing, and the full-atomistic ECEPP/3 potential, (6) Near-native structure selection using a traveling salesman problem-based clustering approach, ICON, and (7) Improved bound generation using chemical shifts of subsets of heavy atoms, generated by SPARTA and CS23D. Computational results of ASTRO-FOLD 2.0 on 47 blind targets of the recently concluded CASP9 experiment are presented.

  8. Adaptive enhanced sampling with a path-variable for the simulation of protein folding and aggregation

    Science.gov (United States)

    Peter, Emanuel K.

    2017-12-01

    In this article, we present a novel adaptive enhanced sampling molecular dynamics (MD) method for the accelerated simulation of protein folding and aggregation. We introduce a path-variable L based on the un-biased momenta p and displacements dq for the definition of the bias s applied to the system and derive 3 algorithms: general adaptive bias MD, adaptive path-sampling, and a hybrid method which combines the first 2 methodologies. Through the analysis of the correlations between the bias and the un-biased gradient in the system, we find that the hybrid methodology leads to an improved force correlation and acceleration in the sampling of the phase space. We apply our method on SPC/E water, where we find a conservation of the average water structure. We then use our method to sample dialanine and the folding of TrpCage, where we find a good agreement with simulation data reported in the literature. Finally, we apply our methodologies on the initial stages of aggregation of a hexamer of Alzheimer's amyloid β fragment 25-35 (Aβ 25-35) and find that transitions within the hexameric aggregate are dominated by entropic barriers, while we speculate that especially the conformation entropy plays a major role in the formation of the fibril as a rate limiting factor.

  9. The Speed Limit of Protein Folding: Alpha-Helix Initiation Modeled and Observed

    Science.gov (United States)

    Lin, Milo; Mohammed, Omar; Zewail, Ahmed

    2011-03-01

    As a primary event of protein folding, alpha-helix initiation is the starting point of macromolecular complexity. In this work, an analytic coarse-grained model which predicts the initiation rate as a function of temperature, is presented. Helix initiation was measured via ultrafast temperature-jump fluorescence refolding experiments on two penta-peptides, and the measured rates agreed well with those of the model. In addition, the temporal separation of rate-limiting diffusion from fast annealing stipulated by the model was confirmed via ensemble-converging all-atom molecular dynamics simulations, which reproduced both the diffusion and the picosecond annealing processes and rates observed experimentally. Some of these results were published in: Mohammed OF, Jas GS, Lin MM, Ma H, Zewail AH (2009) Primary peptide folding dynamics observed with ultrafast temperature jump. Angew Chem 48: 5628-5632. We are grateful to the National Science Foundation and the National Institutes of Health for funding of this research at Caltech. M.M.L. acknowledges financial support from the Krell Institute and the US Department of Energy for a graduate fellowship.

  10. Unconventional Targeting of a Thiol Peroxidase to the Mitochondrial Intermembrane Space Facilitates Oxidative Protein Folding

    Directory of Open Access Journals (Sweden)

    Paraskevi Kritsiligkou

    2017-03-01

    Full Text Available Thiol peroxidases are conserved hydrogen peroxide scavenging and signaling molecules that contain redox-active cysteine residues. We show here that Gpx3, the major H2O2 sensor in yeast, is present in the mitochondrial intermembrane space (IMS, where it serves a compartment-specific role in oxidative metabolism. The IMS-localized Gpx3 contains an 18-amino acid N-terminally extended form encoded from a non-AUG codon. This acts as a mitochondrial targeting signal in a pathway independent of the hitherto known IMS-import pathways. Mitochondrial Gpx3 interacts with the Mia40 oxidoreductase in a redox-dependent manner and promotes efficient Mia40-dependent oxidative protein folding. We show that cells lacking Gpx3 have aberrant mitochondrial morphology, defective protein import capacity, and lower inner membrane potential, all of which can be rescued by expression of a mitochondrial-only form of Gpx3. Together, our data reveal a novel role for Gpx3 in mitochondrial redox regulation and protein homeostasis.

  11. Combining classifiers generated by multi-gene genetic programming for protein fold recognition using genetic algorithm.

    Science.gov (United States)

    Bardsiri, Mahshid Khatibi; Eftekhari, Mahdi; Mousavi, Reza

    2015-01-01

    In this study the problem of protein fold recognition, that is a classification task, is solved via a hybrid of evolutionary algorithms namely multi-gene Genetic Programming (GP) and Genetic Algorithm (GA). Our proposed method consists of two main stages and is performed on three datasets taken from the literature. Each dataset contains different feature groups and classes. In the first step, multi-gene GP is used for producing binary classifiers based on various feature groups for each class. Then, different classifiers obtained for each class are combined via weighted voting so that the weights are determined through GA. At the end of the first step, there is a separate binary classifier for each class. In the second stage, the obtained binary classifiers are combined via GA weighting in order to generate the overall classifier. The final obtained classifier is superior to the previous works found in the literature in terms of classification accuracy.

  12. Improvements in Mixing Time and Mixing Uniformity in Devices Designed for Studies of Protein Folding Kinetics

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Shuhuai [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Bakajin, Olgica [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2007-08-01

    Using a microfluidic laminar flow mixer designed for studies of protein folding kinetics, we demonstrate a mixing time of 1 +/- 1 micros with sample consumption on the order of femtomoles. We recognize two limitations of previously proposed designs: (1) size and shape of the mixing region, which limits mixing uniformity and (2) the formation of Dean vortices at high flow rates, which limits the mixing time. We address these limitations by using a narrow shape-optimized nozzle and by reducing the bend of the side channel streamlines. The final design, which combines both of these features, achieves the best performance. We quantified the mixing performance of the different designs by numerical simulation of coupled Navier-Stokes and convection-diffusion equations and experiments using fluorescence resonance energy-transfer (FRET)-labeled DNA.

  13. A comparative method for finding and folding RNA secondary structures within protein-coding regions

    DEFF Research Database (Denmark)

    Pedersen, Jakob Skou; Meyer, Irmtraud Margret; Forsberg, Roald

    2004-01-01

    Existing computational methods for RNA secondary-structure prediction tacitly assume RNA to only encode functional RNA structures. However, experimental studies have revealed that some RNA sequences, e.g. compact viral genomes, can simultaneously encode functional RNA structures as well as proteins...... that RNA-DECODER's parameters can be automatically trained to successfully fold known secondary structures within the HCV genome. We scan the genomes of HCV and polio virus for conserved secondary-structure elements, and analyze performance as a function of available evolutionary information. On known...... secondary structures, RNA-DECODER shows a sensitivity similar to the programs MFOLD, PFOLD and RNAALIFOLD. When scanning the entire genomes of HCV and polio virus for structure elements, RNA-DECODER's results indicate a markedly higher specificity than MFOLD, PFOLD and RNAALIFOLD....

  14. Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions.

    Directory of Open Access Journals (Sweden)

    Andrzej Stanisław Cieplak

    Full Text Available Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of folding and misfolding is limited for want of a physicochemical theory of protein secondary and tertiary structure. Here we demonstrate that electronic configuration and hyperconjugation of the peptide amide bonds ought to be taken into account to advance such a theory. To capture the effect of polarization of peptide linkages on conformational and H-bonding propensity of the polypeptide backbone, we introduce a function of shielding tensors of the Cα atoms. Carrying no information about side chain-side chain interactions, this function nonetheless identifies basic features of the secondary and tertiary structure, establishes sequence correlates of the metamorphic and pH-driven equilibria, relates binding affinities and folding rate constants to secondary structure preferences, and manifests common patterns of backbone density distribution in amyloidogenic regions of Alzheimer's amyloid β and tau, Parkinson's α-synuclein and prions. Based on those findings, a split-intein like mechanism of molecular recognition is proposed to underlie dimerization of Aβ, tau, αS and PrPC, and divergent pathways for subsequent association of dimers are outlined; a related mechanism is proposed to underlie formation of PrPSc fibrils. The model does account for: (i structural features of paranuclei, off-pathway oligomers, non-fibrillar aggregates and fibrils; (ii effects of incubation conditions, point mutations, isoform lengths, small-molecule assembly modulators and

  15. ZnO nanoparticles assist the refolding of denatured green fluorescent protein.

    Science.gov (United States)

    Pandurangan, Muthuraman; Zamany, Ahmad Jawid; Kim, Doo Hwan

    2016-04-01

    Proteins are essential for cellular and biological processes. Proteins are synthesized and fold into the native structure to become active. The inability of a protein molecule to remain in its native conformation is called as protein misfolding, and this is due to several environmental factors. Protein misfolding and aggregation handle several human diseases. Protein misfolding is believed to be one of the causes of several disorders such as cancer, degenerative diseases, and metabolic pathologies. The zinc oxide (ZnO) nanoparticle was significantly promoted refolding of thermally denatured green fluorescent protein (GFP). In the present study, ZnO nanoparticles interaction with GFP was investigated by ultraviolet-visible spectrophotometer, fluorescence spectrophotometer, and dynamic light scattering. Results suggest that the ZnO nanoparticles significantly assist the refolding of denatured GFP. Copyright © 2015 John Wiley & Sons, Ltd.

  16. Mimicking the action of folding chaperones in molecular dynamics simulations : Application to the refinement of homology-based protein structures

    NARCIS (Netherlands)

    Fan, H.; Mark, A.E.

    A novel method for the refinement of misfolded protein structures is proposed in which the properties of the solvent environment are oscillated in order to mimic some aspects of the role of molecular chaperones play in protein folding in vivo. Specifically, the hydrophobicity of the solvent is

  17. A membrane cell for on-line hydrogen/deuterium exchange to study protein folding and protein-protein interactions by mass spectrometry.

    Science.gov (United States)

    Astorga-Wells, Juan; Landreh, Michael; Johansson, Jan; Bergman, Tomas; Jörnvall, Hans

    2011-09-01

    A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.

  18. A Membrane Cell for On-line Hydrogen/Deuterium Exchange to Study Protein Folding and Protein-Protein Interactions by Mass Spectrometry*

    Science.gov (United States)

    Astorga-Wells, Juan; Landreh, Michael; Johansson, Jan; Bergman, Tomas; Jörnvall, Hans

    2011-01-01

    A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation. PMID:21610101

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  20. On the origins of the weak folding cooperativity of a designed ββα ultrafast protein FSD-1.

    Directory of Open Access Journals (Sweden)

    Chun Wu

    Full Text Available FSD-1, a designed small ultrafast folder with a ββα fold, has been actively studied in the last few years as a model system for studying protein folding mechanisms and for testing of the accuracy of computational models. The suitability of this protein to describe the folding of naturally occurring α/β proteins has recently been challenged based on the observation that the melting transition is very broad, with ill-resolved baselines. Using molecular dynamics simulations with the AMBER protein force field (ff96 coupled with the implicit solvent model (IGB = 5, we shed new light into the nature of this transition and resolve the experimental controversies. We show that the melting transition corresponds to the melting of the protein as a whole, and not solely to the helix-coil transition. The breadth of the folding transition arises from the spread in the melting temperatures (from ∼325 K to ∼302 K of the individual transitions: formation of the hydrophobic core, β-hairpin and tertiary fold, with the helix formed earlier. Our simulations initiated from an extended chain accurately predict the native structure, provide a reasonable estimate of the transition barrier height, and explicitly demonstrate the existence of multiple pathways and multiple transition states for folding. Our exhaustive sampling enables us to assess the quality of the Amber ff96/igb5 combination and reveals that while this force field can predict the correct native fold, it nonetheless overstabilizes the α-helix portion of the protein (Tm = ∼387K as well as the denatured structures.

  1. Applications of optical spectroscopy to studies of protein dynamics and folding

    Science.gov (United States)

    Wolanin, Peter Michael

    2000-11-01

    Understanding the dynamics and folding of a protein are challenges which require the application of a wide range of biophysical tools. This thesis describes experiments using two optical spectroscopies: fluorescence microscopy and tryptophan phosphorescence. For many protein systems, it is difficult to gather precise information from ensembles due to heterogeneity and lack of synchronicity for molecular events. A near- field scanning optical microscope (NSOM) developed for single-molecule studies is presented. Probe manufacture, force feedback, scan distance calibration, and the overall NSOM system design are discussed. Single fluorophores were imaged with a resolution below the diffraction limit using metal-coated probes. In addition, the first NSOM images of two-photon induced fluorescence from single fluorophores are presented. Tryptophan room temperature phosphorescence (RTP) lifetimes range from microseconds to seconds and have greater sensitivity to protein dynamics than tryptophan fluorescence. The latter part of this thesis begins with a review of the quantum mechanics of the triplet state in aromatic molecules. Next, measurements of RTP lifetime of ribonuclease T1 as a function of pH, salt, and temperature are presented and discussed. These disagree with results in the literature which suggested a con-elation in ribonuclease T1 between ΔG and the RTP lifetime. Several studies of alkaline phosphatase (AP) are presented. DSC and RTP measurements of apo AP as a function of pH, showed substantially different behavior for the native protein versus immediately after renaturation. The refolding of apo AP to its native state was monitored using RTP, and the pH and salt dependence of the rate supports the hypothesis that formation of an inter-subunit salt bridge is the rate-limiting structural transition. In addition, DSC and RTP measurements of AP with combinations of Me2+ , Zn2+, and PO43- bound are presented. Combining this with direct Mg2+ binding data and the

  2. Mapping unstructured regions and synergistic folding in intrinsically disordered proteins with amide H/D exchange mass spectrometry.

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    Keppel, Theodore R; Howard, Brent A; Weis, David D

    2011-10-11

    Mapping the structured and disordered regions and identifying disorder-to-order transitions are essential to understanding intrinsically disordered proteins (IDPs). One technique that can provide such information is H/D exchange coupled with mass spectrometry (H/D-MS). To explore the feasibility of H/D-MS for mapping disordered and ordered regions in IDPs, we undertook a systematic evaluation of an unstructured protein, a molten globular protein, and the well-folded complex of the two proteins. Most segments of the unstructured protein, ACTR (activator of thyroid and retinoid receptors, NCOA3_HUMAN, residues 1018-1088), exchange at rates consistent with its assignment as an unstructured protein, but there is slight protection in regions that become helical in the ACTR-CBP complex. The molten globular protein, CBP (the nuclear coactivator binding domain of the CREB binding protein, CBP_MOUSE, residues 2059-2117), is moderately protected from exchange, and the protection is nearly uniform across the length of the protein. The uniformity arises because of rapid interconversion between an ensemble of folded conformers and an ensemble of unstructured conformers. Rapid interconversion causes the H/D exchange kinetics to be dominated by exchange by molecules in unstructured conformations. For the folded ACTR-CBP complex, the exchange data provide a qualitatively accurate description of the complex. Our results provide a useful framework to use in the interpretation of H/D-MS data of intrinsically disordered proteins.

  3. Protein folding and unfolding pathways: The role of energy barriers, configurational entropy and internal energy. Comment on "There and back again: Two views on the protein folding puzzle" by Alexei V. Finkelstein et al.

    Science.gov (United States)

    Olivares-Quiroz, L.

    2017-07-01

    In this Review, Finkelstein et al. [1] provide an accurate, descriptive and scientifically solid discussion on a topic that has attracted the attention of the biophysics community during the past decades. The so-called protein folding problem remains as one of the most challenging issues at the interface of Physics and Molecular Biology, not only due to its academic and scientific relevance by itself, but also due to the vast amount of potential practical applications in drug design, molecular engineering and nanosciences in general [2,3]. Unveiling the physical mechanisms underlying protein folding and unfolding has thus certainly become one of the most relevant challenges faced by modern interdisciplinary research [4].

  4. Beyond anchoring: the expanding role of the hendra virus fusion protein transmembrane domain in protein folding, stability, and function.

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    Smith, Everett Clinton; Culler, Megan R; Hellman, Lance M; Fried, Michael G; Creamer, Trevor P; Dutch, Rebecca Ellis

    2012-03-01

    While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion.

  5. The energy landscapes of repeat-containing proteins: topology, cooperativity, and the folding funnels of one-dimensional architectures.

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    Diego U Ferreiro

    2008-05-01

    Full Text Available Repeat-proteins are made up of near repetitions of 20- to 40-amino acid stretches. These polypeptides usually fold up into non-globular, elongated architectures that are stabilized by the interactions within each repeat and those between adjacent repeats, but that lack contacts between residues distant in sequence. The inherent symmetries both in primary sequence and three-dimensional structure are reflected in a folding landscape that may be analyzed as a quasi-one-dimensional problem. We present a general description of repeat-protein energy landscapes based on a formal Ising-like treatment of the elementary interaction energetics in and between foldons, whose collective ensemble are treated as spin variables. The overall folding properties of a complete "domain" (the stability and cooperativity of the repeating array can be derived from this microscopic description. The one-dimensional nature of the model implies there are simple relations for the experimental observables: folding free-energy (DeltaG(water and the cooperativity of denaturation (m-value, which do not ordinarily apply for globular proteins. We show how the parameters for the "coarse-grained" description in terms of foldon spin variables can be extracted from more detailed folding simulations on perfectly funneled landscapes. To illustrate the ideas, we present a case-study of a family of tetratricopeptide (TPR repeat proteins and quantitatively relate the results to the experimentally observed folding transitions. Based on the dramatic effect that single point mutations exert on the experimentally observed folding behavior, we speculate that natural repeat proteins are "poised" at particular ratios of inter- and intra-element interaction energetics that allow them to readily undergo structural transitions in physiologically relevant conditions, which may be intrinsically related to their biological functions.

  6. Kinetic model for the coupling between allosteric transitions in GroEL and substrate protein folding and aggregation.

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    Tehver, Riina; Thirumalai, D

    2008-04-04

    The bacterial chaperonin GroEL and the co-chaperonin GroES assist in the folding of a number of structurally unrelated substrate proteins (SPs). In the absence of chaperonins, SP folds by the kinetic partitioning mechanism (KPM), according to which a fraction of unfolded molecules reaches the native state directly, while the remaining fraction gets trapped in a potentially aggregation-prone misfolded state. During the catalytic reaction cycle, GroEL undergoes a series of allosteric transitions (TR-->R"-->T) triggered by SP capture, ATP binding and hydrolysis, and GroES binding. We developed a general kinetic model that takes into account the coupling between the rates of the allosteric transitions and the folding and aggregation of the SP. Our model, in which the GroEL allosteric rates and SP-dependent folding and aggregation rates are independently varied without prior assumption, quantitatively fits the GroEL concentration-dependent data on the yield of native ribulose bisphosphate carboxylase/oxygenase (Rubisco) as a function of time. The extracted kinetic parameters for the GroEL reaction cycle are consistent with the available values from independent experiments. In addition, we also obtained physically reasonable parameters for the kinetic steps in the reaction cycle that are difficult to measure. If experimental values for GroEL allosteric rates are used, the time-dependent changes in native-state yield at eight GroEL concentrations can be quantitatively fit using only three SP-dependent parameters. The model predicts that the differences in the efficiencies (as measured by yields of the native state) of GroEL, single-ring mutant (SR1), and variants of SR1, in the rescue of mitochondrial malate dehydrogenase, citrate synthase, and Rubisco, are related to the large variations in the allosteric transition rates. We also show that GroEL/S mutants that efficiently fold one SP at the expense of all others are due to a decrease in the rate of a key step in the

  7. The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain.

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    Søren W. Pedersen

    Full Text Available Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.

  8. Green-lighting green fluorescent protein: faster and more efficient folding by eliminating a cis-trans peptide isomerization event.

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    Rosenman, David J; Huang, Yao-ming; Xia, Ke; Fraser, Keith; Jones, Victoria E; Lamberson, Colleen M; Van Roey, Patrick; Colón, Wilfredo; Bystroff, Christopher

    2014-04-01

    Wild-type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis-trans peptide bond isomerization. The only conserved cis-peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X-P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans-peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type. © 2014 The Protein Society.

  9. Structural plasticity of green fluorescent protein to amino acid deletions and fluorescence rescue by folding-enhancing mutations.

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    Liu, Shu-su; Wei, Xuan; Dong, Xue; Xu, Liang; Liu, Jia; Jiang, Biao

    2015-07-25

    Green fluorescent protein (GFP) and its derivative fluorescent proteins (FPs) are among the most commonly used reporter systems for studying gene expression and protein interaction in biomedical research. Most commercially available FPs have been optimized for their oligomerization state to prevent potential structural constraints that may interfere with the native function of fused proteins. Other approach to reducing structural constraints may include minimizing the structure of GFPs. Previous studies in an enhanced GFP variant (EGFP) identified a series of deletions that can retain GFP fluorescence. In this study, we interrogated the structural plasticity of a UV-optimized GFP variant (GFP(UV)) to amino acid deletions, characterized the effects of deletions and explored the feasibility of rescuing the fluorescence of deletion mutants using folding-enhancing mutations. Transposon mutagenesis was used to screen amino acid deletions in GFP that led to fluorescent and nonfluorescent phenotypes. The fluorescent GFP mutants were characterized for their whole-cell fluorescence and fraction soluble. Fluorescent GFP mutants with internal deletions were purified and characterized for their spectral and folding properties. Folding-ehancing mutations were introduced to deletion mutants to rescue their compromised fluorescence. We identified twelve amino acid deletions that can retain the fluorescence of GFP(UV). Seven of these deletions are either at the N- or C- terminus, while the other five are located at internal helices or strands. Further analysis suggested that the five internal deletions diminished the efficiency of protein folding and chromophore maturation. Protein expression under hypothermic condition or incorporation of folding-enhancing mutations could rescue the compromised fluorescence of deletion mutants. In addition, we generated dual deletion mutants that can retain GFP fluorescence. Our results suggested that a "size-minimized" GFP may be developed by

  10. Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space.

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    Stegemann, Björn; Klebe, Gerhard

    2012-02-01

    Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

  11. Conservation of the folding mechanism between designed primordial (βα)8-barrel proteins and their modern descendant.

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    Carstensen, Linn; Sperl, Josef M; Bocola, Marco; List, Felix; Schmid, Franz X; Sterner, Reinhard

    2012-08-01

    The (βα)(8)-barrel is among the most ancient, frequent, and versatile enzyme structures. It was proposed that modern (βα)(8)-barrel proteins have evolved from an ancestral (βα)(4)-half-barrel by gene duplication and fusion. We explored whether the mechanism of protein folding has remained conserved during this long-lasting evolutionary process. For this purpose, potential primordial (βα)(8)-barrel proteins were constructed by the duplication of a (βα)(4) element of a modern (βα)(8)-barrel protein, imidazole glycerol phosphate synthase (HisF), followed by the optimization of the initial construct. The symmetric variant Sym1 was less stable than HisF and its crystal structure showed disorder in the contact regions between the half-barrels. The next generation variant Sym2 was more stable than HisF, and the contact regions were well resolved. Remarkably, both artificial (βα)(8)-barrels show the same refolding mechanism as HisF and other modern (βα)(8)-barrel proteins. Early in folding, they all equilibrate rapidly with an off-pathway species. On the productive folding path, they form closely related intermediates and reach the folded state with almost identical rates. The high energy barrier that synchronizes folding is thus conserved. The strong differences in stability between these proteins develop only after this barrier and lead to major changes in the unfolding rates. We conclude that the refolding mechanism of (βα)(8)-barrel proteins is robust. It evolved early and, apparently, has remained conserved upon the diversification of sequences and functions that have taken place within this large protein family.

  12. BIOSYNTHESIS, CHARACTERIZATION AND APPLICATION OF TIO2 NANOPARTICLES IN BIOCATALYSIS AND PROTEIN FOLDING

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    Razi Ahmad,

    2013-08-01

    Full Text Available The nano-TiO2 was synthesized using Lactobacillus sp. and characterized by XRD and TEM. The X-ray diffraction showed that TiO2 nanoparticles were crystalline in nature. TEM images revealed that these particles are irregular in shape with an average particle size of 50–100 nm. The biosynthesized nanoparticles were used for the immobilization and refolding of thermally inactivated alpha amylase enzyme. The enzyme after adsorption on TiO2 nanoparticles retained 71% of enzyme activity. The immobilized enzyme was found to be thermally more stable as compared to the free enzyme. When the enzyme was heated to 60°C for 60 min the free enzyme loses all of its activity whereas the adsorbed enzyme retained 82% of its activity.The adsorbed/immobilized protein could be reused five times without any loss in enzyme activity. The operational stability data also shows that after immobilization the stability of alpha amylase increases. To study the nanoparticles-protein interaction, alpha amylase enzyme was inactivated by heating at 60°C for 1 hour. The thermally inactivated alpha amylase when incubated with the biosynthesized TiO2 nanoparticles regains nearly 65% activity after 2.0 hour. Thus TiO2 nanoparticles assist in refolding of the enzyme.

  13. Multiphase Simulated Annealing Based on Boltzmann and Bose-Einstein Distribution Applied to Protein Folding Problem.

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    Frausto-Solis, Juan; Liñán-García, Ernesto; Sánchez-Hernández, Juan Paulo; González-Barbosa, J Javier; González-Flores, Carlos; Castilla-Valdez, Guadalupe

    2016-01-01

    A new hybrid Multiphase Simulated Annealing Algorithm using Boltzmann and Bose-Einstein distributions (MPSABBE) is proposed. MPSABBE was designed for solving the Protein Folding Problem (PFP) instances. This new approach has four phases: (i) Multiquenching Phase (MQP), (ii) Boltzmann Annealing Phase (BAP), (iii) Bose-Einstein Annealing Phase (BEAP), and (iv) Dynamical Equilibrium Phase (DEP). BAP and BEAP are simulated annealing searching procedures based on Boltzmann and Bose-Einstein distributions, respectively. DEP is also a simulated annealing search procedure, which is applied at the final temperature of the fourth phase, which can be seen as a second Bose-Einstein phase. MQP is a search process that ranges from extremely high to high temperatures, applying a very fast cooling process, and is not very restrictive to accept new solutions. However, BAP and BEAP range from high to low and from low to very low temperatures, respectively. They are more restrictive for accepting new solutions. DEP uses a particular heuristic to detect the stochastic equilibrium by applying a least squares method during its execution. MPSABBE parameters are tuned with an analytical method, which considers the maximal and minimal deterioration of problem instances. MPSABBE was tested with several instances of PFP, showing that the use of both distributions is better than using only the Boltzmann distribution on the classical SA.

  14. Dynamic hydration shell restores Kauzmann's 1959 explanation of how the hydrophobic factor drives protein folding

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    Baldwin, Robert L.

    2014-01-01

    Kauzmann's explanation of how the hydrophobic factor drives protein folding is reexamined. His explanation said that hydrocarbon hydration shells are formed, possibly of clathrate water, and they explain why hydrocarbons have uniquely low solubilities in water. His explanation was not universally accepted because of skepticism about the clathrate hydration shell. A revised version is given here in which a dynamic hydration shell is formed by van der Waals (vdw) attraction, as proposed in 1985 by Jorgensen et al. [Jorgensen WL, Gao J, Ravimohan C (1985) J Phys Chem 89:3470–3473]. The vdw hydration shell is implicit in theories of hydrophobicity that contain the vdw interaction between hydrocarbon C and water O atoms. To test the vdw shell model against the known hydration energetics of alkanes, the energetics should be based on the Ben-Naim standard state (solute transfer between fixed positions in the gas and liquid phases). Then the energetics are proportional to n, the number of water molecules correlated with an alkane by vdw attraction, given by the simulations of Jorgensen et al. The energetics show that the decrease in entropy upon hydration is the root cause of hydrophobicity; it probably results from extensive ordering of water molecules in the vdw shell. The puzzle of how hydrophobic free energy can be proportional to nonpolar surface area when the free energy is unfavorable and the only known interaction (the vdw attraction) is favorable, is resolved by finding that the unfavorable free energy is produced by the vdw shell. PMID:25157156

  15. S-glutathionylation of cryptic cysteines enhances titin elasticity by blocking protein folding

    Science.gov (United States)

    Alegre-Cebollada, Jorge; Kosuri, Pallav; Giganti, David; Eckels, Edward; Rivas-Pardo, Jaime Andrés; Hamdani, Nazha; Warren, Chad M.; Solaro, R. John; Linke, Wolfgang A.; Fernández, Julio M.

    2014-01-01

    The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole, and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric oxide signaling cascade that increases cardiac muscle elasticity. However, it is unknown how S-glutathionylation may regulate the elasticity of titin and cardiac tissue. Here we show that mechanical unfolding of titin immunoglobulin (Ig) domains exposes buried cysteine residues, which then can be S-glutathionylated. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of the parent Ig domain as well as its ability to fold. Both effects favor a more extensible state of titin. Furthermore we demonstrate that S-glutathionylation of cryptic cysteines in titin mediates mechano-chemical modulation of the elasticity of human cardiomyocytes. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity. PMID:24630725

  16. Protein P7 of the cystovirus φ6 is located at the three-fold axis of the unexpanded procapsid.

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    Garrett Katz

    Full Text Available The objective of this study was to determine the location of protein P7, the RNA packaging factor, in the procapsid of the φ6 cystovirus. A comparison of cryo-electron microscopy high-resolution single particle reconstructions of the φ6 complete unexpanded procapsid, the protein P2-minus procapsid (P2 is the RNA directed RNA-polymerase, and the P7-minus procapsid, show that prior to RNA packaging the P7 protein is located near the three-fold axis of symmetry. Difference maps highlight the precise position of P7 and demonstrate that in P7-minus particles the P2 proteins are less localized with reduced densities at the three-fold axes. We propose that P7 performs the mechanical function of stabilizing P2 on the inner protein P1 shell which ensures that entering viral single-stranded RNA is replicated.

  17. Early Events, Kinetic Intermediates and the Mechanism of Protein Folding in Cytochrome c

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    David S. Kliger

    2009-04-01

    Full Text Available Kinetic studies of the early events in cytochrome c folding are reviewed with a focus on the evidence for folding intermediates on the submillisecond timescale. Evidence from time-resolved absorption, circular dichroism, magnetic circular dichroism, fluorescence energy and electron transfer, small-angle X-ray scattering and amide hydrogen exchange studies on the t £ 1 ms timescale reveals a picture of cytochrome c folding that starts with the ~ 1-ms conformational diffusion dynamics of the unfolded chains. A fractional population of the unfolded chains collapses on the 1 – 100 ms timescale to a compact intermediate IC containing some native-like secondary structure. Although the existence and nature of IC as a discrete folding intermediate remains controversial, there is extensive high time-resolution kinetic evidence for the rapid formation of IC as a true intermediate, i.e., a metastable state separated from the unfolded state by a discrete free energy barrier. Final folding to the native state takes place on millisecond and longer timescales, depending on the presence of kinetic traps such as heme misligation and proline mis-isomerization. The high folding rates observed in equilibrium molten globule models suggest that IC may be a productive folding intermediate. Whether it is an obligatory step on the pathway to the high free energy barrier associated with millisecond timescale folding to the native state, however, remains to be determined.

  18. A first-principles model of early evolution: emergence of gene families, species, and preferred protein folds.

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    Konstantin B Zeldovich

    2007-07-01

    Full Text Available In this work we develop a microscopic physical model of early evolution where phenotype--organism life expectancy--is directly related to genotype--the stability of its proteins in their native conformations-which can be determined exactly in the model. Simulating the model on a computer, we consistently observe the "Big Bang" scenario whereby exponential population growth ensues as soon as favorable sequence-structure combinations (precursors of stable proteins are discovered. Upon that, random diversity of the structural space abruptly collapses into a small set of preferred proteins. We observe that protein folds remain stable and abundant in the population at timescales much greater than mutation or organism lifetime, and the distribution of the lifetimes of dominant folds in a population approximately follows a power law. The separation of evolutionary timescales between discovery of new folds and generation of new sequences gives rise to emergence of protein families and superfamilies whose sizes are power-law distributed, closely matching the same distributions for real proteins. On the population level we observe emergence of species--subpopulations that carry similar genomes. Further, we present a simple theory that relates stability of evolving proteins to the sizes of emerging genomes. Together, these results provide a microscopic first-principles picture of how first-gene families developed in the course of early evolution.

  19. A single-molecule perspective on the role of solvent hydrogen bonds in protein folding and chemical reactions.

    Science.gov (United States)

    Dougan, Lorna; Koti, Ainavarapu Sri Rama; Genchev, Georgi; Lu, Hui; Fernandez, Julio M

    2008-12-22

    We present an array of force spectroscopy experiments that aim to identify the role of solvent hydrogen bonds in protein folding and chemical reactions at the single-molecule level. In our experiments we control the strength of hydrogen bonds in the solvent environment by substituting water (H(2)O) with deuterium oxide (D(2)O). Using a combination of force protocols, we demonstrate that protein unfolding, protein collapse, protein folding and a chemical reaction are affected in different ways by substituting H(2)O with D(2)O. We find that D(2)O molecules form an integral part of the unfolding transition structure of the immunoglobulin module of human cardiac titin, I27. Strikingly, we find that D(2)O is a worse solvent than H(2)O for the protein I27, in direct contrast with the behaviour of simple hydrocarbons. We measure the effect of substituting H(2)O with D(2)O on the force dependent rate of reduction of a disulphide bond engineered within a single protein. Altogether, these experiments provide new information on the nature of the underlying interactions in protein folding and chemical reactions and demonstrate the power of single-molecule techniques to identify the changes induced by a small change in hydrogen bond strength.

  20. Approaching the thermodynamic view of protein folding through the reproduction of Anfinsen's experiment by undergraduate physical biochemistry students.

    Science.gov (United States)

    Fernandez-Reche, Andres; Cobos, Eva S; Luque, Irene; Ruiz-Sanz, Javier; Martinez, Jose C

    2018-01-04

    In 1972 Christian B. Anfinsen received the Nobel Prize in Chemistry for "…his work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation." The understanding of this principle is crucial for physical biochemistry students, since protein folding studies, bio-computing sciences and protein design approaches are founded on such a well-demonstrated connection. Herein, we describe a detailed and easy-to-follow experiment to reproduce the most relevant assays carried out at Anfinsen's laboratory in the 60s. This experiment provides students with a platform to interpret by themselves the structural and kinetic experiments conceived to understand the protein folding problem. In addition, this three-day experiment brings students a nice opportunity for protein manipulation as well as for the setting up of spectroscopic and chromatographic techniques. © 2018 by The International Union of Biochemistry and Molecular Biology, 2018. © 2018 The International Union of Biochemistry and Molecular Biology.

  1. The Role of Short-Chain Conjugated Poly-(R-3-Hydroxybutyrate (cPHB in Protein Folding

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    Rosetta N. Reusch

    2013-05-01

    Full Text Available Poly-(R-3-hydroxybutyrate (PHB, a linear polymer of R-3-hydroxybutyrate (R-3HB, is a fundamental constituent of biological cells. Certain prokaryotes accumulate PHB of very high molecular weight (10,000 to >1,000,000 residues, which is segregated within granular deposits in the cytoplasm; however, all prokaryotes and all eukaryotes synthesize PHB of medium-chain length (~100–200 residues which resides within lipid bilayers or lipid vesicles, and PHB of short-chain length (<12 residues which is conjugated to proteins (cPHB, primarily proteins in membranes and organelles. The physical properties of cPHB indicate it plays important roles in the targeting and folding of cPHB-proteins. Here we review the occurrence, physical properties and molecular characteristics of cPHB, and discuss its influence on the folding and structure of outer membrane protein A (OmpA of Escherichia coli.

  2. The Role of Short-Chain Conjugated Poly-(R)-3-Hydroxybutyrate (cPHB) in Protein Folding

    Science.gov (United States)

    Reusch, Rosetta N.

    2013-01-01

    Poly-(R)-3-hydroxybutyrate (PHB), a linear polymer of R-3-hydroxybutyrate (R-3HB), is a fundamental constituent of biological cells. Certain prokaryotes accumulate PHB of very high molecular weight (10,000 to >1,000,000 residues), which is segregated within granular deposits in the cytoplasm; however, all prokaryotes and all eukaryotes synthesize PHB of medium-chain length (~100–200 residues) which resides within lipid bilayers or lipid vesicles, and PHB of short-chain length (<12 residues) which is conjugated to proteins (cPHB), primarily proteins in membranes and organelles. The physical properties of cPHB indicate it plays important roles in the targeting and folding of cPHB-proteins. Here we review the occurrence, physical properties and molecular characteristics of cPHB, and discuss its influence on the folding and structure of outer membrane protein A (OmpA) of Escherichia coli. PMID:23702844

  3. Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core.

    Science.gov (United States)

    Gates, Zachary P; Baxa, Michael C; Yu, Wookyung; Riback, Joshua A; Li, Hui; Roux, Benoît; Kent, Stephen B H; Sosnick, Tobin R

    2017-02-28

    The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

  4. Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Kemin; Cao, Nan; Cheng, Bokun; Joachimiak, Andrzej; Tse-Dinh, Yuk-Ching

    2016-01-16

    The DNA topoisomerase I enzyme of Mycobacterium tuberculosis (MtTOP1) is essential for the viability of the organism and survival in a murine model. This topoisomerase is being pursued as a novel target for the discovery of new therapeutic agents for the treatment of drug-resistant tuberculosis. In this study, we succeeded in obtaining a structure of MtTOP1 by first predicting that the C-terminal region of MtTOP1 contains four repeated domains that do not involve the Zn-binding tetracysteine motifs seen in the C-terminal domains of Escherichia coli topoisomerase I. A construct (amino acids A2-T704), MtTOP1-704t, that includes the N-terminal domains (D1-D4) and the first predicted C-terminal domain (D5) of MtTOP1 was expressed and found to retain DNA cleavage-religation activity and catalyze single-stranded DNA catenation. MtTOP1-704t was crystallized, and a structure of 2.52 angstrom resolution limit was obtained. The structure of the MtTOP1 N-terminal domains has features that have not been observed in other previously available bacterial topoisomerase I crystal structures. The first C-terminal domain D5 forms a novel protein fold of a four-stranded antiparallel beta-sheet stabilized by a crossing-over alpha-helix. Since there is only one type IA topoisomerase present in Mycobacteriaceae and related Actinobacteria, this subfamily of type IA topoisomerase may be required for multiple functions in DNA replication, transcription, recombination, and repair. The unique structural features observed for MtTOP1 may allow these topoisomerase I enzymes to carry out physiological functions associated with topoisomerase III enzyme in other bacteria.

  5. BPI-fold (BPIF) containing/plunc protein expression in human fetal major and minor salivary glands.

    Science.gov (United States)

    Alves, Daniel Berretta Moreira; Bingle, Lynne; Bingle, Colin David; Lourenço, Silvia Vanessa; Silva, Andréia Aparecida; Pereira, Débora Lima; Vargas, Pablo Agustin

    2017-01-16

    The aim of this study was to determine expression, not previously described, of PLUNC (palate, lung, and nasal epithelium clone) (BPI-fold containing) proteins in major and minor salivary glands from very early fetal tissue to the end of the second trimester and thus gain further insight into the function of these proteins. Early fetal heads, and major and minor salivary glands were collected retrospectively and glands were classified according to morphodifferentiation stage. Expression of BPI-fold containing proteins was localized through immunohistochemistry. BPIFA2, the major BPI-fold containing protein in adult salivary glands, was detected only in the laryngeal pharynx; the lack of staining in salivary glands suggested salivary expression is either very late in development or is only in adult tissues. Early expression of BPIFA1 was seen in the trachea and nasal cavity with salivary gland expression only seen in late morphodifferentiation stages. BPIFB1 was seen in early neural tissue and at later stages in submandibular and sublingual glands. BPIFA1 is significantly expressed in early fetal oral tissue but BPIFB1 has extremely limited expression and the major salivary BPIF protein (BPIFA2) is not produced in fetal development. Further studies, with more sensitive techniques, will confirm the expression pattern and enable a better understanding of embryonic BPIF protein function.

  6. Enzyme assisted protein extraction from rapeseed, soybean, and microalgae meals

    NARCIS (Netherlands)

    Sari, Y.W.; Bruins, M.E.; Sanders, J.P.M.

    2013-01-01

    Oilseed meals that are by-products from oil production are potential resources for protein. The aim of this work is to investigate the use of enzymes in assisting in the extraction of protein from different oilseed meals, namely rapeseed, soybean, and microalgae meals. In addition, microalgae

  7. Interactions of carbohydrates and proteins by fluorophore-assisted ...

    Indian Academy of Sciences (India)

    A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions is demonstrated by fluorophore-assisted carbohydrate electrophoresis (FACE). The procedure is simple and the cost is low. The advantage of this method is that carbohydrate-protein interactions can be easily displayed by FACE, ...

  8. Interactions of main chain in folding and self assembly of unfolded protein structure: Enquiries with a serine solubilized nonapeptide

    Directory of Open Access Journals (Sweden)

    Kinshuk Raj Srivastava

    2014-06-01

    Full Text Available Interactions of the protein main chain are probed for their role in folding and self-assembly. The interactions are assessed with serine nonapeptide Ac-(Ser-Ala4-Ser-NH2 in poly-L and alternating-L,D structure variations. Being a neutral molecule, Serine nonapeptide has been found to display not only folding-unfolding equilibrium, but also association-dissociation equilibrium as a function of solvent and concentration. Thus scrutiny of intra- and inter-molecular interactions have been undertaken in water, methanol, and DMSO solvents. In water, poly-L peptide displays a PPII-helix conformation which unfolds to extended β-conformation with increase of temperature, apparently in a two-state equilibrium. Poly-L peptide at high concentration and on transfer to the low polarity solvent, methanol, displays ordering as a β-hairpin. This implies folding of the peptide by self assembly. Self assembly and ordering possibly as double-stranded β-helix is also evidence for alternating-L,D peptide. Both isomers were observed to be unfolded in high polarity solvent DMSO. Dynamic light scattering suggests that assembly in both isomers may involve large size aggregates. The results have established that folding and self-assembly can be coupled equilibria dependent upon solute structure, concentration, and solvent. The interactions of the protein main chain involved in folding and self assembly of unfolded structure are illuminated and have been discussed.

  9. A novel plant protein disulfide isomerase family homologous to animal P5 - molecular cloning and characterization as a functional protein for folding of soybean seed-storage proteins.

    Science.gov (United States)

    Wadahama, Hiroyuki; Kamauchi, Shinya; Nakamoto, Yumi; Nishizawa, Keito; Ishimoto, Masao; Kawada, Teruo; Urade, Reiko

    2008-02-01

    The protein disulfide isomerase is known to play important roles in the folding of nascent polypeptides and in the formation of disulfide bonds in the endoplasmic reticulum (ER). In this study, we cloned a gene of a novel protein disulfide isomerase family from soybean leaf (Glycine max L. Merrill. cv Jack) mRNA. The cDNA encodes a protein called GmPDIM. It is composed of 438 amino acids, and its sequence and domain structure are similar to that of animal P5. Recombinant GmPDIM expressed in Escherichia coli displayed an oxidative refolding activity on denatured RNase A. The genomic sequence of GmPDIM was also cloned and sequenced. Comparison of the soybean sequence with sequences from Arabidopsis thaliana and Oryza sativa showed significant conservation of the exon/intron structure. Consensus sequences within the promoters of the GmPDIM genes contained a cis-acting regulatory element for the unfolded protein response, and other regulatory motifs required for seed-specific expression. We observed that expression of GmPDIM was upregulated under ER-stress conditions, and was expressed ubiquitously in soybean tissues such as the cotyledon. It localized to the lumen of the ER. Data from co-immunoprecipitation experiments suggested that GmPDIM associated non-covalently with proglycinin, a precursor of the seed-storage protein glycinin. In addition, GmPDIM associated with the alpha' subunit of beta-conglycinin, a seed-storage protein in the presence of tunicamycin. These results suggest that GmPDIM may play a role in the folding of storage proteins and functions not only as a thiol-oxidoredactase, but also as molecular chaperone.

  10. BCL::Fold - De Novo Prediction of Complex and Large Protein Topologies by Assembly of Secondary Structure Elements

    Science.gov (United States)

    Karakaş, Mert; Woetzel, Nils; Staritzbichler, Rene; Alexander, Nathan; Weiner, Brian E.; Meiler, Jens

    2012-01-01

    Computational de novo protein structure prediction is limited to small proteins of simple topology. The present work explores an approach to extend beyond the current limitations through assembling protein topologies from idealized α-helices and β-strands. The algorithm performs a Monte Carlo Metropolis simulated annealing folding simulation. It optimizes a knowledge-based potential that analyzes radius of gyration, β-strand pairing, secondary structure element (SSE) packing, amino acid pair distance, amino acid environment, contact order, secondary structure prediction agreement and loop closure. Discontinuation of the protein chain favors sampling of non-local contacts and thereby creation of complex protein topologies. The folding simulation is accelerated through exclusion of flexible loop regions further reducing the size of the conformational search space. The algorithm is benchmarked on 66 proteins with lengths between 83 and 293 amino acids. For 61 out of these proteins, the best SSE-only models obtained have an RMSD100 below 8.0 Å and recover more than 20% of the native contacts. The algorithm assembles protein topologies with up to 215 residues and a relative contact order of 0.46. The method is tailored to be used in conjunction with low-resolution or sparse experimental data sets which often provide restraints for regions of defined secondary structure. PMID:23173050

  11. A discriminative method for protein remote homology detection and fold recognition combining Top-n-grams and latent semantic analysis

    Directory of Open Access Journals (Sweden)

    Lin Lei

    2008-12-01

    Full Text Available Abstract Background Protein remote homology detection and fold recognition are central problems in bioinformatics. Currently, discriminative methods based on support vector machine (SVM are the most effective and accurate methods for solving these problems. A key step to improve the performance of the SVM-based methods is to find a suitable representation of protein sequences. Results In this paper, a novel building block of proteins called Top-n-grams is presented, which contains the evolutionary information extracted from the protein sequence frequency profiles. The protein sequence frequency profiles are calculated from the multiple sequence alignments outputted by PSI-BLAST and converted into Top-n-grams. The protein sequences are transformed into fixed-dimension feature vectors by the occurrence times of each Top-n-gram. The training vectors are evaluated by SVM to train classifiers which are then used to classify the test protein sequences. We demonstrate that the prediction performance of remote homology detection and fold recognition can be improved by combining Top-n-grams and latent semantic analysis (LSA, which is an efficient feature extraction technique from natural language processing. When tested on superfamily and fold benchmarks, the method combining Top-n-grams and LSA gives significantly better results compared to related methods. Conclusion The method based on Top-n-grams significantly outperforms the methods based on many other building blocks including N-grams, patterns, motifs and binary profiles. Therefore, Top-n-gram is a good building block of the protein sequences and can be widely used in many tasks of the computational biology, such as the sequence alignment, the prediction of domain boundary, the designation of knowledge-based potentials and the prediction of protein binding sites.

  12. Dependence of α-helical and β-sheet amino acid propensities on the overall protein fold type

    Directory of Open Access Journals (Sweden)

    Fujiwara Kazuo

    2012-08-01

    Full Text Available Abstract Background A large number of studies have been carried out to obtain amino acid propensities for α-helices and β-sheets. The obtained propensities for α-helices are consistent with each other, and the pair-wise correlation coefficient is frequently high. On the other hand, the β-sheet propensities obtained by several studies differed significantly, indicating that the context significantly affects β-sheet propensity. Results We calculated amino acid propensities for α-helices and β-sheets for 39 and 24 protein folds, respectively, and addressed whether they correlate with the fold. The propensities were also calculated for exposed and buried sites, respectively. Results showed that α-helix propensities do not differ significantly by fold, but β-sheet propensities are diverse and depend on the fold. The propensities calculated for exposed sites and buried sites are similar for α-helix, but such is not the case for the β-sheet propensities. We also found some fold dependence on amino acid frequency in β-strands. Folds with a high Ser, Thr and Asn content at exposed sites in β-strands tend to have a low Leu, Ile, Glu, Lys and Arg content (correlation coefficient = −0.90 and to have flat β-sheets. At buried sites in β-strands, the content of Tyr, Trp, Gln and Ser correlates negatively with the content of Val, Ile and Leu (correlation coefficient = −0.93. "All-β" proteins tend to have a higher content of Tyr, Trp, Gln and Ser, whereas "α/β" proteins tend to have a higher content of Val, Ile and Leu. Conclusions The α-helix propensities are similar for all folds and for exposed and buried residues. However, β-sheet propensities calculated for exposed residues differ from those for buried residues, indicating that the exposed-residue fraction is one of the major factors governing amino acid composition in β-strands. Furthermore, the correlations we detected suggest that amino acid composition is related to folding

  13. Protein structure prediction of CASP5 comparative modeling and fold recognition targets using consensus alignment approach and 3D assessment.

    Science.gov (United States)

    Ginalski, Krzysztof; Rychlewski, Leszek

    2003-01-01

    For the fifth round of Critical Assessment of Techniques for Protein Structure Prediction (CASP5) all comparative modeling (CM) and fold recognition (FR) target proteins were modeled using a combination of consensus alignment strategy and 3D assessment. A large number and broad variety of prediction targets, with sequence identity between each modeled domain and the related known structure, ranging from 6 to 49%, represented all difficulty levels in comparative modeling and fold recognition. The critical steps in modeling, selection of template(s) and generation of sequence-to-structure alignment, were based on the results of secondary structure prediction and tertiary fold recognition carried out using the Meta Server coupled with the 3D-Jury system. The main idea behind the modeling procedure was to select the most common alignment variants provided by individual servers, as well as to generate several alternatives for questionable regions and to evaluate them in 3D by building corresponding molecular models. Analysis of fold-specific features and sequence conservation patterns for the target family was also widely used at this stage. For both CM and FR targets remote homologs of known structure were clearly recognized by the 3D-Jury system. In the analogous fold recognition subcategory, the correct fold was identified for five out of eight domains. The average alignment accuracy for FR models (48%) was far less than for CM predictions (80%). These finding, coupled with the observation that in the majority of cases the submitted models were not closer to the experimental structure than their best templates, indicate that, especially for difficult targets, there is still ample room for improvement. Copyright 2003 Wiley-Liss, Inc.

  14. A critical comparison of coarse-grained structure-based approaches and atomic models of protein folding.

    Science.gov (United States)

    Hu, Jie; Chen, Tao; Wang, Moye; Chan, Hue Sun; Zhang, Zhuqing

    2017-05-31

    Structure-based coarse-grained Gō-like models have been used extensively in deciphering protein folding mechanisms because of their simplicity and tractability. Meanwhile, explicit-solvent molecular dynamics (MD) simulations with physics-based all-atom force fields have been applied successfully to simulate folding/unfolding transitions for several small, fast-folding proteins. To explore the degree to which coarse-grained Gō-like models and their extensions to incorporate nonnative interactions are capable of producing folding processes similar to those in all-atom MD simulations, here we systematically compare the computed unfolded states, transition states, and transition paths obtained using coarse-grained models and all-atom explicit-solvent MD simulations. The conformations in the unfolded state in common Gō models are more extended, and are thus more in line with experiment, than those from all-atom MD simulations. Nevertheless, the structural features of transition states obtained by the two types of models are largely similar. In contrast, the folding transition paths are significantly more sensitive to modeling details. In particular, when common Gō-like models are augmented with nonnative interactions, the predicted dimensions of the unfolded conformations become similar to those computed using all-atom MD. With this connection, the large deviations of all-atom MD from simple diffusion theory are likely caused in part by the presence of significant nonnative effects in folding processes modelled by current atomic force fields. The ramifications of our findings to the application of coarse-grained modeling to more complex biomolecular systems are discussed.

  15. The alpha/beta-Hydrolase Fold 3DM Database (ABHDB) as a Tool for Protein Engineering

    NARCIS (Netherlands)

    Kourist, R.; Jochens, H.; Bartsch, S.; Kuipers, R.K.P.; Padhi, S.K.; Gall, M.; Bottcher, D.; Joosten, H.J.; Bornscheuer, U.T.

    2010-01-01

    Aligning the haystack to expose the needle: The 3DM method was used to generate a comprehensive database of the a/ß-hydrolase fold enzyme superfamily. This database facilitates the analysis of structure–function relationships and enables novel insights into this superfamily to be made. In addition

  16. Allosteric effects in coiled-coil proteins folding and lanthanide-ion binding.

    Science.gov (United States)

    Samiappan, Manickasundaram; Alasibi, Samaa; Cohen-Luria, Rivka; Shanzer, Abraham; Ashkenasy, Gonen

    2012-10-07

    Peptide sequences modified with lanthanide-chelating groups at their N-termini, or at their lysine side chains, were synthesized, and new Ln(III) complexes were characterized. We show that partial folding of the conjugates to form trimer coiled coil structures induces coordination of lanthanides to the ligand, which in turn further stabilizes the 3D structure.

  17. Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins

    Directory of Open Access Journals (Sweden)

    Lee Dae-Hee

    2009-03-01

    Full Text Available Abstract Background Escherichia coli has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in E. coli frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant E. coli. Here, we describe the successful use of the immobilized folding machineries for in vitro refolding with the examples of high yield refolding of a ribonuclease A (RNase A and cyclohexanone monooxygenase (CHMO. Results We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191–345 of GroEL and two foldases (DsbA and human peptidyl-prolyl cis-trans isomerase by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively. Conclusion The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of E. coli inclusion bodies in high yield with biological function.

  18. Basic Tilted Helix Bundle – A new protein fold in human FKBP25/FKBP3 and HectD1

    Energy Technology Data Exchange (ETDEWEB)

    Helander, Sara; Montecchio, Meri [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden); Lemak, Alexander [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Farès, Christophe [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Almlöf, Jonas [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden); Li, Yanjun [Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Yee, Adelinda [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Arrowsmith, Cheryl H. [Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7 (Canada); Northeast Structural Genomics Consortium, Toronto, Ontario (Canada); Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Dhe-Paganon, Sirano [Structural Genomics Consortium, University of Toronto, 101 College St, Toronto, Ontario M5G 1L7 (Canada); Sunnerhagen, Maria, E-mail: maria.sunnerhagen@liu.se [Department of Physics, Chemistry and Biology, Division of Chemistry, Linköping University, SE-58183 Linköping (Sweden)

    2014-04-25

    Highlights: • We describe the structure of a novel fold in FKBP25 and HectD. • The new fold is named the Basic Tilted Helix Bundle (BTHB) domain. • A conserved basic surface patch is presented, suggesting a functional role. - Abstract: In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP25{sub 1–73}, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.

  19. Protein recovery from enzyme-assisted aqueous extraction of soybean.

    Science.gov (United States)

    Campbell, Kerry A; Glatz, Charles E

    2010-01-01

    Enzyme-assisted aqueous oil extraction from soybean is a "green" alternative to hexane extraction that must realize potential revenues from a value-added protein co-product. Three technologies were investigated to recover protein from the skim fraction of an aqueous extraction process. Ultrafiltration achieved overall protein yields between 60% and 64%, with solids protein content of 70%, and was effective in reducing stachyose content, with fluxes between 4 and 10 L/m(2) hr. Protein content was limited because of high retention of lipids and the loss of polypeptides below 13.6 kDa. Isoelectric precipitation was effective in recovering the minimally hydrolyzed proteins of skim, with a protein content of 70%, again limited by lipid content. However, protein recovery was only 30% because of the greater solubility of the hydrolyzed proteins. Recovery by the alternative of protein capture on dextran-grafted agarose quaternary-amine expanded bed adsorption resins decreased with decreasing polypeptide molecular weight. Proteins with molecular mass greater than 30 kDa exhibited slow adsorption rates. Expanded bed adsorption was most effective for recovery of proteins with molecular weight between 30 and 12 kDa. Overall, adsorption protein yields were between 14% and 17%.

  20. Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics.

    Science.gov (United States)

    Aliye, Naser; Fabbretti, Attilio; Lupidi, Giulio; Tsekoa, Tsepo; Spurio, Roberto

    2015-02-01

    A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.

  1. Use of multiple picosecond high-mass molecular dynamics simulations to predict crystallographic B-factors of folded globular proteins.

    Science.gov (United States)

    Pang, Yuan-Ping

    2016-09-01

    Predicting crystallographic B-factors of a protein from a conventional molecular dynamics simulation is challenging, in part because the B-factors calculated through sampling the atomic positional fluctuations in a picosecond molecular dynamics simulation are unreliable, and the sampling of a longer simulation yields overly large root mean square deviations between calculated and experimental B-factors. This article reports improved B-factor prediction achieved by sampling the atomic positional fluctuations in multiple picosecond molecular dynamics simulations that use uniformly increased atomic masses by 100-fold to increase time resolution. Using the third immunoglobulin-binding domain of protein G, bovine pancreatic trypsin inhibitor, ubiquitin, and lysozyme as model systems, the B-factor root mean square deviations (mean ± standard error) of these proteins were 3.1 ± 0.2-9 ± 1 Å2 for Cα and 7.3 ± 0.9-9.6 ± 0.2 Å2 for Cγ, when the sampling was done for each of these proteins over 20 distinct, independent, and 50-picosecond high-mass molecular dynamics simulations with AMBER forcefield FF12MC or FF14SB. These results suggest that sampling the atomic positional fluctuations in multiple picosecond high-mass molecular dynamics simulations may be conducive to a priori prediction of crystallographic B-factors of a folded globular protein.

  2. Aspergillus niger protein estA defines a new class of fungal esterases within the alfa/beta hydrolase fold superfamily of proteins

    NARCIS (Netherlands)

    Bourne, Y.; Hasper, A.A.; Chahinian, H.; Juin, M.; Graaff, de L.H.

    2004-01-01

    From the fungus Aspergillus niger, we identified a new gene encoding protein EstA, a member of the alpha/beta-hydrolase fold superfamily but of unknown substrate specificity. EstA was overexpressed and its crystal structure was solved by molecular replacement using a lipaseacetylcholinesterase

  3. Carbon-deuterium bonds as non-perturbative infrared probes of protein dynamics, electrostatics, heterogeneity, and folding.

    Science.gov (United States)

    Zimmermann, Jörg; Romesberg, Floyd E

    2014-01-01

    Vibrational spectroscopy is uniquely able to characterize protein dynamics and microenvironmental heterogeneity because it possesses an inherently high temporal resolution and employs probes of ultimately high structural resolution-the bonds themselves. The use of carbon-deuterium (C-D) bonds as vibrational labels circumvents the spectral congestion that otherwise precludes the use of vibrational spectroscopy to proteins and makes the observation of single vibrations within a protein possible while being wholly non-perturbative. Thus, C-D probes can be used to site-specifically characterize conformational heterogeneity and thermodynamic stability. C-D probes are also uniquely useful in characterizing the electrostatic microenvironment experienced by a specific residue side chain or backbone due to its effect on the C-D absorption frequency. In this chapter we describe the experimental procedures required to use C-D bonds and FT IR spectroscopy to characterize protein dynamics, structural and electrostatic heterogeneity, ligand binding, and folding.

  4. ReFOLD: a server for the refinement of 3D protein models guided by accurate quality estimates.

    Science.gov (United States)

    Shuid, Ahmad N; Kempster, Robert; McGuffin, Liam J

    2017-04-10

    ReFOLD is a novel hybrid refinement server with integrated high performance global and local Accuracy Self Estimates (ASEs). The server attempts to identify and to fix likely errors in user supplied 3D models of proteins via successive rounds of refinement. The server is unique in providing output for multiple alternative refined models in a way that allows users to quickly visualize the key residue locations, which are likely to have been improved. This is important, as global refinement of a full chain model may not always be possible, whereas local regions, or individual domains, can often be much improved. Thus, users may easily compare the specific regions of the alternative refined models in which they are most interested e.g. key interaction sites or domains. ReFOLD was used to generate hundreds of alternative refined models for the CASP12 experiment, boosting our group's performance in the main tertiary structure prediction category. Our successful refinement of initial server models combined with our built-in ASEs were instrumental to our second place ranking on Template Based Modeling (TBM) and Free Modeling (FM)/TBM targets. The ReFOLD server is freely available at: http://www.reading.ac.uk/bioinf/ReFOLD/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Folding DNA into a lipid-conjugated nano-barrel for controlled reconstitution of membrane proteins.

    Science.gov (United States)

    Dong, Yuanchen; Chen, Shuobing; Zhang, Shijian; Sodroski, Joseph; Yang, Zhongqiang; Liu, Dongsheng; Mao, Youdong

    2017-12-20

    Building upon DNA origami technology, we introduce a method to reconstitute a single membrane protein into a self-assembled DNA nano-barrel that scaffolds a nanodisc-like lipid environment. Compared with the membrane-scaffolding-protein nanodisc technique, our approach gives rise to defined stoichiometry, controlled sizes, as well as enhanced stability and homogeneity in membrane protein reconstitution. We further demonstrate potential applications of the DNA nano-barrels in the structural analysis of membrane proteins. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations

    Science.gov (United States)

    Baxa, Michael C.; Haddadian, Esmael J.; Jumper, John M.; Freed, Karl F.; Sosnick, Tobin R.

    2014-01-01

    The loss of conformational entropy is a major contribution in the thermodynamics of protein folding. However, accurate determination of the quantity has proven challenging. We calculate this loss using molecular dynamic simulations of both the native protein and a realistic denatured state ensemble. For ubiquitin, the total change in entropy is TΔSTotal = 1.4 kcal⋅mol−1 per residue at 300 K with only 20% from the loss of side-chain entropy. Our analysis exhibits mixed agreement with prior studies because of the use of more accurate ensembles and contributions from correlated motions. Buried side chains lose only a factor of 1.4 in the number of conformations available per rotamer upon folding (ΩU/ΩN). The entropy loss for helical and sheet residues differs due to the smaller motions of helical residues (TΔShelix−sheet = 0.5 kcal⋅mol−1), a property not fully reflected in the amide N-H and carbonyl C=O bond NMR order parameters. The results have implications for the thermodynamics of folding and binding, including estimates of solvent ordering and microscopic entropies obtained from NMR. PMID:25313044

  7. Hydrophobic and Ionic-Interactions in Bulk and Confined Water with Implications for Collapse and Folding of Proteins

    Science.gov (United States)

    Vaitheeswaran, S.; Chen, Jie; Thirumalai, D.

    2011-10-01

    Water and water-mediated interactions determine the thermodynamics and kinetics of protein folding, protein aggregation and self-assembly in confined spaces. To obtain insights into the role of water in the context of folding problems, we describe computer simulations of a few related model systems. The dynamics of collapse of eicosane shows that upon expulsion of water the linear hydrocarbon chain adopts an ordered helical hairpin structure with 1.5 turns. The structure of dimer of eicosane molecules has two well ordered helical hairpins that are stacked perpendicular to each other. As a prelude to studying folding in confined spaces we used simulations to understand changes in hydrophobic and ionic interactions in nano-sized water droplets. Solvation of hydrophobic and charged species change drastically in nano-scale water droplets. Hydrophobic species are localized at the boundary. The tendency of ions to be at the boundary where water density is low increases as the charge density decreases. The interactions between hydrophobic, polar, and charged residue are also profoundly altered in confined spaces. Using the results of computer simulations and accounting for loss of chain entropy upon confinement we argue and then demonstrate, using simulations in explicit water, that ordered states of generic amphiphilic peptide sequences should be stabilized in cylindrical nanopores.

  8. Rise-time of FRET-acceptor fluorescence tracks protein folding

    NARCIS (Netherlands)

    Lindhoud, S.; Westphal, A.H.; Van Mierlo, C.P.M.; Visser, A.J.W.G.; Borst, J.W.

    2014-01-01

    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no

  9. Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

    NARCIS (Netherlands)

    Lindhoud, S.; Westphal, A.H.; Mierlo, van C.P.M.; Visser, A.J.W.G.; Borst, J.W.

    2014-01-01

    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no

  10. Lipids assist the membrane insertion of a BAM-independent outer membrane protein.

    Science.gov (United States)

    Huysmans, Gerard H M; Guilvout, Ingrid; Chami, Mohamed; Nickerson, Nicholas N; Pugsley, Anthony P

    2015-10-14

    Like several other large, multimeric bacterial outer membrane proteins (OMPs), the assembly of the Klebsiella oxytoca OMP PulD does not rely on the universally conserved β-barrel assembly machinery (BAM) that catalyses outer membrane insertion. The only other factor known to interact with PulD prior to or during outer membrane targeting and assembly is the cognate chaperone PulS. Here, in vitro translation-transcription coupled PulD folding demonstrated that PulS does not act during the membrane insertion of PulD, and engineered in vivo site-specific cross-linking between PulD and PulS showed that PulS binding does not prevent membrane insertion. In vitro folding kinetics revealed that PulD is atypical compared to BAM-dependent OMPs by inserting more rapidly into membranes containing E. coli phospholipids than into membranes containing lecithin. PulD folding was fast in diC14:0-phosphatidylethanolamine liposomes but not diC14:0-phosphatidylglycerol liposomes, and in diC18:1-phosphatidylcholine liposomes but not in diC14:1-phosphatidylcholine liposomes. These results suggest that PulD efficiently exploits the membrane composition to complete final steps in insertion and explain how PulD can assemble independently of any protein-assembly machinery. Lipid-assisted assembly in this manner might apply to other large OMPs whose assembly is BAM-independent.

  11. Structure of the Noncatalytic Domains and Global Fold of the Protein Disulfide Isomerase ERp72

    Energy Technology Data Exchange (ETDEWEB)

    Kozlov, G.; Määttänen, P; Schrag, J; Hura, G; Gabrielli, L; Cygler, M; Thomas, D; Gehring, K

    2009-01-01

    Protein disulfide isomerases are a family of proteins that catalyze the oxidation and isomerization of disulfide bonds in newly synthesized proteins in the endoplasmic reticulum. The family includes general enzymes such as PDI that recognize unfolded proteins, and others that are selective for specific classes of proteins. Here, we report the X-ray crystal structure of central non-catalytic domains of a specific isomerase, ERp72 (also called CaBP2 and protein disulfide-isomerase A4) from Rattus norvegicus. The structure reveals strong similarity to ERp57, a PDI-family member that interacts with the lectin-like chaperones calnexin and calreticulin but, unexpectedly, ERp72 does not interact with calnexin as shown by isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. Small-angle X-ray scattering (SAXS) of ERp72 was used to develop models of the full-length protein using both rigid body refinement and ab initio simulated annealing of dummy atoms. The two methods show excellent agreement and define the relative positions of the five thioredoxin-like domains of ERp72 and potential substrate or chaperone binding sites.

  12. Design of self-assembling transmembrane helical bundles to elucidate principles required for membrane protein folding and ion transport.

    Science.gov (United States)

    Joh, Nathan H; Grigoryan, Gevorg; Wu, Yibing; DeGrado, William F

    2017-08-05

    Ion transporters and channels are able to identify and act on specific substrates among myriads of ions and molecules critical to cellular processes, such as homeostasis, cell signalling, nutrient influx and drug efflux. Recently, we designed Rocker, a minimalist model for Zn2+/H+ co-transport. The success of this effort suggests that de novo membrane protein design has now come of age so as to serve a key approach towards probing the determinants of membrane protein folding, assembly and function. Here, we review general principles that can be used to design membrane proteins, with particular reference to helical assemblies with transport function. We also provide new functional and NMR data that probe the dynamic mechanism of conduction through Rocker.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'. © 2017 The Author(s).

  13. The protein folds as platonic forms: new support for the pre-Darwinian conception of evolution by natural law.

    Science.gov (United States)

    Denton, Michael J; Marshall, Craig J; Legge, Michael

    2002-12-07

    Before the Darwinian revolution many biologists considered organic forms to be determined by natural law like atoms or crystals and therefore necessary, intrinsic and immutable features of the world order, which will occur throughout the cosmos wherever there is life. The search for the natural determinants of organic form-the celebrated "Laws of Form"-was seen as one of the major tasks of biology. After Darwin, this Platonic conception of form was abandoned and natural selection, not natural law, was increasingly seen to be the main, if not the exclusive, determinant of organic form. However, in the case of one class of very important organic forms-the basic protein folds-advances in protein chemistry since the early 1970s have revealed that they represent a finite set of natural forms, determined by a number of generative constructional rules, like those which govern the formation of atoms or crystals, in which functional adaptations are clearly secondary modifications of primary "givens of physics." The folds are evidently determined by natural law, not natural selection, and are "lawful forms" in the Platonic and pre-Darwinian sense of the word, which are bound to occur everywhere in the universe where the same 20 amino acids are used for their construction. We argue that this is a major discovery which has many important implications regarding the origin of proteins, the origin of life and the fundamental nature of organic form. We speculate that it is unlikely that the folds will prove to be the only case in nature where a set of complex organic forms is determined by natural law, and suggest that natural law may have played a far greater role in the origin and evolution of life than is currently assumed.

  14. Exploring fold space preferences of new-born and ancient protein superfamilies.

    Directory of Open Access Journals (Sweden)

    Hannah Edwards

    Full Text Available The evolution of proteins is one of the fundamental processes that has delivered the diversity and complexity of life we see around ourselves today. While we tend to define protein evolution in terms of sequence level mutations, insertions and deletions, it is hard to translate these processes to a more complete picture incorporating a polypeptide's structure and function. By considering how protein structures change over time we can gain an entirely new appreciation of their long-term evolutionary dynamics. In this work we seek to identify how populations of proteins at different stages of evolution explore their possible structure space. We use an annotation of superfamily age to this space and explore the relationship between these ages and a diverse set of properties pertaining to a superfamily's sequence, structure and function. We note several marked differences between the populations of newly evolved and ancient structures, such as in their length distributions, secondary structure content and tertiary packing arrangements. In particular, many of these differences suggest a less elaborate structure for newly evolved superfamilies when compared with their ancient counterparts. We show that the structural preferences we report are not a residual effect of a more fundamental relationship with function. Furthermore, we demonstrate the robustness of our results, using significant variation in the algorithm used to estimate the ages. We present these age estimates as a useful tool to analyse protein populations. In particularly, we apply this in a comparison of domains containing greek key or jelly roll motifs.

  15. Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells

    DEFF Research Database (Denmark)

    Schelhaas, Mario; Malmström, Johan; Pelkmans, Lucas

    2007-01-01

    Cell entry of Simian Virus 40 (SV40) involves caveolar/lipid raft-mediated endocytosis, vesicular transport to the endoplasmic reticulum (ER), translocation into the cytosol, and import into the nucleus. We analyzed the effects of ER-associated processes and factors on infection and on isolated...... 12 of 72 VP1 pentamers. Cryo-electron tomography indicated that loss of interchain disulfides coupled with calcium depletion induces selective dissociation of the 12 vertex pentamers, a step likely to mimic uncoating of the virus in the cytosol. Thus, the virus utilizes the protein folding machinery...

  16. Coupled binding-bending-folding: The complex conformational dynamics of protein-DNA binding studied by atomistic molecular dynamics simulations.

    Science.gov (United States)

    van der Vaart, Arjan

    2015-05-01

    Protein-DNA binding often involves dramatic conformational changes such as protein folding and DNA bending. While thermodynamic aspects of this behavior are understood, and its biological function is often known, the mechanism by which the conformational changes occur is generally unclear. By providing detailed structural and energetic data, molecular dynamics simulations have been helpful in elucidating and rationalizing protein-DNA binding. This review will summarize recent atomistic molecular dynamics simulations of the conformational dynamics of DNA and protein-DNA binding. A brief overview of recent developments in DNA force fields is given as well. Simulations have been crucial in rationalizing the intrinsic flexibility of DNA, and have been instrumental in identifying the sequence of binding events, the triggers for the conformational motion, and the mechanism of binding for a number of important DNA-binding proteins. Molecular dynamics simulations are an important tool for understanding the complex binding behavior of DNA-binding proteins. With recent advances in force fields and rapid increases in simulation time scales, simulations will become even more important for future studies. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.

  17. The Mitochondrial Disulfide Relay System: Roles in Oxidative Protein Folding and Beyond

    Directory of Open Access Journals (Sweden)

    Manuel Fischer

    2013-01-01

    Full Text Available Disulfide bond formation drives protein import of most proteins of the mitochondrial intermembrane space (IMS. The main components of this disulfide relay machinery are the oxidoreductase Mia40 and the sulfhydryl oxidase Erv1/ALR. Their precise functions have been elucidated in molecular detail for the yeast and human enzymes in vitro and in intact cells. However, we still lack knowledge on how Mia40 and Erv1/ALR impact cellular and organism physiology and whether they have functions beyond their role in disulfide bond formation. Here we summarize the principles of oxidation-dependent protein import mediated by the mitochondrial disulfide relay. We proceed by discussing recently described functions of Mia40 in the hypoxia response and of ALR in influencing mitochondrial morphology and its importance for tissue development and embryogenesis. We also include a discussion of the still mysterious function of Erv1/ALR in liver regeneration.

  18. Data-assisted protein structure modeling by global optimization in CASP12.

    Science.gov (United States)

    Joo, Keehyoung; Heo, Seungryong; Joung, InSuk; Hong, Seung Hwan; Lee, Sung Jong; Lee, Jooyoung

    2018-01-17

    In CASP12, 2 types of data-assisted protein structure modeling were experimented. Either SAXS experimental data or cross-linking experimental data was provided for a selected number of CASP12 targets that the CASP12 predictor could utilize for better protein structure modeling. We devised 2 separate energy terms for SAXS data and cross-linking data to drive the model structures into more native-like structures that satisfied the given experimental data as much as possible. In CASP11, we successfully performed protein structure modeling using simulated sparse and ambiguously assigned NOE data and/or correct residue-residue contact information, where the only energy term that folded the protein into its native structure was the term which was originated from the given experimental data. However, the 2 types of experimental data provided in CASP12 were far from being sufficient enough to fold the target protein into its native structure because SAXS data provides only the overall shape of the molecule and the cross-linking contact information provides only very low-resolution distance information. For this reason, we combined the SAXS or cross-linking energy term with our regular modeling energy function that includes both the template energy term and the de novo energy terms. By optimizing the newly formulated energy function, we obtained protein models that fit better with provided SAXS data than the X-ray structure of the target. However, the improvement of the model relative to the 1 modeled without the SAXS data, was not significant. Consistent structural improvement was achieved by incorporating cross-linking data into the protein structure modeling. © 2018 Wiley Periodicals, Inc.

  19. The analysis Arabidopsis thaliana overexpressing a 14kDa self-folding protein [abstract

    Science.gov (United States)

    A recent study in banana identified a 14kDa protein that has been hypothesized to function in regulating the nucleation and growth of the needle-shaped crystals of calcium oxalate that accumulate within the tissues of this plant. To gain further insight in to the functional role of this 14 kDa prote...

  20. A simple lattice model that captures protein folding, aggregation and amyloid formation

    NARCIS (Netherlands)

    Abeln, S.; Vendruscolo, M.; Dobson, C.M.; Frenkel, D.

    2014-01-01

    The ability of many proteins to convert from their functional soluble state to amyloid fibrils can be attributed to intermolecular beta strand formation. Such amyloid formation is associated with neurodegenerative disorders like Alzheimer's and Parkinson's. Molecular modelling can play a key role in

  1. GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Marzec, Michal; Eletto, Davide; Argon, Yair

    2012-01-01

    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, necessitated by the conditions in the endoplasmic reticulum. GRP94's mode of action varies from the general HSP90 theme...

  2. Prediction of optimal folding routes of proteins that satisfy the principle of lowest entropy loss: dynamic contact maps and optimal control.

    Directory of Open Access Journals (Sweden)

    Yaman Arkun

    Full Text Available An optimization model is introduced in which proteins try to evade high energy regions of the folding landscape, and prefer low entropy loss routes during folding. We make use of the framework of optimal control whose convenient solution provides practical and useful insight into the sequence of events during folding. We assume that the native state is available. As the protein folds, it makes different set of contacts at different folding steps. The dynamic contact map is constructed from these contacts. The topology of the dynamic contact map changes during the course of folding and this information is utilized in the dynamic optimization model. The solution is obtained using the optimal control theory. We show that the optimal solution can be cast into the form of a Gaussian Network that governs the optimal folding dynamics. Simulation results on three examples (CI2, Sso7d and Villin show that folding starts by the formation of local clusters. Non-local clusters generally require the formation of several local clusters. Non-local clusters form cooperatively and not sequentially. We also observe that the optimal controller prefers "zipping" or small loop closure steps during folding. The folding routes predicted by the proposed method bear strong resemblance to the results in the literature.

  3. Structure-Based Phylogeny as a Diagnostic for Functional Characterization of Proteins with a Cupin Fold

    OpenAIRE

    Garima Agarwal; Malligarjunan Rajavel; Balasubramanian Gopal; Narayanaswamy Srinivasan

    2009-01-01

    Background: The members of cupin superfamily exhibit large variations in their sequences, functions, organization of domains, quaternary associations and the nature of bound metal ion, despite having a conserved beta-barrel structural scaffold. Here, an attempt has been made to understand structure-function relationships among the members of this diverse superfamily and identify the principles governing functional diversity. The cupin superfamily also contains proteins for which the s...

  4. Computational protein design with electrostatic focusing: experimental characterization of a conditionally folded helical domain with a reduced amino acid alphabet.

    Science.gov (United States)

    Suárez-Diez, Maria; Pujol, Anaïs M; Matzapetakis, Manolis; Jaramillo, Alfonso; Iranzo, Olga

    2013-07-01

    Automated methodologies to design synthetic proteins from first principles use energy computations to estimate the ability of the sequences to adopt a targeted structure. This approach is still far from systematically producing native-like sequences, due, most likely, to inaccuracies when modeling the interactions between the protein and its aqueous environment. This is particularly challenging when engineering small protein domains (with less polar pair interactions than with the solvent). We have re-designed a three-helix bundle, domain B, using a fixed backbone and a four amino acid alphabet. We have enlarged the rotamer library with conformers that increase the weight of electrostatic interactions within the design process without altering the energy function used to compute the folding free energy. Our synthetic sequences show less than 15% similarity to any Swissprot sequence. We have characterized our sequences in different solvents using circular dichroism and nuclear magnetic resonance. The targeted structure achieved is dependent on the solvent used. This method can be readily extended to larger domains. Our method will be useful for the engineering of proteins that become active only in a given solvent and for designing proteins in the context of hydrophobic solvents, an important fraction of the situations in the cell. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Acrylonitrile Quenching of Trp Phosphorescence in Proteins: A Probe of the Internal Flexibility of the Globular Fold

    Science.gov (United States)

    Strambini, Giovanni B.; Gonnelli, Margherita

    2010-01-01

    Quenching of Trp phosphorescence in proteins by diffusion of solutes of various molecular sizes unveils the frequency-amplitude of structural fluctuations. To cover the sizes gap between O2 and acrylamide, we examined the potential of acrylonitrile to probe conformational flexibility of proteins. The distance dependence of the through-space acrylonitrile quenching rate was determined in a glass at 77 K, with the indole analog 2-(3-indoyl) ethyl phenyl ketone. Intensity and decay kinetics data were fitted to a rate, k(r) = k0 exp[−(r − r0)/re], with an attenuation length re = 0.03 nm and a contact rate k0 = 3.6 × 1010 s−1. At ambient temperature, the bimolecular quenching rate constant (kq) was determined for a series of proteins, appositely selected to test the importance of factors such as the degree of Trp burial and structural rigidity. Relative to kq = 1.9 × 109 M−1s−1 for free Trp in water, in proteins kq ranged from 6.5 × 106 M−1s−1 for superficial sites to 1.3 × 102 M−1s−1 for deep cores. The short-range nature of the interaction and the direct correlation between kq and structural flexibility attest that in the microsecond-second timescale of phosphorescence acrylonitrile readily penetrates even compact protein cores and exhibits significant sensitivity to variations in dynamical structure of the globular fold. PMID:20682273

  6. Structural diversity of polyoxomolybdate clusters along the three-fold axis of the molybdenum storage protein.

    Science.gov (United States)

    Poppe, Juliane; Warkentin, Eberhard; Demmer, Ulrike; Kowalewski, Björn; Dierks, Thomas; Schneider, Klaus; Ermler, Ulrich

    2014-09-01

    The molybdenum storage protein (MoSto) can store more than 100 Mo or W atoms as discrete polyoxometalate (POM) clusters. Here, we describe the three POM cluster sites along the threefold axis of the protein complex based on four X-ray structures with slightly different polyoxomolybdate compositions between 1.35 and 2 Å resolution. In contrast to the Moα-out binding site occupied by an Mo3 cluster, the Moα-in and Moβ binding sites contain rather weak and non-uniform electron density for the Mo atoms (but clearly identifiable by anomalous data), suggesting the presence of POM cluster ensembles and/or degradation products of larger aggregates. The "Moα-in cluster ensemble" was interpreted as an antiprism-like Mo6 species superimposed with an Mo7 pyramide and the "Moβ cluster ensemble" as an Mo13 cluster (present mostly in a degraded form) composed of a pyramidal Mo7 and a Mo3 building block linked by three spatially separated MoOx units. Inside the ball-shaped Mo13 cluster sits an occluded central atom, perhaps a metal ion. POM cluster formation at the Moα-in and Moβ sites appears to be driven by filtering out and binding/protecting self-assembled transient species complementary to the protein template. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Redesigning the type II' β-turn in green fluorescent protein to type I': implications for folding kinetics and stability.

    Science.gov (United States)

    Madan, Bharat; Sokalingam, Sriram; Raghunathan, Govindan; Lee, Sun-Gu

    2014-10-01

    Both Type I' and Type II' β-turns have the same sense of the β-turn twist that is compatible with the β-sheet twist. They occur predominantly in two residue β-hairpins, but the occurrence of Type I' β-turns is two times higher than Type II' β-turns. This suggests that Type I' β-turns may be more stable than Type II' β-turns, and Type I' β-turn sequence and structure can be more favorable for protein folding than Type II' β-turns. Here, we redesigned the native Type II' β-turn in GFP to Type I' β-turn, and investigated its effect on protein folding and stability. The Type I' β-turns were designed based on the statistical analysis of residues in natural Type I' β-turns. The substitution of the native "GD" sequence of i+1 and i+2 residues with Type I' preferred "(N/D)G" sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β-turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β-turns in natural β-hairpins can be further optimized by converting the sequence to Type I'. © 2014 Wiley Periodicals, Inc.

  8. The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding.

    Science.gov (United States)

    Patterson, Melissa A; Bandyopadhyay, Anannya; Devaraneni, Prasanna K; Woodward, Josha; Rooney, LeeAnn; Yang, Zhongying; Skach, William R

    2015-11-27

    Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Differential evolution for protein folding optimization based on a three-dimensional AB off-lattice model.

    Science.gov (United States)

    Bošković, Borko; Brest, Janez

    2016-10-01

    This paper presents a differential evolution algorithm that is adapted for the protein folding optimization on a three-dimensional AB off-lattice model. The proposed algorithm is based on a self-adaptive differential evolution that improves the algorithm efficiency and reduces the number of control parameters. A mutation strategy for the fast convergence is used inside the algorithm. A temporal locality is used in order to speed up the algorithm convergence additionally and to find amino-acid conformations with the lowest free energy values. Within this mechanism a new vector is calculated when the trial vector is better than the corresponding vector from the population. This new vector is likely better than the trial vector and this accelerates convergence speed. Because of the fast convergence the algorithm has some chance to be trapped into the local optima. To mitigate this problem the algorithm includes reinitialization. The proposed algorithm was tested on amino-acid sequences that are used frequently in literature. The obtained results show that the proposed algorithm is superior to the algorithms from the literature and the obtained amino-acid sequences have significantly lower free energy values. Graphical Abstract Protein folding optimization on a three-dimensional AB off-lattice model using the differential evolution algorithm.

  10. Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography.

    Science.gov (United States)

    Dong, Xiao-Yan; Chen, Li-Jun; Sun, Yan

    2009-07-03

    This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and beta-cyclodextrin (beta-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein-CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing beta-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6-0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.

  11. Synthesis of PAF, an antifungal protein from P. chrysogenum, by native chemical ligation: native disulfide pattern and fold obtained upon oxidative refolding.

    Science.gov (United States)

    Váradi, Györgyi; Tóth, Gábor K; Kele, Zoltán; Galgóczy, László; Fizil, Ádám; Batta, Gyula

    2013-09-16

    The folding of disulfide proteins is of considerable interest because knowledge of this may influence our present understanding of protein folding. However, sometimes even the disulfide pattern cannot be unequivocally determined by the available experimental techniques. For example, the structures of a few small antifungal proteins (PAF, AFP) have been disclosed recently using NMR spectroscopy but with some ambiguity in the actual disulfide pattern. For this reason, we carried out the chemical synthesis of PAF. Probing different approaches, the oxidative folding of the synthetic linear PAF yielded a folded protein that has identical structure and antifungal activity as the native PAF. In contrast, unfolded linear PAF was inactive, a result that may have implications concerning its redox state in the mode of action.

  12. Probing Protein Folding Kinetics with High-resolution, Stabilized Optical Tweezers

    Science.gov (United States)

    Wong, Wesley; Halvorsen, Ken

    2009-03-01

    Single-molecule techniques provide a powerful means of exploring molecular transitions such as the unfolding and refolding of a protein. However, the quantification of bi-directional transitions and near-equilibrium phenomena poses unique challenges, and is often limited by the detection resolution and long-term stability of the instrument. We have developed unique optical tweezers methods that address these problems, including an interference-based method for high-resolution 3D bead tracking (˜1 nm laterally, ˜0.3 nm vertically, at > 100 Hz), and a continuous autofocus system that stabilizes the trap height to within 1-2 nm longterm [1,2]. We have used our instruments to quantify the force-dependent unfolding and refolding kinetics of single protein domains (e.g. spectrin in collaboration with E. Evans). These single-molecule studies are presented, together with the accompanying probabilistic analysis that we have developed. References: 1. W.P. Wong, V. Heinrich, E. Evans, Mat. Res. Soc. Symp. Proc., 790, P5.1-P5.10 (2004). 2. V. Heinrich, W.P. Wong, K. Halvorsen, E. Evans, Langmuir, 24, 1194-1203 (2008).

  13. The Structure of DdrB from Deinococcus: a New Fold for Single-stranded DNA Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Sugiman-Marangos, S.; Junop, M

    2010-01-01

    Deinococcus spp. are renowned for their amazing ability to recover rapidly from severe genomic fragmentation as a result of exposure to extreme levels of ionizing radiation or desiccation. Despite having been originally characterized over 50 years ago, the mechanism underlying this remarkable repair process is still poorly understood. Here, we report the 2.8 {angstrom} structure of DdrB, a single-stranded DNA (ssDNA) binding protein unique to Deinococcus spp. that is crucial for recovery following DNA damage. DdrB forms a pentameric ring capable of binding single-stranded but not double-stranded DNA. Unexpectedly, the crystal structure reveals that DdrB comprises a novel fold that is structurally and topologically distinct from all other single-stranded binding (SSB) proteins characterized to date. The need for a unique ssDNA binding function in response to severe damage, suggests a distinct role for DdrB which may encompass not only standard SSB protein function in protection of ssDNA, but also more specialized roles in protein recruitment or DNA architecture maintenance. Possible mechanisms of DdrB action in damage recovery are discussed.

  14. Residue contacts predicted by evolutionary covariance extend the application of ab initio molecular replacement to larger and more challenging protein folds

    Directory of Open Access Journals (Sweden)

    Felix Simkovic

    2016-07-01

    Full Text Available For many protein families, the deluge of new sequence information together with new statistical protocols now allow the accurate prediction of contacting residues from sequence information alone. This offers the possibility of more accurate ab initio (non-homology-based structure prediction. Such models can be used in structure solution by molecular replacement (MR where the target fold is novel or is only distantly related to known structures. Here, AMPLE, an MR pipeline that assembles search-model ensembles from ab initio structure predictions (`decoys', is employed to assess the value of contact-assisted ab initio models to the crystallographer. It is demonstrated that evolutionary covariance-derived residue–residue contact predictions improve the quality of ab initio models and, consequently, the success rate of MR using search models derived from them. For targets containing β-structure, decoy quality and MR performance were further improved by the use of a β-strand contact-filtering protocol. Such contact-guided decoys achieved 14 structure solutions from 21 attempted protein targets, compared with nine for simple Rosetta decoys. Previously encountered limitations were superseded in two key respects. Firstly, much larger targets of up to 221 residues in length were solved, which is far larger than the previously benchmarked threshold of 120 residues. Secondly, contact-guided decoys significantly improved success with β-sheet-rich proteins. Overall, the improved performance of contact-guided decoys suggests that MR is now applicable to a significantly wider range of protein targets than were previously tractable, and points to a direct benefit to structural biology from the recent remarkable advances in sequencing.

  15. Protein preconcentration using nanofractures generated by nanoparticle-assisted electric breakdown at junction gaps.

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    Chun-Ping Jen

    Full Text Available Sample preconcentration is an important step that increases the accuracy of subsequent detection, especially for samples with extremely low concentrations. Due to the overlapping of electrical double layers in the nanofluidic channel, the concentration polarization effect can be generated by applying an electric field. Therefore, a nonlinear electrokinetic flow is induced, which results in the fast accumulation of proteins in front of the induced ionic depletion zone, the so-called exclusion-enrichment effect. Nanofractures were created in this work to preconcentrate proteins via the exclusion-enrichment effect. The protein sample was driven by electroosmotic flow and accumulated at a specific location. The preconcentration chip for proteins was fabricated using simple standard soft lithography with a polydimethylsiloxane replica. Nanofractures were formed by utilizing nanoparticle-assisted electric breakdown. The proposed method for nanofracture formation that utilizes nanoparticle deposition at the junction gap between microchannels greatly decreases the required electric breakdown voltage. The experimental results indicate that a protein sample with an extremely low concentration of 1 nM was concentrated to 1.5×10(4-fold in 60 min using the proposed chip.

  16. Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells.

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    Daisuke Ishibashi

    Full Text Available Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrPSc in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrPSc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrPSc degradation were evaluated. The accumulation of PrPSc in cells persistently infected with the prion strain Fukuoka-1 (FK, derived from a patient with Gerstmann-Sträussler-Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrPSc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrPSc from either the 22L or the Chandler strain, indicating that the degradation of PrPSc in host cells might be strain-dependent.

  17. Protein Folding and Aggregation into Amyloid: The Interference by Natural Phenolic Compounds

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    Massimo Stefani

    2013-06-01

    Full Text Available Amyloid aggregation is a hallmark of several degenerative diseases affecting the brain or peripheral tissues, whose intermediates (oligomers, protofibrils and final mature fibrils display different toxicity. Consequently, compounds counteracting amyloid aggregation have been investigated for their ability (i to stabilize toxic amyloid precursors; (ii to prevent the growth of toxic oligomers or speed that of fibrils; (iii to inhibit fibril growth and deposition; (iv to disassemble preformed fibrils; and (v to favor amyloid clearance. Natural phenols, a wide panel of plant molecules, are one of the most actively investigated categories of potential amyloid inhibitors. They are considered responsible for the beneficial effects of several traditional diets being present in green tea, extra virgin olive oil, red wine, spices, berries and aromatic herbs. Accordingly, it has been proposed that some natural phenols could be exploited to prevent and to treat amyloid diseases, and recent studies have provided significant information on their ability to inhibit peptide/protein aggregation in various ways and to stimulate cell defenses, leading to identify shared or specific mechanisms. In the first part of this review, we will overview the significance and mechanisms of amyloid aggregation and aggregate toxicity; then, we will summarize the recent achievements on protection against amyloid diseases by many natural phenols.

  18. In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.

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    Dref C De Moura

    Full Text Available The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L. plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.

  19. Methionine sulfoxides on prion protein Helix-3 switch on the alpha-fold destabilization required for conversion.

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    Giorgio Colombo

    Full Text Available BACKGROUND: The conversion of the cellular prion protein (PrP(C into the infectious form (PrP(Sc is the key event in prion induced neurodegenerations. This process is believed to involve a multi-step conformational transition from an alpha-helical (PrP(C form to a beta-sheet-rich (PrP(Sc state. In addition to the conformational difference, PrP(Sc exhibits as covalent signature the sulfoxidation of M213. To investigate whether such modification may play a role in the misfolding process we have studied the impact of methionine oxidation on the dynamics and energetics of the HuPrP(125-229 alpha-fold. METHODOLOGY/PRINCIPAL FINDINGS: Using molecular dynamics simulation, essential dynamics, correlated motions and signal propagation analysis, we have found that substitution of the sulfur atom of M213 by a sulfoxide group impacts on the stability of the native state increasing the flexibility of regions preceding the site of the modification and perturbing the network of stabilizing interactions. Together, these changes favor the population of alternative states which maybe essential in the productive pathway of the pathogenic conversion. These changes are also observed when the sulfoxidation is placed at M206 and at both, M206 and M213. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the sulfoxidation of Helix-3 methionines might be the switch for triggering the initial alpha-fold destabilization required for the productive pathogenic conversion.

  20. Essential roles of protein-solvent many-body correlation in solvent-entropy effect on protein folding and denaturation: Comparison between hard-sphere solvent and water

    Energy Technology Data Exchange (ETDEWEB)

    Oshima, Hiraku; Kinoshita, Masahiro, E-mail: kinoshit@iae.kyoto-u.ac.jp [Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2015-04-14

    In earlier works, we showed that the entropic effect originating from the translational displacement of water molecules plays the pivotal role in protein folding and denaturation. The two different solvent models, hard-sphere solvent and model water, were employed in theoretical methods wherein the entropic effect was treated as an essential factor. However, there were similarities and differences in the results obtained from the two solvent models. In the present work, to unveil the physical origins of the similarities and differences, we simultaneously consider structural transition, cold denaturation, and pressure denaturation for the same protein by employing the two solvent models and considering three different thermodynamic states for each solvent model. The solvent-entropy change upon protein folding/unfolding is decomposed into the protein-solvent pair (PA) and many-body (MB) correlation components using the integral equation theories. Each component is further decomposed into the excluded-volume (EV) and solvent-accessible surface (SAS) terms by applying the morphometric approach. The four physically insightful constituents, (PA, EV), (PA, SAS), (MB, EV), and (MB, SAS), are thus obtained. Moreover, (MB, SAS) is discussed by dividing it into two factors. This all-inclusive investigation leads to the following results: (1) the protein-water many-body correlation always plays critical roles in a variety of folding/unfolding processes; (2) the hard-sphere solvent model fails when it does not correctly reproduce the protein-water many-body correlation; (3) the hard-sphere solvent model becomes problematic when the dependence of the many-body correlation on the solvent number density and temperature is essential: it is not quite suited to studies on cold and pressure denaturating of a protein; (4) when the temperature and solvent number density are limited to the ambient values, the hard-sphere solvent model is usually successful; and (5) even at the ambient

  1. Genetic Code Optimization for Cotranslational Protein Folding: Codon Directional Asymmetry Correlates with Antiparallel Betasheets, tRNA Synthetase Classes

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    Hervé Seligmann

    2017-01-01

    Full Text Available A new codon property, codon directional asymmetry in nucleotide content (CDA, reveals a biologically meaningful genetic code dimension: palindromic codons (first and last nucleotides identical, codon structure XZX are symmetric (CDA = 0, codons with structures ZXX/XXZ are 5′/3′ asymmetric (CDA = −1/1; CDA = −0.5/0.5 if Z and X are both purines or both pyrimidines, assigning negative/positive (−/+ signs is an arbitrary convention. Negative/positive CDAs associate with (a Fujimoto's tetrahedral codon stereo-table; (b tRNA synthetase class I/II (aminoacylate the 2′/3′ hydroxyl group of the tRNA's last ribose, respectively; and (c high/low antiparallel (not parallel betasheet conformation parameters. Preliminary results suggest CDA-whole organism associations (body temperature, developmental stability, lifespan. Presumably, CDA impacts spatial kinetics of codon-anticodon interactions, affecting cotranslational protein folding. Some synonymous codons have opposite CDA sign (alanine, leucine, serine, and valine, putatively explaining how synonymous mutations sometimes affect protein function. Correlations between CDA and tRNA synthetase classes are weaker than between CDA and antiparallel betasheet conformation parameters. This effect is stronger for mitochondrial genetic codes, and potentially drives mitochondrial codon-amino acid reassignments. CDA reveals information ruling nucleotide-protein relations embedded in reversed (not reverse-complement sequences (5′-ZXX-3′/5′-XXZ-3′.

  2. Acrylonitrile quenching of trp phosphorescence in proteins: a probe of the internal flexibility of the globular fold.

    Science.gov (United States)

    Strambini, Giovanni B; Gonnelli, Margherita

    2010-08-04

    Quenching of Trp phosphorescence in proteins by diffusion of solutes of various molecular sizes unveils the frequency-amplitude of structural fluctuations. To cover the sizes gap between O(2) and acrylamide, we examined the potential of acrylonitrile to probe conformational flexibility of proteins. The distance dependence of the through-space acrylonitrile quenching rate was determined in a glass at 77 K, with the indole analog 2-(3-indoyl) ethyl phenyl ketone. Intensity and decay kinetics data were fitted to a rate, k(r) =k(0) exp[-(r -r(0))/r(e)], with an attenuation length r(e) = 0.03 nm and a contact rate k(0) = 3.6 x 10(10) s(-1). At ambient temperature, the bimolecular quenching rate constant (kq) was determined for a series of proteins, appositely selected to test the importance of factors such as the degree of Trp burial and structural rigidity. Relative to kq = 1.9 x 10(9) M(-1)s(-1) for free Trp in water, in proteins kq ranged from 6.5 x 10(6) M(-1)s(-1) for superficial sites to 1.3 x 10(2) M(-1)s(-1) for deep cores. The short-range nature of the interaction and the direct correlation between kq and structural flexibility attest that in the microsecond-second timescale of phosphorescence acrylonitrile readily penetrates even compact protein cores and exhibits significant sensitivity to variations in dynamical structure of the globular fold. 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Alternative splice variants in TIM barrel proteins from human genome correlate with the structural and evolutionary modularity of this versatile protein fold.

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    Adrián Ochoa-Leyva

    Full Text Available After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα8 barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα2, (βα4, (βα5, (βα6, and (βα7 subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to

  4. Interaction of Alzheimer's A beta peptide with an engineered binding protein--thermodynamics and kinetics of coupled folding-binding.

    Science.gov (United States)

    Hoyer, Wolfgang; Härd, Torleif

    2008-04-25

    The oligomerization and aggregation of the amyloid-beta (A beta) peptide, a cleavage product of the amyloid precursor protein predominantly 40 or 42 amino acids in length, has been implicated in the pathogenesis of Alzheimer's disease. The identification of A beta-binding agents, e.g., antibodies or peptides, constitutes a promising therapeutic approach. However, the amount of structural and biophysical data on the underlying A beta interactions is currently very limited. We have earlier determined the structure of A beta (1-40) in complex with the affibody protein Z(A beta 3), a selected binding protein based on a three-helix bundle scaffold (Z domain). Z(A beta 3) is a dimer of affibody subunits linked via a disulfide bridge involving a selected cysteine mutation at position 28. Z(A beta 3) binds to the central and C-terminal part of A beta (residues 17-36), which adopts a beta-hairpin conformation in the complex. Here we present a detailed biophysical analysis of the Z(A beta 3):A beta (1-40) interaction, employing NMR, circular dichroism spectroscopy, 8-anilino-1-naphthalenesulfonic acid and tyrosine fluorescence, size-exclusion chromatography, thermal denaturation profiles and isothermal titration calorimetry. We conclude that (i) free Z(A beta 3) is characterized by conformational exchange and the loss of helix 1 of the three-helix bundle scaffold; (ii) a high-energy barrier is associated with the conversion of an initial Z(A beta 3):A beta (1-40) recognition complex into the native complex structure, entailing slow binding kinetics; (iii) both A beta and Z(A beta 3) fold upon binding, which, e.g., becomes manifest in the binding thermodynamics that feature a large negative change in heat capacity; (iv) the C28-disulfide does not merely afford dimerization, but its impact on the binding interfaces of the affibody subunits and A beta is a prerequisite for tight binding. The extensive folding coupled to binding observed here likely constitutes an obligate

  5. Taxonomic distribution, repeats, and functions of the S1 domain-containing proteins as members of the OB-fold family.

    Science.gov (United States)

    Deryusheva, Evgeniia I; Machulin, Andrey V; Selivanova, Olga M; Galzitskaya, Oxana V

    2017-04-01

    Proteins of the nucleic acid-binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB-fold in protein structures. Here, we have analyzed the superfamily of nucleic acid-binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA-binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA-binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain-containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB-fold is a distinctive feature of S1 domain-containing proteins. Proteins from the other families and superfamilies have either one OB-fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain-containing proteins. Proteins 2017; 85:602-613. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  6. COMPARISON OF THE HEMOCYANIN BETA-BARREL WITH OTHER GREEK KEY BETA-BARRELS - POSSIBLE IMPORTANCE OF THE BETA-ZIPPER IN PROTEIN-STRUCTURE AND FOLDING

    NARCIS (Netherlands)

    HAZES, B; HOL, WGJ

    The Greek key beta-barrel topology is a folding motif observed in many proteins of widespread evolutionary origin. The arthropodan hemocyanins also have such a Greek key beta-barrel, which forms the core of the third domain of this protein. The hemocyanin beta-barrel was found to be structurally

  7. Penicillin-binding protein folding is dependent on the PrsA peptidyl-prolyl cis-trans isomerase in Bacillus subtilis

    NARCIS (Netherlands)

    Hyyrylainen, Hanne-Leena; Marciniak, Bogumila C.; Dahncke, Kathleen; Pietiainen, Milla; Courtin, Pascal; Vitikainen, Marika; Seppala, Raili; Otto, Andreas; Becher, Doerte; Chapot-Chartier, Marie-Pierre; Kuipers, Oscar P.; Kontinen, Vesa P.; Hyyryläinen, Hanne-Leena; Pietiäinen, Milla

    P>The PrsA protein is a membrane-anchored peptidyl-prolyl cis-trans isomerase in Bacillus subtilis and most other Gram-positive bacteria. It catalyses the post-translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this

  8. Changing folding and binding stability in a viral coat protein: a comparison between substitutions accessible through mutation and those fixed by natural selection.

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    Craig R Miller

    Full Text Available Previous studies have shown that most random amino acid substitutions destabilize protein folding (i.e. increase the folding free energy. No analogous studies have been carried out for protein-protein binding. Here we use a structure-based model of the major coat protein in a simple virus, bacteriophage φX174, to estimate the free energy of folding of a single coat protein and binding of five coat proteins within a pentameric unit. We confirm and extend previous work in finding that most accessible substitutions destabilize both protein folding and protein-protein binding. We compare the pool of accessible substitutions with those observed among the φX174-like wild phage and in experimental evolution with φX174. We find that observed substitutions have smaller effects on stability than expected by chance. An analysis of adaptations at high temperatures suggests that selection favors either substitutions with no effect on stability or those that simultaneously stabilize protein folding and slightly destabilize protein binding. We speculate that these mutations might involve adjusting the rate of capsid assembly. At normal laboratory temperature there is little evidence of directional selection. Finally, we show that cumulative changes in stability are highly variable; sometimes they are well beyond the bounds of single substitution changes and sometimes they are not. The variation leads us to conclude that phenotype selection acts on more than just stability. Instances of larger cumulative stability change (never via a single substitution despite their availability lead us to conclude that selection views stability at a local, not a global, level.

  9. Changing folding and binding stability in a viral coat protein: a comparison between substitutions accessible through mutation and those fixed by natural selection.

    Science.gov (United States)

    Miller, Craig R; Lee, Kuo Hao; Wichman, Holly A; Ytreberg, F Marty

    2014-01-01

    Previous studies have shown that most random amino acid substitutions destabilize protein folding (i.e. increase the folding free energy). No analogous studies have been carried out for protein-protein binding. Here we use a structure-based model of the major coat protein in a simple virus, bacteriophage φX174, to estimate the free energy of folding of a single coat protein and binding of five coat proteins within a pentameric unit. We confirm and extend previous work in finding that most accessible substitutions destabilize both protein folding and protein-protein binding. We compare the pool of accessible substitutions with those observed among the φX174-like wild phage and in experimental evolution with φX174. We find that observed substitutions have smaller effects on stability than expected by chance. An analysis of adaptations at high temperatures suggests that selection favors either substitutions with no effect on stability or those that simultaneously stabilize protein folding and slightly destabilize protein binding. We speculate that these mutations might involve adjusting the rate of capsid assembly. At normal laboratory temperature there is little evidence of directional selection. Finally, we show that cumulative changes in stability are highly variable; sometimes they are well beyond the bounds of single substitution changes and sometimes they are not. The variation leads us to conclude that phenotype selection acts on more than just stability. Instances of larger cumulative stability change (never via a single substitution despite their availability) lead us to conclude that selection views stability at a local, not a global, level.

  10. BPI Fold-Containing Family A Member 2/Parotid Secretory Protein Is an Early Biomarker of AKI.

    Science.gov (United States)

    Kota, Satya K; Pernicone, Elizabeth; Leaf, David E; Stillman, Isaac E; Waikar, Sushrut S; Kota, Savithri Balasubramanian

    2017-12-01

    AKI is a major cause of morbidity and mortality and an important contributor to the development and progression of CKD. Molecular biomarkers that improve the detection and prognostication of AKI are therefore required. We assessed the utility as such of BPI fold-containing family A member 2 (BPIFA2), also known as parotid secretory protein, which we identified via a multiplex quantitative proteomics screen of acutely injured murine kidneys. In physiologic conditions, BPIFA2 is expressed specifically in the parotid glands and is abundant in salivary secretions. In our study, AKI induced Bpifa2 expression in the kidneys of mice within 3 hours. Furthermore, we detected BPIFA2 protein in plasma and urine in these models as early as 6 hours after injury. However, renal injury did not induce the expression of Bpifa2 in mice lacking Nur77 , an immediate early gene expressed in the kidneys during AKI. Notably, patients with AKI had higher blood and urine levels of BPIFA2 than did healthy individuals. Together, our results reveal that BPIFA2 is a potential early biomarker of AKI. Copyright © 2017 by the American Society of Nephrology.

  11. Bactericidal/Permeability-increasing protein fold-containing family member A1 in airway host protection and respiratory disease.

    Science.gov (United States)

    Britto, Clemente J; Cohn, Lauren

    2015-05-01

    Bactericidal/permeability-increasing protein fold-containing family member A1 (BPIFA1), formerly known as SPLUNC1, is one of the most abundant proteins in respiratory secretions and has been identified with increasing frequency in studies of pulmonary disease. Its expression is largely restricted to the respiratory tract, being highly concentrated in the upper airways and proximal trachea. BPIFA1 is highly responsive to airborne pathogens, allergens, and irritants. BPIFA1 actively participates in host protection through antimicrobial, surfactant, airway surface liquid regulation, and immunomodulatory properties. Its expression is modulated in multiple lung diseases, including cystic fibrosis, chronic obstructive pulmonary disease, respiratory malignancies, and idiopathic pulmonary fibrosis. However, the role of BPIFA1 in pulmonary pathogenesis remains to be elucidated. This review highlights the versatile properties of BPIFA1 in antimicrobial protection and its roles as a sensor of environmental exposure and regulator of immune cell function. A greater understanding of the contribution of BPIFA1 to disease pathogenesis and activity may clarify if BPIFA1 is a biomarker and potential drug target in pulmonary disease.

  12. Impact of P-Site tRNA and antibiotics on ribosome mediated protein folding: studies using the Escherichia coli ribosome.

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    Surojit Mondal

    Full Text Available BACKGROUND: The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3' -CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA also displays chaperoning activity. RESULTS: The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin and macrolide antibiotics (erythromycin and josamycin on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3'-CCA end of P/P-site tRNA with the PTC is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA to be important for its chaperoning ability. CONCLUSION: Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.

  13. Symmetric GroEL:GroES2 complexes are the protein-folding functional form of the chaperonin nanomachine.

    Science.gov (United States)

    Yang, Dong; Ye, Xiang; Lorimer, George H

    2013-11-12

    Using calibrated FRET, we show that the simultaneous occupancy of both rings of GroEL by ATP and GroES occurs, leading to the rapid formation of symmetric GroEL:GroES2 "football" particles regardless of the presence or absence of substrate protein (SP). In the absence of SP, these symmetric particles revert to asymmetric GroEL:GroES1 "bullet" particles. The breakage of GroES symmetry requires the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry. These asymmetric particles are both persistent and dynamic; they turnover via the asymmetric cycle. When challenged with SP, however, they revert to symmetric particles within a second. In the presence of SP, the symmetric particles are also persistent and dynamic. They turn over via the symmetric cycle. Under these conditions, the stochastic hydrolysis of ATP and the breakage of nucleotide symmetry also occur within the ensemble of particles. However, on account of SP-catalyzed ADP/ATP exchange, GroES symmetry is rapidly restored. The residence time of both GroES and SP on functional GroEL is reduced to ∼1 s, enabling many more iterations than was previously believed possible, consistent with the iterative annealing mechanism. This result is inconsistent with currently accepted models. Using a foldable SP, we show that as the SP folds to the native state and the population of unfolded SP declines, the population of symmetric particles reverts to asymmetric particles in parallel, a result that is consistent with the former being the folding functional form.

  14. Comparative analysis of the folding dynamics and kinetics of an engineered knotted protein and its variants derived from HP0242 of Helicobacter pylori

    Science.gov (United States)

    Wang, Liang-Wei; Liu, Yu-Nan; Lyu, Ping-Chiang; Jackson, Sophie E.; Hsu, Shang-Te Danny

    2015-09-01

    Understanding the mechanism by which a polypeptide chain thread itself spontaneously to attain a knotted conformation has been a major challenge in the field of protein folding. HP0242 is a homodimeric protein from Helicobacter pylori with intertwined helices to form a unique pseudo-knotted folding topology. A tandem HP0242 repeat has been constructed to become the first engineered trefoil-knotted protein. Its small size renders it a model system for computational analyses to examine its folding and knotting pathways. Here we report a multi-parametric study on the folding stability and kinetics of a library of HP0242 variants, including the trefoil-knotted tandem HP0242 repeat, using far-UV circular dichroism and fluorescence spectroscopy. Equilibrium chemical denaturation of HP0242 variants shows the presence of highly populated dimeric and structurally heterogeneous folding intermediates. Such equilibrium folding intermediates retain significant amount of helical structures except those at the N- and C-terminal regions in the native structure. Stopped-flow fluorescence measurements of HP0242 variants show that spontaneous refolding into knotted structures can be achieved within seconds, which is several orders of magnitude faster than previously observed for other knotted proteins. Nevertheless, the complex chevron plots indicate that HP0242 variants are prone to misfold into kinetic traps, leading to severely rolled-over refolding arms. The experimental observations are in general agreement with the previously reported molecular dynamics simulations. Based on our results, kinetic folding pathways are proposed to qualitatively describe the complex folding processes of HP0242 variants.

  15. N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

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    Etai Jacob

    2013-04-01

    Full Text Available Computational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain.

  16. The conserved Candida albicans CA3427 gene product defines a new family of proteins exhibiting the generic periplasmic binding protein structural fold.

    Directory of Open Access Journals (Sweden)

    Sébastien Santini

    Full Text Available Nosocomial diseases due to Candida albicans infections are in constant rise in hospitals, where they cause serious complications to already fragile intensive care patients. Antifungal drug resistance is fast becoming a serious issue due to the emergence of strains resistant to currently available antifungal agents. Thus the urgency to identify new potential protein targets, the function and structure of which may guide the development of new antifungal drugs. In this context, we initiated a comparative genomics study in search of promising protein coding genes among the most conserved ones in reference fungal genomes. The CA3427 gene was selected on the basis of its presence among pathogenic fungi contrasting with its absence in the non pathogenic Saccharomyces cerevisiae. We report the crystal 3D-structure of the Candida albicans CA3427 protein at 2.1 Å resolution. The combined analysis of its sequence and structure reveals a structural fold originally associated with periplasmic binding proteins. The CA3427 structure highlights a binding site located between the two protein domains, corresponding to a sequence segment conserved among fungi. Two crystal forms of CA3427 were found, suggesting that the presence or absence of a ligand at the proposed binding site might trigger a "Venus flytrap" motion, coupled to the previously described activity of bacterial periplasmic binding proteins. The conserved binding site defines a new subfamily of periplasmic binding proteins also found in many bacteria of the bacteroidetes division, in a choanoflagellate (a free-living unicellular and colonial flagellate eukaryote and in a placozoan (the closest multicellular relative of animals. A phylogenetic analysis suggests that this gene family originated in bacteria before its horizontal transfer to an ancestral eukaryote prior to the radiation of fungi. It was then lost by the Saccharomycetales which include Saccharomyces cerevisiae.

  17. Ancylostoma ceylanicum Excretory-Secretory Protein 2 Adopts a Netrin-Like Fold and Defines a Novel Family of Nematode Proteins

    Energy Technology Data Exchange (ETDEWEB)

    K Kucera; L Harrison; M Cappello; Y Modis

    2011-12-31

    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 {angstrom} 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-likefold. 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.

  18. SAAFEC: Predicting the Effect of Single Point Mutations on Protein Folding Free Energy Using a Knowledge-Modified MM/PBSA Approach.

    Science.gov (United States)

    Getov, Ivan; Petukh, Marharyta; Alexov, Emil

    2016-04-07

    Folding free energy is an important biophysical characteristic of proteins that reflects the overall stability of the 3D structure of macromolecules. Changes in the amino acid sequence, naturally occurring or made in vitro, may affect the stability of the corresponding protein and thus could be associated with disease. Several approaches that predict the changes of the folding free energy caused by mutations have been proposed, but there is no method that is clearly superior to the others. The optimal goal is not only to accurately predict the folding free energy changes, but also to characterize the structural changes induced by mutations and the physical nature of the predicted folding free energy changes. Here we report a new method to predict the Single Amino Acid Folding free Energy Changes (SAAFEC) based on a knowledge-modified Molecular Mechanics Poisson-Boltzmann (MM/PBSA) approach. The method is comprised of two main components: a MM/PBSA component and a set of knowledge based terms delivered from a statistical study of the biophysical characteristics of proteins. The predictor utilizes a multiple linear regression model with weighted coefficients of various terms optimized against a set of experimental data. The aforementioned approach yields a correlation coefficient of 0.65 when benchmarked against 983 cases from 42 proteins in the ProTherm database. the webserver can be accessed via http://compbio.clemson.edu/SAAFEC/.

  19. The fumarate sensor DcuS: progress in rapid protein fold elucidation by combining protein structure prediction methods with NMR spectroscopy

    Science.gov (United States)

    Meiler, Jens; Baker, David

    2005-04-01

    We illustrate how moderate resolution protein structures can be rapidly obtained by interlinking computational prediction methodologies with un- or partially assigned NMR data. To facilitate the application of our recently described method of ranking and subsequent refining alternative structural models using unassigned NMR data [Proc. Natl. Acad. Sci. USA 100 (2003) 15404] for such "structural genomics"-type experiments it is combined with protein models from several prediction techniques, enhanced to utilize partial assignments, and applied on a protein with an unknown structure and fold. From the original NMR spectra obtained for the 140 residue fumarate sensor DcuS, 1100 1H, 13C, and 15N chemical shift signals, 3000 1H- 1H NOESY cross peak intensities, and 209 backbone residual dipolar couplings were extracted and used to rank models produced by de novo structure prediction and comparative modeling methods. The ranking proceeds in two steps: first, an optimal assignment of the NMR peaks to atoms is found for each model independently, and second, the models are ranked based on the consistency between the NMR data and the model assuming these optimal assignments. The low-resolution model selected using this ranking procedure had the correct overall fold and a global backbone RMSD of 6.0Å, and was subsequently refined to 3.7 Å RMSD. With the incorporation of a small number of NOE and residual dipolar coupling constraints available very early in the traditional spectral assignment process, a model with an RMSD of 2.8 Å could rapidly be built. The ability to generate moderate resolution models within days of NMR data collection should facilitate large scale NMR structure determination efforts.

  20. Gradual disordering of the native state on a slow two-state folding protein monitored by single-molecule fluorescence spectroscopy and NMR.

    Science.gov (United States)

    Campos, Luis A; Sadqi, Mourad; Liu, Jianwei; Wang, Xiang; English, Douglas S; Muñoz, Victor

    2013-10-24

    Theory predicts that folding free energy landscapes are intrinsically malleable and as such are expected to respond to perturbations in topographically complex ways. Structural changes upon perturbation have been observed experimentally for unfolded ensembles, folding transition states, and fast downhill folding proteins. However, the native state of proteins that fold in a two-state fashion is conventionally assumed to be structurally invariant during unfolding. Here we investigate how the native and unfolded states of the chicken α-spectrin SH3 domain (a well characterized slow two-state folder) change in response to chemical denaturants and/or temperature. We can resolve the individual properties of the two end-states across the chemical unfolding transition employing single-molecule fluorescence spectroscopy (SM-FRET) and across the thermal unfolding transition by NMR because SH3 folds-unfolds in the slow chemical exchange regime. Our results demonstrate that α-spectrin SH3 unfolds in a canonical way in the sense that it converts between the native state and an unfolded ensemble that expands in response to chemical denaturants. However, as conditions become increasingly destabilizing, the native state also expands gradually, and a large fraction of its native intramolecular hydrogen bonds break up. This gradual disordering of the native state takes place in times shorter than the 100 μs resolution of our SM-FRET experiments. α-Spectrin SH3 thus showcases the extreme plasticity of folding landscapes, which extends to the native state of slow two-state proteins. Our results point to the idea that folding mechanisms under physiological conditions might be quite different from those obtained by linear extrapolation from denaturing conditions. Furthermore, they highlight a pressing need for re-evaluating the conventional procedures for analyzing and interpreting folding experiments, which may be based on too-simplistic assumptions.

  1. The UlaG protein family defines novel structural and functional motifs grafted on an ancient RNase fold

    Directory of Open Access Journals (Sweden)

    Coll Miquel

    2011-09-01

    Full Text Available Abstract Background Bacterial populations are highly successful at colonizing new habitats and adapting to changing environmental conditions, partly due to their capacity to evolve novel virulence and metabolic pathways in response to stress conditions and to shuffle them by horizontal gene transfer (HGT. A common theme in the evolution of new functions consists of gene duplication followed by functional divergence. UlaG, a unique manganese-dependent metallo-β-lactamase (MBL enzyme involved in L-ascorbate metabolism by commensal and symbiotic enterobacteria, provides a model for the study of the emergence of new catalytic activities from the modification of an ancient fold. Furthermore, UlaG is the founding member of the so-called UlaG-like (UlaGL protein family, a recently established and poorly characterized family comprising divalent (and perhaps trivalent metal-binding MBLs that catalyze transformations on phosphorylated sugars and nucleotides. Results Here we combined protein structure-guided and sequence-only molecular phylogenetic analyses to dissect the molecular evolution of UlaG and to study its phylogenomic distribution, its relatedness with present-day UlaGL protein sequences and functional conservation. Phylogenetic analyses indicate that UlaGL sequences are present in Bacteria and Archaea, with bona fide orthologs found mainly in mammalian and plant-associated Gram-negative and Gram-positive bacteria. The incongruence between the UlaGL tree and known species trees indicates exchange by HGT and suggests that the UlaGL-encoding genes provided a growth advantage under changing conditions. Our search for more distantly related protein sequences aided by structural homology has uncovered that UlaGL sequences have a common evolutionary origin with present-day RNA processing and metabolizing MBL enzymes widespread in Bacteria, Archaea, and Eukarya. This observation suggests an ancient origin for the UlaGL family within the broader trunk

  2. Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography.

    Science.gov (United States)

    Vemula, Sandeep; Dedaniya, Akshay; Thunuguntla, Rahul; Mallu, Maheswara Reddy; Parupudi, Pavani; Ronda, Srinivasa Reddy

    2015-01-30

    Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0M) and arginine (0.6M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6×10(8)IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. ASTRO-FOLD: A Combinatorial and Global Optimization Framework for Ab Initio Prediction of Three-Dimensional Structures of Proteins from the Amino Acid Sequence

    Science.gov (United States)

    Klepeis, J. L.; Floudas, C. A.

    2003-01-01

    The field of computational biology has been revolutionized by recent advances in genomics. The completion of a number of genome projects, including that of the human genome, has paved the way toward a variety of challenges and opportunities in bioinformatics and biological systems engineering. One of the first challenges has been the determination of the structures of proteins encoded by the individual genes. This problem, which represents the progression from sequence to structure (genomics to structural genomics), has been widely known as the structure-prediction-in-protein-folding problem. We present the development and application of ASTRO-FOLD, a novel and complete approach for the ab initio prediction of protein structures given only the amino acid sequences of the proteins. The approach exhibits many novel components and the merits of its application are examined for a suite of protein systems, including a number of targets from several critical-assessment-of-structure-prediction experiments. PMID:14507680

  4. A Method for Extracting the Free Energy Surface and Conformational Dynamics of Fast-Folding Proteins from Single Molecule Photon Trajectories

    Science.gov (United States)

    2015-01-01

    Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics. PMID:25988351

  5. Force generation by titin folding.

    Science.gov (United States)

    Mártonfalvi, Zsolt; Bianco, Pasquale; Naftz, Katalin; Ferenczy, György G; Kellermayer, Miklós

    2017-07-01

    Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titin's globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force-dependent folding mechanisms, here we manipulated skeletal-muscle titin molecules with high-resolution optical tweezers. In force-clamp mode, after quenching the force (force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H-bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically-coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten-globule states. Monte-Carlo simulations incorporating a compact molten-globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten-globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles. © 2017 The Protein Society.

  6. Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids.

    Science.gov (United States)

    Aydemir, Fikret; Salganik, Maxim; Resztak, Justyna; Singh, Jasbir; Bennett, Antonette; Agbandje-McKenna, Mavis; Muzyczka, Nicholas

    2016-08-15

    We previously reported that an amino acid substitution, Y704A, near the 2-fold interface of adeno-associated virus (AAV) was defective for transcription of the packaged genome (M. Salganik, F. Aydemir, H. J. Nam, R. McKenna, M. Agbandje-McKenna, and N. Muzyczka, J Virol 88:1071-1079, 2013, doi: http://dx.doi.org/10.1128/JVI.02093-13). In this report, we have characterized the defect in 6 additional capsid mutants located in a region ∼30 Å in diameter on the surface of the AAV type 2 (AAV2) capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3 to 6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA postinfection. In addition, wild-type (wt) capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody-bound wild-type capsids, were able to enter the cell, travel to the nucleus, uncoat, and synthesize a second strand but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in the transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2-fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome that it delivers to the nucleus. Many viruses package enzymes within their capsids that assist in expressing their genomes postinfection, e.g., retroviruses. A number of nonenveloped viruses, including AAV, carry proteases that are needed for capsid maturation or for capsid modification during infection. We describe here what appears to be the first example of

  7. The ribosome regulates flavodoxin folding

    OpenAIRE

    Houwman, Joseline A.

    2017-01-01

    During and after their translation by the ribosome, folding of polypeptides to biologically active proteins is of vital importance for all living organisms. Gaining knowledge about nascent chain folding is required to enhance our understanding of protein folding in the cell. This in turn allows us to obtain insights into factors responsible for protein misfolding, aggregation, and, potentially, for numerous devastating pathologies. In Chapter 1 the model protein flavodoxin is introduced. Also...

  8. Using the Computer Game "FoldIt" to Entice Students to Explore External Representations of Protein Structure in a Biochemistry Course for Nonmajors

    Science.gov (United States)

    Farley, Peter C.

    2013-01-01

    This article describes a novel approach to teaching novice Biochemistry students visual literacy skills and understanding of some aspects of protein structure using the internet resource FoldIt and a worksheet based on selected Introductory Puzzles from this computer game. In responding to a questionnaire, students indicated that they (94%)…

  9. Biophysical Analysis of the N-Terminal Domain from the Human Protein Phosphatase 1 Nuclear Targeting Subunit PNUTS Suggests an Extended Transcription Factor TFIIS-Like Fold.

    Science.gov (United States)

    Zacharchenko, Thomas; Barsukov, Igor; Rigden, Daniel J; Bennett, Daimark; Mayans, Olga

    2016-10-01

    Human protein phosphatase 1 nuclear targeting subunit (PNUTS) plays critical roles in DNA repair, cell growth and survival. The N-terminal domain of PNUTS mediates interactions with Tox4 and the phosphatase and tensin homolog PTEN, which are essential for the roles of this protein. To study this N-terminal domain, we have established its recombinant overproduction in E. coli utilizing NusA fusion. Upon removal of the tag, the remaining PNUTS sample is soluble and highly pure. We have characterized the domain using circular dichroism and nuclear magnetic resonance and analyzed its sequence using bioinformatics. All data agree in suggesting that the PNUTS N-terminal segment adopts a compact, globular fold rich in α-helical content, where the folded fraction is substantially larger than the previously annotated fold. We conclude that this domain adopts a single fold, likely being an extended form of the transcription factor S-II leucine/tryptophan conserved-motif. Thermal denaturation yielded a melting temperature of ~49.5 °C, confirming the stability of the fold. These findings pave the way for the molecular characterization of functional interactions mediated by the N-terminal region of PNUTS.

  10. Temperature-dependent Hammond behavior in a protein-folding reaction: analysis of transition-state movement and ground-state effects.

    Science.gov (United States)

    Taskent, Humeyra; Cho, Jae-Hyun; Raleigh, Daniel P

    2008-05-02

    Characterization of the transition-state ensemble and the nature of the free-energy barrier for protein folding are areas of intense activity and some controversy. A key issue that has emerged in recent years is the width of the free-energy barrier and the susceptibility of the transition state to movement. Here we report denaturant-induced and temperature-dependent folding studies of a small mixed alpha-beta protein, the N-terminal domain of L9 (NTL9). The folding of NTL9 was determined using fluorescence-detected stopped-flow fluorescence measurements conducted at seven different temperatures between 11 and 40 degrees C. Plots of the log of the observed first-order rate constant versus denaturant concentration, "chevron plots," displayed the characteristic V shape expected for two-state folding. There was no hint of deviation from linearity even at the lowest denaturant concentrations. The relative position of the transition state, as judged by the Tanford beta parameter, beta(T), shifts towards the native state as the temperature is increased. Analysis of the temperature dependence of the kinetic and equilibrium m values indicates that the effect is due to significant movement of the transition state and also includes a contribution from temperature-dependent ground-state effects. Analysis of the Leffler plots, plots of Delta G versus Delta G degrees, and their cross-interaction parameters confirms the transition-state movement. Since the protein is destabilized at high temperature, the shift represents a temperature-dependent Hammond effect. This provides independent confirmation of a recent theoretical prediction. The magnitude of the temperature-denaturant cross-interaction parameter is larger for NTL9 than has been reported for the few other cases studied. The implications for temperature-dependent studies of protein folding are discussed.

  11. Penicillin-binding protein folding is dependent on the PrsA peptidyl-prolyl cis-trans isomerase in Bacillus subtilis.

    Science.gov (United States)

    Hyyryläinen, Hanne-Leena; Marciniak, Bogumila C; Dahncke, Kathleen; Pietiäinen, Milla; Courtin, Pascal; Vitikainen, Marika; Seppala, Raili; Otto, Andreas; Becher, Dörte; Chapot-Chartier, Marie-Pierre; Kuipers, Oscar P; Kontinen, Vesa P

    2010-07-01

    Summary The PrsA protein is a membrane-anchored peptidyl-prolyl cis-trans isomerase in Bacillus subtilis and most other Gram-positive bacteria. It catalyses the post-translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispensability. We could construct a viable prsA null mutant in the presence of a high concentration of magnesium. Various changes in cell morphology in the absence of PrsA suggested that PrsA is involved in the biosynthesis of the cylindrical lateral wall. Consistently, four penicillin-binding proteins (PBP2a, PBP2b, PBP3 and PBP4) were unstable in the absence of PrsA, while muropeptide analysis revealed a 2% decrease in the peptidoglycan cross-linkage index. Misfolded PBP2a was detected in PrsA-depleted cells, indicating that PrsA is required for the folding of this PBP either directly or indirectly. Furthermore, strongly increased uniform staining of cell wall with a fluorescent vancomycin was observed in the absence of PrsA. We also demonstrated that PrsA is a dimeric or oligomeric protein which is localized at distinct spots organized in a helical pattern along the cell membrane. These results suggest that PrsA is essential for normal growth most probably as PBP folding is dependent on this PPIase.

  12. F-Type Lectins: A Highly Diversified Family of Fucose-Binding Proteins with a Unique Sequence Motif and Structural Fold, Involved in Self/Non-Self-Recognition

    Directory of Open Access Journals (Sweden)

    Gerardo R. Vasta

    2017-11-01

    Full Text Available The F-type lectin (FTL family is one of the most recent to be identified and structurally characterized. Members of the FTL family are characterized by a fucose recognition domain [F-type lectin domain (FTLD] that displays a novel jellyroll fold (“F-type” fold and unique carbohydrate- and calcium-binding sequence motifs. This novel lectin family comprises widely distributed proteins exhibiting single, double, or greater multiples of the FTLD, either tandemly arrayed or combined with other structurally and functionally distinct domains, yielding lectin subunits of pleiotropic properties even within a single species. Furthermore, the extraordinary variability of FTL sequences (isoforms that are expressed in a single individual has revealed genetic mechanisms of diversification in ligand recognition that are unique to FTLs. Functions of FTLs in self/non-self-recognition include innate immunity, fertilization, microbial adhesion, and pathogenesis, among others. In addition, although the F-type fold is distinctive for FTLs, a structure-based search revealed apparently unrelated proteins with minor sequence similarity to FTLs that displayed the FTLD fold. In general, the phylogenetic analysis of FTLD sequences from viruses to mammals reveals clades that are consistent with the currently accepted taxonomy of extant species. However, the surprisingly discontinuous distribution of FTLDs within each taxonomic category suggests not only an extensive structural/functional diversification of the FTLs along evolutionary lineages but also that this intriguing lectin family has been subject to frequent gene duplication, secondary loss, lateral transfer, and functional co-option.

  13. Chromophore-assisted light inactivation of HaloTag fusion proteins labeled with eosin in living cells.

    Science.gov (United States)

    Takemoto, Kiwamu; Matsuda, Tomoki; McDougall, Mark; Klaubert, Dieter H; Hasegawa, Akira; Los, Georgyi V; Wood, Keith V; Miyawaki, Atsushi; Nagai, Takeharu

    2011-05-20

    Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resolution. This technology, however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen ((1)O(2)) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more production of ((1)O(2)) than fluorescein and about 5-fold efficiency in CALI of β-galactosidase by using an eosin-labeled anti-β-galactosidase antibody compared with the fluorescein-labeled one. To covalently label target protein with eosin, we synthesize a membrane-permeable eosin ligand for HaloTag technology, demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-γ and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomolecule inactivation that is applicable to many cell biological assays in living cells.

  14. Novel membrane frizzled-related protein gene mutation as cause of posterior microphthalmia resulting in high hyperopia with macular folds

    NARCIS (Netherlands)

    Wasmann, Rosemarie A.; Wassink-Ruiter, Jolien S. Klein; Sundin, Olof H.; Morales, Elisa; Verheij, Joke B. G. M.; Pott, Jan Willem R.

    Abstract. Purpose: We present a genetic and clinical analysis of two sisters, 3 and 4 years of age, with nanophthalmos and macular folds. Methods: Ophthalmological examination, general paediatric examination and molecular genetic analysis of the MFRP gene were performed in both affected siblings.

  15. The Protein Disulfide Isomerase of Botrytis cinerea: An ER Protein Involved in Protein Folding and Redox Homeostasis Influences NADPH Oxidase Signaling Processes

    Directory of Open Access Journals (Sweden)

    Robert Marschall

    2017-05-01

    Full Text Available Botrytis cinerea is a filamentous plant pathogen, which infects hundreds of plant species; within its lifestyle, the production of reactive oxygen species (ROS and a balanced redox homeostasis are essential parameters. The pathogen is capable of coping with the plant’s oxidative burst and even produces its own ROS to enhance the plant’s oxidative burst. Highly conserved NADPH oxidase (Nox complexes produce the reactive molecules. The membrane-associated complexes regulate a large variety of vegetative and pathogenic processes. Besides their commonly accepted function at the plasma membrane, recent studies reveal that Nox complexes are also active at the membrane of the endoplasmic reticulum. In this study, we identified the essential ER protein BcPdi1 as new interaction partner of the NoxA complex in B. cinerea. Mutants that lack this ER chaperone display overlapping phenotypes to mutants of the NoxA signaling pathway. The protein appears to be involved in all major developmental processes, such as the formation of sclerotia, conidial anastomosis tubes and infection cushions (IC’s and is needed for full virulence. Moreover, expression analyses and reporter gene studies indicate that BcPdi1 affects the redox homeostasis and unfolded protein response (UPR-related genes. Besides the close association between BcPdi1 and BcNoxA, interaction studies provide evidence that the ER protein might likewise be involved in Ca2+ regulated processes. Finally, we were able to show that the potential key functions of the protein BcPdi1 might be affected by its phosphorylation state.

  16. Contact prediction for beta and alpha-beta proteins using integer linear optimization and its impact on the first principles 3D structure prediction method ASTRO-FOLD.

    Science.gov (United States)

    Rajgaria, R; Wei, Y; Floudas, C A

    2010-06-01

    An integer linear optimization model is presented to predict residue contacts in beta, alpha + beta, and alpha/beta proteins. The total energy of a protein is expressed as sum of a C(alpha)-C(alpha) distance dependent contact energy contribution and a hydrophobic contribution. The model selects contact that assign lowest energy to the protein structure as satisfying a set of constraints that are included to enforce certain physically observed topological information. A new method based on hydrophobicity is proposed to find the beta-sheet alignments. These beta-sheet alignments are used as constraints for contacts between residues of beta-sheets. This model was tested on three independent protein test sets and CASP8 test proteins consisting of beta, alpha + beta, alpha/beta proteins and it was found to perform very well. The average accuracy of the predictions (separated by at least six residues) was approximately 61%. The average true positive and false positive distances were also calculated for each of the test sets and they are 7.58 A and 15.88 A, respectively. Residue contact prediction can be directly used to facilitate the protein tertiary structure prediction. This proposed residue contact prediction model is incorporated into the first principles protein tertiary structure prediction approach, ASTRO-FOLD. The effectiveness of the contact prediction model was further demonstrated by the improvement in the quality of the protein structure ensemble generated using the predicted residue contacts for a test set of 10 proteins.

  17. Contact Prediction for Beta and Alpha-Beta Proteins Using Integer Linear Optimization and its Impact on the First Principles 3D Structure Prediction Method ASTRO-FOLD

    Science.gov (United States)

    Rajgaria, R.; Wei, Y.; Floudas, C. A.

    2010-01-01

    An integer linear optimization model is presented to predict residue contacts in β, α + β, and α/β proteins. The total energy of a protein is expressed as sum of a Cα – Cα distance dependent contact energy contribution and a hydrophobic contribution. The model selects contacts that assign lowest energy to the protein structure while satisfying a set of constraints that are included to enforce certain physically observed topological information. A new method based on hydrophobicity is proposed to find the β-sheet alignments. These β-sheet alignments are used as constraints for contacts between residues of β-sheets. This model was tested on three independent protein test sets and CASP8 test proteins consisting of β, α + β, α/β proteins and was found to perform very well. The average accuracy of the predictions (separated by at least six residues) was approximately 61%. The average true positive and false positive distances were also calculated for each of the test sets and they are 7.58 Å and 15.88 Å, respectively. Residue contact prediction can be directly used to facilitate the protein tertiary structure prediction. This proposed residue contact prediction model is incorporated into the first principles protein tertiary structure prediction approach, ASTRO-FOLD. The effectiveness of the contact prediction model was further demonstrated by the improvement in the quality of the protein structure ensemble generated using the predicted residue contacts for a test set of 10 proteins. PMID:20225257

  18. Interrogating and predicting tolerated sequence diversity in protein folds: application to E. elaterium trypsin inhibitor-II cystine-knot miniprotein.

    Directory of Open Access Journals (Sweden)

    Jennifer L Lahti

    2009-09-01

    Full Text Available Cystine-knot miniproteins (knottins are promising molecular scaffolds for protein engineering applications. Members of the knottin family have multiple loops capable of displaying conformationally constrained polypeptides for molecular recognition. While previous studies have illustrated the potential of engineering knottins with modified loop sequences, a thorough exploration into the tolerated loop lengths and sequence space of a knottin scaffold has not been performed. In this work, we used the Ecballium elaterium trypsin inhibitor II (EETI as a model member of the knottin family and constructed libraries of EETI loop-substituted variants with diversity in both amino acid sequence and loop length. Using yeast surface display, we isolated properly folded EETI loop-substituted clones and applied sequence analysis tools to assess the tolerated diversity of both amino acid sequence and loop length. In addition, we used covariance analysis to study the relationships between individual positions in the substituted loops, based on the expectation that correlated amino acid substitutions will occur between interacting residue pairs. We then used the results of our sequence and covariance analyses to successfully predict loop sequences that facilitated proper folding of the knottin when substituted into EETI loop 3. The sequence trends we observed in properly folded EETI loop-substituted clones will be useful for guiding future protein engineering efforts with this knottin scaffold. Furthermore, our findings demonstrate that the combination of directed evolution with sequence and covariance analyses can be a powerful tool for rational protein engineering.

  19. Folding simulations for proteins with diverse topologies are accessible in days with a physics-based force field and implicit solvent.

    Science.gov (United States)

    Nguyen, Hai; Maier, James; Huang, He; Perrone, Victoria; Simmerling, Carlos

    2014-10-08

    The millisecond time scale needed for molecular dynamics simulations to approach the quantitative study of protein folding is not yet routine. One approach to extend the simulation time scale is to perform long simulations on specialized and expensive supercomputers such as Anton. Ideally, however, folding simulations would be more economical while retaining reasonable accuracy, and provide feedback on structure, stability and function rapidly enough if partnered directly with experiment. Approaches to this problem typically involve varied compromises between accuracy, precision, and cost; the goal here is to address whether simple implicit solvent models have become sufficiently accurate for their weaknesses to be offset by their ability to rapidly provide much more precise conformational data as compared to explicit solvent. We demonstrate that our recently developed physics-based model performs well on this challenge, enabling accurate all-atom simulated folding for 16 of 17 proteins with a variety of sizes, secondary structure, and topologies. The simulations were carried out using the Amber software on inexpensive GPUs, providing ∼1 μs/day per GPU, and >2.5 ms data presented here. We also show that native conformations are preferred over misfolded structures for 14 of the 17 proteins. For the other 3, misfolded structures are thermodynamically preferred, suggesting opportunities for further improvement.

  20. SDS-assisted protein transport through solid-state nanopores.

    Science.gov (United States)

    Restrepo-Pérez, Laura; John, Shalini; Aksimentiev, Aleksei; Joo, Chirlmin; Dekker, Cees

    2017-08-17

    Using nanopores for single-molecule sequencing of proteins - similar to nanopore-based sequencing of DNA - faces multiple challenges, including unfolding of the complex tertiary structure of the proteins and enforcing their unidirectional translocation through nanopores. Here, we combine molecular dynamics (MD) simulations with single-molecule experiments to investigate the utility of SDS (Sodium Dodecyl Sulfate) to unfold proteins for solid-state nanopore translocation, while simultaneously endowing them with a stronger electrical charge. Our simulations and experiments prove that SDS-treated proteins show a considerable loss of the protein structure during the nanopore translocation. Moreover, SDS-treated proteins translocate through the nanopore in the direction prescribed by the electrophoretic force due to the negative charge impaired by SDS. In summary, our results suggest that SDS causes protein unfolding while facilitating protein translocation in the direction of the electrophoretic force; both characteristics being advantageous for future protein sequencing applications using solid-state nanopores.

  1. Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-kappaB rather than apoptosis.

    Directory of Open Access Journals (Sweden)

    Stephen C Graham

    2008-08-01

    Full Text Available Vaccinia virus (VACV, the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-kappaB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 A and 2.7 A resolution, respectively. Strikingly, both these proteins adopt a Bcl-2-like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2-like proteins. Unlike cellular and viral Bcl-2-like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2-like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2-like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2-like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2-like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways.

  2. Co-solute assistance in refolding of recombinant proteins | Gerami ...

    African Journals Online (AJOL)

    Prokaryotic expression system is the most widely used host for the production of recombinant proteins but inclusion body formation is a major bottleneck in the production of recombinant proteins in prokaryotic cells, especially in Escherichia coli. In vitro refolding of inclusion body into the the proteins with native ...

  3. Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH–YopH chaperone–effector complex

    Energy Technology Data Exchange (ETDEWEB)

    Vujanac, Milos; Stebbins, C. Erec, E-mail: stebbins@rockefeller.edu [The Rockefeller University, New York, NY 10065 (United States)

    2013-04-01

    The structure of a SycH–YopH chaperone–effector complex from Yersinia reveals the bacterial state of a protein that adopts different folds in the host and pathogen environments. Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH–YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called β-motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the β-motif. Biochemical studies establish that the β-motif is critical to the stability of this complex. Finally, since previous work has shown that the N-terminal portion of YopH adopts a globular fold that is functional in the host cell, aspects of how this polypeptide adopts radically different folds in the host and in the bacterial environments are analysed.

  4. Adsorption behavior of acidic and basic proteins onto citrate-coated Au surfaces correlated to their native fold, stability, and pI.

    Science.gov (United States)

    Glomm, Wilhelm R; Halskau, Øyvind; Hanneseth, Ann-Mari D; Volden, Sondre

    2007-12-27

    The adsorption of eight different proteins (alpha-lactalbumin (types I and III), bovine serum albumin, hemoglobin, myoglobin, cytochrome c, alpha-casein, and lysozyme) onto a model anionic surface was performed at equivalent bulk (solvent, ionic strength, pH) and surface conditions. Adsorption was monitored on a quartz crystal microbalance with dissipation monitoring (QCM-D) with citrate-coated gold surfaces as adsorbents and has been correlated to native fold stability determined from near- and far-UV circular dichroism (CD) measurements. The proteins studied here were chosen based on their pI and documented knowledge about their structural stability and flexibility. Protein adsorption was found to be independent of global protein charge. Rather, binding occurs through oppositely charged patches on protein and surface. Moreover, data indicate that there is a correlation between secondary and tertiary structure stability and the adsorption characteristics at interfaces. Also, protein surface coverage, layer thickness, and flexibility can be tuned as a function of deposition method. This is discussed in terms of adsorption/spreading kinetics and intermolecular (protein-surface and protein-protein) interactions. Adsorption to surfaces can induce formation of supramolecular structures such as micelles (in the case of alpha-Cas) and multilayers (as for Hb). In the case of alpha-casein, this phenomenon depends on the deposition method and protein concentration. When ranking the surface coverage for proteins added in excess, the order is Lyz < Cyt c < Mb < BSA < alpha-La I < alpha-Cas < alpha-La III < Hb, which can be correlated to the proteins ability to form supramolecular structures (alpha-Cas, Hb), overall conformational flexibilities, and ability to form stable intermediates.

  5. Disordered Cold Regulated15 Proteins Protect Chloroplast Membranes during Freezing through Binding and Folding, But Do Not Stabilize Chloroplast Enzymes in Vivo1[W][OPEN

    Science.gov (United States)

    Thalhammer, Anja; Bryant, Gary; Sulpice, Ronan; Hincha, Dirk K.

    2014-01-01

    Freezing can severely damage plants, limiting geographical distribution of natural populations and leading to major agronomical losses. Plants native to cold climates acquire increased freezing tolerance during exposure to low nonfreezing temperatures in a process termed cold acclimation. This involves many adaptative responses, including global changes in metabolite content and gene expression, and the accumulation of cold-regulated (COR) proteins, whose functions are largely unknown. Here we report that the chloroplast proteins COR15A and COR15B are necessary for full cold acclimation in Arabidopsis (Arabidopsis thaliana). They protect cell membranes, as indicated by electrolyte leakage and chlorophyll fluorescence measurements. Recombinant COR15 proteins stabilize lactate dehydrogenase during freezing in vitro. However, a transgenic approach shows that they have no influence on the stability of selected plastidic enzymes in vivo, although cold acclimation results in increased enzyme stability. This indicates that enzymes are stabilized by other mechanisms. Recombinant COR15 proteins are disordered in water, but fold into amphipathic α-helices at high osmolyte concentrations in the presence of membranes, a condition mimicking molecular crowding induced by dehydration during freezing. X-ray scattering experiments indicate protein-membrane interactions specifically under such crowding conditions. The COR15-membrane interactions lead to liposome stabilization during freezing. Collectively, our data demonstrate the requirement for COR15 accumulation for full cold acclimation of Arabidopsis. The function of these intrinsically disordered proteins is the stabilization of chloroplast membranes during freezing through a folding and binding mechanism, but not the stabilization of chloroplastic enzymes. This indicates a high functional specificity of these disordered plant proteins. PMID:25096979

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

    Science.gov (United States)

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

    2015-09-01

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

  7. The canonical twin-arginine translocase components are not required for secretion of folded green fluorescent protein from the ancestral strain of Bacillus subtilis.

    Science.gov (United States)

    Snyder, Anthony J; Mukherjee, Sampriti; Glass, J Kyle; Kearns, Daniel B; Mukhopadhyay, Suchetana

    2014-05-01

    Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.

  8. Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

    Science.gov (United States)

    Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

    2015-10-01

    The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

  9. SDS-assisted protein transport through solid-state nanopores

    NARCIS (Netherlands)

    Restrepo Perez, L.; John, Shalini; Aksimentiev, Aleksei; Joo, C.; Dekker, C.

    2017-01-01

    Using nanopores for single-molecule sequencing of proteins – similar to nanopore-based sequencing of DNA – faces multiple challenges, including unfolding of the complex tertiary structure of the proteins and enforcing their unidirectional translocation through nanopores. Here, we combine molecular

  10. Physiological effects of over-expressing compartment-specific components of the protein folding machinery in xylose-fermenting Saccharomyces cerevisiae.

    Science.gov (United States)

    Bergdahl, Basti; Gorwa-Grauslund, Marie F; van Niel, Ed W J

    2014-04-23

    Efficient utilization of both glucose and xylose is necessary for a competitive ethanol production from lignocellulosic materials. Although many advances have been made in the development of xylose-fermenting strains of Saccharomyces cerevisiae, the productivity remains much lower compared to glucose. Previous transcriptional analyses of recombinant xylose-fermenting strains have mainly focused on central carbon metabolism. Very little attention has been given to other fundamental cellular processes such as the folding of proteins. Analysis of previously measured transcript levels in a recombinant XR/XDH-strain showed a wide down-regulation of genes targeted by the unfolded protein response during xylose fermentation. Under anaerobic conditions the folding of proteins is directly connected with fumarate metabolism and requires two essential enzymes: FADH2-dependent fumarate reductase (FR) and Ero1p. In this study we tested whether these enzymes impair the protein folding process causing the very slow growth of recombinant yeast strains on xylose under anaerobic conditions. Four strains over-expressing the cytosolic (FRD1) or mitochondrial (OSM1) FR genes and ERO1 in different combinations were constructed. The growth and fermentation performance was evaluated in defined medium as well as in a complex medium containing glucose and xylose. Over-expression of FRD1, alone or in combination with ERO1, did not have any significant effect on xylose fermentation in any medium used. Over-expression of OSM1, on the other hand, led to a diversion of carbon from glycerol to acetate and a decrease in growth rate by 39% in defined medium and by 25% in complex medium. Combined over-expression of OSM1 and ERO1 led to the same diversion of carbon from glycerol to acetate and had a stronger detrimental effect on the growth in complex medium. Increasing the activities of the FR enzymes and Ero1p is not sufficient to increase the anaerobic growth on xylose. So additional components of

  11. Expression of factor H binding protein in meningococcal strains can vary at least 15-fold and is genetically determined

    National Research Council Canada - National Science Library

    Massimiliano Biagini; Marco Spinsanti; Gabriella De Angelis; Sara Tomei; Ilaria Ferlenghi; Maria Scarselli; Fabio Rigat; Nicola Messuti; Alessia Biolchi; Alessandro Muzzi; Giulia Anderloni; Brunella Brunelli; Elena Cartocci; Francesca Buricchi; Chiara Tani; Maria Stella; Monica Moschioni; Elena Del Tordello; Annalisa Colaprico; Silvana Savino; Marzia M. Giuliani; Isabel Delany; Mariagrazia Pizza; Paolo Costantino; Nathalie Norais; Rino Rappuoli; Vega Masignani

    2016-01-01

    Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB...

  12. Detection of initiation sites in protein folding of the four helix bundle ACBP by chemical shift analysis

    DEFF Research Database (Denmark)

    Modig, K.; Jürgensen, Vibeke Würtz; Lindorff-Larsen, K.

    2007-01-01

    A simple alternative method for obtaining "random coil" chemical shifts by intrinsic referencing using the protein's own peptide sequence is presented. These intrinsic random coil backbone shifts were then used to calculate secondary chemical shifts, that provide important information on the resi......A simple alternative method for obtaining "random coil" chemical shifts by intrinsic referencing using the protein's own peptide sequence is presented. These intrinsic random coil backbone shifts were then used to calculate secondary chemical shifts, that provide important information...

  13. Heterologous expression, protein folding and antibody recognition of a neurotoxin from the Mexican coral snake Micrurus laticorallis.

    Science.gov (United States)

    Clement, Herlinda; Flores, Vianey; De la Rosa, Guillermo; Zamudio, Fernando; Alagon, Alejandro; Corzo, Gerardo

    2016-01-01

    The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. HisrMlat1 shows increased biological activities

  14. Microwave-assisted Weak Acid Hydrolysis of Proteins

    Directory of Open Access Journals (Sweden)

    Miyeong Seo

    2012-06-01

    Full Text Available Myoglobin was hydrolyzed by microwave-assisted weak acid hydrolysis with 2% formic acid at 37 oC, 50 oC, and100 oC for 1 h. The most effective hydrolysis was observed at 100 oC. Hydrolysis products were investigated using matrixassistedlaser desorption/ionization time-of-flight mass spectrometry. Most cleavages predominantly occurred at the C-termini ofaspartyl residues. For comparison, weak acid hydrolysis was also performed in boiling water for 20, 40, 60, and 120 min. A 60-min weak acid hydrolysis in boiling water yielded similar results as a 60-min microwave-assisted weak acid hydrolysis at100 oC. These results strongly suggest that microwave irradiation has no notable enhancement effect on acid hydrolysis of proteinsand that temperature is the major factor that determines the effectiveness of weak acid hydrolysis.

  15. Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing

    DEFF Research Database (Denmark)

    Teilum, Kaare; Maki, Kosuke; Kragelund, Birthe B

    2002-01-01

    state. The kinetic data are fully accounted for by three-state mechanisms with either on- or off-pathway intermediates. The intermediate accumulates to a maximum population of 40%, and its stability depends only weakly on denaturant concentration, which is consistent with a marginally stable ensemble...... energy transfer. Although the folding of ACBP was initially described as a concerted two-state process, the tryptophan fluorescence measurements revealed a previously unresolved phase with a time constant tau = 80 micros, indicating formation of an intermediate with only slightly enhanced fluorescence...... showed a major increase in tryptophan-dansyl fluorescence energy transfer, indicating formation of a partially collapsed ensemble of states on the 100-micros time scale. A subsequent decrease in dansyl fluorescence is attributed to intramolecular quenching of donor fluorescence on formation of the native...

  16. Three-Dimensional Protein Fold Determination from Backbone Amide Pseudocontact Shifts Generated by Lanthanide Tags at Multiple Sites

    KAUST Repository

    Yagi, Hiromasa

    2013-06-01

    Site-specific attachment of paramagnetic lanthanide ions to a protein generates pseudocontact shifts (PCS) in the nuclear magnetic resonance (NMR) spectra of the protein that are easily measured as changes in chemical shifts. By labeling the protein with lanthanide tags at four different sites, PCSs are observed for most amide protons and accurate information is obtained about their coordinates in three-dimensional space. The approach is demonstrated with the chaperone ERp29, for which large differences have been reported between X-ray and NMR structures of the C-terminal domain, ERp29-C. The results unambiguously show that the structure of rat ERp29-C in solution is similar to the crystal structure of human ERp29-C. PCSs of backbone amides were the only structural restraints required. Because these can be measured for more dilute protein solutions than other NMR restraints, the approach greatly widens the range of proteins amenable to structural studies in solution. © 2013 Elsevier Ltd. All rights reserved.

  17. Utilizing dynamic light scattering as a process analytical technology for protein folding and aggregation monitoring in vaccine manufacturing.

    Science.gov (United States)

    Yu, Zhou; Reid, Jennifer C; Yang, Yan-Ping

    2013-12-01

    Protein aggregation is a common challenge in the manufacturing of biological products. It is possible to minimize the extent of aggregation through timely measurement and in-depth characterization of aggregation. In this study, we demonstrated the use of dynamic light scattering (DLS) to monitor inclusion body (IB) solubilization, protein refolding, and aggregation near the production line of a recombinant protein-based vaccine candidate. Our results were in good agreement with those measured by size-exclusion chromatography. DLS was also used to characterize the mechanism of aggregation. As DLS is a quick, nonperturbing technology, it can potentially be used as an at-line process analytical technology to ensure complete IB solubilization and aggregate-free refolding. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

  18. A strategy for finding classes of minima on a hypersurface: implications for approaches to the protein folding problem.

    Science.gov (United States)

    Head-Gordon, T; Stillinger, F H; Arrecis, J

    1991-12-15

    Locating the native structure of a given protein is a task made difficult by the complexity of the potential energy hypersurface and by the huge number of local minima it contains. We have explored a strategy (the "antlion" method) for hypersurface modification that suppresses all minima but that of the native structure. Transferrable penalty functions with general applicability for modifying a hypersurface to retain the desired minimum are identified, and two blocked oligopeptides (alanine dipeptide and tetrapeptide) are used for specific numerical illustration of the dramatic simplification that ensues. In addition, an intermediary role for neural networks to manage some aspects of the antlion strategy applied to large polypeptides and proteins is introduced.

  19. Structure of the N-terminal domain of the protein Expansion: an ‘Expansion’ to the Smad MH2 fold

    Energy Technology Data Exchange (ETDEWEB)

    Beich-Frandsen, Mads; Aragón, Eric [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Llimargas, Marta [Institut de Biologia Molecular de Barcelona, IBMB–CSIC, Baldiri Reixac 10, 08028 Barcelona (Spain); Benach, Jordi [ALBA Synchrotron, BP 1413, km 3.3, Cerdanyola del Vallès (Spain); Riera, Antoni [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Universitat de Barcelona, Martí i Franqués 1-11, 08028 Barcelona (Spain); Pous, Joan [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Platform of Crystallography IBMB–CSIC, Baldiri Reixac 10, 08028 Barcelona (Spain); Macias, Maria J., E-mail: maria.macias@irbbarcelona.org [Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain); Catalan Institution for Research and Advanced Studies (ICREA), Passeig Lluís Companys 23, 08010 Barcelona (Spain)

    2015-04-01

    Expansion is a modular protein that is conserved in protostomes. The first structure of the N-terminal domain of Expansion has been determined at 1.6 Å resolution and the new Nα-MH2 domain was found to belong to the Smad/FHA superfamily of structures. Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.

  20. Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Holst, B; Tachibana, C; Winther, Jakob R.

    1997-01-01

    active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important...

  1. Indicator Amino Acid-Derived Estimate of Dietary Protein Requirement for Male Bodybuilders on a Nontraining Day Is Several-Fold Greater than the Current Recommended Dietary Allowance.

    Science.gov (United States)

    Bandegan, Arash; Courtney-Martin, Glenda; Rafii, Mahroukh; Pencharz, Paul B; Lemon, Peter Wr

    2017-05-01

    Background: Despite a number of studies indicating increased dietary protein needs in bodybuilders with the use of the nitrogen balance technique, the Institute of Medicine (2005) has concluded, based in part on methodologic concerns, that "no additional dietary protein is suggested for healthy adults undertaking resistance or endurance exercise."Objective: The aim of the study was to assess the dietary protein requirement of healthy young male bodybuilders ( with ≥3 y training experience) on a nontraining day by measuring the oxidation of ingested l-[1-13C]phenylalanine to 13CO2 in response to graded intakes of protein [indicator amino acid oxidation (IAAO) technique].Methods: Eight men (means ± SDs: age, 22.5 ± 1.7 y; weight, 83.9 ± 11.6 kg; 13.0% ± 6.3% body fat) were studied at rest on a nontraining day, on several occasions (4-8 times) each with protein intakes ranging from 0.1 to 3.5 g · kg-1 · d-1, for a total of 42 experiments. The diets provided energy at 1.5 times each individual's measured resting energy expenditure and were isoenergetic across all treatments. Protein was fed as an amino acid mixture based on the protein pattern in egg, except for phenylalanine and tyrosine, which were maintained at constant amounts across all protein intakes. For 2 d before the study, all participants consumed 1.5 g protein · kg-1 · d-1 On the study day, the protein requirement was determined by identifying the breakpoint in the F13CO2 with graded amounts of dietary protein [mixed-effects change-point regression analysis of F13CO2 (labeled tracer oxidation in breath)].Results: The Estimated Average Requirement (EAR) of protein and the upper 95% CI RDA for these young male bodybuilders were 1.7 and 2.2 g · kg-1 · d-1, respectively.Conclusion: These IAAO data suggest that the protein EAR and recommended intake for male bodybuilders at rest on a nontraining day exceed the current recommendations of the Institute of Medicine by ∼2.6-fold. This trial was

  2. A disorder-induced domino-like destabilization mechanism governs the folding and functional dynamics of the repeat protein IκBα.

    Directory of Open Access Journals (Sweden)

    Srinivasan Sivanandan

    Full Text Available The stability of the repeat protein IκBα, a transcriptional inhibitor in mammalian cells, is critical in the functioning of the NF-κB signaling module implicated in an array of cellular processes, including cell growth, disease, immunity and apoptosis. Structurally, IκBα is complex, with both ordered and disordered regions, thus posing a challenge to the available computational protocols to model its conformational behavior. Here, we introduce a simple procedure to model disorder in systems that undergo binding-induced folding that involves modulation of the contact map guided by equilibrium experimental observables in combination with an Ising-like Wako-Saitô-Muñoz-Eaton model. This one-step procedure alone is able to reproduce a variety of experimental observables, including ensemble thermodynamics (scanning calorimetry, pre-transitions, m-values and kinetics (roll-over in chevron plot, intermediates and their identity, and is consistent with hydrogen-deuterium exchange measurements. We further capture the intricate distance-dynamics between the domains as measured by single-molecule FRET by combining the model predictions with simple polymer physics arguments. Our results reveal a unique mechanism at work in IκBα folding, wherein disorder in one domain initiates a domino-like effect partially destabilizing neighboring domains, thus highlighting the effect of symmetry-breaking at the level of primary sequences. The offshoot is a multi-state and a dynamic conformational landscape that is populated by increasingly partially folded ensembles upon destabilization. Our results provide, in a straightforward fashion, a rationale to the promiscuous binding and short intracellular half-life of IκBα evolutionarily engineered into it through repeats with variable stabilities and expand the functional repertoire of disordered regions in proteins.

  3. Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

    Science.gov (United States)

    Martínez-Alonso, Mónica; Villaverde, Antonio

    2010-01-01

    Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

  4. Differential Rates of Protein Folding and Cellular Trafficking for the Hendra Virus F and G Proteins: Implications for F-G Complex Formation ▿

    OpenAIRE

    Whitman, Shannon D.; Smith, Everett Clinton; Dutch, Rebecca Ellis

    2009-01-01

    Hendra virus F protein-promoted membrane fusion requires the presence of the viral attachment protein, G. However, events leading to the association of these glycoproteins remain unclear. Results presented here demonstrate that Hendra virus G undergoes slower secretory pathway trafficking than is observed for Hendra virus F. This slowed trafficking is not dependent on the G protein cytoplasmic tail, the presence of the G receptor ephrin B2, or interaction with other viral proteins. Instead, H...

  5. Folding of the RNA recognition motif (RRM) domains of the amyotrophic lateral sclerosis (ALS)-linked protein TDP-43 reveals an intermediate state.

    Science.gov (United States)

    Mackness, Brian C; Tran, Meme T; McClain, Shannan P; Matthews, C Robert; Zitzewitz, Jill A

    2014-03-21

    Pathological alteration of TDP-43 (TAR DNA-binding protein-43), a protein involved in various RNA-mediated processes, is a hallmark feature of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Fragments of TDP-43, composed of the second RNA recognition motif (RRM2) and the disordered C terminus, have been observed in cytoplasmic inclusions in sporadic amyotrophic lateral sclerosis cases, suggesting that conformational changes involving RRM2 together with the disordered C terminus play a role in aggregation and toxicity. The biophysical data collected by CD and fluorescence spectroscopies reveal a three-state equilibrium unfolding model for RRM2, with a partially folded intermediate state that is not observed in RRM1. Strikingly, a portion of RRM2 beginning at position 208, which mimics a cleavage site observed in patient tissues, increases the population of this intermediate state. Mutually stabilizing interactions between the domains in the tethered RRM1 and RRM2 construct reduce the population of the intermediate state and enhance DNA/RNA binding. Despite the high sequence homology of the two domains, a network of large hydrophobic residues in RRM2 provides a possible explanation for the increased stability of RRM2 compared with RRM1. The cluster analysis suggests that the intermediate state may play a functional role by enhancing access to the nuclear export signal contained within its sequence. The intermediate state may also serve as a molecular hazard linking productive folding and function with pathological misfolding and aggregation that may contribute to disease.

  6. Novel BAFF-receptor antibody to natively folded recombinant protein eliminates drug resistant human B-cell malignancies in vivo.

    Science.gov (United States)

    Qin, Hong; Wei, Guowei; Sakamaki, Ippei; Dong, Zhenyuan; Cheng, Wesley A; Smith, D Lynne; Wen, Feng; Sun, Han; Kim, Kunhwa; Cha, Soung-Chul; Bover, Laura; Neelapu, Sattva S; Kwak, Larry W

    2017-11-27

    Monoclonal antibodies (mAbs) such as anti-CD20 rituximab, are proven therapies in B-cell malignancies, yet many patients develop resistance. Novel therapies against alternative targets are needed to circumvent resistance mechanisms. We sought to generate mAbs against human B-cell activating factor receptor (BAFF-R/TNFRSF13C), which has not yet been targeted successfully for cancer therapy. Novel mAbs were generated against BAFF-R, expressed as a natively folded cell-surface immunogen on mouse fibroblast cells. Chimeric BAFF-R mAbs were developed and assessed for in vitro and in vivo monotherapy cytotoxicity. The chimeric mAbs were tested against human B-cell tumor lines, primary patient samples, and drug-resistant tumors. Chimeric antibodies bound with high affinity to multiple human malignant B-cell lines and induced potent antibody-dependent cellular cytotoxicity (ADCC) against multiple subtypes of human lymphoma and leukemia, including primary tumors from patients who had relapsed after anti-CD20 therapy. Chimeric antibodies also induced ADCC against ibrutinib-resistant and rituximab-insensitive CD20-deficient variant lymphomas, respectively. Importantly, they demonstrated remarkable in vivo growth inhibition of drug-resistant tumor models in immunodeficient mice. Our method generated novel anti-BAFF-R antibody therapeutics with remarkable single-agent antitumor effects. We propose that these antibodies represent an effective new strategy for targeting and treating drug resistant B-cell malignancies and warrant further development. Copyright ©2017, American Association for Cancer Research.

  7. Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: Functional convergence of a common protein fold

    Energy Technology Data Exchange (ETDEWEB)

    Casados-Vázquez, Luz E.; Lara-González, Samuel; Brieb, Luis G. (LNGB-Mexico)

    2012-04-18

    Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight {beta}-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.

  8. Stability Mechanisms of Laccase Isoforms using a Modified FoldX Protocol Applicable to Widely Different Proteins

    DEFF Research Database (Denmark)

    Christensen, Niels J.; Kepp, Kasper P.

    2013-01-01

    A recent computational protocol that accurately predicts and rationalizes protein multisite mutant stabilities has been extended to handle widely different isoforms of laccases. We apply the protocol to four isoenzymes of Trametes versicolor laccase (TvL) with variable lengths (498–503 residues...... with experimental distributions of stability effects from mutation. The residues causing the differential stability of the four isoforms are consistent with a range of compiled laccase wild type data, suggesting that we may have identified general drivers of laccase stability. Several sites near Cu, notably 79, 241...

  9. Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements.

    Science.gov (United States)

    Ogburn, Ryenne N; Jin, Lorrain; Meng, He; Fitzgerald, Michael C

    2017-11-03

    The proteins in an MCF-7 cell line were probed for tamoxifen (TAM) and n-desmethyl tamoxifen (NDT) induced stability changes using the Stability of Proteins from Rates of Oxidation (SPROX) technique in combination with two different quantitative proteomics strategies, including one based on SILAC and one based on isobaric mass tags. Over 1000 proteins were assayed for TAM- and NDT-induced protein stability changes, and a total of 163 and 200 protein hits were identified in the TAM and NDT studies, respectively. A subset of 27 high-confidence protein hits were reproducibly identified with both proteomics strategies and/or with multiple peptide probes. One-third of the high-confidence hits have previously established experimental links to the estrogen receptor, and nearly all of the high-confidence hits have established links to breast cancer. One high-confidence protein hit that has known estrogen receptor binding properties, Y-box binding protein 1 (YBX1), was further validated as a direct binding target of TAM using both the SPROX and pulse proteolysis techniques. Proteins with TAM- and/or NDT-induced expression level changes were also identified in the SILAC-SPROX experiments. These proteins with expression level changes included only a small fraction of those with TAM- and/or NDT-induced stability changes.

  10. Vapor treatment of electrospray droplets: evidence for the folding of initially denatured proteins on the sub-millisecond time-scale.

    Science.gov (United States)

    Kharlamova, Anastasia; DeMuth, J Corinne; McLuckey, Scott A

    2012-01-01

    The exposure of electrospray droplets generated from either highly acidic or highly basic solutions to basic or acidic vapors, respectively, admitted into the counter-current drying gas, has been shown to lead to significant changes in the observed charge state distributions of proteins. In both cases, distributions of charge states changed from relatively high charge states, indicative of largely denatured proteins, to lower charge state distributions that are more consistent with native protein conformations. Ubiquitin, cytochrome c, myoglobin, and carbonic anhydrase were used as model systems. In some cases, bimodal distributions were observed that are not noted under any solution pH conditions. The extent to which changes in charge state distributions occur depends upon the initial solution pH and the pK(a) or pK(b) of the acidic or basic reagent, respectively. The evolution of charged droplets in the sampling region of the mass spectrometer inlet aperture, where the vapor exposure takes place, occurs within roughly 1 ms. The observed changes in the spectra, therefore, are a function of the magnitude of the pH change as well as the rates at which the proteins can respond to this change. The exposure of electrospray droplets in this fashion may provide means for accessing transient folding states for further characterization by mass spectrometry. © American Society for Mass Spectrometry, 2011

  11. The N terminus of Myxococcus xanthus CarA repressor is an autonomously folding domain that mediates physical and functional interactions with both operator DNA and antirepressor protein.

    Science.gov (United States)

    Pérez-Marín, Mari Cruz; López-Rubio, Jose Juan; Murillo, Francisco Jose; Elías-Arnanz, Montserrat; Padmanabhan, S

    2004-08-06

    Expression of the Myxococcus xanthus carB operon, which encodes the majority of the enzymes involved in light-induced carotenogenesis, is down-regulated in the dark by the CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves repression of carB by physically interacting with CarA to dis-mantle CarA-DNA complexes. Here, we demonstrate that the N- and C-terminal portions of CarA are organized as distinct structural and functional domains. Specifically, we show that the 78 N-terminal residues of CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro, sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter binding, and this is relieved by CarS. In vivo, substitution of the gene carA by that for CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background. However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual function domain that orchestrates specific DNA-protein and protein-protein interactions and, when overexpressed, can be functionally competent in vivo.

  12. Differential rates of protein folding and cellular trafficking for the Hendra virus F and G proteins: implications for F-G complex formation.

    Science.gov (United States)

    Whitman, Shannon D; Smith, Everett Clinton; Dutch, Rebecca Ellis

    2009-09-01

    Hendra virus F protein-promoted membrane fusion requires the presence of the viral attachment protein, G. However, events leading to the association of these glycoproteins remain unclear. Results presented here demonstrate that Hendra virus G undergoes slower secretory pathway trafficking than is observed for Hendra virus F. This slowed trafficking is not dependent on the G protein cytoplasmic tail, the presence of the G receptor ephrin B2, or interaction with other viral proteins. Instead, Hendra virus G was found to undergo intrinsically slow oligomerization within the endoplasmic reticulum. These results suggest that the critical F-G interactions occur only after the initial steps of synthesis and cellular transport.

  13. RNA folding: structure prediction, folding kinetics and ion electrostatics.

    Science.gov (United States)

    Tan, Zhijie; Zhang, Wenbing; Shi, Yazhou; Wang, Fenghua

    2015-01-01

    Beyond the "traditional" functions such as gene storage, transport and protein synthesis, recent discoveries reveal that RNAs have important "new" biological functions including the RNA silence and gene regulation of riboswitch. Such functions of noncoding RNAs are strongly coupled to the RNA structures and proper structure change, which naturally leads to the RNA folding problem including structure prediction and folding kinetics. Due to the polyanionic nature of RNAs, RNA folding structure, stability and kinetics are strongly coupled to the ion condition of solution. The main focus of this chapter is to review the recent progress in the three major aspects in RNA folding problem: structure prediction, folding kinetics and ion electrostatics. This chapter will introduce both the recent experimental and theoretical progress, while emphasize the theoretical modelling on the three aspects in RNA folding.

  14. A Phosphorylation Tag for Uranyl Mediated Protein Purification and Photo Assisted Tag Removal

    DEFF Research Database (Denmark)

    Zhang, Qiang; Jørgensen, Thomas. J. D.; Nielsen, Peter E

    2014-01-01

    enables target protein purification from an E. coli extract by immobilized uranyl affinity chromatography. Subsequently, the tag can be efficiently removed by UV-irradiation assisted uranyl photocleavage. We therefore suggest that the divalent uranyl ion (UO22+) may provide a dual function in protein......Most protein purification procedures include an affinity tag fused to either the N or C-terminal end of the protein of interest as well as a procedure for tag removal. Tag removal is not straightforward and especially tag removal from the C-terminal end is a challenge due to the characteristics...... of enzymes available for this purpose. In the present study, we demonstrate the utility of the divalent uranyl ion in a new procedure for protein purification and tag removal. By employment of a GFP (green florescence protein) recombinant protein we show that uranyl binding to a phosphorylated C-terminal tag...

  15. Towards plant protein refinery: Review on protein extraction using alkalo and potential enzymatic assistance

    NARCIS (Netherlands)

    Sari, Y.W.; Mulder, W.J.; Sanders, J.P.M.; Bruins, M.E.

    2015-01-01

    The globally increasing protein demands require additional resources to those currently available. Furthermore, the optimal usage of protein fractions from both traditional and new protein resources, such as algae and leaves, is essential. Here, we present an overview on alkaline plant protein

  16. Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H. Eric (Van Andel); (NU Singapore)

    2012-02-21

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 {angstrom} crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  17. Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors.

    Science.gov (United States)

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H Eric

    2011-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  18. A one-dimensional free energy surface does not account for two-probe folding kinetics of protein alpha(3)D.

    Science.gov (United States)

    Liu, Feng; Dumont, Charles; Zhu, Yongjin; DeGrado, William F; Gai, Feng; Gruebele, Martin

    2009-02-14

    We present fluorescence-detected measurements of the temperature-jump relaxation kinetics of the designed three-helix bundle protein alpha(3)D taken under solvent conditions identical to previous infrared-detected kinetics. The fluorescence-detected rate is similar to the IR-detected rate only at the lowest temperature where we could measure it (326 K). The fluorescence-detected rate decreases by a factor of 3 over the 326-344 K temperature range, whereas the IR-detected rate remains nearly constant over the same range. To investigate this probe dependence, we tested an extensive set of physically reasonable one-dimensional (1D) free energy surfaces by Langevin dynamics simulation. The simulations included coordinate- and temperature-dependent roughness, diffusion coefficients, and IR/fluorescence spectroscopic signatures. None of these can reproduce the IR and fluorescence data simultaneously, forcing us to the conclusion that a 1D free energy surface cannot accurately describe the folding of alpha(3)D. This supports the hypothesis that alpha(3)D has a multidimensional free energy surface conducive to downhill folding at 326 K, and that it is already an incipient downhill folder with probe-dependent kinetics near its melting point.

  19. Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Shiva Kumar

    Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR. Crystal structures of a number of Class B GPCR extracellular domains (ECD bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  20. Processed Meat Protein and Heat-Stable Peptide Marker Identification Using Microwave-Assisted Tryptic Digestion

    Directory of Open Access Journals (Sweden)

    Magdalena Montowska

    2016-01-01

    Full Text Available New approaches to rapid examination of proteins and peptides in complex food matrices are of great interest to the community of food scientists. The aim of the study is to examine the influence of microwave irradiation on the acceleration of enzymatic cleavage and enzymatic digestion of denatured proteins in cooked meat of five species (cattle, horse, pig, chicken and turkey and processed meat products (coarsely minced, smoked, cooked and semi-dried sausages. Severe protein aggregation occurred not only in heated meat under harsh treatment at 190 °C but also in processed meat products. All the protein aggregates were thoroughly hydrolyzed aft er 1 h of trypsin treatment with short exposure times of 40 and 20 s to microwave irradiation at 138 and 303 W. There were much more missed cleavage sites observed in all microwave-assisted digestions. Despite the incompleteness of microwave-assisted digestion, six unique peptide markers were detected, which allowed unambiguous identification of processed meat derived from the examined species. Although the microwave-assisted tryptic digestion can serve as a tool for rapid and high-throughput protein identification, great caution and pre-evaluation of individual samples is recommended in protein quantitation.

  1. Bacillus systems exporting folded proteins and folding exported proteins

    NARCIS (Netherlands)

    Goosens, Vivianne Jacoba

    2013-01-01

    Belangrijk eiwit ontdekt voor eiwittranslocatie Het eiwit QcrA wordt, voor vervoer over het celmembraan, in gevouwen vorm (met toegevoegde cofactoren) getransporteerd. Dat concludeert Vivianne Goosens in haar promotieonderzoek. Zij onderzocht twee manieren van eiwittranslocatie: één waarbij de

  2. Efficient ASK-assisted system for expression and purification of plant F-box proteins.

    Science.gov (United States)

    Li, Haiou; Yao, Ruifeng; Ma, Sui; Hu, Shuai; Li, Suhua; Wang, Yupei; Yan, Chun; Xie, Daoxin; Yan, Jianbin

    2017-09-05

    Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  3. Visualized and precise design of artificial small RNAs for regulating T7 RNA polymerase and enhancing recombinant protein folding in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yujia Zhao

    2016-12-01

    Full Text Available Small non-coding RNAs (sRNAs have received much attention in recent years due to their unique biological properties, which can efficiently and specifically tune target gene expressions in bacteria. Inspired by natural sRNAs, recent works have proposed the use of artificial sRNAs (asRNAs as genetic tools to regulate desired gene that has been applied in several fields, such as metabolic engineering and bacterial physiology studies. However, the rational design of asRNAs is still a challenge. In this study, we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs. T7 expression system was one of the most useful recombinant protein expression systems. However, it was deeply limited by the formation of inclusion body. To settle this problem, we designed a series of asRNAs to inhibit the T7 RNA polymerase (Gene1 expression to balance the rate between transcription and folding of recombinant protein. Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E. coli, the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.

  4. Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD). K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein

    DEFF Research Database (Denmark)

    O'Reilly, Linda P; Andresen, Brage S; Engel, Paul C

    2005-01-01

    the gene for K364R was overexpressed in Escherichia coli, the synthesized mutant protein only exhibited activity when the gene for chaperonin GroELS was co-overexpressed. Levels of activity correlated with the amounts of native MCAD protein visible in western blots. The R256T mutant, by contrast, displayed...... no activity either with or without chaperonin, but in this case a strong MCAD protein band was seen in the western blots throughout. The proteins were also purified, and the enzyme function and thermostability investigated. The K364R protein showed only moderate kinetic impairment, whereas the R256T protein...... was again totally inactive. Neither mutant showed marked depletion of FAD. The pure K364R protein was considerably less thermostable than wild-type MCAD. Western blots indicated that, although the R256T mutant protein is less thermostable than normal MCAD, it is much more stable than K364R. Though...

  5. The evolutionary history of protein fold families and proteomes confirms that the archaeal ancestor is more ancient than the ancestors of other superkingdoms

    Directory of Open Access Journals (Sweden)

    Kim Kyung Mo

    2012-01-01

    Full Text Available Abstract Background The entire evolutionary history of life can be studied using myriad sequences generated by genomic research. This includes the appearance of the first cells and of superkingdoms Archaea, Bacteria, and Eukarya. However, the use of molecular sequence information for deep phylogenetic analyses is limited by mutational saturation, differential evolutionary rates, lack of sequence site independence, and other biological and technical constraints. In contrast, protein structures are evolutionary modules that are highly conserved and diverse enough to enable deep historical exploration. Results Here we build phylogenies that describe the evolution of proteins and proteomes. These phylogenetic trees are derived from a genomic census of protein domains defined at the fold family (FF level of structural classification. Phylogenomic trees of FF structures were reconstructed from genomic abundance levels of 2,397 FFs in 420 proteomes of free-living organisms. These trees defined timelines of domain appearance, with time spanning from the origin of proteins to the present. Timelines are divided into five different evolutionary phases according to patterns of sharing of FFs among superkingdoms: (1 a primordial protein world, (2 reductive evolution and the rise of Archaea, (3 the rise of Bacteria from the common ancestor of Bacteria and Eukarya and early development of the three superkingdoms, (4 the rise of Eukarya and widespread organismal diversification, and (5 eukaryal diversification. The relative ancestry of the FFs shows that reductive evolution by domain loss is dominant in the first three phases and is responsible for both the diversification of life from a universal cellular ancestor and the appearance of superkingdoms. On the other hand, domain gains are predominant in the last two phases and are responsible for organismal diversification, especially in Bacteria and Eukarya. Conclusions The evolution of functions that are

  6. Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism.

    Science.gov (United States)

    Zybailov, Boris; Friso, Giulia; Kim, Jitae; Rudella, Andrea; Rodríguez, Verenice Ramírez; Asakura, Yukari; Sun, Qi; van Wijk, Klaas J

    2009-08-01

    The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate

  7. Frustration, function and folding.

    Science.gov (United States)

    Ferreiro, Diego U; Komives, Elizabeth A; Wolynes, Peter G

    2017-11-01

    Natural protein molecules are exceptional polymers. Encoded in apparently random strings of amino-acids, these objects perform clear physical tasks that are rare to find by simple chance. Accurate folding, specific binding, powerful catalysis, are examples of basic chemical activities that the great majority of polypeptides do not display, and are thought to be the outcome of the natural history of proteins. Function, a concept genuine to Biology, is at the core of evolution and often conflicts with the physical constraints. Locating the frustration between discrepant goals in a recurrent system leads to fundamental insights about the chances and necessities that shape the encoding of biological information. Copyright © 2017 Elsevier Ltd. All rights reserved.