WorldWideScience

Sample records for assisted protein folding

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

    Science.gov (United States)

    Kleinschmidt, Jörg H

    2015-09-01

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

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

    Science.gov (United States)

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

    2016-02-16

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

  3. Work Done by Titin Protein Folding Assists Muscle Contraction

    Directory of Open Access Journals (Sweden)

    Jaime Andrés Rivas-Pardo

    2016-02-01

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

  4. Discovery of Proteomic Code with mRNA Assisted Protein Folding

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

  6. 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...

  7. Protein folding and wring resonances

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren

    1997-01-01

    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...

  8. Protein folding, protein homeostasis, and cancer

    Institute of Scientific and Technical Information of China (English)

    John H. Van Drie

    2011-01-01

    Proteins fold into their functional 3-dimensional structures from a linear amino acid sequence. In vitro this process is spontaneous; while in vivo it is orchestrated by a specialized set of proteins, called chaperones. Protein folding is an ongoing cellular process, as cellular proteins constantly undergo synthesis and degradation. Here emerging links between this process and cancer are reviewed. This perspective both yields insights into the current struggle to develop novel cancer chemotherapeutics and has implications for future chemotherapy discovery.

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

    Science.gov (United States)

    Smeller, László

    2016-07-01

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

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

    DEFF Research Database (Denmark)

    Ellgaard, Lars; McCaul, Nicholas; Chatsisvili, Anna;

    2016-01-01

    and the variety of ER-specific protein modifications. Here, we review chaperone-assisted co- and post-translational folding and assembly in the ER and underline the influence of protein modifications on these processes. We emphasize how method development has helped advance the field by allowing researchers...... 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....

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

    Science.gov (United States)

    Dyson, H Jane; Wright, Peter E

    2017-01-17

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

  12. Structural features of protein folding nuclei.

    Science.gov (United States)

    Garbuzynskiy, S O; Kondratova, M S

    2008-03-05

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

  13. Evolutionary computer programming of protein folding and structure predictions.

    Science.gov (United States)

    Nölting, Bengt; Jülich, Dennis; Vonau, Winfried; Andert, Karl

    2004-07-07

    In order to understand the mechanism of protein folding and to assist the rational de-novo design of fast-folding, non-aggregating and stable artificial enzymes it is very helpful to be able to simulate protein folding reactions and to predict the structures of proteins and other biomacromolecules. Here, we use a method of computer programming called "evolutionary computer programming" in which a program evolves depending on the evolutionary pressure exerted on the program. In the case of the presented application of this method on a computer program for folding simulations, the evolutionary pressure exerted was towards faster finding deep minima in the energy landscape of protein folding. Already after 20 evolution steps, the evolved program was able to find deep minima in the energy landscape more than 10 times faster than the original program prior to the evolution process.

  14. Accelerated molecular dynamics simulations of protein folding.

    Science.gov (United States)

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

    2015-07-30

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

  15. 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)

  16. Kinetics and Thermodynamics of Membrane Protein Folding

    Directory of Open Access Journals (Sweden)

    Ernesto A. Roman

    2014-03-01

    Full Text Available Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

  17. The robustness and innovability of protein folds.

    Science.gov (United States)

    Tóth-Petróczy, Agnes; Tawfik, Dan S

    2014-06-01

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

  18. A Survey of Protein Fold Recognition Algorithms

    Directory of Open Access Journals (Sweden)

    M. S. Abual-Rub

    2008-01-01

    Full Text Available Problem statement: Predicting the tertiary structure of proteins from their linear sequence is really a big challenge in biology. This challenge is related to the fact that the traditional computational methods are not powerful enough to search for the correct structure in the huge conformational space. This inadequate capability of the computational methods, however, is a major obstacle in facing this problem. Trying to solve the problem of the protein fold recognition, most of the researchers have examined the use of the protein threading technique. This problem is known as NP-hard; researchers have used various methods such as neural networks, Monte Carlo, support vector machine and genetic algorithms to solve it. Some researchers tried the use of the parallel evolutionary methods for protein fold recognition but it is less well known. Approach: We reviewed various algorithms that have been developed for protein structure prediction by threading and fold recognition. Moreover, we provided a survey of parallel evolutionary methods for protein fold recognition. Results: The findings of this survey showed that evolutionary methods can be used to resolve the protein fold recognition problem. Conclusion: There are two aspects of protein fold recognition problem: First is the computational difficulty and second is that current energy functions are still not accurate enough to calculate the free energy of a given conformation.

  19. Stochastic Resonance in Protein Folding Dynamics.

    Science.gov (United States)

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

    2016-05-04

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

  20. Protein Folding: A New Geometric Analysis

    OpenAIRE

    Simmons, Walter A.; Joel L. Weiner

    2008-01-01

    A geometric analysis of protein folding, which complements many of the models in the literature, is presented. We examine the process from unfolded strand to the point where the strand becomes self-interacting. A central question is how it is possible that so many initial configurations proceed to fold to a unique final configuration. We put energy and dynamical considerations temporarily aside and focus upon the geometry alone. We parameterize the structure of an idealized protein using the ...

  1. 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.

  2. Exploring the mechanisms of protein folding

    CERN Document Server

    Xu, Ji; Ren, Ying; Li, Jinghai

    2013-01-01

    Neither of the two prevalent theories, namely thermodynamic stability and kinetic stability, provides a comprehensive understanding of protein folding. The thermodynamic theory is misleading because it assumes that free energy is the exclusive dominant mechanism of protein folding, and attributes the structural transition from one characteristic state to another to energy barriers. Conversely, the concept of kinetic stability overemphasizes dominant mechanisms that are related to kinetic factors. This article explores the stability condition of protein structures from the viewpoint of meso-science, paying attention to the compromise in the competition between minimum free energy and other dominant mechanisms. Based on our study of complex systems, we propose that protein folding is a meso-scale, dissipative, nonlinear and non-equilibrium process that is dominated by the compromise between free energy and other dominant mechanisms such as environmental factors. Consequently, a protein shows dynamic structures,...

  3. Structural characteristics of novel protein folds.

    Directory of Open Access Journals (Sweden)

    Narcis Fernandez-Fuentes

    2010-04-01

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

  4. 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......-range excitations, `wring' modes, that play an important role in protein denaturation and stability. Energy can be pumped into these excitations, either thermally or by an external force....

  5. 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.

  6. Microfluidic mixers for studying protein folding.

    Science.gov (United States)

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

    2012-04-10

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

  7. Explicit solvent molecular dynamics simulations of chaperonin-assisted rhodanese folding

    Institute of Scientific and Technical Information of China (English)

    Ying Ren; Jian Gao; Ji Xu; Wei Ge; Jinghai Li

    2009-01-01

    Chaperonins are known to facilitate the productive folding of numerous misfolded proteins, Despite their established importance, the mechanism of chaperonin-assisted protein folding remains unknown. In the present article, all-atom explicit solvent molecular dynamics (MD) simulations have been performed for the first time on rhodanese folding in a series of cavity-size and cavity-charge chaperonin mutants. A compromise between stability and flexibility of chaperonin structure during the substrate folding has been observed and the key factors affecting this dynamic process are discussed.

  8. 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.

  9. Towards a systematic classification of protein folds

    DEFF Research Database (Denmark)

    Lindgård, Per-Anker; Bohr, Henrik

    1997-01-01

    in the usual protein data base coordinate format can be transformed into the proposed chain representation. Taking into account hydrophobic forces we have found a mechanism for the formation of domains with a unique fold containing predicted magic numbers {4,6,9,12,16,18,...} of secondary structures...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    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...

  11. 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.

  12. Substrate protein folds while it is bound to the ATP-independent chaperone Spy.

    Science.gov (United States)

    Stull, Frederick; Koldewey, Philipp; Humes, Julia R; Radford, Sheena E; Bardwell, James C A

    2016-01-01

    Chaperones assist in the folding of many proteins in the cell. Although the most well-studied chaperones use cycles of ATP binding and hydrolysis to assist in protein folding, a number of chaperones have been identified that promote folding in the absence of high-energy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we characterized the kinetic mechanism of substrate folding by the small ATP-independent chaperone Spy from Escherichia coli. Spy rapidly associates with its substrate, immunity protein 7 (Im7), thereby eliminating Im7's potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while it remains bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while being continuously bound to a chaperone.

  13. The role of ascorbate in protein folding.

    Science.gov (United States)

    Szarka, András; Lőrincz, Tamás

    2014-05-01

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

  14. A simple theory of protein folding kinetics

    CERN Document Server

    Pande, Vijay S

    2010-01-01

    We present a simple model of protein folding dynamics that captures key qualitative elements recently seen in all-atom simulations. The goals of this theory are to serve as a simple formalism for gaining deeper insight into the physical properties seen in detailed simulations as well as to serve as a model to easily compare why these simulations suggest a different kinetic mechanism than previous simple models. Specifically, we find that non-native contacts play a key role in determining the mechanism, which can shift dramatically as the energetic strength of non-native interactions is changed. For protein-like non-native interactions, our model finds that the native state is a kinetic hub, connecting the strength of relevant interactions directly to the nature of folding kinetics.

  15. Analysis of protein folds using protein contact networks

    Indian Academy of Sciences (India)

    Pankaj Barah; Somdatta Sinha

    2008-08-01

    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 the biophysical and biochemical functions. Proteins are defined as having a common `fold' if they have major secondary structural elements with same topological connections. It is known that folding mechanisms are largely determined by a protein's topology rather than its interatomic interactions. The native state protein structures can, thus, be modelled, using a graph-theoretical approach, as coarse-grained networks of amino acid residues as `nodes' and the inter-residue interactions/contacts as `links'. Using the network representation of protein structures and their 2D contact maps, we have identified the conserved contact patterns (groups of contacts) representing two typical folds – the EF-hand and the ubiquitin-like folds. Our results suggest that this direct and computationally simple methodology can be used to infer about the presence of specific folds from the protein's contact map alone.

  16. 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.

  17. Structure Characterization of the Folding Intermediates of Proteins

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Although the native state and the fully unfolded state of proteins have been extensively studied, the folding pathway and intermediates in the protein folding process have not been thoroughly investigated.To understand the mechanisms of protein folding, the early intermediates in the protein folding process must be clearly characterized.The present paper is a mini review containing 20 references involving studies on folding intermediates of several proteins.

  18. 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.

  19. Influence of Conformational Entropy on the Protein Folding Rate

    Directory of Open Access Journals (Sweden)

    Oxana V. Galzitskaya

    2010-04-01

    Full Text Available One of the most important questions in molecular biology is what determines folding pathways: native structure or protein sequence. There are many proteins that have similar structures but very different sequences, and a relevant question is whether such proteins have similar or different folding mechanisms. To explain the differences in folding rates of various proteins, the search for the factors affecting the protein folding process goes on. Here, based on known experimental data, and using theoretical modeling of protein folding based on a capillarity model, we demonstrate that the relation between the average conformational entropy and the average energy of contacts per residue, that is the entropy capacity, will determine the possibility of the given chain to fold to a particular topology. The difference in the folding rate for proteins sharing more ball-like and less ball-like folds is the result of differences in the conformational entropy due to a larger surface of the boundary between folded and unfolded phases in the transition state for proteins with a more ball-like fold. The result is in agreement with the experimental folding rates for 67 proteins. Proteins with high or low side chain entropy would have extended unfolded regions and would require some additional agents for complete folding. Such proteins are common in nature, and their structural properties are of biological importance.

  20. Improving Protein Fold Recognition by Deep Learning Networks

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2008-08-01

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

  2. Protein structural codes and nucleation sites for protein folding

    Institute of Scientific and Technical Information of China (English)

    Jiang Fan; Li Nan

    2007-01-01

    One of the long-standing controversial arguments in protein folding is Levinthal's paradox. We have recently proposed a new nucleation hypothesis and shown that the nucleation residues are the most conserved sequences in protein. To avoid the complicated effect of tertiary interactions, we limit our search for structural codes to the nucleation residues. Starting with the hypotheses of secondary structure nucleation and conservation of residues important for folding, we have analysed 762 folds classified as unique by SCOP. Segments of 17 residues around the top 20% conserved amino acids are analysed, resulting in approximately 100 clusters each for the main secondary structure classes of helix,sheet and coil. Helical clusters have the longest correlation range, coils the shortest (four residues). Strong specific sequence-structure correlation is observed for coil but not for helix and sheet, suggesting a mapping relationship between the sequence and the structure for coil. We propose that the central sequences in these clusters form 'structural codes',a useful basis set for identifying nucleation sites, protein fragments stable in isolation, and secondary structural patterns in proteins (particularly turns and loops).

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

    Science.gov (United States)

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

    2009-10-01

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

  4. 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.

  5. 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.

  6. Inferring the Rate-Length Law of Protein Folding

    CERN Document Server

    Lane, Thomas J

    2013-01-01

    We investigate the rate-length scaling law of protein folding, a key undetermined scaling law in the analytical theory of protein folding. We demonstrate that chain length is a dominant factor determining folding times, and that the unambiguous determination of the way chain length corre- lates with folding times could provide key mechanistic insight into the folding process. Four specific proposed laws (power law, exponential, and two stretched exponentials) are tested against one an- other, and it is found that the power law best explains the data. At the same time, the fit power law results in rates that are very fast, nearly unreasonably so in a biological context. We show that any of the proposed forms are viable, conclude that more data is necessary to unequivocally infer the rate-length law, and that such data could be obtained through a small number of protein folding experiments on large protein domains.

  7. 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.

  8. 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

  9. 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...

  10. Protein Folding Pathways Revealed by Essential Dynamics Sampling.

    Science.gov (United States)

    Narzi, Daniele; Daidone, Isabella; Amadei, Andrea; Di Nola, Alfredo

    2008-11-11

    The characterization of the protein folding process represents one of the major challenges in molecular biology. Here, a method to simulate the folding process of a protein to its native state is reported, the essential dynamics sampling (EDS) method, and is successfully applied to detecting the correct folding pathways of two small proteins, the all-β SH3 domain of Src tyrosine kinase transforming protein (SH3) and the α/β B1 domain of streptococcal protein G (GB1). The main idea of the method is that a subset of the natural modes of fluctuation in the native state is key in directing the folding process. A biased molecular dynamics simulation is performed, in which the restrained degrees of freedom are chosen among those obtained by a principal component, or essential dynamics, analysis of the positional fluctuations of the Cα atoms in the native state. Successful folding is obtained if the restraints are applied only to the eigenvectors with lowest eigenvalues, representing the most rigid quasi-constraint motions. If the essential eigenvectors, the ones accounting for most of the variance, are used, folding is not successful. These results clearly show that the eigenvectors with lowest eigenvalues contain the main mechanical information necessary to drive the folding process, while the essential eigenvectors represent the large concerted motions which can occur without folding/unfolding the protein.

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

    Science.gov (United States)

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

    2006-05-15

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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Structure-based prediction of protein-folding transition paths

    CERN Document Server

    Jacobs, William M

    2016-01-01

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

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

    Science.gov (United States)

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

    2005-11-15

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

  18. Review: Protein folding pathology in domestic animals

    Institute of Scientific and Technical Information of China (English)

    GRUYSErik

    2004-01-01

    Fibrillar proteins form structural elements of cells and the extracellular matrix. Pathological lesions of fibrillar microanatomical structures, or secondary fibrillar changes in globular proteins are well known. A special group concerns histologically amorphous deposits, amyloid. The major characteristics of amyloid are: apple green birefringence after Congo red staining of histological sections, and non-branching 7-10nm thick fibrils on electron microscopy revealing a high content of cross beta pleated sheets. About 25 different types of amyloid have been characterised. In animals, AA-amyloid is the most frequent type. Other types of amyloid in animals represent: AIAPP (in cats), AApoAⅠ, AApoAⅡ, localised AL-amyloid, amyloid in odontogenic or mammary tumors and amyloid in the brain. In old dogs Aβ and in sheep APrPsc-amyloid can be encountered. AA-amyloidosis is a systemic disorder with a precursor in blood, acute phase serum amyloid A (SAA). In chronic inflammatory processes AA-amyloid can be deposited. A rapid crystallization of SAA to amyloid fibrils on small beta-sheeted fragments, the ‘amyloid enhancing factor' (AEF), is known and the AEF has been shown to penetrate the enteric barrier. Amyloid fibrils can aggregate from various precursor proteins in vitro in particular at acidic pH and when proteolytic fragments are formed. Molecular chaperones influence this process. Tissue data point to amyloid fibrillogenesis in lysosomes and near cell surfaces. A comparison can be made of the fibrillogenesis in prion diseases and in enhanced AA-amyloidosis. In the reactive form, acute phase SAA is the supply of the precursor protein, whereas in the prion diseases, cell membrane proteins form a structural source. AI3-amyloid in brain tissue of aged dogs showing signs of dementia forms a canine counterpart of senile dementia of the Alzheimer type (ccSDAT) in man. Misfolded proteins remain potential food hazards. Developments concerning prevention of amyloidogenesis

  19. Protein folding pathology in domestic animals

    Institute of Scientific and Technical Information of China (English)

    GRUYS Erik

    2004-01-01

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

  20. Solitons and protein folding: An In Silico experiment

    Science.gov (United States)

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

    2015-10-01

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

  1. 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…

  2. 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...

  3. 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.

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

    Science.gov (United States)

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

    2016-04-26

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Ignatova, Zoya; Gierasch, Lila M

    2007-01-01

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

  7. Super-secondary structures and modeling of protein folds.

    Science.gov (United States)

    Efimov, Alexander V

    2013-01-01

    A characteristic feature of the polypeptide chain is its ability to form a restricted set of commonly occurring folding units composed of two or more elements of secondary structure that are adjacent along the chain. Some of these super-secondary structures exhibit a unique handedness and a unique overall fold irrespective of whether they occur in homologous or nonhomologous proteins. Such super-secondary structures are of particular value since they can be used as starting structures in protein modeling. The larger protein folds can be obtained by stepwise addition of other secondary structural elements to the starting structures taking into account a set of simple rules inferred from known principles of protein structure.

  8. 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...

  9. On the polymer physics origins of protein folding thermodynamics

    Science.gov (United States)

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

    2016-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Giovanna Valentini

    2013-12-01

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

  11. 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...

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

    Science.gov (United States)

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

    2013-10-24

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

  13. Entropic formulation for the protein folding process: hydrophobic stability correlates with folding rates

    CERN Document Server

    Molin, J P Dal

    2016-01-01

    We assume that the protein folding process follows two autonomous steps: the conformational search for the native, mainly ruled by the hydrophobic effect; and, the final adjustment stage, which eventually gives stability to the native. Our main tool of investigation is a 3D lattice model provided with a ten-letter alphabet, the stereochemical model. This model was conceived for Monte Carlo (MC) simulations when one keeps in mind the kinetic behavior of protein-like chains in solution. In order to characterize the folding characteristic time ({\\tau}) by two distinct sampling methods, first we present two sets of 10^{3} MC simulations for a fast protein-like sequence. For these sets of folding times, {\\tau} and {\\tau}_{q} were obtained with the application of the standard Metropolis algorithm (MA), and a modified algorithm (M_{q}A). The results for {\\tau}_{q}reveal two things: i) the hydrophobic chain-solvent interactions plus a set of inter-residues steric constraints are enough to emulate the first stage of t...

  14. Topology, Geometry, and Stability: Protein Folding and Evolution

    CERN Document Server

    Simmons, Walter

    2015-01-01

    The protein folding problem must ultimately be solved on all length scales from the atomic up through a hierarchy of complicated structures. By analyzing the stability of the folding process using physics and mathematics, this paper shows that features without length scales, i.e. topological features, are potentially of central importance. Topology is a natural mathematical tool for the study of shape and we avail ourselves of that tool to examine the relationship between the amino acid sequence and the shapes of protein molecules. We apply what we learn to conjectures about their biological evolution.

  15. A twist on folding: Predicting optimal sequences and optimal folds of simple protein models with the hidden-force algorithm

    CERN Document Server

    Kolossváry, István

    2012-01-01

    We propose a new way of looking at global optimization of off-lattice protein models. We present a dual optimization concept of predicting optimal sequences as well as optimal folds. We validate the utility of the recently introduced hidden-force Monte Carlo optimization algorithm by finding significantly lower energy folds for minimalist protein models than previously reported. Further, we also find the protein sequence that yields the lowest energy fold amongst all sequences for a given chain length and residue mixture. In particular, for protein models with a binary sequence, we show that the sequence-optimized folds form more compact cores than the lowest energy folds of the historically fixed, Fibonacci-series sequences of chain lengths of 13, 21, 34, 55, and 89. We emphasize that while the protein model we used is minimalist, the methodology is applicable to detailed protein models, and sequence optimization may yield novel folds and aid de novo protein design.

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

    Science.gov (United States)

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

    2011-08-01

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

  17. Buttressing a new paradigm in protein folding: experimental tools to distinguish between downhill and multi-state folding mechanisms

    OpenAIRE

    Nagalakshmi, Tiruvarur Sooriyanarayanan

    2014-01-01

    Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biologíoa Molecular. Fecha de lectura: 15-07-2014 Many single-domain proteins fold in milliseconds or longer. However, the advent of fast folding kinetic techniques has permitted to identify many other proteins that fold in the order of (few) microseconds and thus very closely to the folding speed limit. This suggests that the proteins that fold in microsecond timescale either ...

  18. A digitally assisted, signal folding neural recording amplifier.

    Science.gov (United States)

    Chen, Yi; Basu, Arindam; Liu, Lei; Zou, Xiaodan; Rajkumar, Ramamoorthy; Dawe, Gavin Stewart; Je, Minkyu

    2014-08-01

    A novel signal folding and reconstruction scheme for neural recording applications that exploits the 1/f(n) characteristics of neural signals is described in this paper. The amplified output is 'folded' into a predefined range of voltages by using comparison and reset circuits along with the core amplifier. After this output signal is digitized and transmitted, a reconstruction algorithm can be applied in the digital domain to recover the amplified signal from the folded waveform. This scheme enables the use of an analog-to-digital convertor with less number of bits for the same effective dynamic range. It also reduces the transmission data rate of the recording chip. Both of these features allow power and area savings at the system level. Other advantages of the proposed topology are increased reliability due to the removal of pseudo-resistors, lower harmonic distortion and low-voltage operation. An analysis of the reconstruction error introduced by this scheme is presented along with a behavioral model to provide a quick estimate of the post reconstruction dynamic range. Measurement results from two different core amplifier designs in 65 nm and 180 nm CMOS processes are presented to prove the generality of the proposed scheme in the neural recording applications. Operating from a 1 V power supply, the amplifier in 180 nm CMOS has a gain of 54.2 dB, bandwidth of 5.7 kHz, input referred noise of 3.8 μVrms and power dissipation of 2.52 μW leading to a NEF of 3.1 in spike band. It exhibits a dynamic range of 66 dB and maximum SNDR of 43 dB in LFP band. It also reduces system level power (by reducing the number of bits in the ADC by 2) as well as data rate to 80% of a conventional design. In vivo measurements validate the ability of this amplifier to simultaneously record spike and LFP signals.

  19. Protein folding activity and the central dogma of molecular biology

    OpenAIRE

    Pallavi, Ghosh; Dipankar, Chatterji

    2003-01-01

    Biological systems, in general, can function effectively when the products of the system are in proper configuration and harmful effects due to misaggregation are avoided. Folding of proteins and their functional consequences have been a subject of active research since several years now. However it is not clear whether during protein synthesis from genetic message, the same set of rules are employed or participation of new efforts take place. In this review we show that at least in the case ...

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

    Science.gov (United States)

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

    2011-04-20

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

  1. 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.

  2. Functional Subunits of Eukaryotic Chaperonin CCT/TRiC in Protein Folding

    Directory of Open Access Journals (Sweden)

    M. Anaul Kabir

    2011-01-01

    Full Text Available Molecular chaperones are a class of proteins responsible for proper folding of a large number of polypeptides in both prokaryotic and eukaryotic cells. Newly synthesized polypeptides are prone to nonspecific interactions, and many of them make toxic aggregates in absence of chaperones. The eukaryotic chaperonin CCT is a large, multisubunit, cylindrical structure having two identical rings stacked back to back. Each ring is composed of eight different but similar subunits and each subunit has three distinct domains. CCT assists folding of actin, tubulin, and numerous other cellular proteins in an ATP-dependent manner. The catalytic cooperativity of ATP binding/hydrolysis in CCT occurs in a sequential manner different from concerted cooperativity as shown for GroEL. Unlike GroEL, CCT does not have GroES-like cofactor, rather it has a built-in lid structure responsible for closing the central cavity. The CCT complex recognizes its substrates through diverse mechanisms involving hydrophobic or electrostatic interactions. Upstream factors like Hsp70 and Hsp90 also work in a concerted manner to transfer the substrate to CCT. Moreover, prefoldin, phosducin-like proteins, and Bag3 protein interact with CCT and modulate its function for the fine-tuning of protein folding process. Any misregulation of protein folding process leads to the formation of misfolded proteins or toxic aggregates which are linked to multiple pathological disorders.

  3. Fusion-protein-assisted protein crystallization.

    Science.gov (United States)

    Kobe, Bostjan; Ve, Thomas; Williams, Simon J

    2015-07-01

    Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

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

    Science.gov (United States)

    Arkun, Yaman; Gur, Mert

    2012-01-01

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

  5. WeFold: a coopetition for protein structure prediction.

    Science.gov (United States)

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

    2014-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    HART,WILLIAM E.; ISTRAIL,SORIN

    2000-06-01

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

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

    CERN Document Server

    Zhang, Cheng

    2010-01-01

    We report a single-copy tempering method for enhancing sampling in simulating large complex systems. A continuous temperature space random walk is achieved by a Langevin equation, which is guided by a runtime estimate of the thermal average energy through a novel integral identity. We first validated the method in a two-dimensional Ising model and a Lennard-Jones liquid system. Then the method was 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 folded into atomic accuracy: the alpha carbon root mean square deviation of the best folded conformations from the native states are 0.2 A, 0.4 A, and 0.4 A, for trpzip2, trp-cage, and villin headpiece, respectively.

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

    Science.gov (United States)

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

    2016-03-02

    Knowledge of the transfer free energy of amino acids from aqueous solution to a lipid bilayer is essential for understanding membrane protein folding and for predicting membrane protein structure. Here we report a computational approach that can calculate the folding free energy of the transmembrane region of outer membrane β-barrel proteins (OMPs) by combining an empirical energy function with a reduced discrete state space model. We quantitatively analyzed the transfer free energies of 20 amino acid residues at the center of the lipid bilayer of OmpLA. Our results are in excellent agreement with the experimentally derived hydrophobicity scales. We further exhaustively calculated the transfer free energies of 20 amino acids at all positions in the TM region of OmpLA. We found that the asymmetry of the Gram-negative bacterial outer membrane as well as the TM residues of an OMP determine its functional fold in vivo. Our results suggest that the folding process of an OMP is driven by the lipid-facing residues in its hydrophobic core, and its NC-IN topology is determined by the differential stabilities of OMPs in the asymmetrical outer membrane. The folding free energy is further reduced by lipid A and assisted by general depth-dependent cooperativities that exist between polar and ionizable residues. Moreover, context-dependency of transfer free energies at specific positions in OmpLA predict regions important for protein function as well as structural anomalies. Our computational approach is fast, efficient and applicable to any OMP.

  9. 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/

  10. Dissection of SARS Coronavirus Spike Protein into Discrete Folded Fragments

    Institute of Scientific and Technical Information of China (English)

    LI Shuang; CAI Zhen; CHEN Yong; LIN Zhanglin

    2006-01-01

    The spike protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) mediates cell fusion by binding to target cell surface receptors. This paper reports a simple method for dissecting the viral protein and for searching for foldable fragments in a random but systematic manner. The method involves digestion by DNase I to generate a pool of short DNA segments, followed by an additional step of reassembly of these segments to produce a library of DNA fragments with random ends but controllable lengths. To rapidly screen for discrete folded polypeptide fragments, the reassembled gene fragments were further cloned into a vector as N-terminal fusions to a folding reporter gene which was a variant of green fluorescent protein. Two foldable fragments were identified for the SARS-CoV spike protein, which coincide with various anti-SARS peptides derived from the hepated repeat (HR) region 2 of the spike protein. The method should be applicable to other viral proteins to isolate antigen or vaccine candidates, thus providing an alternative to the full-length proteins (subunits) or linear short peptides.

  11. Folding pathways of a helix-turn-helix model protein

    CERN Document Server

    Hoffmann, D

    1997-01-01

    A small model polypeptide represented in atomic detail is folded using Monte Carlo dynamics. The polypeptide is designed to have a native conformation similar to the central part of the helix-turn-helix protein ROP. Starting from a beta-strand conformation or two different loop conformations of the protein glutamine synthetase, six trajectories are generated using the so-called window move in dihedral angle space. This move changes conformations locally and leads to realistic, quasi-continuously evolving trajectories. Four of the six trajectories end in stable native-like conformations. Their folding pathways show a fast initial development of a helix-bend-helix motif, followed by a dynamic behaviour predicted by the diffusion-collision model of Karplus and Weaver. The phenomenology of the pathways is consistent with experimental results.

  12. Emergence of protein fold families through rational design.

    Directory of Open Access Journals (Sweden)

    Feng Ding

    2006-07-01

    Full Text Available Diverse proteins with similar structures are grouped into families of homologs and analogs, if their sequence similarity is higher or lower, respectively, than 20%-30%. It was suggested that protein homologs and analogs originate from a common ancestor and diverge in their distinct evolutionary time scales, emerging as a consequence of the physical properties of the protein sequence space. Although a number of studies have determined key signatures of protein family organization, the sequence-structure factors that differentiate the two evolution-related protein families remain unknown. Here, we stipulate that subtle structural changes, which appear due to accumulating mutations in the homologous families, lead to distinct packing of the protein core and, thus, novel compositions of core residues. The latter process leads to the formation of distinct families of homologs. We propose that such differentiation results in the formation of analogous families. To test our postulate, we developed a molecular modeling and design toolkit, Medusa, to computationally design protein sequences that correspond to the same fold family. We find that analogous proteins emerge when a backbone structure deviates only 1-2 angstroms root-mean-square deviation from the original structure. For close homologs, core residues are highly conserved. However, when the overall sequence similarity drops to approximately 25%-30%, the composition of core residues starts to diverge, thereby forming novel families of protein homologs. This direct observation of the formation of protein homologs within a specific fold family supports our hypothesis. The conservation of amino acids in designed sequences recapitulates that of the naturally occurring sequences, thereby validating our computational design methodology.

  13. Evidence that bilayer bending rigidity affects membrane protein folding.

    Science.gov (United States)

    Booth, P J; Riley, M L; Flitsch, S L; Templer, R H; Farooq, A; Curran, A R; Chadborn, N; Wright, P

    1997-01-07

    The regeneration kinetics of the integral membrane protein bacteriorhodopsin have been investigated in a lipid-based refolding system. Previous studies on bacteriorhodopsin regeneration have involved detergent-based systems, and in particular mixed dimyristoylphosphatidylcholine (DMPC)/CHAPS micelles. Here, we show that the short chain lipid dihexanoylphosphatidylcholine (DHPC) can be substituted for the detergent CHAPS and that bacteriorhodopsin can be regenerated to high yield in mixed DMPC/DHPC micelles. Bacteriorhodopsin refolding kinetics are measured in the mixed DMPC/DHPC micelles. Rapid, stopped flow mixing is employed to initiate refolding of denatured bacterioopsin in SDS micelles with mixed DMPC/DHPC micelles and time-resolved fluorescence spectroscopy to follow changes in protein fluorescence during folding. Essentially identical refolding kinetics are observed for mixed DMPC/CHAPS and mixed DMPC/DHPC micelles. Only one second-order retinal/apoprotein reaction is identified, in which retinal binds to a partially folded apoprotein intermediate, and the free energy of this retinal binding reaction is found to be the same in both types of mixed micelles. Formation of the partially folded apoprotein intermediate is a rate-limiting step in protein folding and appears to be biexponential. Both apparent rate constants are found to be dependent on the relative proportion of DMPC present in the mixed DMPC/DHPC micelles as well as on the pH of the aqueous phase. Increasing the DMPC concentration should increase the bending rigidity of the amphiphilic bilayer, and this is found to slow the rate of formation of the partially folded apoprotein intermediate. Increasing the mole fraction of DMPC from 0.3 to 0.6 slows the two apparent rate constants associated with formation of this intermediate from 0.29 and 0.031 to 0.11 and 0.013 s-1, respectively. Formation of the intermediate also slows with increasing pH, from 0.11 and 0.013 s-1 at pH 6 to 0.033 and 0.0053 s-1 at

  14. Benchmarking consensus model quality assessment for protein fold recognition

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

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

  16. Predictive energy landscapes for folding membrane protein assemblies

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

    Waldo, Geoffrey S.

    2007-09-18

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

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

    Science.gov (United States)

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

    2015-06-30

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

  19. Exploring energy landscapes of protein folding and aggregation.

    Science.gov (United States)

    Mousseau, Normand; Derreumaux, Philippe

    2008-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Alex Kentsis

    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. Heuristic algorithm for off-lattice protein folding problem

    Institute of Scientific and Technical Information of China (English)

    CHEN Mao; HUANG Wen-qi

    2006-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Ruben A. Abagyan

    2000-06-01

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

  3. 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

    2008-12-01

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

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

    Science.gov (United States)

    Buijsman, W.; Sheinman, M.

    2014-02-01

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

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

    Science.gov (United States)

    Buijsman, W; Sheinman, M

    2014-02-01

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

  6. 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

    , 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....

  7. 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.

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

    Science.gov (United States)

    Muñoz, Victor; Cerminara, Michele

    2016-09-01

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

  9. 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

  10. 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.

  11. 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.

  12. Protein folding: the optically induced electronic excitations model

    Energy Technology Data Exchange (ETDEWEB)

    Jeknic-Dugic, J [Department of Physics, Faculty of Science, Nis (Serbia)], E-mail: jjeknic@pmf.ni.ac.yu

    2009-07-15

    The large-molecules conformational transitions problem (the 'protein folding problem') is an open issue of vivid current science research work of fundamental importance for a number of modern science disciplines as well as for nanotechnology. Here, we elaborate the recently proposed quantum-decoherence-based approach to the issue. First, we emphasize a need for detecting the elementary quantum mechanical processes (whose combinations may give a proper description of the realistic experimental situations) and then we design such a model. As distinct from the standard approach that deals with the conformation system, we investigate the optically induced transitions in the molecule electrons system that, in effect, may give rise to a conformation change in the molecule. Our conclusion is that such a model may describe the comparatively slow conformational transitions.

  13. 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.

  14. Procollagen triple helix assembly: an unconventional chaperone-assisted folding paradigm.

    Science.gov (United States)

    Makareeva, Elena; Leikin, Sergey

    2007-10-10

    Fibers composed of type I collagen triple helices form the organic scaffold of bone and many other tissues, yet the energetically preferred conformation of type I collagen at body temperature is a random coil. In fibers, the triple helix is stabilized by neighbors, but how does it fold? The observations reported here reveal surprising features that may represent a new paradigm for folding of marginally stable proteins. We find that human procollagen triple helix spontaneously folds into its native conformation at 30-34 degrees C but not at higher temperatures, even in an environment emulating Endoplasmic Reticulum (ER). ER-like molecular crowding by nonspecific proteins does not affect triple helix folding or aggregation of unfolded chains. Common ER chaperones may prevent aggregation and misfolding of procollagen C-propeptide in their traditional role of binding unfolded polypeptide chains. However, such binding only further destabilizes the triple helix. We argue that folding of the triple helix requires stabilization by preferential binding of chaperones to its folded, native conformation. Based on the triple helix folding temperature measured here and published binding constants, we deduce that HSP47 is likely to do just that. It takes over 20 HSP47 molecules to stabilize a single triple helix at body temperature. The required 50-200 microM concentration of free HSP47 is not unusual for heat-shock chaperones in ER, but it is 100 times higher than used in reported in vitro experiments, which did not reveal such stabilization.

  15. Probing folding free energy landscape of small proteins through minimalistic models: Folding of HP-36 and -amyloid

    Indian Academy of Sciences (India)

    Arnab Mukherjee; Biman Bagchi

    2003-10-01

    Folding dynamics and energy landscape picture of protein conformations of HP-36 and -amyloid (A) are investigated by extensive Brownian dynamics simulations, where the inter amino acid interactions are given by a minimalistic model (MM) we recently introduced [J. Chem. Phys. 118 4733 (2003)]. In this model, a protein is constructed by taking two atoms for each amino acid. One atom represents the backbone C atom, while the other mimics the whole side chain residue. Sizes and interactions of the side residues are all different and specific to a particular amino acid. The effect of water-mediated folding is mapped into the MM by suitable choice of interaction parameters of the side residues obtained from the amino acid hydropathy scale. A new non-local helix potential is incorporated to generate helices at the appropriate positions in a protein. Simulations have been done by equilibrating the protein at high temperature followed by a sudden quench. The subsequent folding is monitored to observe the dynamics of topological contacts (topo), relative contact order parameter (RCO), and the root mean square deviation (RMSD) from the realprotein native structure. The folded structures of different model proteins (HP-36 and ) resemble their respective real native state rather well. The dynamics of folding shows multistage decay, with an initial hydrophobic collapse followed by a long plateau. Analysis of topo and RCO correlates the late stage folding with rearrangement of the side chain residues, particularly those far apart in the sequence. The long plateau also signifies large entropic free energy barrier near the native state, as predicted from theories of protein folding.

  16. 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.

  17. Efficient conformational space exploration in ab initio protein folding simulation.

    Science.gov (United States)

    Ullah, Ahammed; Ahmed, Nasif; Pappu, Subrata Dey; Shatabda, Swakkhar; Ullah, A Z M Dayem; Rahman, M Sohel

    2015-08-01

    Ab initio protein folding simulation largely depends on knowledge-based energy functions that are derived from known protein structures using statistical methods. These knowledge-based energy functions provide us with a good approximation of real protein energetics. However, these energy functions are not very informative for search algorithms and fail to distinguish the types of amino acid interactions that contribute largely to the energy function from those that do not. As a result, search algorithms frequently get trapped into the local minima. On the other hand, the hydrophobic-polar (HP) model considers hydrophobic interactions only. The simplified nature of HP energy function makes it limited only to a low-resolution model. In this paper, we present a strategy to derive a non-uniform scaled version of the real 20×20 pairwise energy function. The non-uniform scaling helps tackle the difficulty faced by a real energy function, whereas the integration of 20×20 pairwise information overcomes the limitations faced by the HP energy function. Here, we have applied a derived energy function with a genetic algorithm on discrete lattices. On a standard set of benchmark protein sequences, our approach significantly outperforms the state-of-the-art methods for similar models. Our approach has been able to explore regions of the conformational space which all the previous methods have failed to explore. Effectiveness of the derived energy function is presented by showing qualitative differences and similarities of the sampled structures to the native structures. Number of objective function evaluation in a single run of the algorithm is used as a comparison metric to demonstrate efficiency.

  18. 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.

  19. How the folding rates of two- and multistate proteins depend on the amino acid properties.

    Science.gov (United States)

    Huang, Jitao T; Huang, Wei; Huang, Shanran R; Li, Xin

    2014-10-01

    Proteins fold by either two-state or multistate kinetic mechanism. We observe that amino acids play different roles in different mechanism. Many residues that are easy to form regular secondary structures (α helices, β sheets and turns) can promote the two-state folding reactions of small proteins. Most of hydrophilic residues can speed up the multistate folding reactions of large proteins. Folding rates of large proteins are equally responsive to the flexibility of partial amino acids. Other properties of amino acids (including volume, polarity, accessible surface, exposure degree, isoelectric point, and phase transfer energy) have contributed little to folding kinetics of the proteins. Cysteine is a special residue, it triggers two-state folding reaction and but inhibits multistate folding reaction. These findings not only provide a new insight into protein structure prediction, but also could be used to direct the point mutations that can change folding rate.

  20. Numerical Simulation of Folding and Unfolding of Proteins

    CERN Document Server

    Kouza, Maksim

    2013-01-01

    The thesis examines in detail the folding and unfolding processes of a number of proteins including hbSBD, DDLNF4, single and multi Ubiquitin. Using simplified coarse-grained off-lattice Go model and CD experiments we have shown the two-state behavior of hbSBD protein. It was shown that refolding pathways of single Ubiquitin depend on what end is anchored to the surface. Namely, the fixation of the N-terminal changes refolding pathways but anchoring the C-terminal leaves them unchanged. Interestingly, the end fixation has no effect on multi-domain Ubiquitin. Using the Go modeling and all-atom models with explicit water, we have studied the mechanical unfolding mechanism of DDFLN4 in detail. We predict that, contrary to the AFM experiments, an additional unfolding peak would occur at the end-to-end $\\Delta R \\approx 1.5 $nm in the force-extension curve. Our study reveals the important role of non-native interactions which are responsible for a peak located at $\\Delta R \\approx 22 $nm. This peak can not be enco...

  1. Folding Behaviour for Proteins BBL and E3BD with Gō-like Models

    Institute of Scientific and Technical Information of China (English)

    ZUO Guang-Hong; ZHANG Jian; WANG Jun; WANG Wei

    2005-01-01

    @@ Folding behaviour of protein BBL and its homologue domain E3BD are studied by using an off-lattice Gō-like model. It is found that the folding behaviours of these two proteins are different. Protein BBL folds in a downhill manner, which is consistent with experiments. In contrast, protein E3BD folds cooperatively and has a bimodal distribution of the Q values (the similarity to the native state). By analysing the native structures of the two proteins, it is found that the difference in folding behaviours can be attributed to the different structural features described by the number of nonlocal contacts per residue.

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

    Science.gov (United States)

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

    2010-08-10

    Addition of iron salts to chaotrope-denatured aporubredoxin (apoRd) leads to nearly quantitative recovery of its single Fe(SCys)(4) site and native protein structure without significant dilution of the chaotrope. This "high-chaotrope" approach was used to examine iron binding and protein folding events using stopped-flow UV-vis absorption and CD spectroscopies. With a 100-fold molar excess of ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFe(III)Rd structure was recovered in >90% yield with a t(1/2) of Ser iron ligand variants support formation of an unfolded-Fe(SCys)(3) complex between steps 1 and 2, which we propose is the key nucleation event that pulls together distal regions of the protein chain. These results show that folding of chaotrope-denatured apoRd is iron-nucleated and driven by extraordinarily rapid formation of the Fe(SCys)(4) site from an essentially random coil apoprotein. This high-chaotrope, multispectroscopy approach could clarify folding pathways of other [M(SCys)(3)]- or [M(SCys)(4)]-containing proteins.

  3. 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.

  4. 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.

  5. Modeling of folds and folding pathways for some protein families of (α + β)- and (α/β)-classes.

    Science.gov (United States)

    Gordeev, Alexey B; Efimov, Alexander V

    2013-01-01

    In this paper, updated structural trees for α/β-proteins containing five- and seven-segment (α/β)-motifs are represented. Novel structural motifs occurring in some families of (α + β)- and (α/β)-proteins are also characterized. Databases of these proteins have been compiled from the Protein Data Bank (PDB) and Structural Classification of Proteins (SCOP) and the corresponding structural trees have been constructed. The classification of these proteins has been developed and organized as an extension of the PCBOST database, which is available at http://strees.protres.ru . In total, the updated Protein Classification Based on Structural Trees database contains 11 structural trees, 106 levels, 635 folds, 4911 proteins and domains, and 14,202 PDB entries.

  6. 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.

  7. New light on protein folding: Unraveling folding and unfolding mechanisms using time-resolved and two-dimensional vibrational spectroscopy

    NARCIS (Netherlands)

    H. Meuzelaar

    2015-01-01

    How a protein folds from its one-dimensional sequence of amino acids into its three-dimensional, functional structure on biologically relevant time scales (typically on the micro- to millisecond time scale) is one of the most challenging questions currently investigated in several scientific discipl

  8. Transferable coarse-grained potential for $\\textit{de novo}$ protein folding and design

    CERN Document Server

    Coluzza, Ivan

    2014-01-01

    Protein folding and design are major biophysical problems, the solution of which would lead to important applications especially in medicine. Here a novel protein model capable of simultaneously provide quantitative protein design and folding is introduced. With computer simulations it is shown that, for a large set of real protein structures, the model produces designed sequences with similar physical properties to the corresponding natural occurring sequences. The designed sequences are not yet fully realistic and require further experimental testing. For an independent set of proteins, notoriously difficult to fold, the correct folding of both the designed and the natural sequences is also demonstrated. The folding properties are characterized by free energy calculations. which not only are consistent among natural and designed proteins, but we also show a remarkable precision when the folded structures are compared to the experimentally determined ones. Ultimately, this novel coarse-grained protein model ...

  9. Residual ordered structure in denatured proteins and the problem of protein folding.

    Science.gov (United States)

    Basharov, Mahmud A

    2012-02-01

    Structural characteristics of numerous globular proteins in the denatured state have been reviewed using literature data. Recent more precise experiments show that in contrast to the conventional standpoint, proteins under strongly denaturing conditions do not unfold completely and adopt a random coil state, but contain significant residual ordered structure. These results cast doubt on the basis of the conventional approach representing the process of protein folding as a spontaneous transition of a polypeptide chain from the random coil state to the unique globular structure. The denaturation of proteins is explained in terms of the physical properties of proteins such as stability, conformational change, elasticity, irreversible denaturation, etc. The spontaneous renaturation of some denatured proteins most probably is merely the manifestation of the physical properties (e.g., the elasticity) of the proteins per se, caused by the residual structure present in the denatured state. The pieces of the ordered structure might be the centers of the initiation of renaturation, where the restoration of the initial native conformation of denatured proteins begins. Studies on the denaturation of proteins hardly clarify how the proteins fold into the native conformation during the successive residue-by-residue elongation of the polypeptide chain on the ribosome.

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

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Yingang, E-mail: fengyg@qibebt.ac.cn [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Song, Xiaxia [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Lin, Jinzhong [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xuan, Jinsong [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Cui, Qiu [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Wang, Jinfeng [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

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

  11. 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.

  12. Discrete Nonlinear Schrodinger Equation, Solitons and Organizing Principles for Protein Folding

    CERN Document Server

    Molkenthin, Nora; Niemi, Antti J

    2010-01-01

    We introduce a novel generalization of the discrete nonlinear Schr\\"odinger equation. It supports solitons that describe how proteins fold. As an example we scrutinize the villin headpiece HP35, an archetypal protein for testing both experimental and theoretical approaches to protein folding. Using explicit soliton profiles we construct its carbon backbone with an unprecedented accuracy.

  13. 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…

  14. 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

    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...... demonstrated using a complex insertion mutant library of TNF-alpha, from which different folding competent mutant proteins were uncovered....

  15. A spatio-temporal mining approach towards summarizing and analyzing protein folding trajectories

    Directory of Open Access Journals (Sweden)

    Ucar Duygu

    2007-04-01

    Full Text Available Abstract Understanding the protein folding mechanism remains a grand challenge in structural biology. In the past several years, computational theories in molecular dynamics have been employed to shed light on the folding process. Coupled with high computing power and large scale storage, researchers now can computationally simulate the protein folding process in atomistic details at femtosecond temporal resolution. Such simulation often produces a large number of folding trajectories, each consisting of a series of 3D conformations of the protein under study. As a result, effectively managing and analyzing such trajectories is becoming increasingly important. In this article, we present a spatio-temporal mining approach to analyze protein folding trajectories. It exploits the simplicity of contact maps, while also integrating 3D structural information in the analysis. It characterizes the dynamic folding process by first identifying spatio-temporal association patterns in contact maps, then studying how such patterns evolve along a folding trajectory. We demonstrate that such patterns can be leveraged to summarize folding trajectories, and to facilitate the detection and ordering of important folding events along a folding path. We also show that such patterns can be used to identify a consensus partial folding pathway across multiple folding trajectories. Furthermore, we argue that such patterns can capture both local and global structural topology in a 3D protein conformation, thereby facilitating effective structural comparison amongst conformations. We apply this approach to analyze the folding trajectories of two small synthetic proteins-BBA5 and GSGS (or Beta3S. We show that this approach is promising towards addressing the above issues, namely, folding trajectory summarization, folding events detection and ordering, and consensus partial folding pathway identification across trajectories.

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

    Science.gov (United States)

    Inouye, M; Fu, X; Shinde, U

    2001-04-01

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

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

    Science.gov (United States)

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

    2015-11-13

    Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a (13)C-labeled retinal cofactor and extensively (13)C-(15)N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications.

  18. Coarse-grained models of protein folding: toy models or predictive tools?

    Science.gov (United States)

    Clementi, Cecilia

    2008-02-01

    Coarse-grained models are emerging as a practical alternative to all-atom simulations for the characterization of protein folding mechanisms over long time scales. While a decade ago minimalist toy models were mainly designed to test general hypotheses on the principles regulating protein folding, the latest coarse-grained models are increasingly realistic and can be used to characterize quantitatively the detailed folding mechanism of specific proteins. The ability of such models to reproduce the essential features of folding dynamics suggests that each single atomic degree of freedom is not by itself particularly relevant to folding and supports a statistical mechanical approach to characterize folding transitions. When combined with more refined models and with experimental studies, the systematic investigation of protein systems and complexes using coarse-grained models can advance our theoretical understanding of the actual organizing principles that emerge from the complex network of interactions among protein atomic constituents.

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

    CERN Document Server

    Buijsman, Wouter

    2012-01-01

    Various biological sensory systems exhibit a response to the relative change of the stimulus, often reffered to as fold-change detection. Here, we present a mechanism consisting of two interacting proteins, able to detect a fold-change effectively. This mechanism, in contrast to other proposed mechanisms, does not consume chemical energy and is not subject to transcriptional and translational noise. We show by analytical and numerical calculations that the mechanism can have a fast, precise and efficient response for parameters that are relevant to eukaryotic cells.

  20. 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...

  1. Curvature of the energy landscape and folding of model proteins.

    Science.gov (United States)

    Mazzoni, Lorenzo N; Casetti, Lapo

    2006-11-24

    We study the geometric properties of the energy landscape of coarse-grained, off-lattice models of polymers by endowing the configuration space with a suitable metric, depending on the potential energy function, such that the dynamical trajectories are the geodesics of the metric. Using numerical simulations, we show that the fluctuations of the curvature clearly mark the folding transition, and that this quantity allows to distinguish between polymers having a proteinlike behavior (i.e., that fold to a unique configuration) and polymers which undergo a hydrophobic collapse but do not have a folding transition. These geometrical properties are defined by the potential energy without requiring any prior knowledge of the native configuration.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

  4. Thermodynamics of downhill folding: multi-probe analysis of PDD, a protein that folds over a marginal free energy barrier.

    Science.gov (United States)

    Naganathan, Athi N; Muñoz, Victor

    2014-07-31

    Downhill folding proteins fold in microseconds by crossing a very low or no free energy barrier (energy landscape in detail using multiprobe thermodynamic experiments. This type of thermodynamic behavior has been investigated in depth on the protein BBL, an example of extreme (one-state) downhill folding in which there is no free energy barrier at any condition, including the denaturation midpoint. However, an open question is, to what extent is such thermodynamic behavior observed on less extreme downhill folders? Here we perform a multiprobe spectroscopic characterization of the microsecond folder PDD, a structural and functional homologue of BBL that folds within the downhill regime, but is not an example of one-state downhill folding; rather at the denaturation midpoint PDD folds by crossing an incipient free energy barrier. Model-free analysis of the unfolding curves from four different spectroscopic probes together with differential scanning calorimetry reveals a dispersion of ∼9 K in the apparent melting temperature and also marked differences in unfolding broadness (from ∼50 to ∼130 kJ mol(-1) when analyzed with a two-state model), confirming that such properties are also observed on less extreme downhill folders. We subsequently perform a global quantitative analysis of the unfolding data of PDD using the same ME statistical mechanical model that was used before for the BBL domain. The analysis shows that this simple model captures all of the features observed on the unfolding of PDD (i.e., the intensity and temperature dependence of the different spectroscopic signals). From the model we estimate a free energy landscape for PDD in which the maximal thermodynamic barrier (i.e., at the denaturation midpoint) is only ∼0.5 RT, consistent with previous independent estimates. Our results highlight that multiprobe unfolding experiments in equilibrium combined with statistical mechanical modeling provide important insights into the structural events that

  5. Protein Folding Simulations in a Deformed Energy Landscape

    OpenAIRE

    Hansmann, Ulrich H. E.

    2000-01-01

    A modified version of stochastic tunneling, a recently introduced global optimization technique, is introduced as a new generalized-ensemble technique and tested for a benchmark peptide, Met-enkephalin. It is demonstrated that the new technique allows to evaluate folding properties of this peptide

  6. 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

    2017-03-13

    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.

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

    Science.gov (United States)

    Weikl, Thomas R; Dill, Ken A

    2003-06-01

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

  8. 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 folding reaction under different conditions, as seen for the PDZ protein domain family. We have here analyzed the folding kinetics of two PDZ domains and found that a previously undetected third transition state is rate limiting under conditions that stabilize the native state relative to the denatured...

  9. How a spatial arrangement of secondary structure elements is dispersed in the universe of protein folds.

    Directory of Open Access Journals (Sweden)

    Shintaro Minami

    Full Text Available It has been known that topologically different proteins of the same class sometimes share the same spatial arrangement of secondary structure elements (SSEs. However, the frequency by which topologically different structures share the same spatial arrangement of SSEs is unclear. It is important to estimate this frequency because it provides both a deeper understanding of the geometry of protein folds and a valuable suggestion for predicting protein structures with novel folds. Here we clarified the frequency with which protein folds share the same SSE packing arrangement with other folds, the types of spatial arrangement of SSEs that are frequently observed across different folds, and the diversity of protein folds that share the same spatial arrangement of SSEs with a given fold, using a protein structure alignment program MICAN, which we have been developing. By performing comprehensive structural comparison of SCOP fold representatives, we found that approximately 80% of protein folds share the same spatial arrangement of SSEs with other folds. We also observed that many protein pairs that share the same spatial arrangement of SSEs belong to the different classes, often with an opposing N- to C-terminal direction of the polypeptide chain. The most frequently observed spatial arrangement of SSEs was the 2-layer α/β packing arrangement and it was dispersed among as many as 27% of SCOP fold representatives. These results suggest that the same spatial arrangements of SSEs are adopted by a wide variety of different folds and that the spatial arrangement of SSEs is highly robust against the N- to C-terminal direction of the polypeptide chain.

  10. The folding type of a protein is relevant to the amino acid composition

    OpenAIRE

    Nakashima, Hiroshi; Nishikawa, Ken; Ooi, Tatsuo

    1986-01-01

    The folding types of 135 proteins, the three-dimensional structures of which are known, were analyzed in terms of the amino acid composition. The amino acid composition of a protein was expressed as a point in a multidimensional space spanned with 20 axes, on which the corresponding contents of 20 amino acids in the protein were represented. The distribution pattern of proteins in this composition space was examined in relation to five folding types, , ß, /ß, +ß, and irregular type. The resul...

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

    DEFF Research Database (Denmark)

    Højgaard, Casper; Kofoed, Christian; Espersen, Roall;

    2016-01-01

    Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable ...

  12. Cleavage Mapping the Topology of Protein Folding Intermediates

    Science.gov (United States)

    2007-11-02

    investigate the changes that occur in two of these mutants. V66L has a greatly lowered m value while that of A90S is substantially increased (5...stability of the folded state of nuclease. The cleavage technique will be used to investigate the changes that occur in two of these mutants. V66L...Connecticut, 06520 3Instituto de Qufmica y Fisicoquimica Biolögicas, Facultad de Farmacia y Bioqufmica (UBA-CONICET), Buenos Aires, Argentina 4

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

    DEFF Research Database (Denmark)

    Højgaard, Casper; Kofoed, Christian; Espersen, Roall;

    2016-01-01

    Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable...... 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...

  14. 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.

  15. Importance of native-state topology for determining the folding rate of two-state proteins.

    Science.gov (United States)

    Gromiha, M Michael

    2003-01-01

    Understanding the relationship between amino acid sequences and folding rate of proteins is a challenging task similar to protein folding problem. In this work, we have analyzed the relative importance of protein sequence and structure for predicting the protein folding rates in terms of amino acid properties and contact distances, respectively. We found that the parameters derived with protein sequence (physical-chemical, energetic, and conformational properties of amino acid residues) show very weak correlation (|r| proteins, indicating that the sequence information alone is not sufficient to understand the folding rates of two-state proteins. However, the maximum positive correlation obtained for the properties, number of medium-range contacts, and alpha-helical tendency reveals the importance of local interactions to initiate protein folding. On the other hand, a remarkable correlation (r varies from -0.74 to -0.88) has been obtained between structural parameters (contact order, long-range order, and total contact distance) and protein folding rates. Further, we found that the secondary structure content and solvent accessibility play a marginal role in determining the folding rates of two-state proteins. Multiple regression analysis carried out with the combination of three properties, beta-strand tendency, enthalpy change, and total contact distance improved the correlation to 0.92 with protein folding rates. The relative importance of existing methods along with multiple-regression model proposed in this work will be discussed. Our results demonstrate that the native-state topology is the major determinant for the folding rates of two-state proteins.

  16. 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.

  17. Folding of Aggregated Proteins to Functionally Active Form

    Science.gov (United States)

    2006-06-01

    proteins. In general, these expression systems can be divided into three groups on the basis of the host used: bacterial, insect or yeast, and...translational modifications, and the high cost of production [1]. Yeast or insect cells typically provide faster and cheaper systems for protein production...of reducing agents such as DTT, reduced glutathione or phosphine derivatives like Tris(2-carboxyethyl)pho- sphine (TCEP). Proteins are typically

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

    Science.gov (United States)

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

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

  19. Funnels, Pathways and the Energy Landscape of Protein Folding A Synthesis

    CERN Document Server

    Bryngelson, J D; Socci, N D; Wolynes, P G

    1995-01-01

    The understanding, and even the description of protein folding is impeded by the complexity of the process. Much of this complexity can be described and understood by taking a statistical approach to the energetics of protein conformation, that is, to the energy landscape. The statistical energy landscape approach explains when and why unique behaviors, such as specific folding pathways, occur in some proteins and more generally explains the distinction between folding processes common to all sequences and those peculiar to individual sequences. This approach also gives new, quantitative insights into the interpretation of experiments and simulations of protein folding thermodynamics and kinetics. Specifically, the picture provides simple explanations for folding as a two-state first-order phase transition, for the origin of metastable collapsed unfolded states and for the curved Arrhenius plots observed in both laboratory experiments and discrete lattice simulations. The relation of these quantitative ideas ...

  20. Inversion of the balance between hydrophobic and hydrogen bonding interactions in protein folding and aggregation.

    Directory of Open Access Journals (Sweden)

    Anthony W Fitzpatrick

    2011-10-01

    Full Text Available Identifying the forces that drive proteins to misfold and aggregate, rather than to fold into their functional states, is fundamental to our understanding of living systems and to our ability to combat protein deposition disorders such as Alzheimer's disease and the spongiform encephalopathies. We report here the finding that the balance between hydrophobic and hydrogen bonding interactions is different for proteins in the processes of folding to their native states and misfolding to the alternative amyloid structures. We find that the minima of the protein free energy landscape for folding and misfolding tend to be respectively dominated by hydrophobic and by hydrogen bonding interactions. These results characterise the nature of the interactions that determine the competition between folding and misfolding of proteins by revealing that the stability of native proteins is primarily determined by hydrophobic interactions between side-chains, while the stability of amyloid fibrils depends more on backbone intermolecular hydrogen bonding interactions.

  1. 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

    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...

  2. 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.

  3. Ring Separation Highlights the Protein-Folding Mechanism Used by the Phage EL-Encoded Chaperonin.

    Science.gov (United States)

    Molugu, Sudheer K; Hildenbrand, Zacariah L; Morgan, David Gene; Sherman, Michael B; He, Lilin; Georgopoulos, Costa; Sernova, Natalia V; Kurochkina, Lidia P; Mesyanzhinov, Vadim V; Miroshnikov, Konstantin A; Bernal, Ricardo A

    2016-04-05

    Chaperonins are ubiquitous, ATP-dependent protein-folding molecular machines that are essential for all forms of life. Bacteriophage φEL encodes its own chaperonin to presumably fold exceedingly large viral proteins via profoundly different nucleotide-binding conformations. Our structural investigations indicate that ATP likely binds to both rings simultaneously and that a misfolded substrate acts as the trigger for ATP hydrolysis. More importantly, the φEL complex dissociates into two single rings resulting from an evolutionarily altered residue in the highly conserved ATP-binding pocket. Conformational changes also more than double the volume of the single-ring internal chamber such that larger viral proteins are accommodated. This is illustrated by the fact that φEL is capable of folding β-galactosidase, a 116-kDa protein. Collectively, the architecture and protein-folding mechanism of the φEL chaperonin are significantly different from those observed in group I and II chaperonins.

  4. Statistical Analysis of Native Contact Formation in the Folding of Designed Model Proteins

    OpenAIRE

    Tiana, G.; R. A. Broglia(University of Milano, INFN Milano and University of Copenhagen)

    2000-01-01

    The time evolution of the formation probability of native bonds has been studied for designed sequences which fold fast into the native conformation. From this analysis a clear hierarchy of bonds emerge a) local, fast forming highly stable native bonds built by some of the most strongly interacting amino acids of the protein, b) non-local bonds formed late in the folding process, in coincidence with the folding nucleus, and involving essentially the same strongly interacting amino acids alrea...

  5. Improvement of the relative entropy based protein folding method

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The "relative entropy" has been used as a minimization function to predict the tertiary structure of a protein backbone, and good results have been obtained. However, in our previous work, the ensemble average of the contact potential was estimated by an approximate calculation. In order to improve the theoretical integrity of the relative-entropy-based method, a new theoretical calculation method of the ensemble average of the contact potential was presented in this work, which is based on the thermodynamic perturbation theory. Tests of the improved algorithm were performed on twelve small proteins. The root mean square deviations of the predicted versus the native structures from Protein Data Bank range from 0.40 to 0.60 nm. Compared with the previous approximate values, the average prediction accuracy is improved by 0.04 nm.

  6. Improvement of the relative entropy based protein folding method

    Institute of Scientific and Technical Information of China (English)

    QI LiSheng; SU JiGuo; CHEN WeiZu; WANG CunXin

    2009-01-01

    The "relative entropy" has been used as a minimization function to predict the tertiary structure of a protein backbone, and good results have been obtained. However, in our previous work, the ensemble average of the contact potential was estimated by an approximate calculation. In order to improve the theoretical integrity of the relative-entropy-based method, a new theoretical calculation method of the ensemble average of the contact potential was presented in this work, which is based on the thermodynamic perturbation theory. Testa of the improved algorithm were performed on twelve small proteins. The root mean square deviations of the predicted versus the native structures from Protein Data Bank range from 0.40 to 0.60 nm. Compared with the previous approximate values, the average prediction accuracy is improved by 0.04 nm.

  7. Folding and stability of helical bundle proteins from coarse-grained models.

    Science.gov (United States)

    Kapoor, Abhijeet; Travesset, Alex

    2013-07-01

    We develop a coarse-grained model where solvent is considered implicitly, electrostatics are included as short-range interactions, and side-chains are coarse-grained to a single bead. The model depends on three main parameters: hydrophobic, electrostatic, and side-chain hydrogen bond strength. The parameters are determined by considering three level of approximations and characterizing the folding for three selected proteins (training set). Nine additional proteins (containing up to 126 residues) as well as mutated versions (test set) are folded with the given parameters. In all folding simulations, the initial state is a random coil configuration. Besides the native state, some proteins fold into an additional state differing in the topology (structure of the helical bundle). We discuss the stability of the native states, and compare the dynamics of our model to all atom molecular dynamics simulations as well as some general properties on the interactions governing folding dynamics.

  8. BiP Clustering Facilitates Protein Folding in the Endoplasmic Reticulum

    Science.gov (United States)

    Robinson, Anne S.; Petzold, Linda

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-07-01

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

  10. Protein folding and translocation : single-molecule investigations

    NARCIS (Netherlands)

    Leeuwen, Rudolphus Gerardus Henricus van

    2006-01-01

    This thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a protein that was being unfolded and refolded using our optical tweezers setup. Our measurements clearly

  11. 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

  12. Understanding the influence of codon translation rates on cotranslational protein folding.

    Science.gov (United States)

    O'Brien, Edward P; Ciryam, Prajwal; Vendruscolo, Michele; Dobson, Christopher M

    2014-05-20

    Protein domains can fold into stable tertiary structures while they are synthesized by the ribosome in a process known as cotranslational folding. If a protein does not fold cotranslationally, however, it has the opportunity to do so post-translationally, that is, after the nascent chain has been fully synthesized and released from the ribosome. The rate at which a ribosome adds an amino acid encoded by a particular codon to the elongating nascent chain can vary significantly and is called the codon translation rate. Recent experiments have illustrated the profound impact that codon translation rates can have on the cotranslational folding process and the acquisition of function by nascent proteins. Synonymous codon mutations in an mRNA molecule change the chemical identity of a codon and its translation rate without changing the sequence of the synthesized protein. This change in codon translation rate can, however, cause a nascent protein to malfunction as a result of cotranslational misfolding. In some situations, such dysfunction can have profound implications; for example, it can alter the substrate specificity of an ABC transporter protein, resulting in patients who are nonresponsive to chemotherapy treatment. Thus, codon translation rates are crucial in coordinating protein folding in a cellular environment and can affect downstream cellular processes that depend on the proper functioning of newly synthesized proteins. As the importance of codon translation rates makes clear, a necessary aspect of fully understanding cotranslational folding lies in considering the kinetics of the process in addition to its thermodynamics. In this Account, we examine the contributions that have been made to elucidating the mechanisms of cotranslational folding by using the theoretical and computational tools of chemical kinetics, molecular simulations, and systems biology. These efforts have extended our ability to understand, model, and predict the influence of codon

  13. 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.

  14. 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

  15. The folding type of a protein is relevant to the amino acid composition.

    Science.gov (United States)

    Nakashima, H; Nishikawa, K; Ooi, T

    1986-01-01

    The folding types of 135 proteins, the three-dimensional structures of which are known, were analyzed in terms of the amino acid composition. The amino acid composition of a protein was expressed as a point in a multidimensional space spanned with 20 axes, on which the corresponding contents of 20 amino acids in the protein were represented. The distribution pattern of proteins in this composition space was examined in relation to five folding types, alpha, beta, alpha/beta, alpha + beta, and irregular type. The results show that amino acid compositions of the alpha, beta, and alpha/beta types are located in different regions in the composition space, thus allowing distinct separation of proteins depending on the folding types. The points representing proteins of the alpha + beta and irregular types, however, are widely scattered in the space, and the existing regions overlap with those of the other folding types. A simple method of utilizing the "distance" in the space was found to be convenient for classification of proteins into the five folding types. The assignment of the folding type with this method gave an accuracy of 70% in the coincidence with the experimental data.

  16. Influence of denatured and intermediate states of folding on protein aggregation.

    Science.gov (United States)

    Fawzi, Nicolas L; Chubukov, Victor; Clark, Louis A; Brown, Scott; Head-Gordon, Teresa

    2005-04-01

    We simulate the aggregation thermodynamics and kinetics of proteins L and G, each of which self-assembles to the same alpha/beta [corrected] topology through distinct folding mechanisms. We find that the aggregation kinetics of both proteins at an experimentally relevant concentration exhibit both fast and slow aggregation pathways, although a greater proportion of protein G aggregation events are slow relative to those of found for protein L. These kinetic differences are correlated with the amount and distribution of intrachain contacts formed in the denatured state ensemble (DSE), or an intermediate state ensemble (ISE) if it exists, as well as the folding timescales of the two proteins. Protein G aggregates more slowly than protein L due to its rapidly formed folding intermediate, which exhibits native intrachain contacts spread across the protein, suggesting that certain early folding intermediates may be selected for by evolution due to their protective role against unwanted aggregation. Protein L shows only localized native structure in the DSE with timescales of folding that are commensurate with the aggregation timescale, leaving it vulnerable to domain swapping or nonnative interactions with other chains that increase the aggregation rate. Folding experiments that characterize the structural signatures of the DSE, ISE, or the transition state ensemble (TSE) under nonaggregating conditions should be able to predict regions where interchain contacts will be made in the aggregate, and to predict slower aggregation rates for proteins with contacts that are dispersed across the fold. Since proteins L and G can both form amyloid fibrils, this work also provides mechanistic and structural insight into the formation of prefibrillar species.

  17. Role of local and nonlocal interactions in folding and misfolding of globular proteins

    Science.gov (United States)

    Kumar, Adesh; Baruah, Anupaul; Biswas, Parbati

    2017-02-01

    A Monte Carlo simulation based sequence design method is proposed to study the role of the local and the nonlocal interactions with varying secondary structure content in protein folding, misfolding and unfolding. A statistical potential is developed from the compilation of a data set of proteins, which accounts for the respective contribution of local and the nonlocal interactions. Sequences are designed through a combination of positive and negative design by a Monte Carlo simulation in the sequence space. The weights of the local and the nonlocal interactions are tuned appropriately to study the role of the local and the nonlocal interactions in the folding, unfolding and misfolding of the designed sequences. Results suggest that the nonlocal interactions are the primary determinant of protein folding while the local interactions may be required but not always necessary. The nonlocal interactions mainly guide the polypeptide chain to form compact structures but do not differentiate between the native-like conformations, while the local interactions stabilize the target conformation against the native-like competing conformations. The study concludes that the local interactions govern the fold-misfold transition, while the nonlocal interactions regulate the fold-unfold transition of proteins. However, for proteins with predominantly β-sheet content, the nonlocal interactions control both fold-misfold and fold-unfold transitions.

  18. Probing possible downhill folding: native contact topology likely places a significant constraint on the folding cooperativity of proteins with approximately 40 residues.

    Science.gov (United States)

    Badasyan, Artem; Liu, Zhirong; Chan, Hue Sun

    2008-12-12

    Experiments point to appreciable variations in folding cooperativity among natural proteins with approximately 40 residues, indicating that the behaviors of these proteins are valuable for delineating the contributing factors to cooperative folding. To explore the role of native topology in a protein's propensity to fold cooperatively and how native topology might constrain the degree of cooperativity achievable by a given set of physical interactions, we compared folding/unfolding kinetics simulated using three classes of native-centric C(alpha) chain models with different interaction schemes. The approach was applied to two homologous 45-residue fragments from the peripheral subunit-binding domain family and a 39-residue fragment of the N-terminal domain of ribosomal protein L9. Free-energy profiles as functions of native contact number were computed to assess the heights of thermodynamic barriers to folding. In addition, chevron plots of folding/unfolding rates were constructed as functions of native stability to facilitate comparison with available experimental data. Although common Gō-like models with pairwise Lennard-Jones-type interactions generally fold less cooperatively than real proteins, the rank ordering of cooperativity predicted by these models is consistent with experiment for the proteins investigated, showing increasing folding cooperativity with increasing nonlocality of a protein's native contacts. Models that account for water-expulsion (desolvation) barriers and models with many-body (nonadditive) interactions generally entail higher degrees of folding cooperativity indicated by more linear model chevron plots, but the rank ordering of cooperativity remains unchanged. A robust, experimentally valid rank ordering of model folding cooperativity independent of the multiple native-centric interaction schemes tested here argues that native topology places significant constraints on how cooperatively a protein can fold.

  19. Detecting Selection on Protein Stability through Statistical Mechanical Models of Folding and Evolution

    Directory of Open Access Journals (Sweden)

    Ugo Bastolla

    2014-03-01

    Full Text Available The properties of biomolecules depend both on physics and on the evolutionary process that formed them. These two points of view produce a powerful synergism. Physics sets the stage and the constraints that molecular evolution has to obey, and evolutionary theory helps in rationalizing the physical properties of biomolecules, including protein folding thermodynamics. To complete the parallelism, protein thermodynamics is founded on the statistical mechanics in the space of protein structures, and molecular evolution can be viewed as statistical mechanics in the space of protein sequences. In this review, we will integrate both points of view, applying them to detecting selection on the stability of the folded state of proteins. We will start discussing positive design, which strengthens the stability of the folded against the unfolded state of proteins. Positive design justifies why statistical potentials for protein folding can be obtained from the frequencies of structural motifs. Stability against unfolding is easier to achieve for longer proteins. On the contrary, negative design, which consists in destabilizing frequently formed misfolded conformations, is more difficult to achieve for longer proteins. The folding rate can be enhanced by strengthening short-range native interactions, but this requirement contrasts with negative design, and evolution has to trade-off between them. Finally, selection can accelerate functional movements by favoring low frequency normal modes of the dynamics of the native state that strongly correlate with the functional conformation change.

  20. Equilibrium distribution from distributed computing (simulations of protein folding).

    Science.gov (United States)

    Scalco, Riccardo; Caflisch, Amedeo

    2011-05-19

    Multiple independent molecular dynamics (MD) simulations are often carried out starting from a single protein structure or a set of conformations that do not correspond to a thermodynamic ensemble. Therefore, a significant statistical bias is usually present in the Markov state model generated by simply combining the whole MD sampling into a network whose nodes and links are clusters of snapshots and transitions between them, respectively. Here, we introduce a depth-first search algorithm to extract from the whole conformation space network the largest ergodic component, i.e., the subset of nodes of the network whose transition matrix corresponds to an ergodic Markov chain. For multiple short MD simulations of a globular protein (as in distributed computing), the steady state, i.e., stationary distribution determined using the largest ergodic component, yields more accurate free energy profiles and mean first passage times than the original network or the ergodic network obtained by imposing detailed balance by means of symmetrization of the transition counts.

  1. 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

    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...... 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......, 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....

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

    Science.gov (United States)

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

    2012-03-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2002-05-01

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

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

    Science.gov (United States)

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

    2016-01-28

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

  6. The Emerging Roles of Early Protein Folding Events in the Secretory Pathway in the Development of Neurodegenerative Maladies

    Science.gov (United States)

    Dubnikov, Tatyana; Cohen, Ehud

    2017-01-01

    Although, protein aggregation and deposition are unifying features of various neurodegenerative disorders, recent studies indicate that different mechanisms can lead to the development of the same malady. Among these, failure in early protein folding and maturation emerge as key mechanistic events that lead to the manifestation of a myriad of illnesses including Alzheimer's disease and prion disorders. Here we delineate the cascade of maturation steps that nascent polypeptides undergo in the secretory pathway to become functional proteins, and the chaperones that supervise and assist this process, focusing on the subgroup of proline cis/trans isomerases. We also describe the chaperones whose failure was found to be an underlying event that initiates the run-up toward neurodegeneration as well as chaperones whose activity impairs protein homeostasis (proteostasis) and thus, promotes the manifestation of these maladies. Finally, we discuss the roles of aggregate deposition sites in the cellular attempt to maintain proteostasis and point at potential targets for therapeutic interventions. PMID:28223916

  7. A protein block based fold recognition method for the annotation of twilight zone sequences.

    Science.gov (United States)

    Suresh, V; Ganesan, K; Parthasarathy, S

    2013-03-01

    The description of protein backbone was recently improved with a group of structural fragments called Structural Alphabets instead of the regular three states (Helix, Sheet and Coil) secondary structure description. Protein Blocks is one of the Structural Alphabets used to describe each and every region of protein backbone including the coil. According to de Brevern (2000) the Protein Blocks has 16 structural fragments and each one has 5 residues in length. Protein Blocks fragments are highly informative among the available Structural Alphabets and it has been used for many applications. Here, we present a protein fold recognition method based on Protein Blocks for the annotation of twilight zone sequences. In our method, we align the predicted Protein Blocks of a query amino acid sequence with a library of assigned Protein Blocks of 953 known folds using the local pair-wise alignment. The alignment results with z-value ≥ 2.5 and P-value ≤ 0.08 are predicted as possible folds. Our method is able to recognize the possible folds for nearly 35.5% of the twilight zone sequences with their predicted Protein Block sequence obtained by pb_prediction, which is available at Protein Block Export server.

  8. Protein fold determination from sparse distance restraints: The restrained generic protein direct Monte Carlo method

    Energy Technology Data Exchange (ETDEWEB)

    Debe, D.A.; Carlson, M.J.; Chan, S.I; Goddard, W.A. III [California Inst. of Tech., Pasadena, CA (United States); Sadanobu, Jiro [Teijin Limited, Iwakuni, Yamaguchi (Japan). Polymer and Materials Research Labs.

    1999-04-15

    The authors present the generate-and-select hierarchy for tertiary protein structure prediction. The foundation of this hierarchy is the Restrained Generic Protein (RGP) Direct Monte Carlo method. The RGP method is a highly efficient off-lattice residue buildup procedure that can quickly generate the complete set of topologies that satisfy a very small number of interresidue distance restraints. For three restraints uniformly distributed in a 72-residue protein, the authors demonstrate that the size of this set is {approximately}10{sup 4}. The RGP method can generate this set of structures in less than 1 h using a Silicon Graphics R10000 single processor workstation. Following structure generation, a simple criterion that measures the burial of hydrophobic and hydrophilic residues can reliably select a reduced set of {approximately}10{sup 2} structures that contains the native topology. A minimization of the structures in the reduced set typically ranks the native topology in the five lowest energy folds. Thus, using this hierarchical approach, the authors suggest that de novo prediction of moderate resolution globular protein structure can be achieved in just a few hours on a single processor workstation.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

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

    2014-07-01

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

  11. Chapter 3: A fluorescent window into protein folding and aggregation in cells.

    Science.gov (United States)

    Ignatova, Zoya; Gierasch, Lila M

    2008-01-01

    Evolutionary selective pressures have tuned the efficiency of the protein-folding reaction in the crowded complex environment in the cell. Nevertheless, the fidelity of folding is imperfect, leading to off-pathway intermolecular interactions that compete with proper folding and to consequent formation of thermodynamically stable aggregates. Such aggregates constitute the histopathological hallmarks of many neurodegenerative pathologies. Yet, most of the approaches to characterize protein folding and/or misfolding are limited to in vitro conditions. Here, we describe a strategy to directly monitor the behavior of a protein in prokaryotic and eukaryotic cells. The method is based on incorporation of structurally non-perturbing, specific binding motifs for a bis-arsenical fluoroscein dye, FlAsH, in sites that result in distinct dye fluorescence signals for the folded and unfolded states of the protein under study. Our approach has been developed using as a case study the predominantly beta-sheet intracellular lipid-binding protein, cellular retinoic acid-binding protein, alone or as a chimera fused to the exon 1-encoded fragment of huntingtin, which harbors a polyglutamine repeat tract. We have designed protocols to label this protein in vivo and to monitor the resulting fluorescence signal, which reports on any misfolding transition and formation of aggregates, yielding quantitatively interpretable data.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, P. J.; Lai, S. K., E-mail: sklai@coll.phy.ncu.edu.tw [Complex Liquids Laboratory, Department of Physics, National Central University, Chungli 320 Taiwan (China); Molecular Science and Technology Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan (China); Cheong, S. A. [Division of Physics and Applied Physics, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371 (Singapore)

    2014-05-28

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

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

    Science.gov (United States)

    González-Charro, Vicente; Rey, Antonio

    2014-09-01

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

  14. 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.

  15. Atomistic protein folding simulations on the submillisecond time scale using worldwide distributed computing.

    Science.gov (United States)

    Pande, Vijay S; Baker, Ian; Chapman, Jarrod; Elmer, Sidney P; Khaliq, Siraj; Larson, Stefan M; Rhee, Young Min; Shirts, Michael R; Snow, Christopher D; Sorin, Eric J; Zagrovic, Bojan

    2003-01-01

    Atomistic simulations of protein folding have the potential to be a great complement to experimental studies, but have been severely limited by the time scales accessible with current computer hardware and algorithms. By employing a worldwide distributed computing network of tens of thousands of PCs and algorithms designed to efficiently utilize this new many-processor, highly heterogeneous, loosely coupled distributed computing paradigm, we have been able to simulate hundreds of microseconds of atomistic molecular dynamics. This has allowed us to directly simulate the folding mechanism and to accurately predict the folding rate of several fast-folding proteins and polymers, including a nonbiological helix, polypeptide alpha-helices, a beta-hairpin, and a three-helix bundle protein from the villin headpiece. Our results demonstrate that one can reach the time scales needed to simulate fast folding using distributed computing, and that potential sets used to describe interatomic interactions are sufficiently accurate to reach the folded state with experimentally validated rates, at least for small proteins.

  16. Protein Folding under Mediation of Ordering Water: an Off-Lattice Gō-Like Model Study

    Institute of Scientific and Technical Information of China (English)

    ZUO Guang-Hong; HU Jun; FANG Hai-Ping

    2007-01-01

    @@ Water plays an important role in the structure and function of biomolecules. Water confined at the nanoscale usually exhibits phenomena not seen in bulk water, including the ice-like ordering structure on the surfaces of many substrates. We investigate the behaviour of protein folding in which the proteins are asssumed in an environment with ordering water by using of an off-lattice Gō-like model. It is found that in the physiological temperature, both the folding rate and the thermodynamic stability of the protein are greatly promoted by the existence of ordering of water.

  17. The role of proline in the prevention of aggregation during protein folding in vitro.

    Science.gov (United States)

    Kumar, T K; Samuel, D; Jayaraman, G; Srimathi, T; Yu, C

    1998-10-01

    Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glycine and ethylene glycol fail to exhibit the 'aggregation-blockade' role shown by proline. Results of viscosity and ANS fluorescence (1-anilino-8-naphthalene sulphonic acid) experiments suggest that proline at high concentrations forms an ordered supramolecular assembly. Based on these results, it is proposed that proline behaves as a protein folding chaperone due to the formation of an ordered, amphipathic supramolecular assembly. To our knowledge, this is the first report wherein proline is proposed as a protein folding aid.

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

    Science.gov (United States)

    Martínez, Leandro

    2014-01-01

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

  19. 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.

  20. 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 dive...

  1. 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.

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    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. Transition Pathway and Its Free-Energy Profile: A Protocol for Protein Folding Simulations

    Directory of Open Access Journals (Sweden)

    In-Ho Lee

    2013-08-01

    Full Text Available We propose a protocol that provides a systematic definition of reaction coordinate and related free-energy profile as the function of temperature for the protein-folding simulation. First, using action-derived molecular dynamics (ADMD, we investigate the dynamic folding pathway model of a protein between a fixed extended conformation and a compact conformation. We choose the pathway model to be the reaction coordinate, and the folding and unfolding processes are characterized by the ADMD step index, in contrast to the common a priori reaction coordinate as used in conventional studies. Second, we calculate free-energy profile as the function of temperature, by employing the replica-exchange molecular dynamics (REMD method. The current method provides efficient exploration of conformational space and proper characterization of protein folding/unfolding dynamics from/to an arbitrary extended conformation. We demonstrate that combination of the two simulation methods, ADMD and REMD, provides understanding on molecular conformational changes in proteins. The protocol is tested on a small protein, penta-peptide of met-enkephalin. For the neuropeptide met-enkephalin system, folded, extended, and intermediate sates are well-defined through the free-energy profile over the reaction coordinate. Results are consistent with those in the literature.

  5. Statistical analysis of native contact formation in the folding of designed model proteins

    Science.gov (United States)

    Tiana, Guido; Broglia, Ricardo A.

    2001-02-01

    The time evolution of the formation probability of native bonds has been studied for designed sequences which fold fast into the native conformation. From this analysis a clear hierarchy of bonds emerge: (a) local, fast forming highly stable native bonds built by some of the most strongly interacting amino acids of the protein; (b) nonlocal bonds formed late in the folding process, in coincidence with the folding nucleus, and involving essentially the same strongly interacting amino acids already participating in the fast bonds; (c) the rest of the native bonds whose behavior is subordinated, to a large extent, to that of the strong local and nonlocal native contacts.

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

    Science.gov (United States)

    Guerriero, Christopher J; Brodsky, Jeffrey L

    2012-04-01

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

  7. A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter*

    OpenAIRE

    2014-01-01

    Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase...

  8. Protein folding, unfolding and aggregation. Pressure induced intermediate states on the refolding pathway of horseradish peroxidase

    Science.gov (United States)

    Smeller, László; Fidy, Judit; Heremans, Karel

    2004-04-01

    We studied the refolding and aggregation of pressure unfolded proteins. Horseradish peroxidase was found to be very stable and no partially folded intermediates were populated during the refolding. However, the removal of the haem group or the Ca2+ ions or reduction of the disulfide bridge destabilized the protein, resulting in a significant amount of aggregation prone intermediate conformation. Substitution of the haem for fluorescent porphyrin however did not influence the refolding of the protein.

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

    Science.gov (United States)

    Cetinbaş, Murat; Shakhnovich, Eugene I

    2013-01-01

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

  10. 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.

  11. 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.

  12. Protein folds recognized by an intelligent predictor based-on evolutionary and structural information.

    Science.gov (United States)

    Cheung, Ngaam J; Ding, Xue-Ming; Shen, Hong-Bin

    2016-02-05

    Protein fold recognition is an important and essential step in determining tertiary structure of a protein in biological science. In this study, a model termed NiRecor is developed for recognizing protein folds based on artificial neural networks incorporated in an adaptive heterogeneous particle swarm optimizer. The main contribution of NiRecor is that it is a data-driven and highly-performing predictor without manually tuning control parameters for different data sets. In biological science, since evolutionary- and structure-based information of amino acid sequences is greatly important in determination of tertiary structure of a protein, accordingly, in NiRecor we employ two different feature sets, which involve position specific scoring matrix and secondary structure prediction matrix, to predict the structural classes of protein folds. The experimental results demonstrate the proposed method is powerful in predicting protein folds with higher precisions by improvements of 1.1 ∼7.8 percentages on three benchmark datasets by comparing with several existing predictors.

  13. Role of N-terminal region of Escherichia coli maltodextrin glucosidase in folding and function of the protein.

    Science.gov (United States)

    Pastor, Ashutosh; Singh, Amit K; Shukla, Prakash K; Equbal, Md Javed; Malik, Shikha T; Singh, Tej P; Chaudhuri, Tapan K

    2016-09-01

    Maltodextrin glucosidase (MalZ) hydrolyses short malto-oligosaccharides from the reducing end releasing glucose and maltose in Escherichia coli. MalZ is a highly aggregation prone protein and molecular chaperonins GroEL and GroES assist in the folding of this protein to a substantial level. The N-terminal region of this enzyme appears to be a unique domain as seen in sequence comparison studies with other amylases as well as through homology modelling. The sequence and homology model analysis show a probability of disorder in the N-Terminal region of MalZ. The crystal structure of this enzyme has been reported in the present communication. Based on the crystallographic structure, it has been interpreted that the N-terminal region of the enzyme (Met1-Phe131) might be unstructured or flexible. To understand the role of the N-terminal region of MalZ in its enzymatic activity, and overall stability, a truncated version (Ala111-His616) of MalZ was created. The truncated version failed to fold into an active enzyme both in E. coli cytosol and in vitro even with the assistance of chaperonins GroEL and GroES. Furthermore, the refolding effort of N-truncated MalZ in the presence of isolated N-terminal domain didn't succeed. Our studies suggest that while the structural rigidity or orientation of the N-terminal region of the MalZ protein may not be essential for its stability and function, but the said domain is likely to play an important role in the formation of the native structure of the protein when present as an integral part of the protein.

  14. 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.

  15. 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+).

  16. Supercoiled DNA folded by nonhistone proteins in cultured mouse carcinoma cells.

    Science.gov (United States)

    Nakane, M; Ide, T; Anzai, K; Ohara, S; Andoh, T

    1978-07-01

    Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with gamma-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.

  17. Topology-based modeling of intrinsically disordered proteins: balancing intrinsic folding and intermolecular interactions.

    Science.gov (United States)

    Ganguly, Debabani; Chen, Jianhan

    2011-04-01

    Coupled binding and folding is frequently involved in specific recognition of so-called intrinsically disordered proteins (IDPs), a newly recognized class of proteins that rely on a lack of stable tertiary fold for function. Here, we exploit topology-based Gō-like modeling as an effective tool for the mechanism of IDP recognition within the theoretical framework of minimally frustrated energy landscape. Importantly, substantial differences exist between IDPs and globular proteins in both amino acid sequence and binding interface characteristics. We demonstrate that established Gō-like models designed for folded proteins tend to over-estimate the level of residual structures in unbound IDPs, whereas under-estimating the strength of intermolecular interactions. Such systematic biases have important consequences in the predicted mechanism of interaction. A strategy is proposed to recalibrate topology-derived models to balance intrinsic folding propensities and intermolecular interactions, based on experimental knowledge of the overall residual structure level and binding affinity. Applied to pKID/KIX, the calibrated Gō-like model predicts a dominant multistep sequential pathway for binding-induced folding of pKID that is initiated by KIX binding via the C-terminus in disordered conformations, followed by binding and folding of the rest of C-terminal helix and finally the N-terminal helix. This novel mechanism is consistent with key observations derived from a recent NMR titration and relaxation dispersion study and provides a molecular-level interpretation of kinetic rates derived from dispersion curve analysis. These case studies provide important insight into the applicability and potential pitfalls of topology-based modeling for studying IDP folding and interaction in general.

  18. Protein folding of the SAP domain, a naturally occurring two-helix bundle.

    Science.gov (United States)

    Dodson, Charlotte A; Arbely, Eyal

    2015-07-01

    The SAP domain from the Saccharomyces cerevisiae Tho1 protein is comprised of just two helices and a hydrophobic core and is one of the smallest proteins whose folding has been characterised. Φ-value analysis revealed that Tho1 SAP folds through a transition state where helix 1 is the most extensively formed element of secondary structure and flickering native-like core contacts from Leu35 are also present. The contacts that contribute most to native state stability of Tho1 SAP are not formed in the transition state.

  19. A Branch and Bound Algorithm for the Protein Folding Problem in the HP Lattice Model

    Institute of Scientific and Technical Information of China (English)

    Mao Chen; Wen-Qi Huang

    2005-01-01

    A branch and bound algorithm is proposed for the two-dimensional protein folding problem in the HP lattice model. In this algorithm, the benefit of each possible location of hydrophobic monomers is evaluated and only promising nodes are kept for further branching at each level. The proposed algorithm is compared with other well-known methods for 10 benchmark sequences with lengths ranging from 20 to 100 monomers. The results indicate that our method is a very efficient and promising tool for the protein folding problem.

  20. Purification and characterization of oligonucleotide binding (OB)-fold protein from medicinal plant Tinospora cordifolia.

    Science.gov (United States)

    Amir, Mohd; Haque, Md Anzarul; Wahiduzzaman; Dar, Mohammad Aasif; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2016-01-01

    The oligonucleotide binding fold (OB-fold) is a small structural motif present in many proteins. It is originally named for its oligonucleotide or oligosaccharide binding properties. These proteins have been identified as essential for replication, recombination and repair of DNA. We have successfully purified a protein contains OB-fold from the stem of Tinospora cordifolia, a medicinal plants of north India. Stems were crushed and centrifuged, and fraction obtained at 60% ammonium sulphate was extensively dialyzed and applied to the weak anion exchange chromatography on Hi-Trap DEAE-FF in 50mM Tris-HCl buffer at pH 8.0. Eluted fractions were concentrated and applied to gel filtration column to get pure protein. We observed a single band of 20-kDa on SDS-PAGE. Finally, the protein was identified as OB-fold by MALDI-TOF. The purified OB-fold protein was characterized for its secondary structural elements using circular dichroism (CD) in the far-UV region. Generally the OB-fold has a characteristic feature as five-stranded beta-sheet coiled to form a closed beta- barrel. To estimate its chemical stability, guanidinium chloride-induced denaturation curve was followed by observing changes in the far-UV CD as a function of the denaturant concentration. Analysis of this denaturation curve gave values of 8.90±0.25kcalmol(-1) and 3.78±0.18M for ΔGD° (Gibbs free energy change at 25°C) and Cm (midpoint of denaturation), respectively. To determine heat stability parameters of OB-fold protein, differential scanning calorimetry was performed. Calorimetric values of ΔGD°, Tm (midpoint of denaturation), ΔHm (enthalpy change at Tm), and ΔCp (constant-pressure heat capacity change) are 9.05±0.27kcalmol(-1), 85.2±0,3°C, 105±4kcalmol(-1) and 1.6±0.08kcalmol(-1)K(-1). This is the first report on the isolation, purification and characterization of OB-fold protein from a medicinal plant T. cordifolia.

  1. Dynamical Coupling of Intrinsically Disordered Proteins and Their Hydration Water: Comparison with Folded Soluble and Membrane Proteins

    Science.gov (United States)

    Gallat, F.-X.; Laganowsky, A.; Wood, K.; Gabel, F.; van Eijck, L.; Wuttke, J.; Moulin, M.; Härtlein, M.; Eisenberg, D.; Colletier, J.-P.; Zaccai, G.; Weik, M.

    2012-01-01

    Hydration water is vital for various macromolecular biological activities, such as specific ligand recognition, enzyme activity, response to receptor binding, and energy transduction. Without hydration water, proteins would not fold correctly and would lack the conformational flexibility that animates their three-dimensional structures. Motions in globular, soluble proteins are thought to be governed to a certain extent by hydration-water dynamics, yet it is not known whether this relationship holds true for other protein classes in general and whether, in turn, the structural nature of a protein also influences water motions. Here, we provide insight into the coupling between hydration-water dynamics and atomic motions in intrinsically disordered proteins (IDP), a largely unexplored class of proteins that, in contrast to folded proteins, lack a well-defined three-dimensional structure. We investigated the human IDP tau, which is involved in the pathogenic processes accompanying Alzheimer disease. Combining neutron scattering and protein perdeuteration, we found similar atomic mean-square displacements over a large temperature range for the tau protein and its hydration water, indicating intimate coupling between them. This is in contrast to the behavior of folded proteins of similar molecular weight, such as the globular, soluble maltose-binding protein and the membrane protein bacteriorhodopsin, which display moderate to weak coupling, respectively. The extracted mean square displacements also reveal a greater motional flexibility of IDP compared with globular, folded proteins and more restricted water motions on the IDP surface. The results provide evidence that protein and hydration-water motions mutually affect and shape each other, and that there is a gradient of coupling across different protein classes that may play a functional role in macromolecular activity in a cellular context. PMID:22828339

  2. 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...

  3. Translational diffusion of hydration water correlates with functional motions in folded and intrinsically disordered proteins.

    Science.gov (United States)

    Schirò, Giorgio; Fichou, Yann; Gallat, Francois-Xavier; Wood, Kathleen; Gabel, Frank; Moulin, Martine; Härtlein, Michael; Heyden, Matthias; Colletier, Jacques-Philippe; Orecchini, Andrea; Paciaroni, Alessandro; Wuttke, Joachim; Tobias, Douglas J; Weik, Martin

    2015-01-01

    Hydration water is the natural matrix of biological macromolecules and is essential for their activity in cells. The coupling between water and protein dynamics has been intensively studied, yet it remains controversial. Here we combine protein perdeuteration, neutron scattering and molecular dynamics simulations to explore the nature of hydration water motions at temperatures between 200 and 300 K, across the so-called protein dynamical transition, in the intrinsically disordered human protein tau and the globular maltose binding protein. Quasi-elastic broadening is fitted with a model of translating, rotating and immobile water molecules. In both experiment and simulation, the translational component markedly increases at the protein dynamical transition (around 240 K), regardless of whether the protein is intrinsically disordered or folded. Thus, we generalize the notion that the translational diffusion of water molecules on a protein surface promotes the large-amplitude motions of proteins that are required for their biological activity.

  4. 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

  5. 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.

  6. Does alpha-helix folding necessarily provide an energy source for the protein-lipid binding?

    Science.gov (United States)

    Gursky, Olga

    2007-01-01

    Lipid-induced alpha-helix folding, which occurs in many lipid surface-binding proteins and peptides such as apolipoproteins and synucleins, has been proposed to provide an energy source for protein-lipid interactions. We propose that in a system comprised of a phospholipid surface and a small polypeptide that is unfolded in solution and binds reversibly to lipid surface, helical folding involves expenditure of free energy as compared to a similar polypeptide that is alpha-helical in solution. This is a consequence of the entropic cost of helix folding that is illustrated in a simple thermodynamic model and exemplifies the general "key-into-lock" paradigm of protein-ligand binding. Even though this simple model does not explicitly address the protein-induced lipid re-arrangement and may not directly apply to large proteins that undergo significant tertiary structural changes upon lipid binding, it suggests that the notion of helix folding as an energy source for lipid binding should be treated with caution.

  7. 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.

  8. Estimation of protein folding free energy barriers from calorimetric data by multi-model Bayesian analysis.

    Science.gov (United States)

    Naganathan, Athi N; Perez-Jimenez, Raul; Muñoz, Victor; Sanchez-Ruiz, Jose M

    2011-10-14

    The realization that folding free energy barriers can be small enough to result in significant population of the species at the barrier top has sprouted in several methods to estimate folding barriers from equilibrium experiments. Some of these approaches are based on fitting the experimental thermogram measured by differential scanning calorimetry (DSC) to a one-dimensional representation of the folding free-energy surface (FES). Different physical models have been used to represent the FES: (1) a Landau quartic polynomial as a function of the total enthalpy, which acts as an order parameter; (2) the projection onto a structural order parameter (i.e. number of native residues or native contacts) of the free energy of all the conformations generated by Ising-like statistical mechanical models; and (3) mean-field models that define conformational entropy and stabilization energy as functions of a continuous local order parameter. The fundamental question that emerges is how can we obtain robust, model-independent estimates of the thermodynamic folding barrier from the analysis of DSC experiments. Here we address this issue by comparing the performance of various FES models in interpreting the thermogram of a protein with a marginal folding barrier. We chose the small α-helical protein PDD, which folds-unfolds in microseconds crossing a free energy barrier previously estimated as ~1 RT. The fits of the PDD thermogram to the various models and assumptions produce FES with a consistently small free energy barrier separating the folded and unfolded ensembles. However, the fits vary in quality as well as in the estimated barrier. Applying Bayesian probabilistic analysis we rank the fit performance using a statistically rigorous criterion that leads to a global estimate of the folding barrier and its precision, which for PDD is 1.3 ± 0.4 kJ mol(-1). This result confirms that PDD folds over a minor barrier consistent with the downhill folding regime. We have further

  9. Simplified protein models can rival all atom simulations in predicting folding pathways and structure

    Science.gov (United States)

    Adhikari, Aashish N.; Freed, Karl F.; Sosnick, Tobin R.

    2014-01-01

    We demonstrate the ability of simultaneously determining a protein’s folding pathway and structure using a properly formulated model without prior knowledge of the native structure. Our model employs a natural coordinate system for describing proteins and a search strategy inspired by the observation that real proteins fold in a sequential fashion by incrementally stabilizing native-like substructures or "foldons". Comparable folding pathways and structures are obtained for the twelve proteins recently studied using atomistic molecular dynamics simulations [K. Lindorff-Larsen, S. Piana, R.O. Dror, D. E. Shaw, Science 334, 517 (2011)], with our calculations running several orders of magnitude faster. We find that native-like propensities in the unfolded state do not necessarily determine the order of structure formation, a departure from a major conclusion of the MD study. Instead, our results support a more expansive view wherein intrinsic local structural propensities may be enhanced or overridden in the folding process by environmental context. The success of our search strategy validates it as an expedient mechanism for folding both in silico and in vivo. PMID:23889448

  10. 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.

  11. A Differential Evolution Approach for Protein Folding Using a Lattice Model

    Institute of Scientific and Technical Information of China (English)

    Heitor Silverio Lopes; Reginaldo Bitello

    2007-01-01

    Protein folding is a relevant computational problem in Bioinformatics, for which many heuristic algorithms have been proposed. This work presents a methodology for the application of differential evolution (DE) to the problem of protein folding, using the bi-dimensional hydrophobic-polar model. DE is a relatively recent evolutionary algorithm, and has been used successfully in several engineering optimization problems, usually with continuous variables. We introduce the concept of genotype-phenotype mapping in DE in order to provide a mapping between the real-valued vector and an actual folding. The methodology is detailed and several experiments with benchmarks are done. We compared the results with other similar implementations. The proposed DE has shown to be competitive, statistically consistent and very promising.

  12. 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.

  13. 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.

  14. Folding of small knotted proteins: Insights from a mean field coarse-grained model

    Energy Technology Data Exchange (ETDEWEB)

    Najafi, Saeed; Potestio, Raffaello, E-mail: potestio@mpip-mainz.mpg.de [Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz (Germany)

    2015-12-28

    A small but relevant number of proteins whose native structure is known features nontrivial topology, i.e., they are knotted. Understanding the process of folding from a swollen unknotted state to the biologically relevant native conformation is, for these proteins, particularly difficult, due to their rate-limiting topological entanglement. To shed some light into this conundrum, we introduced a structure-based coarse-grained model of the protein, where the information about the folded conformation is encoded in bonded angular interactions only, which do not favor the formation of native contacts. A stochastic search scheme in parameter space is employed to identify a set of interactions that maximizes the probability to attain the knotted state. The optimal knotting pathways of the two smallest knotted proteins, obtained through this approach, are consistent with the results derived by means of coarse-grained as well as full atomistic simulations.

  15. Cooperative folding of intrinsically disordered domains drives assembly of a strong elongated protein

    Science.gov (United States)

    Gruszka, Dominika T.; Whelan, Fiona; Farrance, Oliver E.; Fung, Herman K. H.; Paci, Emanuele; Jeffries, Cy M.; Svergun, Dmitri I.; Baldock, Clair; Baumann, Christoph G.; Brockwell, David J.; Potts, Jennifer R.; Clarke, Jane

    2015-06-01

    Bacteria exploit surface proteins to adhere to other bacteria, surfaces and host cells. Such proteins need to project away from the bacterial surface and resist significant mechanical forces. SasG is a protein that forms extended fibrils on the surface of Staphylococcus aureus and promotes host adherence and biofilm formation. Here we show that although monomeric and lacking covalent cross-links, SasG maintains a highly extended conformation in solution. This extension is mediated through obligate folding cooperativity of the intrinsically disordered E domains that couple non-adjacent G5 domains thermodynamically, forming interfaces that are more stable than the domains themselves. Thus, counterintuitively, the elongation of the protein appears to be dependent on the inherent instability of its domains. The remarkable mechanical strength of SasG arises from tandemly arrayed `clamp' motifs within the folded domains. Our findings reveal an elegant minimal solution for the assembly of monomeric mechano-resistant tethers of variable length.

  16. Determination of Protein Folding Intermediate Structures Consistent with Data from Oxidative Footprinting Mass Spectrometry.

    Science.gov (United States)

    Heinkel, Florian; Gsponer, Jörg

    2016-01-29

    The mapping of folding landscapes remains an important challenge in protein chemistry. Pulsed oxidative labeling of exposed residues and their detection via mass spectrometry provide new means of taking time-resolved "snapshots" of the structural changes that occur during protein folding. However, such experiments have been so far only interpreted qualitatively. Here, we report the detailed structural interpretation of mass spectrometry data from fast photochemical oxidation of proteins (FPOP) experiments at atomic resolution in a biased molecular dynamics approach. We are able to calculate structures of the early folding intermediate of the model system barstar that are fully consistent with FPOP data and Φ values. Furthermore, structures calculated with both FPOP data and Φ values are significantly less compact and have fewer helical residues than intermediate structures calculated with Φ values only. This improves the agreement with the experimental β-Tanford value and CD measurements. The restraints that we introduce facilitate the structural interpretation of FPOP data and provide new means for refined structure calculations of transiently sampled states on protein folding landscapes.

  17. The Histone Database: an integrated resource for histones and histone fold-containing proteins.

    Science.gov (United States)

    Mariño-Ramírez, Leonardo; Levine, Kevin M; Morales, Mario; Zhang, Suiyuan; Moreland, R Travis; Baxevanis, Andreas D; Landsman, David

    2011-01-01

    Eukaryotic chromatin is composed of DNA and protein components-core histones-that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins.

  18. Small-World Effect of Complex Network and Its Application toProtein Folding

    Institute of Scientific and Technical Information of China (English)

    卢全国; 陈宝方; 彭华魁; 祖巧红

    2004-01-01

    The famous "six letters" experiment carried out by Milgram demonstrated the existence of small-world effect in a complex network. One vertex tends to be connected to another by a shortest path through network because of the small-world effect. This paper uses the small-world effect to study protein folding pathway.

  19. Catalysis of Protein Folding by Chaperones Accelerates Evolutionary Dynamics in Adapting Cell Populations

    OpenAIRE

    Murat Cetinbaş; Shakhnovich, Eugene I.

    2013-01-01

    Although molecular chaperones are essential components of protein homeostatic machinery, their mechanism of action and impact on adaptation and evolutionary dynamics remain controversial. Here we developed a physics-based ab initio multi-scale model of a living cell for population dynamics simulations to elucidate the effect of chaperones on adaptive evolution. The 6-loci genomes of model cells encode model proteins, whose folding and interactions in cellular milieu can be evaluated exactly f...

  20. Probing the Folding-Unfolding Transition of a Thermophilic Protein, MTH1880.

    Directory of Open Access Journals (Sweden)

    Heeyoun Kim

    Full Text Available The folding mechanism of typical proteins has been studied widely, while our understanding of the origin of the high stability of thermophilic proteins is still elusive. Of particular interest is how an atypical thermophilic protein with a novel fold maintains its structure and stability under extreme conditions. Folding-unfolding transitions of MTH1880, a thermophilic protein from Methanobacterium thermoautotrophicum, induced by heat, urea, and GdnHCl, were investigated using spectroscopic techniques including circular dichorism, fluorescence, NMR combined with molecular dynamics (MD simulations. Our results suggest that MTH1880 undergoes a two-state N to D transition and it is extremely stable against temperature and denaturants. The reversibility of refolding was confirmed by spectroscopic methods and size exclusion chromatography. We found that the hyper-stability of the thermophilic MTH1880 protein originates from an extensive network of both electrostatic and hydrophobic interactions coordinated by the central β-sheet. Spectroscopic measurements, in combination with computational simulations, have helped to clarify the thermodynamic and structural basis for hyper-stability of the novel thermophilic protein MTH1880.

  1. New fungal defensin-like peptides provide evidence for fold change of proteins in evolution

    Science.gov (United States)

    Wu, Yucheng; Gao, Bin

    2016-01-01

    Defensins containing a consensus cystine framework, Cys[1]…Cys[2]X3Cys[3]…Cys[4]… Cys[5]X1Cys[6] (X, any amino acid except Cys; …, variable residue numbers), are extensively distributed in a variety of multicellular organisms (plants, fungi and invertebrates) and essentially involved in immunity as microbicidal agents. This framework is a prerequisite for forming the cysteine-stabilized α-helix and β-sheet (CSαβ) fold, in which the two invariant motifs, Cys[2]X3Cys[3]/Cys[5]X1Cys[6], are key determinants of fold formation. By using a computational genomics approach, we identified a large superfamily of fungal defensin-like peptides (fDLPs) in the phytopathogenic fungal genus – Zymoseptoria, which includes 132 structurally typical and 63 atypical members. These atypical fDLPs exhibit an altered cystine framework and accompanying fold change associated with their secondary structure elements and disulfide bridge patterns, as identified by protein structure modelling. Despite this, they definitely are homologous with the typical fDLPs in view of their precise gene structure conservation and identical precursor organization. Sequence and structural analyses combined with functional data suggest that most of Zymoseptoria fDLPs might have lost their antimicrobial activity. The present study provides a clear example of fold change in the evolution of proteins and is valuable in establishing remote homology among peptide superfamily members with different folds. PMID:27913751

  2. 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/.

  3. Re-entrant-Groove-Assisted VLS Growth of Boron Carbide Five-Fold Twinned Nanowires

    Institute of Scientific and Technical Information of China (English)

    FU Xin; JIANG Jun; LIU Chao; YU Zhi-Yang; Steffan LEA; YUAN Jun

    2009-01-01

    We report a preferential growth of boron carbide nanowires with a Eve-fold twinned internal structure.The nanowires are found to grow catalytically via iron boron nanoparticles,but unusually the catalytic particle is in contact with the low-energy surfaces of boron carbide with V-shaped contact lines.We propose that this catalytical growth may be caused by preferential nucleation at the re-entrant grooves due to the twinning planes,followed by rapid spreading of atomic steps.This is consistent with the observed temperature dependence of the five-fold twinned nanowire growth.

  4. Enhanced Protein Fold Prediction Method Through a Novel Feature Extraction Technique.

    Science.gov (United States)

    Wei, Leyi; Liao, Minghong; Gao, Xing; Zou, Quan

    2015-09-01

    Information of protein 3-dimensional (3D) structures plays an essential role in molecular biology, cell biology, biomedicine, and drug design. Protein fold prediction is considered as an immediate step for deciphering the protein 3D structures. Therefore, protein fold prediction is one of fundamental problems in structural bioinformatics. Recently, numerous taxonomic methods have been developed for protein fold prediction. Unfortunately, the overall prediction accuracies achieved by existing taxonomic methods are not satisfactory although much progress has been made. To address this problem, we propose a novel taxonomic method, called PFPA, which is featured by combining a novel feature set through an ensemble classifier. Particularly, the sequential evolution information from the profiles of PSI-BLAST and the local and global secondary structure information from the profiles of PSI-PRED are combined to construct a comprehensive feature set. Experimental results demonstrate that PFPA outperforms the state-of-the-art predictors. To be specific, when tested on the independent testing set of a benchmark dataset, PFPA achieves an overall accuracy of 73.6%, which is the leading accuracy ever reported. Moreover, PFPA performs well without significant performance degradation on three updated large-scale datasets, indicating the robustness and generalization of PFPA. Currently, a webserver that implements PFPA is freely available on http://121.192.180.204:8080/PFPA/Index.html.

  5. Generic framework for mining cellular automata models on protein-folding simulations.

    Science.gov (United States)

    Diaz, N; Tischer, I

    2016-05-13

    Cellular automata model identification is an important way of building simplified simulation models. In this study, we describe a generic architectural framework to ease the development process of new metaheuristic-based algorithms for cellular automata model identification in protein-folding trajectories. Our framework was developed by a methodology based on design patterns that allow an improved experience for new algorithms development. The usefulness of the proposed framework is demonstrated by the implementation of four algorithms, able to obtain extremely precise cellular automata models of the protein-folding process with a protein contact map representation. Dynamic rules obtained by the proposed approach are discussed, and future use for the new tool is outlined.

  6. Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance.

    Science.gov (United States)

    Bazzacco, Paola; Billon-Denis, Emmanuelle; Sharma, K Shivaji; Catoire, Laurent J; Mary, Sophie; Le Bon, Christel; Point, Elodie; Banères, Jean-Louis; Durand, Grégory; Zito, Francesca; Pucci, Bernard; Popot, Jean-Luc

    2012-02-21

    Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.

  7. 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.

  8. Distance-dependent hydrophobic-hydrophobic contacts in protein folding simulations.

    Science.gov (United States)

    Onofrio, Angelo; Parisi, Giovanni; Punzi, Giuseppe; Todisco, Simona; Di Noia, Maria Antonietta; Bossis, Fabrizio; Turi, Antonio; De Grassi, Anna; Pierri, Ciro Leonardo

    2014-09-21

    Successful prediction of protein folding from an amino acid sequence is a challenge in computational biology. In order to reveal the geometric constraints that drive protein folding, highlight those constraints kept or missed by distinct lattices and for establishing which class of intra- and inter-secondary structure element interactions is the most relevant for the correct folding of proteins, we have calculated inter-alpha carbon distances in a set of 42 crystal structures consisting of mainly helix, sheet or mixed conformations. The inter-alpha carbon distances were also calculated in several lattice "hydrophobic-polar" models built from the same protein set. We found that helix structures are more prone to form "hydrophobic-hydrophobic" contacts than beta-sheet structures. At a distance lower than or equal to 3.8 Å (very short-range interactions), "hydrophobic-hydrophobic" contacts are almost absent in the native structures, while they are frequent in all the analyzed lattice models. At distances in-between 3.8 and 9.5 Å (short-/medium-range interactions), the best performing lattice for reproducing mainly helix structures is the body-centered-cubic lattice. If protein structures contain sheet portions, lattice performances get worse, with few exceptions observed for double-tetrahedral and body-centered-cubic lattices. Finally, we can observe that ab initio protein folding algorithms, i.e. those based on the employment of lattices and Monte Carlo simulated annealings, can be improved simply and effectively by preventing the generation of "hydrophobic-hydrophobic" contacts shorter than 3.8 Å, by monitoring the "hydrophobic-hydrophobic/polar-polar" contact ratio in short-/medium distance ranges and by using preferentially a body-centered-cubic lattice.

  9. In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide-isomerase (DsbA).

    Science.gov (United States)

    Wunderlich, M; Glockshuber, R

    1993-11-25

    The formation of disulfide bonds in Escherichia coli is catalyzed by periplasmic protein disulfide-isomerase (DsbA). When the alpha-amylase/trypsin inhibitor from Ragi, a protein containing five intramolecular disulfide bridges, is secreted into the periplasm of E. coli, large amounts of misfolded inhibitor with incomplete or incorrect disulfides are accumulated. Folding of the inhibitor in the periplasm is not improved when DsbA is coexpressed and cosecreted. However, an up to 14-fold increase in correctly folded inhibitor is observed by co-expression of DsbA in conjugation with the addition of reduced glutathione to the growth medium. This peptide acts as a disulfide-shuffling reagent and can pass the outer membrane of E. coli. Since the influence of DsbA on the folding yield of the inhibitor is reduced in the presence of oxidized glutathione, the in vivo function of DsbA appears to be dependent on the ratio between oxidizing and reducing thiol equivalents in the periplasm. The high stability of thiol reagents against air oxidation during growth of E. coli allows the investigation of oxidative protein folding in vivo under controlled, thiol-dependent redox conditions.

  10. Mycobacterial PE_PGRS Proteins Contain Calcium-Binding Motifs with Parallel β-roll Folds

    Institute of Scientific and Technical Information of China (English)

    Nandita; Bachhawat; Balvinder; Singh

    2007-01-01

    The PE_PGRS family of proteins unique to mycobacteria is demonstrated to con- rain multiple calcium-binding and glycine-rich sequence motifs GGXGXD/NXUX. This sequence repeat constitutes a calcium-binding parallel/3-roll or parallel β-helix structure and is found in RTX toxins secreted by many Gram-negative bacteria. It is predicted that the highly homologous PE_PGRS proteins containing multiple copies of the nona-peptide motif could fold into similar calcium-binding structures. The implication of the predicted calcium-binding property of PE_PGRS proteins in the Ught of macrophage-pathogen interaction and pathogenesis is presented.

  11. Characterizing folding funnels with replica exchange Wang-Landau simulation of lattice proteins.

    Science.gov (United States)

    Shi, Guangjie; Wüst, Thomas; Landau, David P

    2016-11-01

    We have studied the folding of ribonuclease A by mapping it onto coarse-grained lattice protein models. With replica exchange Wang-Landau sampling, we calculated the free energy vs end-to-end distance as a function of temperature. A mapping to the famous hydrophobic-polar (HP) model shows a relatively shallow folding funnel and flat free energy minimum, reflecting the high degeneracy of the ground state. In contrast, extending the HP model with an additional "neutral" monomer type (i.e., a mapping to the three-letter H0P model) has a well developed, rough free energy funnel with a low degeneracy ground state. In both cases, folding funnels are asymmetric with temperature dependent shape.

  12. Characterizing folding funnels with replica exchange Wang-Landau simulation of lattice proteins

    Science.gov (United States)

    Shi, Guangjie; Wüst, Thomas; Landau, David P.

    2016-11-01

    We have studied the folding of ribonuclease A by mapping it onto coarse-grained lattice protein models. With replica exchange Wang-Landau sampling, we calculated the free energy vs end-to-end distance as a function of temperature. A mapping to the famous hydrophobic-polar (HP) model shows a relatively shallow folding funnel and flat free energy minimum, reflecting the high degeneracy of the ground state. In contrast, extending the HP model with an additional "neutral" monomer type (i.e., a mapping to the three-letter H0P model) has a well developed, rough free energy funnel with a low degeneracy ground state. In both cases, folding funnels are asymmetric with temperature dependent shape.

  13. How the diffusivity profile reduces the arbitrariness of protein folding free energies

    CERN Document Server

    Hinczewski, Michael; Dzubiella, Joachim; Netz, Roland R

    2010-01-01

    The concept of a protein diffusing in its free energy folding landscape has been fruitful for both theory and experiment. Yet the choice of the reaction coordinate (RC) introduces an undesirable degree of arbitrariness into the problem. We analyze extensive simulation data of an alpha-helix in explicit water solvent as it stochastically folds and unfolds. The free energy profiles for different RCs exhibit significant variation, some having an activation barrier, others not. We show that this variation has little effect on the predicted folding kinetics if the diffusivity profiles are properly taken into account. This kinetic quasi-universality is rationalized by an RC rescaling, which, due to the reparameterization invariance of the Fokker-Planck equation, allows the combination of free energy and diffusivity effects into a single function, the rescaled free energy profile. This rescaled free energy indeed shows less variation among different RCs than the bare free energy and diffusivity profiles separately d...

  14. Protein folding: understanding the role of water and the low Reynolds number environment as the peptide chain emerges from the ribosome and folds.

    Science.gov (United States)

    Sen, Siddhartha; Voorheis, H Paul

    2014-12-21

    The mechanism of protein folding during early stages of the process has three determinants. First, moving water molecules obey the rules of low Reynolds number physics without an inertial component. Molecular movement is instantaneous and size insensitive. Proteins emerging from the ribosome move and rotate without an external force if they change shape, forming and propagating helical structures that increases translocational efficiency. Forward motion ceases when the shape change or propelling force ceases. Second, application of quantum field theory to water structure predicts the spontaneous formation of low density coherent units of fixed size that expel dissolved atmospheric gases. Structured water layers with both coherent and non-coherent domains, form a sheath around the new protein. The surface of exposed hydrophobic amino acids is protected from water contact by small nanobubbles of dissolved atmospheric gases, 5 or 6 molecules on average, that vibrate, attracting even widely separated resonating nanobubbles. This force results from quantum effects, appearing only when the system is within and interacts with an oscillating electromagnetic field. The newly recognized quantum force sharply bends the peptide and is part of a dynamic field determining the pathway of protein folding. Third, the force initiating the tertiary folding of proteins arises from twists at the position of each hydrophobic amino acid, that minimizes surface exposure of the hydrophobic amino acids and propagates along the protein. When the total bend reaches 360°, the leading segment of water sheath intersects the trailing segment. This steric self-intersection expels water from overlapping segments of the sheath and by Newton׳s second law moves the polypeptide chain in an opposite direction. Consequently, with very few exceptions that we enumerate and discuss, tertiary structures are absent from proteins without hydrophobic amino acids, which control the early stages of protein

  15. Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins.

    Science.gov (United States)

    Shenkarev, Zakhar O; Lyukmanova, Ekaterina N; Butenko, Ivan O; Petrovskaya, Lada E; Paramonov, Alexander S; Shulepko, Mikhail A; Nekrasova, Oksana V; Kirpichnikov, Mikhail P; Arseniev, Alexander S

    2013-02-01

    Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.

  16. Protein-fold recognition using an improved single-source K diverse shortest paths algorithm.

    Science.gov (United States)

    Lhota, John; Xie, Lei

    2016-04-01

    Protein structure prediction, when construed as a fold recognition problem, is one of the most important applications of similarity search in bioinformatics. A new protein-fold recognition method is reported which combines a single-source K diverse shortest path (SSKDSP) algorithm with Enrichment of Network Topological Similarity (ENTS) algorithm to search a graphic feature space generated using sequence similarity and structural similarity metrics. A modified, more efficient SSKDSP algorithm is developed to improve the performance of graph searching. The new implementation of the SSKDSP algorithm empirically requires 82% less memory and 61% less time than the current implementation, allowing for the analysis of larger, denser graphs. Furthermore, the statistical significance of fold ranking generated from SSKDSP is assessed using ENTS. The reported ENTS-SSKDSP algorithm outperforms original ENTS that uses random walk with restart for the graph search as well as other state-of-the-art protein structure prediction algorithms HHSearch and Sparks-X, as evaluated by a benchmark of 600 query proteins. The reported methods may easily be extended to other similarity search problems in bioinformatics and chemoinformatics. The SSKDSP software is available at http://compsci.hunter.cuny.edu/~leixie/sskdsp.html.

  17. 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.

  18. Discrete Frenet frame, inflection point solitons, and curve visualization with applications to folded proteins

    Science.gov (United States)

    Hu, Shuangwei; Lundgren, Martin; Niemi, Antti J.

    2011-06-01

    We develop a transfer matrix formalism to visualize the framing of discrete piecewise linear curves in three-dimensional space. Our approach is based on the concept of an intrinsically discrete curve. This enables us to more effectively describe curves that in the limit where the length of line segments vanishes approach fractal structures in lieu of continuous curves. We verify that in the case of differentiable curves the continuum limit of our discrete equation reproduces the generalized Frenet equation. In particular, we draw attention to the conceptual similarity between inflection points where the curvature vanishes and topologically stable solitons. As an application we consider folded proteins, their Hausdorff dimension is known to be fractal. We explain how to employ the orientation of Cβ carbons of amino acids along a protein backbone to introduce a preferred framing along the backbone. By analyzing the experimentally resolved fold geometries in the Protein Data Bank we observe that this Cβ framing relates intimately to the discrete Frenet framing. We also explain how inflection points (a.k.a. soliton centers) can be located in the loops and clarify their distinctive rôle in determining the loop structure of folded proteins.

  19. Differential scanning calorimetry as a tool for protein folding and stability.

    Science.gov (United States)

    Johnson, Christopher M

    2013-03-01

    Differential scanning calorimetry measures the heat capacity of states and the excess heat associated with transitions that can be induced by temperature change. The integral of the excess heat capacity is the enthalpy for this process. Despite this potentially intimidating sounding physical chemistry background, DSC has found almost universal application in studying biological macromolecules. In the case of proteins, DSC can be used to determine equilibrium thermodynamic stability and folding mechanism but can also be used in a more qualitative manner screening for thermal stability as an indicator for, ligand binding, pharmaceutical formulation or conditions conducive to crystal growth. DSC usually forms part of a wider biophysical characterisation of the biological system of interest and so the literature is diverse and difficult to categorise for the technique in isolation. This review therefore describes the potential uses of DSC in studying protein folding and stability, giving brief examples of applications from the recent literature. There have also been some interesting developments in the use of DSC to determine barrier heights for fast folding proteins and in studying complex protein mixtures such as human plasma that are considered in more detail.

  20. Specificity of the initial collapse in the folding of the cold shock protein.

    Science.gov (United States)

    Magg, Christine; Kubelka, Jan; Holtermann, Georg; Haas, Elisha; Schmid, Franz X

    2006-07-28

    The two-state folding reaction of the cold shock protein from Bacillus caldolyticus (Bc-Csp) is preceded by a rapid chain collapse. A fast shortening of intra-protein distances was revealed by Förster resonance energy transfer (FRET) measurements with protein variants that carried individual pairs of donor and acceptor chromophores at various positions along the polypeptide chain. Here we investigated the specificity of this rapid compaction. Energy transfer experiments that probed the stretching of strand beta2 and the close approach between the strands beta1 and beta2 revealed that the beta1-beta2 hairpin is barely formed in the collapsed form, although it is native-like in the folding transition state of Bc-Csp. The time course of the collapse could not be resolved by pressure or temperature jump experiments, indicating that the collapsed and extended forms are not separated by an energy barrier. The co-solute (NH4)2SO4 stabilizes both native Bc-Csp and the collapsed form, which suggests that the large hydrated SO4(2-) ions are excluded from the surface of the collapsed form in a similar fashion as they are excluded from folded Bc-Csp. Ethylene glycol increases the stability of proteins because it is excluded preferentially from the backbone, which is accessible in the unfolded state. The collapsed form of Bc-Csp resembles the unfolded form in its interaction with ethylene glycol, suggesting that in the collapsed form the backbone is still accessible to water and small molecules. Our results thus rule out that the collapsed form is a folding intermediate with native-like chain topology. It is better described as a mixture of compact conformations that belong to the unfolded state ensemble. However, some of its structural elements are reminiscent of the native protein.

  1. Mis-folding and self-association: opportunities for alternative modes of self-recognition during the folding of TIM barrel proteins

    Science.gov (United States)

    Matthews, C. Robert

    2006-03-01

    The (βα)8 or TIM barrel motif is one of the most common in biology, represented in all three super-kingdoms of life. Detailed thermodynamic and kinetic analysis of the folding reactions of three members of the TIM barrel family of proteins reveal a common propensity to mis-fold to an off-pathway intermediate in less than a few milliseconds. The unfolding of this stopped-flow burst-phase intermediate controls access to an on-pathway intermediate that is highly-populated at moderate denaturant concentrations. Curiously, the equilibrium intermediate for two of the three nominally monomeric proteins spontaneously adopts a dimeric form; the native state of the third also dimerizes at micromolar concentrations. The early mis-folding reactions may reflect the rapid access to non-native folds enabled by the simple, repetitive βα topology of this motif. The propensity of stable forms, either intermediate or native, to form dimers may reflect a segment-swapping mechanism enabled by the modular folding of these single structural domain proteins. Off-pathway intermediates and non-native dimers serve as examples of important, alternative intra- or inter-molecular self-recognition events.

  2. 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.

  3. On the simulation of protein folding by short time scale molecular dynamics and distributed computing.

    Science.gov (United States)

    Fersht, Alan R

    2002-10-29

    There are proposals to overcome the current incompatibilities between the time scales of protein folding and molecular dynamics simulation by using a large number of short simulations of only tens of nanoseconds (distributed computing). According to the principles of first-order kinetic processes, a sufficiently large number of short simulations will include, de facto, a small number of long time scale events that have proceeded to completion. But protein folding is not an elementary kinetic step: folding has a series of early conformational steps that lead to lag phases at the beginning of the kinetics. The presence of these lag phases can bias short simulations toward selecting minor pathways that have fewer or faster lag steps and so miss the major folding pathways. Attempts to circumvent the lags by using loosely coupled parallel simulations that search for first-order transitions are also problematic because of the difficulty of detecting transitions in molecular dynamics simulations. Nevertheless, the procedure of using parallel independent simulations is perfectly valid and quite feasible once the time scale of simulation proceeds past the lag phases into a single exponential region.

  4. Protein folding with implicit crowders: a study of conformational states using the Wang-Landau method.

    Science.gov (United States)

    Hoppe, Travis; Yuan, Jian-Min

    2011-03-10

    In this paper we introduce the idea of the implicit crowding method to study the statistical mechanical behaviors of folding of β-sheet peptides. Using a simple bead-lattice model, we are able to consider, separately, the conformational entropy involving the bond angles along the backbone and the orientational entropy associated with the dihedral angles. We use a Ising-like model to partially account for the dihedral angle entropy and, implicitly, the hydrogen-bond formations. We also compare our results to recent experiments and find good quantitative agreement on the predicted folded fraction. On the basis of the predictions from the scaled particle theory, we investigate changes in the melting temperature of the protein, suggesting crowding enhanced stability for a variant of trpzip hairpin and a slight instability for the larger β-sheet designed proteins.

  5. BCL::MP-fold: Membrane protein structure prediction guided by EPR restraints.

    Science.gov (United States)

    Fischer, Axel W; Alexander, Nathan S; Woetzel, Nils; Karakas, Mert; Weiner, Brian E; Meiler, Jens

    2015-11-01

    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 Å for 27, better than 6 Å for 22, and better than 4 Å for 15 of the 29 proteins, demonstrating the algorithms' ability to sample the native topology. The average enrichment could be improved from 1.3 to 2.5, showing the improved discrimination power by using EPR data.

  6. Integron gene cassettes: a repository of novel protein folds with distinct interaction sites.

    Directory of Open Access Journals (Sweden)

    Visaahini Sureshan

    Full Text Available Mobile gene cassettes captured within integron arrays encompass a vast and diverse pool of genetic novelty. In most cases, functional annotation of gene cassettes directly recovered by cassette-PCR is obscured by their characteristically high sequence novelty. This inhibits identification of those specific functions or biological features that might constitute preferential factors for lateral gene transfer via the integron system. A structural genomics approach incorporating x-ray crystallography has been utilised on a selection of cassettes to investigate evolutionary relationships hidden at the sequence level. Gene cassettes were accessed from marine sediments (pristine and contaminated sites, as well as a range of Vibrio spp. We present six crystal structures, a remarkably high proportion of our survey of soluble proteins, which were found to possess novel folds. These entirely new structures are diverse, encompassing all-α, α+β and α/β fold classes, and many contain clear binding pocket features for small molecule substrates. The new structures emphasise the large repertoire of protein families encoded within the integron cassette metagenome and which remain to be characterised. Oligomeric association is a notable recurring property common to these new integron-derived proteins. In some cases, the protein-protein contact sites utilised in homomeric assembly could instead form suitable contact points for heterogeneous regulator/activator proteins or domains. Such functional features are ideal for a flexible molecular componentry needed to ensure responsive and adaptive bacterial functions.

  7. Extreme Folding

    Science.gov (United States)

    Demaine, Erik

    2012-02-01

    Our understanding of the mathematics and algorithms behind paper folding, and geometric folding in general, has increased dramatically over the past several years. These developments have found a surprisingly broad range of applications. In the art of origami, it has helped spur the technical origami revolution. In engineering and science, it has helped solve problems in areas such as manufacturing, robotics, graphics, and protein folding. On the recreational side, it has led to new kinds of folding puzzles and magic. I will give an overview of the mathematics and algorithms of folding, with a focus on new mathematics and sculpture.

  8. 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.

  9. OPUS-Dom: applying the folding-based method VECFOLD to determine protein domain boundaries.

    Science.gov (United States)

    Wu, Yinghao; Dousis, Athanasios D; Chen, Mingzhi; Li, Jialin; Ma, Jianpeng

    2009-01-30

    In this article, we present a de novo method for predicting protein domain boundaries, called OPUS-Dom. The core of the method is a novel coarse-grained folding method, VECFOLD, which constructs low-resolution structural models from a target sequence by folding a chain of vectors representing the predicted secondary-structure elements. OPUS-Dom generates a large ensemble of folded structure decoys by VECFOLD and labels the domain boundaries of each decoy by a domain parsing algorithm. Consensus domain boundaries are then derived from the statistical distribution of the putative boundaries and three empirical sequence-based domain profiles. OPUS-Dom generally outperformed several state-of-the-art domain prediction algorithms over various benchmark protein sets. Even though each VECFOLD-generated structure contains large errors, collectively these structures provide a more robust delineation of domain boundaries. The success of OPUS-Dom suggests that the arrangement of protein domains is more a consequence of limited coordination patterns per domain arising from tertiary packing of secondary-structure segments, rather than sequence-specific constraints.

  10. Wang-Landau sampling of the interplay between surface adsorption and folding of HP lattice proteins

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying Wai [ORNL; Wuest, Thomas [Swiss Federal Research Institute, Switzerland; Landau, David P [University of Georgia, Athens, GA

    2014-01-01

    Generic features associated with the adsorption of proteins on solid surfaces are reviewed within the framework of the hydrophobic-polar (HP) lattice protein model. The thermodynamic behavior and structural properties of various HP protein sequences interacting with attractive surfaces have been studied using extensive Wang-Landau sampling with different types of surfaces, each of which attracts either: all monomers, only hydrophobic (H) monomers, or only polar (P) monomers, respectively. Consequently, different types of folding behavior occur for varied surface strengths. Analysis of the combined patterns of various structural observables, e.g., the derivatives of the numbers of interaction contacts, together with the specific heat, leads to the identification of fundamental categories of folding and transition hierarchies. We also inferred a connection between the transition categories and the relative surface strengths, i.e., the ratios of the surface attractive strengths to the intra-chain attraction among H monomers. We thus believe that the folding hierarchies and identification scheme are generic for different HP sequences interacting with attractive surfaces, regardless of the chain length, sequence, or surface attraction.

  11. Protein folding modulates the swapped dimerization mechanism of methyl-accepting chemotaxis heme sensors.

    Directory of Open Access Journals (Sweden)

    Marta A Silva

    Full Text Available The periplasmic sensor domains GSU0582 and GSU0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens. Both contain one c-type heme group and their crystal structures revealed that these domains form swapped dimers with a PAS fold formed from the two protein chains. The swapped dimerization of these sensors is related to the mechanism of signal transduction and the formation of the swapped dimer involves significant folding changes and conformational rearrangements within each monomeric component. However, the structural changes occurring during this process are poorly understood and lack a mechanistic framework. To address this issue, we have studied the folding and stability properties of two distinct heme-sensor PAS domains, using biophysical spectroscopies. We observed substantial differences in the thermodynamic stability (ΔG = 14.6 kJ.mol(-1 for GSU0935 and ΔG = 26.3 kJ.mol(-1 for GSU0582, and demonstrated that the heme moiety undergoes conformational changes that match those occurring at the global protein structure. This indicates that sensing by the heme cofactor induces conformational changes that rapidly propagate to the protein structure, an effect which is directly linked to the signal transduction mechanism. Interestingly, the two analyzed proteins have distinct levels of intrinsic disorder (25% for GSU0935 and 13% for GSU0582, which correlate with conformational stability differences. This provides evidence that the sensing threshold and intensity of the propagated allosteric effect is linked to the stability of the PAS-fold, as this property modulates domain swapping and dimerization. Analysis of the PAS-domain shows that disorder segments are found either at the hinge region that controls helix motions or in connecting segments of the β-sheet interface. The latter is known to be widely involved in both intra- and intermolecular interactions, supporting the view that it's folding

  12. 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

  13. 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

    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...... 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......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...

  14. Monte Carlo simulations of the HP model (the "Ising model" of protein folding)

    Science.gov (United States)

    Li, Ying Wai; Wüst, Thomas; Landau, David P.

    2011-09-01

    Using Wang-Landau sampling with suitable Monte Carlo trial moves (pull moves and bond-rebridging moves combined) we have determined the density of states and thermodynamic properties for a short sequence of the HP protein model. For free chains these proteins are known to first undergo a collapse "transition" to a globule state followed by a second "transition" into a native state. When placed in the proximity of an attractive surface, there is a competition between surface adsorption and folding that leads to an intriguing sequence of "transitions". These transitions depend upon the relative interaction strengths and are largely inaccessible to "standard" Monte Carlo methods.

  15. Relaxation rate for an ultrafast folding protein is independent of chemical denaturant concentration.

    Science.gov (United States)

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

    2007-11-28

    The connection between free-energy surfaces and chevron plots has been investigated in a laser temperature jump kinetic study of a small ultrafast folding protein, the 35-residue subdomain from the villin headpiece. Unlike all other proteins that have been studied so far, no measurable dependence of the unfolding/refolding relaxation rate on denaturant concentration was observed over a wide range of guanidinium chloride concentration. Analysis with a simple Ising-like theoretical model shows that this denaturant-invariant relaxation rate can be explained by a large movement of the major free energy barrier, together with a denaturant- and reaction coordinate-dependent diffusion coefficient.

  16. Crystal structure of TruD, a novel pseudouridine synthase with a new protein fold.

    Science.gov (United States)

    Kaya, Yusuf; Del Campo, Mark; Ofengand, James; Malhotra, Arun

    2004-04-30

    TruD, a recently discovered novel pseudouridine synthase in Escherichia coli, is responsible for modifying uridine13 in tRNA(Glu) to pseudouridine. It has little sequence homology with the other 10 pseudouridine synthases in E. coli which themselves have been grouped into four related protein families. Crystal structure determination of TruD revealed a two domain structure consisting of a catalytic domain that differs in sequence but is structurally very similar to the catalytic domain of other pseudouridine synthases and a second large domain (149 amino acids, 43% of total) with a novel alpha/beta fold that up to now has not been found in any other protein.

  17. 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.

  18. 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.

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

    Science.gov (United States)

    Anciano Granadillo, Victor J.; Earley, Jennifer N.; Shuck, Sarah C.; Georgiadis, Millie M.; Fitch, Richard W.; Turchi, John J.

    2010-01-01

    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. PMID:21188165

  20. CASP11--An Evaluation of a Modular BCL::Fold-Based Protein Structure Prediction Pipeline.

    Science.gov (United States)

    Fischer, Axel W; Heinze, Sten; Putnam, Daniel K; Li, Bian; Pino, James C; Xia, Yan; Lopez, Carlos F; Meiler, Jens

    2016-01-01

    In silico prediction of a protein's tertiary structure remains an unsolved problem. The community-wide Critical Assessment of Protein Structure Prediction (CASP) experiment provides a double-blind study to evaluate improvements in protein structure prediction algorithms. We developed a protein structure prediction pipeline employing a three-stage approach, consisting of low-resolution topology search, high-resolution refinement, and molecular dynamics simulation to predict the tertiary structure of proteins from the primary structure alone or including distance restraints either from predicted residue-residue contacts, nuclear magnetic resonance (NMR) nuclear overhauser effect (NOE) experiments, or mass spectroscopy (MS) cross-linking (XL) data. The protein structure prediction pipeline was evaluated in the CASP11 experiment on twenty regular protein targets as well as thirty-three 'assisted' protein targets, which also had distance restraints available. Although the low-resolution topology search module was able to sample models with a global distance test total score (GDT_TS) value greater than 30% for twelve out of twenty proteins, frequently it was not possible to select the most accurate models for refinement, resulting in a general decay of model quality over the course of the prediction pipeline. In this study, we provide a detailed overall analysis, study one target protein in more detail as it travels through the protein structure prediction pipeline, and evaluate the impact of limited experimental data.

  1. Postranslational modifications significantly alter the binding-folding pathways of proteins associating with DNA

    Science.gov (United States)

    Papoian, Garegin

    2012-02-01

    Many important regulators of gene activity are natively disordered, but fully or partially order when they bind to their targets on DNA. Interestingly, the ensembles of disordered states for such free proteins are not structurally featureless, but can qualitatively differ from protein to protein. In particular, in random coil like states the chains are swollen, making relatively few contacts, while in molten globule like states a significant collapse occurs, with ensuing high density of intra-protein interactions. Furthermore, since many DNA binding proteins are positively charged polyelectrolytes, the electrostatic self-repulsion also influences the degree of collapse of the chain and its conformational preferences in the free state and upon binding to DNA. In our work, we have found that the nature of the natively disordered ensemble significantly affects the way the protein folds upon binding to DNA. In particular, we showed that posttranslational modifications of amino acid residues, such as lysine acetylation, can alter the degree of collapse and conformational preferences for a free protein, and also profoundly impact the binding affinity and pathways for the protein DNA association. These trends will be discussed in the context of DNA interacting with various histone tails and the p53 protein.

  2. Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation

    Science.gov (United States)

    Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W.; Voisset, Cécile

    2016-01-01

    6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI+] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI+]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI+] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases. PMID:27633137

  3. Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation.

    Science.gov (United States)

    Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu Hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W; Voisset, Cécile

    2016-09-16

    6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.

  4. Revisiting the NMR structure of the ultrafast downhill folding protein gpW from bacteriophage λ.

    Directory of Open Access Journals (Sweden)

    Lorenzo Sborgi

    Full Text Available GpW is a 68-residue protein from bacteriophage λ that participates in virus head morphogenesis. Previous NMR studies revealed a novel α+β fold for this protein. Recent experiments have shown that gpW folds in microseconds by crossing a marginal free energy barrier (i.e., downhill folding. These features make gpW a highly desirable target for further experimental and computational folding studies. As a step in that direction, we have re-determined the high-resolution structure of gpW by multidimensional NMR on a construct that eliminates the purification tags and unstructured C-terminal tail present in the prior study. In contrast to the previous work, we have obtained a full manual assignment and calculated the structure using only unambiguous distance restraints. This new structure confirms the α+β topology, but reveals important differences in tertiary packing. Namely, the two α-helices are rotated along their main axis to form a leucine zipper. The β-hairpin is orthogonal to the helical interface rather than parallel, displaying most tertiary contacts through strand 1. There also are differences in secondary structure: longer and less curved helices and a hairpin that now shows the typical right-hand twist. Molecular dynamics simulations starting from both gpW structures, and calculations with CS-Rosetta, all converge to our gpW structure. This confirms that the original structure has strange tertiary packing and strained secondary structure. A comparison of NMR datasets suggests that the problems were mainly caused by incomplete chemical shift assignments, mistakes in NOE assignment and the inclusion of ambiguous distance restraints during the automated procedure used in the original study. The new gpW corrects these problems, providing the appropriate structural reference for future work. Furthermore, our results are a cautionary tale against the inclusion of ambiguous experimental information in the determination of protein

  5. Sequence, structure, and cooperativity in folding of elementary protein structural motifs.

    Science.gov (United States)

    Lai, Jason K; Kubelka, Ginka S; Kubelka, Jan

    2015-08-11

    Residue-level unfolding of two helix-turn-helix proteins--one naturally occurring and one de novo designed--is reconstructed from multiple sets of site-specific (13)C isotopically edited infrared (IR) and circular dichroism (CD) data using Ising-like statistical-mechanical models. Several model variants are parameterized to test the importance of sequence-specific interactions (approximated by Miyazawa-Jernigan statistical potentials), local structural flexibility (derived from the ensemble of NMR structures), interhelical hydrogen bonds, and native contacts separated by intervening disordered regions (through the Wako-Saitô-Muñoz-Eaton scheme, which disallows such configurations). The models are optimized by directly simulating experimental observables: CD ellipticity at 222 nm for model proteins and their fragments and (13)C-amide I' bands for multiple isotopologues of each protein. We find that data can be quantitatively reproduced by the model that allows two interacting segments flanking a disordered loop (double sequence approximation) and incorporates flexibility in the native contact maps, but neither sequence-specific interactions nor hydrogen bonds are required. The near-identical free energy profiles as a function of the global order parameter are consistent with expected similar folding kinetics for nearly identical structures. However, the predicted folding mechanism for the two motifs is different, reflecting the order of local stability. We introduce free energy profiles for "experimental" reaction coordinates--namely, the degree of local folding as sensed by site-specific (13)C-edited IR, which highlight folding heterogeneity and contrast its overall, average description with the detailed, local picture.

  6. 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

  7. Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy.

    Science.gov (United States)

    Borgia, Alessandro; Wensley, Beth G; Soranno, Andrea; Nettels, Daniel; Borgia, Madeleine B; Hoffmann, Armin; Pfeil, Shawn H; Lipman, Everett A; Clarke, Jane; Schuler, Benjamin

    2012-01-01

    Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes.

  8. Retinal proteins associated with redox regulation and protein folding play central roles in response to high glucose conditions.

    Science.gov (United States)

    Wang, Ssu-Han; Lee, Wen-Chi; Chou, Hsiu-Chuan

    2015-03-01

    Diabetic retinopathy typically causes poor vision and blindness. A previous study revealed that a high blood glucose concentration induces glycoxidation and weakens the retinal capillaries. Nevertheless, the molecular mechanisms underlying the effects of high blood glucose induced diabetic retinopathy remain to be elucidated. In the present study, we cultured the retinal pigmented epithelial cell line ARPE-19 in mannitol-balanced 5.5, 25, and 100 mM glucose media and investigated protein level alterations. Proteomic analysis revealed significant changes in 137 protein features, of which 124 demonstrated changes in a glucose concentration dependent manner. Several proteins functionally associated with redox regulation, protein folding, or the cytoskeleton are affected by increased glucose concentrations. Additional analyses also revealed that cellular oxidative stress, including endoplasmic reticulum stress, was significantly increased after treatment with high glucose concentrations. However, the mitochondrial membrane potential and cell survival remained unchanged during treatment with high glucose concentrations. To summarize, in this study, we used a comprehensive retinal pigmented epithelial cell based proteomic approach for identifying changes in protein expression associated retinal markers induced by high glucose concentrations. Our results revealed that a high glucose condition can induce cellular oxidative stress and modulate the levels of proteins with functions in redox regulation, protein folding, and cytoskeleton regulation; however, cell viability and mitochondrial integrity are not significantly disturbed under these high glucose conditions.

  9. 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.

  10. Generic folding and transition hierarchies for surface adsorption of hydrophobic-polar lattice model proteins

    Science.gov (United States)

    Li, Ying Wai; Wüst, Thomas; Landau, David P.

    2013-01-01

    The thermodynamic behavior and structural properties of hydrophobic-polar (HP) lattice proteins interacting with attractive surfaces are studied by means of Wang-Landau sampling. Three benchmark HP sequences (48mer, 67mer, and 103mer) are considered with different types of surfaces, each of which attract either all monomers, only hydrophobic (H) monomers, or only polar (P) monomers, respectively. The diversity of folding behavior in dependence of surface strength is discussed. Analyzing the combined patterns of various structural observables, such as, e.g., the derivatives of the numbers of surface contacts, together with the specific heat, we are able to identify generic categories of folding and transition hierarchies. We also infer a connection between these transition categories and the relative surface strengths, i.e., the ratio of the surface attractive strength to the interchain attraction among H monomers. The validity of our proposed classification scheme is reinforced by the analysis of additional benchmark sequences. We thus believe that the folding hierarchies and identification scheme are generic for HP proteins interacting with attractive surfaces, regardless of chain length, sequence, or surface attraction.

  11. Bioinformatical parsing of folding-on-binding proteins reveals their compositional and evolutionary sequence design.

    Science.gov (United States)

    Narasumani, Mohanalakshmi; Harrison, Paul M

    2015-12-18

    Intrinsic disorder occurs when (part of) a protein remains unfolded during normal functioning. Intrinsically-disordered regions can contain segments that 'fold on binding' to another molecule. Here, we perform bioinformatical parsing of human 'folding-on-binding' (FB) proteins, into four subsets: Ordered regions, FB regions, Disordered regions that surround FB regions ('Disordered-around-FB'), and Other-Disordered regions. We examined the composition and evolutionary behaviour (across vertebrate orthologs) of these subsets. From a convergence of three separate analyses, we find that for hydrophobicity, Ordered regions segregate from the other subsets, but the Ordered and FB regions group together as highly conserved, and the Disordered-around-FB and Other-Disordered regions as less conserved (with a lesser significant difference between Ordered and FB regions). FB regions are highly-conserved with net positive charge, whereas Disordered-around-FB have net negative charge and are relatively less hydrophobic than FB regions. Indeed, these Disordered-around-FB regions are excessively hydrophilic compared to other disordered regions generally. We describe how our results point towards a possible compositionally-based steering mechanism of folding-on-binding.

  12. 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.

  13. Estimating free-energy barrier heights for an ultrafast folding protein from calorimetric and kinetic data.

    Science.gov (United States)

    Godoy-Ruiz, Raquel; Henry, Eric R; Kubelka, Jan; Hofrichter, James; Muñoz, Victor; Sanchez-Ruiz, Jose M; Eaton, William A

    2008-05-15

    Differential scanning calorimetry was used to measure the temperature dependence of the absolute heat capacity of the 35-residue subdomain of the villin headpiece, a protein that folds in 5 mus and is therefore assumed to have a small free-energy barrier separating folded and unfolded states. To obtain an estimate of the barrier height from the calorimetric data, two models, a variable-barrier model and an Ising-like model, were used to fit the heat capacity in excess of the folded state over the temperature range 15-125 degrees C. The variable-barrier model is based on an empirical mathematical form for the density of states, with four adjustable parameters and the enthalpy (H) as a reaction coordinate. The Ising-like model is based on the inter-residue contact map of the X-ray structure with exact enumeration of approximately 10(5) possible conformations, with two adjustable parameters in the partition function, and either the fraction of native contacts (Q) or the number of ordered residues (P) as reaction coordinates. The variable-barrier model provides an excellent fit to the data and yields a barrier height at the folding temperature ranging from 0.4 to 1.1 kcal mol(-1), while the Ising-like model provides a less good fit and yields barrier heights of 2.3 +/- 0.1 kcal mol(-1) and 2.1 +/- 0.1 kcal mol(-1) for the Q and P reaction coordinates, respectively. In both models, the barrier to folding increases with increasing temperature. Assuming a sufficiently large activation energy for diffusion on the free-energy surfaces, both models are consistent with the observation of a temperature-independent folding rate in previously published laser temperature-jump experiments. Analysis of this kinetic data, using an approximate form for the pre-exponential factor of Kramers theory and the 70 ns relaxation time for the fast phase that precedes the unfolding/refolding relaxation to determine the diffusion coefficient, results in a barrier height of 1.6 +/- 0.3 kcal mol

  14. A vocabulary of ancient peptides at the origin of folded proteins.

    Science.gov (United States)

    Alva, Vikram; Söding, Johannes; Lupas, Andrei N

    2015-12-14

    The seemingly limitless diversity of proteins in nature arose from only a few thousand domain prototypes, but the origin of these themselves has remained unclear. We are pursuing the hypothesis that they arose by fusion and accretion from an ancestral set of peptides active as co-factors in RNA-dependent replication and catalysis. Should this be true, contemporary domains may still contain vestiges of such peptides, which could be reconstructed by a comparative approach in the same way in which ancient vocabularies have been reconstructed by the comparative study of modern languages. To test this, we compared domains representative of known folds and identified 40 fragments whose similarity is indicative of common descent, yet which occur in domains currently not thought to be homologous. These fragments are widespread in the most ancient folds and enriched for iron-sulfur- and nucleic acid-binding. We propose that they represent the observable remnants of a primordial RNA-peptide world.

  15. HDLs protect pancreatic β-cells against ER stress by restoring protein folding and trafficking.

    Science.gov (United States)

    Pétremand, Jannick; Puyal, Julien; Chatton, Jean-Yves; Duprez, Jessica; Allagnat, Florent; Frias, Miguel; James, Richard W; Waeber, Gérard; Jonas, Jean-Christophe; Widmann, Christian

    2012-05-01

    Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors.

  16. Transient ribosomal attenuation coordinates protein synthesis and co-translational folding.

    Science.gov (United States)

    Zhang, Gong; Hubalewska, Magdalena; Ignatova, Zoya

    2009-03-01

    Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein SufI can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding.

  17. The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum

    DEFF Research Database (Denmark)

    Xiao, Ruoyu; Wilkinson, Bonney; Solovyov, Anton;

    2004-01-01

    and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 +/- 8%) in vivo. These results suggest that PDI and its ER......In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae......, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI...

  18. 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...

  19. Investigating protein structure and folding with coherent two-dimensional infrared spectroscopy

    Science.gov (United States)

    Baiz, Carlos; Peng, Chunte; Reppert, Michael; Jones, Kevin; Tokmakoff, Andrei

    2012-02-01

    We present a new technique to quantitatively determine the secondary structure composition of proteins in solution based on ultrafast two-dimensional infrared (2DIR) spectroscopy. The percentage of residues in alpha-helix, beta-sheet, and unstructured conformations is extracted from a principal component analysis of the measured amide-I 2DIR spectra. We benchmark the method against a library of commercially-available proteins by comparing the predicted structure compositions with the x-ray crystal structures. The new technique offers sub-picosecond time resolution, and can be used to study systems that are difficult to study with conventional methods such as gels, intrinsically disordered peptides, fibers, and aggregates. We use the technique to investigate the structural changes and timescales associated with folding and denaturing of small proteins via equilibrium and transient temperature-jump 2DIR spectroscopy.

  20. Domain-Swapped Dimers of Intracellular Lipid-Binding Proteins: Evidence for Ordered Folding Intermediates.

    Science.gov (United States)

    Assar, Zahra; Nossoni, Zahra; Wang, Wenjing; Santos, Elizabeth M; Kramer, Kevin; McCornack, Colin; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H

    2016-09-06

    Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form.

  1. Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.

    Directory of Open Access Journals (Sweden)

    Florence Guillière

    Full Text Available While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

  2. Discovery of Manassantin A Protein Targets Using Large-Scale Protein Folding and Stability Measurements.

    Science.gov (United States)

    Geer Wallace, M Ariel; Kwon, Do-Yeon; Weitzel, Douglas H; Lee, Chen-Ting; Stephenson, Tesia N; Chi, Jen-Tsan; Mook, Robert A; Dewhirst, Mark W; Hong, Jiyong; Fitzgerald, Michael C

    2016-08-05

    Manassantin A is a natural product that has been shown to have anticancer activity in cell-based assays, but has a largely unknown mode-of-action. Described here is the use of two different energetics-based approaches to identify protein targets of manassantin A. Using the stability of proteins from rates of oxidation technique with an isobaric mass tagging strategy (iTRAQ-SPROX) and the pulse proteolysis technique with a stable isotope labeling with amino acids in cell culture strategy (SILAC-PP), over 1000 proteins in a MDA-MB-231 cell lysate grown under hypoxic conditions were assayed for manassantin A interactions (both direct and indirect). A total of 28 protein hits were identified with manassantin A-induced thermodynamic stability changes. Two of the protein hits (filamin A and elongation factor 1α) were identified using both experimental approaches. The remaining 26 hit proteins were only assayed in either the iTRAQ-SPROX or the SILAC-PP experiment. The 28 potential protein targets of manassantin A identified here provide new experimental avenues along which to explore the molecular basis of manassantin A's mode of action. The current work also represents the first application iTRAQ-SPROX and SILAC-PP to the large-scale analysis of protein-ligand binding interactions involving a potential anticancer drug with an unknown mode-of-action.

  3. Replica-exchange Wang-Landau simulations of the H0P model of protein folding

    Science.gov (United States)

    Shi, Guangjie; Landau, David P.; Wüst, Thomas; Li, Ying Wai Li

    2015-03-01

    The hydrophobic-polar (HP) model has served as a coarse-grained lattice protein folding model attracting scientists from various disciplines. However, simplification into H and P monomers may yield high ground state degeneracies which stands in contrast to the generally unique native states of natural proteins. We propose a simple modification, by introducing a new type of ``neutral'' monomer, 0, i.e. neither hydrophobic nor polar, rendering the model more realistic without increasing the difficulties of sampling significantly. With the newly developed parallel Wang-Landau (replica exchange Wang-Landau) scheme and an innovative method of estimating the ground state degeneracies, we investigated some widely studied HP proteins and their H0P counterparts. Dramatic differences in ground state and thermodynamic properties have been observed, e.g. the estimation of ground state degeneracy for the 46mer is 460,000 for the HP version and only 20 for the H0P mapping. Similarly, the specific heat and structural properties: radius of gyration and etc. show more pronounced signals associated with folding. Supported by NSF.

  4. 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.

  5. Ribosome-based protein folding systems are structurally divergent but functionally universal across biological kingdoms.

    Science.gov (United States)

    Ito, Koreaki

    2005-07-01

    In bacteria, Trigger factor (TF) is the first chaperone that interacts with nascent polypeptides as soon as they emerge from the exit tunnel of the ribosome. TF binds to the ribosomal protein L23 located next to the tunnel exit of the large subunit, with which it forms a cradle-like space embracing the polypeptide exit region. It cooperates with the DnaK Hsp70 chaperone system to ensure correct folding of a number of newly translated cytosolic proteins in Escherichia coli. Whereas TF is exclusively found in prokaryotes and chloroplasts, Saccharomyces cerevisiae, a eukaryotic microorganism, has a three-member Hsp70-J protein complex, Ssb-Ssz-Zuo, which could act as a ribosome-associated folding facilitator. In the work reported in this volume of Molecular Microbiology, Rauch et al. (2005, Mol Microbiol, doi:10.1111/j.1365-2958.2005.04690.x) examined the functional similarity of the ribosome-associated chaperones in prokaryotes and eukaryotes. In spite of the fact that TF and the Hsp70-based triad are structurally unrelated, TF can bind to the yeast ribosome via Rpl25 (the L23 counterpart) and can substitute for some, but not all, of the functions assigned to Ssb-Ssz-Zuo in yeast. The functional conservation of the ribosome-associated chaperones without structural similarity is remarkable and suggests that during evolution nature has employed a common design but divergent components to facilitate folding of polypeptides as they emerge from the ribosomal exit, a fundamental process required for the efficient expression of genetic information.

  6. 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.

  7. Desolvation penalty for burying hydrogen-bonded peptide groups in protein folding.

    Science.gov (United States)

    Baldwin, Robert L

    2010-12-16

    A novel analysis of the enthalpy of protein unfolding is proposed and used to test for a desolvation penalty when hydrogen-bonded peptide groups are desolvated via folding. The unfolding enthalpy has three components, (1) the change when peptide hydrogen bonds are broken and the exposed -CO and -NH groups are solvated, (2) the change when protein-protein van der Waals interactions are broken and replaced by protein-water van der Waals interactions, and (3) the change produced by the hydrophobic interaction when nonpolar groups in the protein interior (represented as a liquid hydrocarbon) are transferred to water. A key feature of the analysis is that the enthalpy change from the hydrophobic interaction goes through 0 at 22 °C according to the liquid hydrocarbon model. Protein unfolding enthalpies are smaller at 22 °C than the enthalpy change for unfolding an alanine peptide helix. Data in the literature indicate that the van der Waals contribution to the unfolding enthalpy is considerably larger than the unfolding enthalpy itself at 22 °C, and therefore, a sizable desolvation penalty is predicted. Such a desolvation penalty was predicted earlier from electrostatic calculations of a stabilizing interaction between water and the hydrogen-bonded peptide group.

  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. Protein folding: complex potential for the driving force in a two-dimensional space of collective variables.

    Science.gov (United States)

    Chekmarev, Sergei F

    2013-10-14

    Using the Helmholtz decomposition of the vector field of folding fluxes in a two-dimensional space of collective variables, a potential of the driving force for protein folding is introduced. The potential has two components. One component is responsible for the source and sink of the folding flows, which represent respectively, the unfolded states and the native state of the protein, and the other, which accounts for the flow vorticity inherently generated at the periphery of the flow field, is responsible for the canalization of the flow between the source and sink. The theoretical consideration is illustrated by calculations for a model β-hairpin protein.

  10. Deuterated protein folds obtained directly from unassigned nuclear overhauser effect data.

    Science.gov (United States)

    Bermejo, Guillermo A; Llinás, Miguel

    2008-03-26

    We demonstrate the feasibility of determining the global fold of a highly deuterated protein from unassigned experimental NMR nuclear Overhauser effect (NOE) data only. The method relies on the calculation of a spatial configuration of covalently unconnected protons-a "cloud"-directly from unassigned distance restraints derived from 13C- and 15N-edited NOESY spectra. Each proton in the cloud, labeled by its chemical shift and that of the directly bound 13C or 15N, is subsequently mapped to specific atoms in the protein. This is achieved via graph-theoretical protocols that search for connectivities in graphs that encode the structural information within the cloud. The peptidyl HN chain is traced by seeking for all possible routes and selecting the one that yields the minimal sum of sequential distances. Complete proton identification in the cloud is achieved by linking the side-chain protons to proximal main-chain HNs via bipartite graph matching. The identified protons automatically yield the NOE assignments, which in turn are used for structure calculation with RosettaNMR, a protocol that incorporates structural bias derived from protein databases. The method, named Sparse-Constraint CLOUDS, was applied to experimental NOESY data on the 58-residue Z domain of staphylococcal protein A. The generated structures are of similar accuracy to those previously reported, which were derived via a conventional approach involving a larger NMR data set. Additional tests were performed on seven reported protein structures of various folds, using restraint lists simulated from the known atomic coordinates.

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

    Directory of Open Access Journals (Sweden)

    Tyo Keith EJ

    2012-03-01

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

  12. Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns.

    Science.gov (United States)

    Yuan, Jie; Zhou, Huifang; Yang, Yicong; Li, Weimin; Wan, Yi; Wang, Lili

    2015-05-01

    Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC.

  13. Extended particle swarm optimisation method for folding protein on triangular lattice.

    Science.gov (United States)

    Guo, Yuzhen; Wu, Zikai; Wang, Ying; Wang, Yong

    2016-02-01

    In this study, the authors studied the protein structure prediction problem by the two-dimensional hydrophobic-polar model on triangular lattice. Particularly the non-compact conformation was modelled to fold the amino acid sequence into a relatively larger triangular lattice, which is more biologically realistic and significant than the compact conformation. Then protein structure prediction problem was abstracted to match amino acids to lattice points. Mathematically, the problem was formulated as an integer programming and they transformed the biological problem into an optimisation problem. To solve this problem, classical particle swarm optimisation algorithm was extended by the single point adjustment strategy. Compared with square lattice, conformations on triangular lattice are more flexible in several benchmark examples. They further compared the authors' algorithm with hybrid of hill climbing and genetic algorithm. The results showed that their method was more effective in finding solution with lower energy and less running time.

  14. Assessing the Outer Membrane Insertion and Folding of Multimeric Transmembrane β-Barrel Proteins.

    Science.gov (United States)

    Leo, Jack C; Oberhettinger, Philipp; Linke, Dirk

    2015-01-01

    In addition to the cytoplasmic membrane, Gram-negative bacteria have a second lipid bilayer, the outer membrane, which is the de facto barrier between the cell and the extracellular milieu. Virtually all integral proteins of the outer membrane form β-barrels, which are inserted into the outer membrane by the BAM complex. Some outer membrane proteins, like the porins and trimeric autotransporter adhesins, are multimeric. In the former case, the porin trimer consists of three individual β-barrels, whereas in the latter, the single autotransporter β-barrel domain is formed by three separate polypeptides. This chapter reviews methods to investigate the folding and membrane insertion of multimeric OMPs and further explains the use of a BamA depletion strain to study the effects of the BAM complex on multimeric OMPs in E. coli.

  15. Assessing Coupled Protein Folding and Binding Through Temperature-Dependent Isothermal Titration Calorimetry.

    Science.gov (United States)

    Sahu, Debashish; Bastidas, Monique; Lawrence, Chad W; Noid, William G; Showalter, Scott A

    2016-01-01

    Broad interest in the thermodynamic driving forces of coupled macromolecular folding and binding is motivated by the prevalence of disorder-to-order transitions observed when intrinsically disordered proteins (IDPs) bind to their partners. Isothermal titration calorimetry (ITC) is one of the few methods available for completely evaluating the thermodynamic parameters describing a protein-ligand binding event. Significantly, when the effective ΔH° for the coupled folding and binding process is determined by ITC in a temperature series, the constant-pressure heat capacity change (ΔCp) associated with these coupled equilibria is experimentally accessible, offering a unique opportunity to investigate the driving forces behind them. Notably, each of these molecular-scale events is often accompanied by strongly temperature-dependent enthalpy changes, even over the narrow temperature range experimentally accessible for biomolecules, making single temperature determinations of ΔH° less informative than typically assumed. Here, we will document the procedures we have adopted in our laboratory for designing, executing, and globally analyzing temperature-dependent ITC studies of coupled folding and binding in IDP interactions. As a biologically significant example, our recent evaluation of temperature-dependent interactions between the disordered tail of FCP1 and the winged-helix domain from Rap74 will be presented. Emphasis will be placed on the use of publically available analysis programs written in MATLAB that facilitate quantification of the thermodynamic forces governing IDP interactions. Although motivated from the perspective of IDPs, the experimental design principles and data fitting procedures presented here are general to the study of most noncooperative ligand binding equilibria.

  16. 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.

  17. Amphipathic polymers: tools to fold integral membrane proteins to their active form.

    Science.gov (United States)

    Pocanschi, Cosmin L; Dahmane, Tassadite; Gohon, Yann; Rappaport, Fabrice; Apell, Hans-Jürgen; Kleinschmidt, Jörg H; Popot, Jean-Luc

    2006-11-28

    Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dimensional structure has proven to be a major challenge. We describe here the use of an amphipathic polymer, amphipol A8-35, as a novel environment that allows both beta-barrel and alpha-helical MPs to fold to their native state, in the absence of detergents or lipids. Amphipols, which are extremely mild surfactants, appear to favor the formation of native intramolecular protein-protein interactions over intermolecular or protein-surfactant ones. The feasibility of the approach is demonstrated using as models OmpA and FomA, two outer membrane proteins from the eubacteria Escherichia coli and Fusobacterium nucleatum, respectively, and bacteriorhodopsin, a light-driven proton pump from the plasma membrane of the archaebacterium Halobacterium salinarium.

  18. Efficient and reproducible folding simulations of the Trp-case protein with multiscale molecular dynamics

    Institute of Scientific and Technical Information of China (English)

    XIA XueFeng; ZHANG Song; HUANG go; ZHOU Yun; SUN ZhiRong

    2008-01-01

    Folding simulations are often time-consuming or highly sensitive to the initial conformation of the simulation even for mini protein like the Trp-cage. Here, we present a multiscale molecular dynamics method which appears to be both efficient and insensitive to the starting conformation based on the testing results from the Trp-cage protein. In this method the simulated system is simultaneously modeled on atoms and coarse-grained particles with incremental coarsening levels. The dynamics of coarse-grained particles are adapted to the recent trajectories of finer-grained particles instead of fixed and parameterized energy functions as used in previous coarse-grained models. In addition, the compositions of coarse-grained particles are allowed to be updated automatically based on the coherence during its history. Starting from the fully extended conformation and other several different conformations of the Trp-cage protein, our method successfully finds out the native-like conformations of the Trp-cage protein in the largest cluster of the trajectories in all of the eight performed simulations within at most 10 ns simulation time. The results show that approaches based on multiscale modeling are promising for ab initio protein structure prediction.

  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. Mutation bias favors protein folding stability in the evolution of small populations.

    Science.gov (United States)

    Mendez, Raul; Fritsche, Miriam; Porto, Markus; Bastolla, Ugo

    2010-05-01

    Mutation bias in prokaryotes varies from extreme adenine and thymine (AT) in obligatory endosymbiotic or parasitic bacteria to extreme guanine and cytosine (GC), for instance in actinobacteria. GC mutation bias deeply influences the folding stability of proteins, making proteins on the average less hydrophobic and therefore less stable with respect to unfolding but also less susceptible to misfolding and aggregation. We study a model where proteins evolve subject to selection for folding stability under given mutation bias, population size, and neutrality. We find a non-neutral regime where, for any given population size, there is an optimal mutation bias that maximizes fitness. Interestingly, this optimal GC usage is small for small populations, large for intermediate populations and around 50% for large populations. This result is robust with respect to the definition of the fitness function and to the protein structures studied. Our model suggests that small populations evolving with small GC usage eventually accumulate a significant selective advantage over populations evolving without this bias. This provides a possible explanation to the observation that most species adopting obligatory intracellular lifestyles with a consequent reduction of effective population size shifted their mutation spectrum towards AT. The model also predicts that large GC usage is optimal for intermediate population size. To test these predictions we estimated the effective population sizes of bacterial species using the optimal codon usage coefficients computed by dos Reis et al. and the synonymous to non-synonymous substitution ratio computed by Daubin and Moran. We found that the population sizes estimated in these ways are significantly smaller for species with small and large GC usage compared to species with no bias, which supports our prediction.

  1. Mutation bias favors protein folding stability in the evolution of small populations.

    Directory of Open Access Journals (Sweden)

    Raul Mendez

    2010-05-01

    Full Text Available Mutation bias in prokaryotes varies from extreme adenine and thymine (AT in obligatory endosymbiotic or parasitic bacteria to extreme guanine and cytosine (GC, for instance in actinobacteria. GC mutation bias deeply influences the folding stability of proteins, making proteins on the average less hydrophobic and therefore less stable with respect to unfolding but also less susceptible to misfolding and aggregation. We study a model where proteins evolve subject to selection for folding stability under given mutation bias, population size, and neutrality. We find a non-neutral regime where, for any given population size, there is an optimal mutation bias that maximizes fitness. Interestingly, this optimal GC usage is small for small populations, large for intermediate populations and around 50% for large populations. This result is robust with respect to the definition of the fitness function and to the protein structures studied. Our model suggests that small populations evolving with small GC usage eventually accumulate a significant selective advantage over populations evolving without this bias. This provides a possible explanation to the observation that most species adopting obligatory intracellular lifestyles with a consequent reduction of effective population size shifted their mutation spectrum towards AT. The model also predicts that large GC usage is optimal for intermediate population size. To test these predictions we estimated the effective population sizes of bacterial species using the optimal codon usage coefficients computed by dos Reis et al. and the synonymous to non-synonymous substitution ratio computed by Daubin and Moran. We found that the population sizes estimated in these ways are significantly smaller for species with small and large GC usage compared to species with no bias, which supports our prediction.

  2. GANDivAWeb: A web server for detecting early folding units ("foldons" from protein 3D structures

    Directory of Open Access Journals (Sweden)

    Krishnan Arun

    2008-03-01

    Full Text Available Abstract Background It has long been known that small regions of proteins tend to fold independently and are then stabilized by interactions between these distinct subunits or modules. Such units, also known as autonomous folding units (AFUs or"foldons" play a key role in protein folding. A knowledge of such early folding units has diverse applications in protein engineering as well as in developing an understanding of the protein folding process. Such AFUs can also be used as model systems in order to study the structural organization of proteins. Results In an earlier work, we had utilized a global network partitioning algorithm to identify modules in proteins. We had shown that these modules correlate well with AFUs. In this work, we have developed a webserver, GANDivAWeb, to identify early folding units or "foldons" in networks using the algorithm described earlier. The website has three functionalities: (a It is able to display information on the modularity of a database of 1420 proteins used in the original work, (b It can take as input an uploaded PDB file, identify the modules using the GANDivA algorithm and email the results back to the user and (c It can take as input an uploaded PDB file and a results file (obtained from functionality (b and display the results using the embedded viewer. The results include the module decomposition of the protein, plots of cartoon representations of the protein colored by module identity and connectivity as well as contour plots of the hydrophobicity and relative accessible surface area (RASA distributions. Conclusion We believe that the GANDivAWeb server, will be a useful tool for scientists interested in the phenomena of protein folding as well as in protein engineering. Our tool not only provides a knowledge of the AFUs through a natural graph partitioning approach but is also able to identify residues that are critical during folding. It is our intention to use this tool to study the topological

  3. 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

  4. Origin of a folded repeat protein from an intrinsically disordered ancestor

    Science.gov (United States)

    Zhu, Hongbo; Sepulveda, Edgardo; Hartmann, Marcus D; Kogenaru, Manjunatha; Ursinus, Astrid; Sulz, Eva; Albrecht, Reinhard; Coles, Murray; Martin, Jörg; Lupas, Andrei N

    2016-01-01

    Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2–5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin. DOI: http://dx.doi.org/10.7554/eLife.16761.001 PMID:27623012

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

    Directory of Open Access Journals (Sweden)

    Frank C Pickard

    2014-07-01

    Full Text Available 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.

  6. 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.

  7. 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

  8. Web-based toolkits for topology prediction of transmembrane helical proteins, fold recognition, structure and binding scoring, folding-kinetics analysis and comparative analysis of domain combinations.

    Science.gov (United States)

    Zhou, Hongyi; Zhang, Chi; Liu, Song; Zhou, Yaoqi

    2005-07-01

    We have developed the following web servers for protein structural modeling and analysis at http://theory.med.buffalo.edu: THUMBUP, UMDHMM(TMHP) and TUPS, predictors of transmembrane helical protein topology based on a mean-burial-propensity scale of amino acid residues (THUMBUP), hidden Markov model (UMDHMM(TMHP)) and their combinations (TUPS); SPARKS 2.0 and SP3, two profile-profile alignment methods, that match input query sequence(s) to structural templates by integrating sequence profile with knowledge-based structural score (SPARKS 2.0) and structure-derived profile (SP3); DFIRE, a knowledge-based potential for scoring free energy of monomers (DMONOMER), loop conformations (DLOOP), mutant stability (DMUTANT) and binding affinity of protein-protein/peptide/DNA complexes (DCOMPLEX & DDNA); TCD, a program for protein-folding rate and transition-state analysis of small globular proteins; and DOGMA, a web-server that allows comparative analysis of domain combinations between plant and other 55 organisms. These servers provide tools for prediction and/or analysis of proteins on the secondary structure, tertiary structure and interaction levels, respectively.

  9. 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.

  10. Alpha-haemoglobin stabilizing protein (AHSP) stabilizes apo-α-haemoglobin in a partially folded state

    Science.gov (United States)

    Krishna Kumar, Kaavya; Dickson, Claire F.; Weiss, Mitchell J.; Mackay, Joel P.; Gell, David A.

    2015-01-01

    SYNOPSIS To produce functional haemoglobin, nascent α-globin (αo) and β-globin (βo) chains must each bind a single haem molecule (to form αh and βh) and interact together to form heterodimers. The precise sequence of binding events is unknown, and it has been suggested that additional factors might enhance the efficiency of Hb folding. The α-haemoglobin stabilizing protein (AHSP) has previously been shown to bind αh and regulate redox activity of the haem iron. Here, we use a combination of classical and dynamic light scattering and NMR spectroscopy to demonstrate that AHSP forms a heterodimeric complex with αo that inhibits αo aggregation and promotes αo folding in the absence of haem. These findings indicate that AHSP may function as an αo-specific chaperone, and suggest an important role for αo in guiding Hb assembly by stabilizing βo and inhibiting off-pathway self-association of βh. PMID:20860551

  11. AHSP (α-haemoglobin-stabilizing protein) stabilizes apo-α-haemoglobin in a partially folded state.

    Science.gov (United States)

    Krishna Kumar, Kaavya; Dickson, Claire F; Weiss, Mitchell J; Mackay, Joel P; Gell, David A

    2010-12-01

    To produce functional Hb (haemoglobin), nascent α-globin (αo) and β-globin (βo) chains must each bind a single haem molecule (to form αh and βh) and interact together to form heterodimers. The precise sequence of binding events is unknown, and it has been suggested that additional factors might enhance the efficiency of Hb folding. AHSP (α-haemoglobin-stabilizing protein) has been shown previously to bind αh and regulate redox activity of the haem iron. In the present study, we used a combination of classical and dynamic light scattering and NMR spectroscopy to demonstrate that AHSP forms a heterodimeric complex with αo that inhibits αo aggregation and promotes αo folding in the absence of haem. These findings indicate that AHSP may function as an αo-specific chaperone, and suggest an important role for αo in guiding Hb assembly by stabilizing βo and inhibiting off-pathway self-association of βh.

  12. Microsecond folding and domain motions of a spider silk protein structural switch.

    Science.gov (United States)

    Ries, Julia; Schwarze, Simone; Johnson, Christopher M; Neuweiler, Hannes

    2014-12-10

    Web spiders rapidly assemble protein monomers, so-called spidroins, into extraordinarily tough silk fibers. The process involves the pH-triggered self-association of the spidroin N-terminal domain (NTD), which contains a structural switch connecting spidroins to supermolecules. Single-molecule spectroscopy can detect conformational heterogeneity that is hidden to conventional methods, but motions of the NTD are beyond the resolution limit. Here, we engineered probes for 1 nm conformational changes based on the phenomenon of fluorescence quenching by photoinduced electron transfer into the isolated NTD of a spidroin from the nursery web spider Euprosthenops australis. Correlation analysis of single-molecule fluorescence fluctuations uncovered site-dependent nanosecond-to-microsecond movement of secondary and tertiary structure. Kinetic amplitudes were most pronounced for helices that are part of the association interface and where structural studies show large displacements between monomeric and dimeric conformations. A single tryptophan at the center of the five-helix bundle toggled conformations in ∼100 μs and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 μs. We deduced a free-energy surface that exhibits native-state ruggedness with apparently similar barrier heights to folding and native motions. Observed equilibrium dynamics within the domain suggest a conformational selection mechanism in the rapid association of spidroins through their NTDs during silk synthesis by web spiders.

  13. Oxidative folding and reductive activities of EhPDI, a protein disulfide isomerase from Entamoeba histolytica.

    Science.gov (United States)

    Mares, Rosa E; Magaña, Paloma D; Meléndez-López, Samuel G; Licea, Alexei F; Cornejo-Bravo, José M; Ramos, Marco A

    2009-09-01

    PDI enzymes are oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. We have previously identified an E. histolytica PDI enzyme (EhPDI) that exhibits oxidase activity in vivo. However, little is known about the specific role of its redox-related structural features on the enzymatic activity. Here, we have studied the in vivo oxidative folding of EhPDI by mutagenic analysis and functional complementation assays as well as the in vitro oxidative folding and reductive activities by comparative kinetics using functional homologues in standard assays. We have found that the active-site cysteine residues of the functional domains (Trx-domains) are essential for catalysis of disulfide bond formation in polypeptides and proteins, such as the bacterial alkaline phosphatase. Furthermore, we have shown that the recombinant EhPDI enzyme has some typical properties of PDI enzymes: oxidase and reductase activities. These activities were comparable to those observed for other functional equivalents, such as bovine PDI or bacterial thioredoxin, under the same experimental conditions. These findings will be helpful for further studies intended to understand the physiological role of EhPDI.

  14. An Evolutionary Strategy for All-Atom Folding of the 60-Amino-Acid Bacterial Ribosomal Protein L20

    Science.gov (United States)

    Schug, A.; Wenzel, W.

    2006-01-01

    We have investigated an evolutionary algorithm for de novo all-atom folding of the bacterial ribosomal protein L20. We report results of two simulations that converge to near-native conformations of this 60-amino-acid, four-helix protein. We observe a steady increase of “native content” in both simulated ensembles and a large number of near-native conformations in their final populations. We argue that these structures represent a significant fraction of the low-energy metastable conformations, which characterize the folding funnel of this protein. These data validate our all-atom free-energy force field PFF01 for tertiary structure prediction of a previously inaccessible structural family of proteins. We also compare folding simulations of the evolutionary algorithm with the basin-hopping technique for the Trp-cage protein. We find that the evolutionary algorithm generates a dynamic memory in the simulated population, which leads to faster overall convergence. PMID:16565067

  15. Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates.

    Science.gov (United States)

    Yu, Hao; Gupta, Amar Nath; Liu, Xia; Neupane, Krishna; Brigley, Angela M; Sosova, Iveta; Woodside, Michael T

    2012-09-04

    Protein folding is described conceptually in terms of diffusion over a configurational free-energy landscape, typically reduced to a one-dimensional profile along a reaction coordinate. In principle, kinetic properties can be predicted directly from the landscape profile using Kramers theory for diffusive barrier crossing, including the folding rates and the transition time for crossing the barrier. Landscape theory has been widely applied to interpret the time scales for protein conformational dynamics, but protein folding rates and transition times have not been calculated directly from experimentally measured free-energy profiles. We characterized the energy landscape for native folding of the prion protein using force spectroscopy, measuring the change in extension of a single protein molecule at high resolution as it unfolded/refolded under tension. Key parameters describing the landscape profile were first recovered from the distributions of unfolding and refolding forces, allowing the diffusion constant for barrier crossing and the transition path time across the barrier to be calculated. The full landscape profile was then reconstructed from force-extension curves, revealing a double-well potential with an extended, partially unfolded transition state. The barrier height and position were consistent with the previous results. Finally, Kramers theory was used to predict the folding rates from the landscape profile, recovering the values observed experimentally both under tension and at zero force in ensemble experiments. These results demonstrate how advances in single-molecule theory and experiment are harnessing the power of landscape formalisms to describe quantitatively the mechanics of folding.

  16. 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.

  17. 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.

  18. From the test tube to the cell: exploring the folding and aggregation of a beta-clam protein.

    Science.gov (United States)

    Ignatova, Zoya; Krishnan, Beena; Bombardier, Jeffrey P; Marcelino, Anna Marie C; Hong, Jiang; Gierasch, Lila M

    2007-01-01

    A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico-chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these processes in vivo. Understanding protein misfolding and aggregation diseases and developing therapeutic strategies to these diseases demand that we gain mechanistic insight into behaviors and misbehaviors of proteins as they fold in vivo. We have developed a fluorescence approach using FlAsH labeling to study the thermodynamics of folding of a model beta-rich protein, cellular retinoic acid binding protein (CRABP) in Escherichia coli cells. The labeling approach has also enabled us to follow aggregation of a modified version of CRABP and chimeras between CRABP and huntingtin exon 1 with its glutamine repeat tract. In this article, we review our recent results using FlAsH labeling to study in-vivo folding and present new observations that hint at fundamental differences between the thermodynamics and kinetics of protein folding in vivo and in vitro.

  19. 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

    Early conformational events during folding of acyl-CoA binding protein (ACBP), an 86-residue alpha-helical protein, were explored by using a continuous-flow mixing apparatus with a dead time of 70 micros to measure changes in intrinsic tryptophan fluorescence and tryptophan-dansyl fluorescence en...

  20. 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.

  1. Bloch spin waves and emergent structure in protein folding with HIV envelope glycoprotein as an example

    Science.gov (United States)

    Dai, Jin; Niemi, Antti J.; He, Jianfeng; Sieradzan, Adam; Ilieva, Nevena

    2016-03-01

    We inquire how structure emerges during the process of protein folding. For this we scrutinize collective many-atom motions during all-atom molecular dynamics simulations. We introduce, develop, and employ various topological techniques, in combination with analytic tools that we deduce from the concept of integrable models and structure of discrete nonlinear Schrödinger equation. The example we consider is an α -helical subunit of the HIV envelope glycoprotein gp41. The helical structure is stable when the subunit is part of the biological oligomer. But in isolation, the helix becomes unstable, and the monomer starts deforming. We follow the process computationally. We interpret the evolving structure both in terms of a backbone based Heisenberg spin chain and in terms of a side chain based XY spin chain. We find that in both cases the formation of protein supersecondary structure is akin the formation of a topological Bloch domain wall along a spin chain. During the process we identify three individual Bloch walls and we show that each of them can be modelled with a precision of tenths to several angstroms in terms of a soliton solution to a discrete nonlinear Schrödinger equation.

  2. Folding in solution of the C-catalytic protein fragment of angiotensin-converting enzyme.

    Science.gov (United States)

    Vamvakas, Sotirios-Spyridon M; Leondiadis, Leondios; Pairas, George; Manessi-Zoupa, Evy; Spyroulias, Georgios A; Cordopatis, Paul

    2009-08-01

    Angiotensin-converting enzyme (ACE) is a key molecule of the renin-angiotensin-aldosterone system which is responsible for the control of blood pressure. For over 30 years it has become the target for fighting off hypertension. Many inhibitors of the enzyme have been synthesized and used widely in medicine despite the lack of ACE structure. The last 5 years the crystal structure of ACE separate domains has been revealed, but in order to understand how the enzyme works it is necessary to study its structure in solution. We present here the cloning, overexpression in Escherichia coli, purification and structural study of the Ala(959) to Ser(1066) region (ACE_C) that corresponds to the C-catalytic domain of human somatic angiotensin-I-converting enzyme. ACE_C was purified under denatured conditions and the yield was 6 mg/l of culture. Circular dichroism (CD) spectroscopy indicated that 1,1,1-trifluoroethanol (TFE) is necessary for the correct folding of the protein fragment. The described procedure can be used for the production of an isotopically labelled ACE(959-1066) protein fragment in order to study its structure in solution by NMR spectroscopy.

  3. Simulation of folding of a small alpha-helical protein in atomistic detail using worldwide-distributed computing.

    Science.gov (United States)

    Zagrovic, Bojan; Snow, Christopher D; Shirts, Michael R; Pande, Vijay S

    2002-11-08

    By employing thousands of PCs and new worldwide-distributed computing techniques, we have simulated in atomistic detail the folding of a fast-folding 36-residue alpha-helical protein from the villin headpiece. The total simulated time exceeds 300 micros, orders of magnitude more than previous simulations of a molecule of this size. Starting from an extended state, we obtained an ensemble of folded structures, which is on average 1.7A and 1.9A away from the native state in C(alpha) distance-based root-mean-square deviation (dRMS) and C(beta) dRMS sense, respectively. The folding mechanism of villin is most consistent with the hydrophobic collapse view of folding: the molecule collapses non-specifically very quickly ( approximately 20ns), which greatly reduces the size of the conformational space that needs to be explored in search of the native state. The conformational search in the collapsed state appears to be rate-limited by the formation of the aromatic core: in a significant fraction of our simulations, the C-terminal phenylalanine residue packs improperly with the rest of the hydrophobic core. We suggest that the breaking of this interaction may be the rate-determining step in the course of folding. On the basis of our simulations we estimate the folding rate of villin to be approximately 5micros. By analyzing the average features of the folded ensemble obtained by simulation, we see that the mean folded structure is more similar to the native fold than any individual folded structure. This finding highlights the need for simulating ensembles of molecules and averaging the results in an experiment-like fashion if meaningful comparison between simulation and experiment is to be attempted. Moreover, our results demonstrate that (1) the computational methodology exists to simulate the multi-microsecond regime using distributed computing and (2) that potential sets used to describe interatomic interactions may be sufficiently accurate to reach the folded state

  4. Importance of hydrophobic cluster formation through long-range contacts in the folding transition state of two-state proteins.

    Science.gov (United States)

    Selvaraj, S; Gromiha, M Michael

    2004-06-01

    Understanding the folding pathways of proteins is a challenging task. The Phi value approach provides a detailed understanding of transition-state structures of folded proteins. In this work, we have computed the hydrophobicity associated with each residue in the folded state of 16 two-state proteins and compared the Phi values of each mutant residue. We found that most of the residues with high Phi value coincide with local maximum in surrounding hydrophobicity, or have nearby residues that show such maximum in hydrophobicity, indicating the importance of hydrophobic interactions in the transition state. We have tested our approach to different structural classes of proteins, such as alpha-helical, SH3 domains of all-beta proteins, beta-sandwich, and alpha/beta proteins, and we observed a good agreement with experimental results. Further, we have proposed a hydrophobic contact network pattern to relate the Phi values with long-range contacts, which will be helpful to understand the transition-state structures of folded proteins. The present approach could be used to identify potential hydrophobic clusters that may form through long-range contacts during the transition state.

  5. 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 withou

  6. Outer membrane β-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA.

    Science.gov (United States)

    Gessmann, Dennis; Chung, Yong Hee; Danoff, Emily J; Plummer, Ashlee M; Sandlin, Clifford W; Zaccai, Nathan R; Fleming, Karen G

    2014-04-22

    Outer membrane β-barrel proteins (OMPs) are crucial for numerous cellular processes in prokaryotes and eukaryotes. Despite extensive studies on OMP biogenesis, it is unclear why OMPs require assembly machineries to fold into their native outer membranes, as they are capable of folding quickly and efficiently through an intrinsic folding pathway in vitro. By investigating the folding of several bacterial OMPs using membranes with naturally occurring Escherichia coli lipids, we show that phosphoethanolamine and phosphoglycerol head groups impose a kinetic barrier to OMP folding. The kinetic retardation of OMP folding places a strong negative pressure against spontaneous incorporation of OMPs into inner bacterial membranes, which would dissipate the proton motive force and undoubtedly kill bacteria. We further show that prefolded β-barrel assembly machinery subunit A (BamA), the evolutionarily conserved, central subunit of the BAM complex, accelerates OMP folding by lowering the kinetic barrier imposed by phosphoethanolamine head groups. Our results suggest that OMP assembly machineries are required in vivo to enable physical control over the spontaneously occurring OMP folding reaction in the periplasm. Mechanistic studies further allowed us to derive a model for BamA function, which explains how OMP assembly can be conserved between prokaryotes and eukaryotes.

  7. A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae.

    Science.gov (United States)

    Pineda-Lucena, Antonio; Liao, Jack C C; Cort, John R; Yee, Adelinda; Kennedy, Michael A; Edwards, Aled M; Arrowsmith, Cheryl H

    2003-05-01

    As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by ORF YML108W from Saccharomyces cerevisiae. YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown. Moreover, this protein does not have sequence similarity to any other protein. The NMR structure of YML108W consists of a four-stranded beta-sheet with strand order 2143 and two alpha-helices, with an overall topology of betabetaalphabetabetaalpha. Strand beta1 runs parallel to beta4, and beta2:beta1 and beta4:beta3 pairs are arranged in an antiparallel fashion. Although this fold belongs to the split betaalphabeta family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds.

  8. Structure–function–folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights

    Indian Academy of Sciences (India)

    P M Krishna Mohan; Ramakrishna V Hosur

    2009-09-01

    The detailed characterization of the structure, dynamics and folding process of a protein is crucial for understanding the biological functions it performs. Modern biophysical and nuclear magnetic resonance (NMR) techniques have provided a way to obtain accurate structural and thermodynamic information on various species populated on the energy landscape of a given protein. In this context, we review here the structure–function–folding relationship of an important protein, namely, dynein light chain protein (DLC8). DLC8, the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions and dissociates into a pure monomer below pH 4. Cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface are anticipated to be playing crucial roles in the variety of functions the protein performs. NMR investigations have provided great insights into these aspects of DLC8 in recent years.

  9. A tri-gram based feature extraction technique using linear probabilities of position specific scoring matrix for protein fold recognition.

    Science.gov (United States)

    Paliwal, Kuldip K; Sharma, Alok; Lyons, James; Dehzangi, Abdollah

    2014-03-01

    In biological sciences, the deciphering of a three dimensional structure of a protein sequence is considered to be an important and challenging task. The identification of protein folds from primary protein sequences is an intermediate step in discovering the three dimensional structure of a protein. This can be done by utilizing feature extraction technique to accurately extract all the relevant information followed by employing a suitable classifier to label an unknown protein. In the past, several feature extraction techniques have been developed but with limited recognition accuracy only. In this study, we have developed a feature extraction technique based on tri-grams computed directly from Position Specific Scoring Matrices. The effectiveness of the feature extraction technique has been shown on two benchmark datasets. The proposed technique exhibits up to 4.4% improvement in protein fold recognition accuracy compared to the state-of-the-art feature extraction techniques.

  10. Analyses of simulations of three-dimensional lattice proteins in comparison with a simplified statistical mechanical model of protein folding.

    Science.gov (United States)

    Abe, H; Wako, H

    2006-07-01

    Folding and unfolding simulations of three-dimensional lattice proteins were analyzed using a simplified statistical mechanical model in which their amino acid sequences and native conformations were incorporated explicitly. Using this statistical mechanical model, under the assumption that only interactions between amino acid residues within a local structure in a native state are considered, the partition function of the system can be calculated for a given native conformation without any adjustable parameter. The simulations were carried out for two different native conformations, for each of which two foldable amino acid sequences were considered. The native and non-native contacts between amino acid residues occurring in the simulations were examined in detail and compared with the results derived from the theoretical model. The equilibrium thermodynamic quantities (free energy, enthalpy, entropy, and the probability of each amino acid residue being in the native state) at various temperatures obtained from the simulations and the theoretical model were also examined in order to characterize the folding processes that depend on the native conformations and the amino acid sequences. Finally, the free energy landscapes were discussed based on these analyses.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Folding and Dimerization of Tick-Borne Encephalitis Virus Envelope Proteins prM and E in the Endoplasmic Reticulum

    OpenAIRE

    Ivo C. Lorenz; Allison, Steven L.; Heinz, Franz X; Helenius, Ari

    2002-01-01

    Flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected c...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Juan Frausto-Solis

    2016-01-01

    Full Text Available 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.

  18. Fast Tree Search for A Triangular Lattice Model of Protein Folding

    Institute of Scientific and Technical Information of China (English)

    Xiaomei Li; Nengchao Wang

    2004-01-01

    Using a triangular lattice model to study the designability of protein folding, we overcame the parity problem of previous cubic lattice model and enumerated all the sequences and compact structures on a simple two-dimensional triangular lattice model of size 4+5+6+5+4. We used two types of amino acids, hydrophobic and polar, to make up the sequences, and achieved 223+212 different sequences excluding the reverse symmetry sequences. The total string number of distinct compact structures was 219,093, excluding reflection symmetry in the self-avoiding path of length 24 triangular lattice model. Based on this model, we applied a fast search algorithm by constructing a cluster tree. The algorithm decreased the computation by computing the objective energy of non-leaf nodes. The parallel experiments proved that the fast tree search algorithm yielded an exponential speed-up in the model of size 4+5+6+5+4. Designability analysis was performed to understand the search result.

  19. High-resolution x-ray crystal structures of the villin headpiece subdomain, an ultrafast folding protein

    Science.gov (United States)

    Chiu, Thang K.; Kubelka, Jan; Herbst-Irmer, Regine; Eaton, William A.; Hofrichter, James; Davies, David R.

    2005-01-01

    The 35-residue subdomain of the villin headpiece (HP35) is a small ultrafast folding protein that is being intensely studied by experiments, theory, and simulations. We have solved the x-ray structures of HP35 and its fastest folding mutant [K24 norleucine (nL)] to atomic resolution and compared their experimentally measured folding kinetics by using laser temperature jump. The structures, which are in different space groups, are almost identical to each other but differ significantly from previously solved NMR structures. Hence, the differences between the x-ray and NMR structures are probably not caused by lattice contacts or crystal/solution differences, but reflect the higher accuracy of the x-ray structures. The x-ray structures reveal important details of packing of the hydrophobic core and some additional features, such as cross-helical H bonds. Comparison of the x-ray structures indicates that the nL substitution produces only local perturbations. Consequently, the finding that the small stabilization by the mutation is completely reflected in an increased folding rate suggests that this region of the protein is as structured in the transition state as in the folded structure. It is therefore a target for engineering to increase the folding rate of the subdomain from ≈0.5 μs–1 for the nL mutant to the estimated theoretical speed limit of ≈3 μs–1. PMID:15894611

  20. Tannin-assisted aggregation of natively unfolded proteins

    Science.gov (United States)

    Zanchi, D.; Narayanan, T.; Hagenmuller, D.; Baron, A.; Guyot, S.; Cabane, B.; Bouhallab, S.

    2008-06-01

    Tannin-protein interactions are essentially physical: hydrophobic and hydrogen-bond-mediated. We explored the tannin-assisted protein aggregation on the case of β-casein, which is a natively unfolded protein known for its ability to form micellar aggregates. We used several tannins with specified length. Our SAXS results show that small tannins increase the number of proteins per micelle, but keeping their size constant. It leads to a tannin-assisted compactization of micelles. Larger tannins, with linear dimensions greater than the crown width of micelles, lead to the aggregation of micelles by a bridging effect. Experimental results can be understood within a model where tannins are treated as effective enhancers of hydrophobic attraction between specific sites in proteins.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Zeldovich, Konstantin B; Chen, Peiqiu; Shakhnovich, Boris E; Shakhnovich, Eugene I

    2007-07-01

    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.

  4. How hydrophobicity and the glycosylation site of glycans affect protein folding and stability: a molecular dynamics simulation.

    Science.gov (United States)

    Lu, Diannan; Yang, Cheng; Liu, Zheng

    2012-01-12

    Glycosylation is one of the most common post-translational modifications in the biosynthesis of protein, but its effect on the protein conformational transitions underpinning folding and stabilization is poorly understood. In this study, we present a coarse-grained off-lattice 46-β barrel model protein glycosylated by glycans with different hydrophobicity and glycosylation sites to examine the effect of glycans on protein folding and stabilization using a Langevin dynamics simulation, in which an H term was proposed as the index of the hydrophobicity of glycan. Compared with its native counterpart, introducing glycans of suitable hydrophobicity (0.1 enthalpy effect. The simulations have shown both the stabilization and the destabilization effects of glycosylation, as experimentally reported in the literature, and provided molecular insight into glycosylated proteins. The understanding of the effects of glycans with different hydrophobicities on the folding and stability of protein, as attempted by the present work, is helpful not only to explain the stabilization and destabilization effect of real glycoproteins but also to design protein-polymer conjugates for biotechnological purposes.

  5. Protein folding pathways and state transitions described by classical equations of motion of an elastic network model.

    Science.gov (United States)

    Williams, Gareth; Toon, Andrew J

    2010-12-01

    Protein topology defined by the matrix of residue contacts has proved to be a fruitful basis for the study of protein dynamics. The widely implemented coarse-grained elastic network model of backbone fluctuations has been used to describe crystallographic temperature factors, allosteric couplings, and some aspects of the folding pathway. In the present study, we develop a model of protein dynamics based on the classical equations of motion of a damped network model (DNM) that describes the folding path from a completely unfolded state to the native conformation through a single-well potential derived purely from the native conformation. The kinetic energy gained through the collapse of the protein chain is dissipated through a friction term in the equations of motion that models the water bath. This approach is completely general and sufficiently fast that it can be applied to large proteins. Folding pathways for various proteins of different classes are described and shown to correlate with experimental observations and molecular dynamics and Monte Carlo simulations. Allosteric transitions between alternative protein structures are also modeled within the DNM through an asymmetric double-well potential.

  6. 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

    2012-03-07

    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 or X-ray crystallography. In these density maps, the general arrangement of secondary structure elements (SSEs) is revealed, whereas 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 SSEs de novo before atomic detail is added using Rosetta. In a benchmark of 27 proteins, the protocol consistently and reproducibly achieves models with root mean square deviation values <3 Å.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. A cytosolic relay of heat shock proteins HSP70-1A and HSP90β monitors the folding trajectory of the serotonin transporter.

    Science.gov (United States)

    El-Kasaby, Ali; Koban, Florian; Sitte, Harald H; Freissmuth, Michael; Sucic, Sonja

    2014-10-17

    Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.

  9. 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

    2017-02-13

    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.

  10. Interaction networks in protein folding via atomic-resolution experiments and long-time-scale molecular dynamics simulations

    DEFF Research Database (Denmark)

    Sborgi, Lorenzo; Verma, Abhinav; Piana, Stefano;

    2015-01-01

    The integration of atomic-resolution experimental and computational methods offers the potential for elucidating key aspects of protein folding that are not revealed by either approach alone. Here, we combine equilibrium NMR measurements of thermal unfolding and long molecular dynamics simulation...

  11. Posttranslational folding of vesicular stomatitis virus G protein in the ER: involvement of noncovalent and covalent complexes

    NARCIS (Netherlands)

    Braakman, L.J.; Silva, A. de; Helenius, A.

    1993-01-01

    In this study, we show that posttranslational folding of Vesicular Stomatitis virus G protein subunits can involve noncovalent, multimeric complexes as transient intermediates. The complexes are heterogeneous in size (4-21S20,W), contain several G glycopolypeptides, and are associated with BiP/GRP78

  12. Denatured-state energy landscapes of a protein structural database reveal the energetic determinants of a framework model for folding.

    Science.gov (United States)

    Wang, Suwei; Gu, Jenny; Larson, Scott A; Whitten, Steven T; Hilser, Vincent J

    2008-09-19

    Position-specific denatured-state thermodynamics were determined for a database of human proteins by use of an ensemble-based model of protein structure. The results of modeling denatured protein in this manner reveal important sequence-dependent thermodynamic properties in the denatured ensembles as well as fundamental differences between the denatured and native ensembles in overall thermodynamic character. The generality and robustness of these results were validated by performing fold-recognition experiments, whereby sequences were matched with their respective folds based on amino acid propensities for the different energetic environments in the protein, as determined through cluster analysis. Correlation analysis between structure and energetic information revealed that sequence segments destined for beta-sheet in the final native fold are energetically more predisposed to a broader repertoire of states than are sequence segments destined for alpha-helix. These results suggest that within the subensemble of mostly unstructured states, the energy landscapes are dominated by states in which parts of helices adopt structure, whereas structure formation for sequences destined for beta-strand is far less probable. These results support a framework model of folding, which suggests that, in general, the denatured state has evolutionarily evolved to avoid low-energy conformations in sequences that ultimately adopt beta-strand. Instead, the denatured state evolved so that sequence segments that ultimately adopt alpha-helix and coil will have a high intrinsic structure formation capability, thus serving as potential nucleation sites.

  13. 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.

  14. Lattice and off-lattice side chain models of protein folding: linear time structure prediction better than 86% of optimal.

    Science.gov (United States)

    Hart, W E; Istrail, S

    1997-01-01

    This paper considers the protein energy minimization problem for lattice and off-lattice protein folding models that explicitly represent side chains. Lattice models of proteins have proven useful tools for reasoning about protein folding in unrestricted continuous space through analogy. This paper provides the first illustration of how rigorous algorithmic analyses of lattice models can lead to rigorous algorithmic analyses of off-lattice models. We consider two side chain models: a lattice model that generalizes the HP model (Dill, 1985) to explicitly represent side chains on the cubic lattice and a new off-lattice model, the HP Tangent Spheres Side Chain model (HP-TSSC), that generalizes this model further by representing the backbone and side chains of proteins with tangent spheres. We describe algorithms with mathematically guaranteed error bounds for both of these models. In particular, we describe a linear time performance guaranteed approximation algorithm for the HP side chain model that constructs conformations whose energy is better than 86% of optimal in a face-centered cubic lattice, and we demonstrate how this provides a better than 70% performance guarantee for the HP-TSSC model. Our analysis provides a mathematical methodology for transferring performance guarantees on lattices to off-lattice models. These results partially answer the open question of Ngo et al. (1994) concerning the complexity of protein folding models that include side chains.

  15. Influences of heterogeneous native contact energy and many-body interactions on the prediction of protein folding mechanisms.

    Science.gov (United States)

    Zhang, Zhuqing; Ouyang, Yanhua; Chen, Tao

    2016-11-16

    Since single-point mutant perturbation has been used to probe protein folding mechanisms in experiments, the ϕ-value has become a critical parameter to infer the transition state (TS) for two-state proteins. Experimentally, large scale analysis has shown a nearly single uniform ϕ-value with normally distributed error from 24 different proteins; moreover, in zero stability conditions, the intrinsic variable ϕ(0) is around 0.36. To explore how and to what extent theoretical models can capture experimental phenomena, we here use structure-based explicit chain coarse-grained models to investigate the influence of single-point mutant perturbation on protein folding for single domain two-state proteins. Our results indicate that uniform, additive contact energetic interactions cannot predict experimental Brønsted plots well. Those points deviate largely from the main data sets in Brønsted plots, are mostly hydrophobic, and are located in N- and C-terminal contacting regions. Heterogenous contact energy, which is dependent on sequence separation, can narrow the point dispersion in a Brønsted plot. Moreover, we demonstrate that combining many-body interactions with heterogeneous native contact energy can present mean ϕ-values consistent with experimental findings, with a comparable distributed error. This indicates that for more accurate elucidation of protein folding mechanisms by residue-level structure-based models, these elements should be considered.

  16. Solvent viscosity dependence of the folding rate of a small protein: distributed computing study.

    Science.gov (United States)

    Zagrovic, Bojan; Pande, Vijay

    2003-09-01

    By using distributed computing techniques and a supercluster of more than 20,000 processors we simulated folding of a 20-residue Trp Cage miniprotein in atomistic detail with implicit GB/SA solvent at a variety of solvent viscosities (gamma). This allowed us to analyze the dependence of folding rates on viscosity. In particular, we focused on the low-viscosity regime (values below the viscosity of water). In accordance with Kramers' theory, we observe approximately linear dependence of the folding rate on 1/gamma for values from 1-10(-1)x that of water viscosity. However, for the regime between 10(-4)-10(-1)x that of water viscosity we observe power-law dependence of the form k approximately gamma(-1/5). These results suggest that estimating folding rates from molecular simulations run at low viscosity under the assumption of linear dependence of rate on inverse viscosity may lead to erroneous results.

  17. BCL::contact-low confidence fold recognition hits boost protein contact prediction and de novo structure determination.

    Science.gov (United States)

    Karakaş, Mert; Woetzel, Nils; Meiler, Jens

    2010-02-01

    Knowledge of all residue-residue contacts within a protein allows determination of the protein fold. Accurate prediction of even a subset of long-range contacts (contacts between amino acids far apart in sequence) can be instrumental for determining tertiary structure. Here we present BCL::Contact, a novel contact prediction method that utilizes artificial neural networks (ANNs) and specializes in the prediction of medium to long-range contacts. BCL::Contact comes in two modes: sequence-based and structure-based. The sequence-based mode uses only sequence information and has individual ANNs specialized for helix-helix, helix-strand, strand-helix, strand-strand, and sheet-sheet contacts. The structure-based mode combines results from 32-fold recognition methods with sequence information to a consensus prediction. The two methods were presented in the 6(th) and 7(th) Critical Assessment of Techniques for Protein Structure Prediction (CASP) experiments. The present work focuses on elucidating the impact of fold recognition results onto contact prediction via a direct comparison of both methods on a joined benchmark set of proteins. The sequence-based mode predicted contacts with 42% accuracy (7% false positive rate), while the structure-based mode achieved 45% accuracy (2% false positive rate). Predictions by both modes of BCL::Contact were supplied as input to the protein tertiary structure prediction program Rosetta for a benchmark of 17 proteins with no close sequence homologs in the protein data bank (PDB). Rosetta created higher accuracy models, signified by an improvement of 1.3 A on average root mean square deviation (RMSD), when driven by the predicted contacts. Further, filtering Rosetta models by agreement with the predicted contacts enriches for native-like fold topologies.

  18. Identification of intermediate species in protein-folding by quantitative analysis of amplitudes in time-domain fluorescence spectroscopy

    Indian Academy of Sciences (India)

    Anoop M Saxena; G Krishnamoorthy; Jayant B Udgaonkar; N Periasamy

    2007-03-01

    In protein-folding studies it is often required to differentiate a system with only two-states, namely the native (N) and unfolded (U) forms of the protein present at any condition of the solvent, from a situation wherein intermediate state(s) could also be present. 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 studies. In contrast to the steady-state methods, time-resolved fluorescence has the capability to reveal the presence of heterogeneity of structural forms due to the `fingerprint’ nature of fluorescence lifetimes of various forms. In this work, we establish this method by quantitative analysis of amplitudes associated with fluorescence lifetimes in multiexponential decays. First, we show that we can estimate, accurately, the relative population of species from two-component mixtures of non-interacting molecules such as fluorescent dyes, peptides and proteins. Subsequently, we demonstrate, by analysing the amplitudes of fluorescence lifetimes which are controlled by fluorescence resonance energy transfer (FRET), that the equilibrium folding-unfolding transition of the small singledomain protein barstar is not a two-step process.

  19. 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

  20. Unfolding and Folding of the Three-Helix Bundle Protein KIX in the Absence of Solvent

    Science.gov (United States)

    Schennach, Moritz; Schneeberger, Eva-Maria; Breuker, Kathrin

    2016-06-01

    Electron capture dissociation was used to probe the structure, unfolding, and folding of KIX ions in the gas phase. At energies for vibrational activation that were sufficiently high to cause loss of small molecules such as NH3 and H2O by breaking of covalent bonds in about 5% of the KIX (M + nH)n+ ions with n = 7-9, only partial unfolding was observed, consistent with our previous hypothesis that salt bridges play an important role in stabilizing the native solution fold after transfer into the gas phase. Folding of the partially unfolded ions on a timescale of up to 10 s was observed only for (M + nH)n+ ions with n = 9, but not n = 7 and n = 8, which we attribute to differences in the distribution of charges within the (M + nH)n+ ions.

  1. Optimized Wang-Landau sampling of lattice polymers: ground state search and folding thermodynamics of HP model proteins.

    Science.gov (United States)

    Wüst, Thomas; Landau, David P

    2012-08-14

    Coarse-grained (lattice-) models have a long tradition in aiding efforts to decipher the physical or biological complexity of proteins. Despite the simplicity of these models, however, numerical simulations are often computationally very demanding and the quest for efficient algorithms is as old as the models themselves. Expanding on our previous work [T. Wüst and D. P. Landau, Phys. Rev. Lett. 102, 178101 (2009)], we present a complete picture of a Monte Carlo method based on Wang-Landau sampling in combination with efficient trial moves (pull, bond-rebridging, and pivot moves) which is particularly suited to the study of models such as the hydrophobic-polar (HP) lattice model of protein folding. With this generic and fully blind Monte Carlo procedure, all currently known putative ground states for the most difficult benchmark HP sequences could be found. For most sequences we could also determine the entire energy density of states and, together with suitably designed structural observables, explore the thermodynamics and intricate folding behavior in the virtually inaccessible low-temperature regime. We analyze the differences between random and protein-like heteropolymers for sequence lengths up to 500 residues. Our approach is powerful both in terms of robustness and speed, yet flexible and simple enough for the study of many related problems in protein folding.

  2. Optimized Wang-Landau sampling of lattice polymers: Ground state search and folding thermodynamics of HP model proteins

    Science.gov (United States)

    Wüst, Thomas; Landau, David P.

    2012-08-01

    Coarse-grained (lattice-) models have a long tradition in aiding efforts to decipher the physical or biological complexity of proteins. Despite the simplicity of these models, however, numerical simulations are often computationally very demanding and the quest for efficient algorithms is as old as the models themselves. Expanding on our previous work [T. Wüst and D. P. Landau, Phys. Rev. Lett. 102, 178101 (2009)], 10.1103/PhysRevLett.102.178101, we present a complete picture of a Monte Carlo method based on Wang-Landau sampling in combination with efficient trial moves (pull, bond-rebridging, and pivot moves) which is particularly suited to the study of models such as the hydrophobic-polar (HP) lattice model of protein folding. With this generic and fully blind Monte Carlo procedure, all currently known putative ground states for the most difficult benchmark HP sequences could be found. For most sequences we could also determine the entire energy density of states and, together with suitably designed structural observables, explore the thermodynamics and intricate folding behavior in the virtually inaccessible low-temperature regime. We analyze the differences between random and protein-like heteropolymers for sequence lengths up to 500 residues. Our approach is powerful both in terms of robustness and speed, yet flexible and simple enough for the study of many related problems in protein folding.

  3. Lattice and off-lattice side chain models of protein folding: Linear time structure prediction better than 86% of optimal

    Energy Technology Data Exchange (ETDEWEB)

    Hart, W.E.; Istrail, S. [Sandia National Labs., Albuquerque, NM (United States). Algorithms and Discrete Mathematics Dept.

    1996-08-09

    This paper considers the protein structure prediction problem for lattice and off-lattice protein folding models that explicitly represent side chains. Lattice models of proteins have proven extremely useful tools for reasoning about protein folding in unrestricted continuous space through analogy. This paper provides the first illustration of how rigorous algorithmic analyses of lattice models can lead to rigorous algorithmic analyses of off-lattice models. The authors consider two side chain models: a lattice model that generalizes the HP model (Dill 85) to explicitly represent side chains on the cubic lattice, and a new off-lattice model, the HP Tangent Spheres Side Chain model (HP-TSSC), that generalizes this model further by representing the backbone and side chains of proteins with tangent spheres. They describe algorithms for both of these models with mathematically guaranteed error bounds. In particular, the authors describe a linear time performance guaranteed approximation algorithm for the HP side chain model that constructs conformations whose energy is better than 865 of optimal in a face centered cubic lattice, and they demonstrate how this provides a 70% performance guarantee for the HP-TSSC model. This is the first algorithm in the literature for off-lattice protein structure prediction that has a rigorous performance guarantee. The analysis of the HP-TSSC model builds off of the work of Dancik and Hannenhalli who have developed a 16/30 approximation algorithm for the HP model on the hexagonal close packed lattice. Further, the analysis provides a mathematical methodology for transferring performance guarantees on lattices to off-lattice models. These results partially answer the open question of Karplus et al. concerning the complexity of protein folding models that include side chains.

  4. 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.

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

    Science.gov (United States)

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

    2016-07-01

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

  6. Protein Folding and Structure Prediction from the Ground Up: The Atomistic Associative Memory, Water Mediated, Structure and Energy Model.

    Science.gov (United States)

    Chen, Mingchen; Lin, Xingcheng; Zheng, Weihua; Onuchic, José N; Wolynes, Peter G

    2016-08-25

    The associative memory, water mediated, structure and energy model (AWSEM) is a coarse-grained force field with transferable tertiary interactions that incorporates local in sequence energetic biases using bioinformatically derived structural information about peptide fragments with locally similar sequences that we call memories. The memory information from the protein data bank (PDB) database guides proper protein folding. The structural information about available sequences in the database varies in quality and can sometimes lead to frustrated free energy landscapes locally. One way out of this difficulty is to construct the input fragment memory information from all-atom simulations of portions of the complete polypeptide chain. In this paper, we investigate this approach first put forward by Kwac and Wolynes in a more complete way by studying the structure prediction capabilities of this approach for six α-helical proteins. This scheme which we call the atomistic associative memory, water mediated, structure and energy model (AAWSEM) amounts to an ab initio protein structure prediction method that starts from the ground up without using bioinformatic input. The free energy profiles from AAWSEM show that atomistic fragment memories are sufficient to guide the correct folding when tertiary forces are included. AAWSEM combines the efficiency of coarse-grained simulations on the full protein level with the local structural accuracy achievable from all-atom simulations of only parts of a large protein. The results suggest that a hybrid use of atomistic fragment memory and database memory in structural predictions may well be optimal for many practical applications.

  7. 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.

  8. 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.

  9. Information and redundancy in the burial folding code of globular proteins within a wide range of shapes and sizes.

    Science.gov (United States)

    Ferreira, Diogo C; van der Linden, Marx G; de Oliveira, Leandro C; Onuchic, José N; de Araújo, Antônio F Pereira

    2016-04-01

    Recent ab initio folding simulations for a limited number of small proteins have corroborated a previous suggestion that atomic burial information obtainable from sequence could be sufficient for tertiary structure determination when combined to sequence-independent geometrical constraints. Here, we use simulations parameterized by native burials to investigate the required amount of information in a diverse set of globular proteins comprising different structural classes and a wide size range. Burial information is provided by a potential term pushing each atom towards one among a small number L of equiprobable concentric layers. An upper bound for the required information is provided by the minimal number of layers L(min) still compatible with correct folding behavior. We obtain L(min) between 3 and 5 for seven small to medium proteins with 50 ≤ Nr ≤ 110 residues while for a larger protein with Nr = 141 we find that L ≥ 6 is required to maintain native stability. We additionally estimate the usable redundancy for a given L ≥ L(min) from the burial entropy associated to the largest folding-compatible fraction of "superfluous" atoms, for which the burial term can be turned off or target layers can be chosen randomly. The estimated redundancy for small proteins with L = 4 is close to 0.8. Our results are consistent with the above-average quality of burial predictions used in previous simulations and indicate that the fraction of approachable proteins could increase significantly with even a mild, plausible, improvement on sequence-dependent burial prediction or on sequence-independent constraints that augment the detectable redundancy during simulations.

  10. The ability to enhance the solubility of its fusion partners is an intrinsic property of maltose-binding protein but their folding is either spontaneous or chaperone-mediated.

    Directory of Open Access Journals (Sweden)

    Sreejith Raran-Kurussi

    Full Text Available Escherichia coli maltose binding protein (MBP is commonly used to promote the solubility of its fusion partners. To investigate the mechanism of solubility enhancement by MBP, we compared the properties of MBP fusion proteins refolded in vitro with those of the corresponding fusion proteins purified under native conditions. We fused five aggregation-prone passenger proteins to 3 different N-terminal tags: His₆-MBP, His₆-GST and His₆. After purifying the 15 fusion proteins under denaturing conditions and refolding them by rapid dilution, we recovered far more of the soluble MBP fusion proteins than their GST- or His-tagged counterparts. Hence, we can reproduce the solubilizing activity of MBP in a simple in vitro system, indicating that no additional factors are required to mediate this effect. We assayed both the soluble fusion proteins and their TEV protease digestion products (i.e., with the N-terminal tag removed for biological activity. Little or no activity was detected for some fusion proteins whereas others were quite active. When the MBP fusions proteins were purified from E. coli under native conditions they were all substantially active. These results indicate that the ability of MBP to promote the solubility of its fusion partners in vitro sometimes, but not always, results in their proper folding. We show that the folding of some passenger proteins is mediated by endogenous chaperones in vivo. Hence, MBP serves as a passive participant in the folding process; passenger proteins either fold spontaneously or with the assistance of chaperones.

  11. Depletion of the chromatin looping proteins CTCF and cohesin causes chromatin compaction: insight into chromatin folding by polymer modelling.

    Directory of Open Access Journals (Sweden)

    Mariliis Tark-Dame

    2014-10-01

    Full Text Available Folding of the chromosomal fibre in interphase nuclei is an important element in the regulation of gene expression. For instance, physical contacts between promoters and enhancers are a key element in cell-type-specific transcription. We know remarkably little about the principles that control chromosome folding. Here we explore the view that intrachromosomal interactions, forming a complex pattern of loops, are a key element in chromosome folding. CTCF and cohesin are two abundant looping proteins of interphase chromosomes of higher eukaryotes. To investigate the role of looping in large-scale (supra Mb folding of human chromosomes, we knocked down the gene that codes for CTCF and the one coding for Rad21, an essential subunit of cohesin. We measured the effect on chromosome folding using systematic 3D fluorescent in situ hybridization (FISH. Results show that chromatin becomes more compact after reducing the concentration of these two looping proteins. The molecular basis for this counter-intuitive behaviour is explored by polymer modelling usingy the Dynamic Loop model (Bohn M, Heermann DW (2010 Diffusion-driven looping provides a consistent framework for chromatin organization. PLoS ONE 5: e12218.. We show that compaction can be explained by selectively decreasing the number of short-range loops, leaving long-range looping unchanged. In support of this model prediction it has recently been shown by others that CTCF and cohesin indeed are responsible primarily for short-range looping. Our results suggest that the local and the overall changes in of chromosome structure are controlled by a delicate balance between short-range and long-range loops, allowing easy switching between, for instance, open and more compact chromatin states.

  12. Geometry of the energy landscape and folding transition in a simple model of a protein.

    Science.gov (United States)

    Mazzoni, Lorenzo N; Casetti, Lapo

    2008-05-01

    A geometric analysis of the global properties of the energy landscape of a minimalistic model of a polypeptide is presented, which is based on the relation between dynamical trajectories and geodesics of a suitable manifold, whose metric is completely determined by the potential energy. We consider different sequences, some with a definite proteinlike behavior, a unique native state and a folding transition, and others undergoing a hydrophobic collapse with no tendency to a unique native state. The global geometry of the energy landscape appears to contain relevant information on the behavior of the various sequences: in particular, the fluctuations of the curvature of the energy landscape, measured by means of numerical simulations, clearly mark the folding transition and allow the proteinlike sequences to be distinguished from the others.

  13. Classification of conformational stability of protein mutants from 3D pseudo-folding graph representation of protein sequences using support vector machines.

    Science.gov (United States)

    Fernández, Michael; Caballero, Julio; Fernández, Leyden; Abreu, Jose Ignacio; Acosta, Gianco

    2008-01-01

    This work reports a novel 3D pseudo-folding graph representation of protein sequences for modeling purposes. Amino acids euclidean distances matrices (EDMs) encode primary structural information. Amino Acid Pseudo-Folding 3D Distances Count (AAp3DC) descriptors, calculated from the EDMs of a large data set of 1363 single protein mutants of 64 proteins, were tested for building a classifier for the signs of the change of thermal unfolding Gibbs free energy change (DeltaDeltaG) upon single mutations. An optimum support vector machine (SVM) with a radial basis function (RBF) kernel well recognized stable and unstable mutants with accuracies over 70% in crossvalidation test. To the best of our knowledge, this result for stable mutant recognition is the highest ever reported for a sequence-based predictor with more than 1000 mutants. Furthermore, the model adequately classified mutations associated to diseases of human prion protein and human transthyretin.

  14. How to fold intricately: using theory and experiments to unravel the properties of knotted proteins

    CERN Document Server

    Jackson, Sophie E; Micheletti, Cristian

    2016-01-01

    Over the years, advances in experimental and computational methods have helped us to understand the role of thermodynamic, kinetic and active (chaperone-aided) effects in coordinating the folding steps required to achieving a knotted native state. Here, we review such developments by paying particular attention to the complementarity of experimental and computational studies. Key open issues that could be tackled with either or both approaches are finally pointed out.

  15. n→π* Interactions in Poly(lactic acid) Suggest a Role in Protein Folding

    OpenAIRE

    Newberry, Robert W; Raines, Ronald T.

    2013-01-01

    Poly(lactic acid) (PLA) is a versatile synthetic polyester. We noted that this depsipeptide analog of polyalanine has a helical structure that resembles a polyproline II helix. Using natural bond orbital analysis, we find that n→π* interactions between sequential ester carbonyl groups contribute 0.44 kcal/mol per monomer to the conformational stability of PLA helices. We conclude that analogous n→π* interactions could direct the folding of a polypeptide chain into a polyproline II helix prior...

  16. 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 Å(2) for Cα and 7.3 ± 0.9-9.6 ± 0.2 Å(2) 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.

  17. MitoNEET Is a Uniquely Folded 2Fe-2S Outer Mitochondrial Membrane Protein Stabilized By Pioglitazone

    Energy Technology Data Exchange (ETDEWEB)

    Paddock, M.L.; Wiley, S.E.; Axelrod, H.L.; Cohen, A.E.; Roy, M.; Abresch, E.C.; Capraro, D.; Murphy, A.N.; Nechushtai, R.; Dixon, J.E.; Jennings, P.A.; /UC, San Diego /SLAC, SSRL /Hebrew U.

    2007-10-19

    Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Angstrom x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the {approx}650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a {beta}-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer.

  18. 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 chime

  19. Scale-free behaviour of amino acid pair interactions in folded proteins

    DEFF Research Database (Denmark)

    Petersen, Steffen B.; Neves-Petersen, Maria Teresa; Mortensen, Rasmus J.

    2012-01-01

    that they are in buried a-helices or b-strands, in a spatial distance of 3.8–4.3A° and in a sequence distance .4 residues. We speculate that the scale free organization of the amino acid pair interactions in the 8D protein structure combined with the clear dominance of pairs of Ala, Ile, Leu and Val is important......The protein structure is a cumulative result of interactions between amino acid residues interacting with each other through space and/or chemical bonds. Despite the large number of high resolution protein structures, the ‘‘protein structure code’’ has not been fully identified. Our manuscript...... presents a novel approach to protein structure analysis in order to identify rules for spatial packing of amino acid pairs in proteins. We have investigated 8706 high resolution non-redundant protein chains and quantified amino acid pair interactions in terms of solvent accessibility, spatial and sequence...

  20. 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.

  1. Location of glycine mutations within a bacterial collagen protein affects degree of disruption of triple-helix folding and conformation.

    Science.gov (United States)

    Cheng, Haiming; Rashid, Shayan; Yu, Zhuoxin; Yoshizumi, Ayumi; Hwang, Eileen; Brodsky, Barbara

    2011-01-21

    The hereditary bone disorder osteogenesis imperfecta is often caused by missense mutations in type I collagen that change one Gly residue to a larger residue and that break the typical (Gly-Xaa-Yaa)(n) sequence pattern. Site-directed mutagenesis in a recombinant bacterial collagen system was used to explore the effects of the Gly mutation position and of the identity of the residue replacing Gly in a homogeneous collagen molecular population. Homotrimeric bacterial collagen proteins with a Gly-to-Arg or Gly-to-Ser replacement formed stable triple-helix molecules with a reproducible 2 °C decrease in stability. All Gly replacements led to a significant delay in triple-helix folding, but a more dramatic delay was observed when the mutation was located near the N terminus of the triple-helix domain. This highly disruptive mutation, close to the globular N-terminal trimerization domain where folding is initiated, is likely to interfere with triple-helix nucleation. A positional effect of mutations was also suggested by trypsin sensitivity for a Gly-to-Arg replacement close to the triple-helix N terminus but not for the same replacement near the center of the molecule. The significant impact of the location of a mutation on triple-helix folding and conformation could relate to the severe consequences of mutations located near the C terminus of type I and type III collagens, where trimerization occurs and triple-helix folding is initiated.

  2. Protein fold recognition with a two-layer method based on SVM-SA, WP-NN and C4.5 (TLM-SNC).

    Science.gov (United States)

    Zangooei, Mohammad Hossein; Jalili, Saeed

    2013-01-01

    The structural knowledge of protein is crucial in understanding its biological role. An effort is made to assign a fold to a given protein in a protein fold recognition problem. A computational Two-Layer Method (TLM) based on the Support Vector Machine (SVM), the Neural Network (NN) and the Decision Tree (C4.5) has been developed in this study for the assignment of a protein sequence to a folding class in SCOP. Prediction accuracy is measured on a dataset and the accuracy of the proposed method is very promising in comparison with other classification methods.

  3. The lid domain of Caenorhabditis elegans Hsc70 influences ATP turnover, cofactor binding and protein folding activity.

    Directory of Open Access Journals (Sweden)

    Li Sun

    Full Text Available Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70 has not been studied to date.We find that CeHsc70 is characterized by a high ATP turnover rate and limited by post-hydrolysis nucleotide exchange. This rate-limiting step is defined by the helical lid domain at the C-terminus. A certain truncation in this domain (CeHsc70-Δ545 reduces the turnover rate and renders the hydrolysis step rate-limiting. The helical lid domain also affects cofactor affinities as the lidless mutant CeHsc70-Δ512 binds more strongly to DNJ-13, forming large protein complexes in the presence of ATP. Despite preserving the ability to hydrolyze ATP and interact with its cofactors DNJ-13 and BAG-1, the truncation of the helical lid domain leads to the loss of all protein folding activity, highlighting the requirement of this domain for the functionality of the nematode's Hsc70 protein.

  4. 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

    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....

  5. 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 correspon

  6. His6 tag-assisted chemical protein synthesis

    Science.gov (United States)

    Bang, Duhee; Kent, Stephen B. H.

    2005-04-01

    To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that His6 tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses. affinity purification | native chemical ligation

  7. Long range correlations and folding angle in polymers with applications to {\\alpha}-helical proteins

    CERN Document Server

    Krokhotin, Andrey; Niemi, Antti J

    2013-01-01

    The conformational complexity of linear polymers far exceeds that of point-like atoms and molecules. Polymers can bend, twist, even become knotted. Thus they may also display a much richer phase structure than point particles. But it is not very easy to characterize the phase of a polymer. Essentially, the only 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, discloses the phase of the polymer. Moreover, 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, a novel tool to identify and scrutinize the phases of polymers. We argue for a mean-field relationship between its values and those of the scaling exponent in the radius of gyration. But unlike in the case of the radius of gyration, the value of the folding angle can be evaluated from a s...

  8. A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter*

    Science.gov (United States)

    El-Kasaby, Ali; Koban, Florian; Sitte, Harald H.; Freissmuth, Michael; Sucic, Sonja

    2014-01-01

    Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay. PMID:25202009

  9. Lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: comparison with detergent micelles, bicelles and liposomes.

    Science.gov (United States)

    Lyukmanova, E N; Shenkarev, Z O; Khabibullina, N F; Kopeina, G S; Shulepko, M A; Paramonov, A S; Mineev, K S; Tikhonov, R V; Shingarova, L N; Petrovskaya, L E; Dolgikh, D A; Arseniev, A S; Kirpichnikov, M P

    2012-03-01

    Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.

  10. TDP-43 Proteinopathies: A New Player in Neurodegenerative Diseases with Defective Protein Folding

    Directory of Open Access Journals (Sweden)

    Suna Lahut

    2012-03-01

    Full Text Available The proteome is the sum of all proteins inside a cell, and proteostasis (protein homeostasis is the stable condition of the proteome. Proteostasis is essential for the cellular and organismal health. Stress, aging and the chronic expression of misfolded proteins challenge the proteostasis machinery and the vitality of the cell. There is increasing evidence that the accumulation of damaged proteins not only has direct consequences on the efficiency and fidelity of cellular processes but, when not corrected, that they initiate a cascade of dysfunction, which in humans is associated with a plethora of diseases of protein conformation, referred to as proteinopathies. Alzheimer’s Disease (AD, Parkinson’s Disease (PD, Huntington’s Disease (HD, Amyotrophic Lateral Sclerosis (ALS, cancer and diabetes, whose frequencies have drastically increased in countries with aging populations, are all consequences of misfolded proteins. This paper focuses on TDP-43, which excelled as a key protein in neurodegenerative processes because of its association with different diseases, especially with ALS and Frontotemporal Lobar Dementia (FTLD, the two best studied examples of TDP-43 proteinopathies.

  11. Symmetric key structural residues in symmetric proteins with beta-trefoil fold.

    Directory of Open Access Journals (Sweden)

    Jianhui Feng

    Full Text Available To understand how symmetric structures of many proteins are formed from asymmetric sequences, the proteins with two repeated beta-trefoil domains in Plant Cytotoxin B-chain family and all presently known beta-trefoil proteins are analyzed by structure-based multi-sequence alignments. The results show that all these proteins have similar key structural residues that are distributed symmetrically in their structures. These symmetric key structural residues are further analyzed in terms of inter-residues interaction numbers and B-factors. It is found that they can be distinguished from other residues and have significant propensities for structural framework. This indicates that these key structural residues may conduct the formation of symmetric structures although the sequences are asymmetric.

  12. Protein sequence comparison and fold recognition: progress and good-practice benchmarking.

    Science.gov (United States)

    Söding, Johannes; Remmert, Michael

    2011-06-01

    Protein sequence comparison methods have grown increasingly sensitive during the last decade and can often identify distantly related proteins sharing a common ancestor some 3 billion years ago. Although cellular function is not conserved so long, molecular functions and structures of protein domains often are. In combination with a domain-centered approach to function and structure prediction, modern remote homology detection methods have a great and largely underexploited potential for elucidating protein functions and evolution. Advances during the last few years include nonlinear scoring functions combining various sequence features, the use of sequence context information, and powerful new software packages. Since progress depends on realistically assessing new and existing methods and published benchmarks are often hard to compare, we propose 10 rules of good-practice benchmarking.

  13. ORION: a web server for protein fold recognition and structure prediction using evolutionary hybrid profiles.

    Science.gov (United States)

    Ghouzam, Yassine; Postic, Guillaume; Guerin, Pierre-Edouard; de Brevern, Alexandre G; Gelly, Jean-Christophe

    2016-06-20

    Protein structure prediction based on comparative modeling is the most efficient way to produce structural models when it can be performed. ORION is a dedicated webserver based on a new strategy that performs this task. The identification by ORION of suitable templates is performed using an original profile-profile approach that combines sequence and structure evolution information. Structure evolution information is encoded into profiles using structural features, such as solvent accessibility and local conformation -with Protein Blocks-, which give an accurate description of the local protein structure. ORION has recently been improved, increasing by 5% the quality of its results. The ORION web server accepts a single protein sequence as input and searches homologous protein structures within minutes. Various databases such as PDB, SCOP and HOMSTRAD can be mined to find an appropriate structural template. For the modeling step, a protein 3D structure can be directly obtained from the selected template by MODELLER and displayed with global and local quality model estimation measures. The sequence and the predicted structure of 4 examples from the CAMEO server and a recent CASP11 target from the 'Hard' category (T0818-D1) are shown as pertinent examples. Our web server is accessible at http://www.dsimb.inserm.fr/ORION/.

  14. Contribution of hydration to protein folding thermodynamics. I. The enthalpy of hydration.

    Science.gov (United States)

    Makhatadze, G I; Privalov, P L

    1993-07-20

    The enthalpy of hydration of polar and non-polar groups upon protein unfolding has been estimated for four globular proteins in the temperature range 5 to 125 degrees C, using structural information on the groups in these proteins exposed to water in the native and unfolded states and volume-corrected calorimetric information on the enthalpy and heat capacity of transfer into water of various model compounds. It has been shown that the enthalpy of hydration of polar groups greatly exceeds the enthalpy of hydration of non-polar groups. At low temperatures both these enthalpies are negative and change in opposite direction with increasing temperature. Subtracting the total enthalpy of hydration of polar and non-polar groups from the calorimetrically determined enthalpy of protein unfolding, the total enthalpy of internal interactions maintaining the native protein structure has been determined. Using thermodynamic information on the sublimation of organic crystals, the total enthalpy was divided into two components: one associated with the interactions between the non-polar groups (van der Waals interaction) and the rest associated with the interactions between polar groups (hydrogen bonding). This made it possible to estimate the overall enthalpies of disruption of contacts between the polar groups with their exposure to water and between the non-polar groups with their exposure to water. It appears that these enthalpies have opposite signs in the temperature range considered and change in opposite directions with increasing temperature. The enthalpy of transfer of non-polar groups from the protein interior into water is negative below 25 degrees C and positive above. The enthalpy of transfer of polar groups from the protein interior into water is positive at low temperatures and becomes negative at higher temperatures. Over the considered temperature range, however, the enthalpy of transfer of non-polar groups dominates. This results in a positive enthalpy of

  15. Defining the nature of thermal intermediate in 3 state folding proteins: apoflavodoxin, a study case.

    Directory of Open Access Journals (Sweden)

    Rebeca García-Fandiño

    Full Text Available The early stages of the thermal unfolding of apoflavodoxin have been determined by using atomistic multi microsecond-scale molecular dynamics (MD simulations complemented with a variety of experimental techniques. Results strongly suggest that the intermediate is reached very early in the thermal unfolding process and that it has the properties of an "activated" form of the native state, where thermal fluctuations in the loops break loop-loop contacts. The unrestrained loops gain then kinetic energy corrupting short secondary structure elements without corrupting the core of the protein. The MD-derived ensembles agree with experimental observables and draw a picture of the intermediate state inconsistent with a well-defined structure and characteristic of a typical partially disordered protein. Our results allow us to speculate that proteins with a well packed core connected by long loops might behave as partially disordered proteins under native conditions, or alternatively behave as three state folders. Small details in the sequence, easily tunable by evolution, can yield to one or the other type of proteins.

  16. Reproducible In-Silico Folding of a Four Helix 60 Amino Acid Protein in a Transferable All-Atom Forcefield

    Science.gov (United States)

    Schug, Alexander

    2005-03-01

    For predicting the protein tertiary structure one approach describes the native state of a protein as the global minimum of an appropiate free-energy forcefield. We have recently developed such a all-atom protein forcefield (PFF01). As major challenge remains the search for the global minimum for which we developed efficient methods. Using these we were able to predict the structure of helical proteins from different families ranging in size from 20 to 60 amino acids starting with random configurations. For the four helix 60 amino acid protein Bacterial Ribosomal Protein L20 (pdb code: 1GYZ) we used a simple client-master model for distributed computing. Starting from a set of random structures three phases of different folding simulations refined this set to a final one with 50 configurations. During this process the amount of native-like structures increased strongly. Six out of the ten structures best in energy approached the native structure within 5 åbackbone rmsd. The conformation with the lowest energy had a backbone rmsd value of 4.6 åtherefore correctly predicting the tertiary structure of 1GYZ.ReferencesA. Schug et al, Phys. Rev. Letters, 91:158102, 2003A. Schug et al, J. Am. Chem. Soc. (in press), 2004

  17. Expression of factor H binding protein in meningococcal strains can vary at least 15-fold and is genetically determined.

    Science.gov (United States)

    Biagini, Massimiliano; Spinsanti, Marco; De Angelis, Gabriella; Tomei, Sara; Ferlenghi, Ilaria; Scarselli, Maria; Rigat, Fabio; Messuti, Nicola; Biolchi, Alessia; Muzzi, Alessandro; Anderloni, Giulia; Brunelli, Brunella; Cartocci, Elena; Buricchi, Francesca; Tani, Chiara; Stella, Maria; Moschioni, Monica; Del Tordello, Elena; Colaprico, Annalisa; Savino, Silvana; Giuliani, Marzia M; Delany, Isabel; Pizza, Mariagrazia; Costantino, Paolo; Norais, Nathalie; Rappuoli, Rino; Masignani, Vega

    2016-03-01

    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). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.

  18. A coarse-grained protein force field for folding and structure prediction.

    Science.gov (United States)

    Maupetit, Julien; Tuffery, P; Derreumaux, Philippe

    2007-11-01

    We have revisited the protein coarse-grained optimized potential for efficient structure prediction (OPEP). The training and validation sets consist of 13 and 16 protein targets. Because optimization depends on details of how the ensemble of decoys is sampled, trial conformations are generated by molecular dynamics, threading, greedy, and Monte Carlo simulations, or taken from publicly available databases. The OPEP parameters are varied by a genetic algorithm using a scoring function which requires that the native structure has the lowest energy, and the native-like structures have energy higher than the native structure but lower than the remote conformations. Overall, we find that OPEP correctly identifies 24 native or native-like states for 29 targets and has very similar capability to the all-atom discrete optimized protein energy model (DOPE), found recently to outperform five currently used energy models.

  19. Cyt toxins produced by Bacillus thuringiensis: a protein fold conserved in several pathogenic microorganisms.

    Science.gov (United States)

    Soberón, Mario; López-Díaz, Jazmin A; Bravo, Alejandra

    2013-03-01

    Bacillus thuringiensis bacteria produce different insecticidal proteins known as Cry and Cyt toxins. Among them the Cyt toxins represent a special and interesting group of proteins. Cyt toxins are able to affect insect midgut cells but also are able to increase the insecticidal damage of certain Cry toxins. Furthermore, the Cyt toxins are able to overcome resistance to Cry toxins in mosquitoes. There is an increasing potential for the use of Cyt toxins in insect control. However, we still need to learn more about its mechanism of action in order to define it at the molecular level. In this review we summarize important aspects of Cyt toxins produced by Bacillus thuringiensis, including current knowledge of their mechanism of action against mosquitoes and also we will present a primary sequence and structural comparison with related proteins found in other pathogenic bacteria and fungus that may indicate that Cyt toxins have been selected by several pathogenic organisms to exert their virulence phenotypes.

  20. Folding and fibril formation of the cell cycle protein Cks1.

    Science.gov (United States)

    Bader, Reto; Seeliger, Markus A; Kelly, Sadie E; Ilag, Leopold L; Meersman, Filip; Limones, Alejandra; Luisi, Ben F; Dobson, Christopher M; Itzhaki, Laura S

    2006-07-07

    The Saccharomyces cerevisiae Cks protein Cks1 has a COOH-terminal glutamine-rich sequence not present in other homologues. Cks proteins domain swap to form dimers but unique to Cks1 is the anti-parallel arrangement of protomers within the dimer. Despite the differences in Cks1 compared with other Cks proteins, we find the domain swapping properties are very similar. However, aggregation of Cks1 occurs by a route distinct from the other Cks proteins studied to date. Cks1 formed fibrillar aggregates at room temperature and neutral pH. During this process, Cks1 underwent proteolytic cleavage at a trypsin-like site into two fragments, the globular Cks domain and the glutamine-rich COOH terminus. At high protein concentrations, the rate of fibril formation was the same as the rate of proteolysis. The dominant species present within the fibrils was the glutamine-rich sequence. Consistent with this result, fibril formation was enhanced by addition of trypsin. Moreover, a truncated variant lacking the glutamine-rich sequence did not form fibrils under the same conditions. A lag phase at low protein concentrations indicates that fibril formation occurs through a nucleation and growth mechanism. The aggregates appear to resemble amyloid fibrils, in that they show the typical cross-beta x-ray diffraction pattern. Moreover, infrared spectroscopy data indicate that the glutamine side chains are hydrogen-bonded along the axis of the fibril. Our results indicate that the proteolytic reaction is the crucial step initiating aggregation and demonstrate that Cks1 is a simple, tunable model system for exploring aggregation mechanisms associated with polyglutamine deposition diseases.

  1. 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.

  2. Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.

    Science.gov (United States)

    Curran, A R; Templer, R H; Booth, P J

    1999-07-20

    Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.

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

    Science.gov (United States)

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

    2016-04-07

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

  4. Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water.

    Science.gov (United States)

    Spolar, R S; Livingstone, J R; Record, M T

    1992-04-28

    This extension of the liquid hydrocarbon model seeks to quantify the thermodynamic contributions to protein stability from the removal of nonpolar and polar surface from water. Thermodynamic data for the transfer of hydrocarbons and organic amides from water to the pure liquid phase are analyzed to obtain contributions to the thermodynamics of folding from the reduction in water-accessible surface area. Although the removal of nonpolar surface makes the dominant contribution to the standard heat capacity change of folding (delta C0fold), here we show that inclusion of the contribution from removal of polar surface allows a quantitative prediction of delta C0fold within the uncertainty of the calorimetrically determined value. Moreover, analysis of the contribution of polar surface area to the enthalpy of transfer of liquid amides provides a means of estimating the contributions from changes in nonpolar and polar surface area as well as other factors to the enthalpy of folding (delta H0fold). In addition to estimates of delta H0fold, this extension of the liquid hydrocarbon model provides a thermodynamic explanation for the observation [Privalov, P. L., & Khechinashvili, N. N. (1974) J. Mol. Biol. 86, 665-684] that the specific enthalpy of folding (cal g-1) of a number of globular proteins converges to a common value at approximately 383 K. Because amounts of nonpolar and polar surface area buried by these proteins upon folding are found to be linear functions of molar mass, estimates of both delta C0fold and delta H0fold may be obtained given only the molar mass of the protein of interest.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. 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.

  6. Protein folding of the HOP model: A parallel Wang—Landau study

    Science.gov (United States)

    Shi, G.; Wüst, T.; Li, Y. W.; Landau, D. P.

    2015-09-01

    We propose a simple modification to the hydrophobic-polar (HP) protein model, by introducing a new type of monomer, “0”, with intermediate hydrophobicity of some amino acids between H and P. With the replica-exchange Wang-Landau sampling method, we investigate some widely studied HP sequences as well as their H0P counterparts and observe that the H0P sequences exhibit dramatically reduced ground state degeneracy and more significant transition signals at low temperature for some thermodynamic properties, such as the specific heat.

  7. Protein folding of the H0P model: A parallel Wang-Landau study

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Guangjie [University of Georgia, Athens, GA; Wuest, Thomas [ETH Zurich, Switzerland; Li, Ying Wai [ORNL; Landau, David P [University of Georgia, Athens, GA

    2015-01-01

    We propose a simple modication to the hydrophobic-polar (HP) protein model, by introducing a new type of monomer, "0", with intermediate hydrophobicity of some amino acids between H and P. With the replica-exchange Wang-Landau sampling method, we investigate some widely studied HP sequences as well as their H0P counterparts and observe that the H0P sequences exhibit dramatically reduced ground state degeneracy and more signicant transition signals at low temperature for some thermodynamic properties, such as the specific heat.

  8. Dislocation, fold and striae of corneal flap with laser assisted in situ keratomileusis after ocular trauma: a case report

    Institute of Scientific and Technical Information of China (English)

    Sang Yanzhi; Liu Xin; Zhong Ming; Zhao Chunyan

    2008-01-01

    Objective: To report the occurrence, management and outcome of late-onset traumatic dehiscence and islocation of laser assisted in situ keratomileusis (LASIK) flaps. Treatment and Results: One patient occurred ate-onset LASIK corneal flap dislocation after ocular trauma 7days after surgery. The flap was lifted, stretched, and epositioned after irrigation and scraping of the stromal bed and the underside of the flap. A bandage contact lens as placed, and topical antibiotic and corticosteroids were given postoperatively. The dislocated corneal flap was successfully repositioned in the case. The dislocated flap was repositioned 7 days after the trauma, and the patient recovered his uncorrected visual acuity (UCVA) of 10/20, 20/20 day 1 and day 20 after the procedure, of 20/20 20 days later and had a well-positioned flap with a clear interface. Diffuse lamellar keratitis developed in the patients hat resolved with the use of topical corticosteroids. Conclusion: Laser in situ keratomileusis corneal flaps are ulnerable to traumatic dehiscence and dislocation, which should be pay more attention to it for us.

  9. In Silico Folding of a Three Helix Protein and Characterization of Its Free-Energy Landscape in an All-Atom Force Field

    Science.gov (United States)

    Herges, T.; Wenzel, W.

    2005-01-01

    We report the reproducible first-principles folding of the 40 amino-acid, three-helix headpiece of the HIV accessory protein in a recently developed all-atom free-energy force field. Six of 20 simulations using an adapted basin-hopping method converged to better than 3Å backbone rms deviation to the experimental structure. Using over 60 000 low-energy conformations of this protein, we constructed a decoy tree that completely characterizes its folding funnel.

  10. In-silico folding of a three helix protein and characterization of its free-energy landscape in an all-atom forcefield

    CERN Document Server

    Herges, T

    2003-01-01

    We report the reproducible first-principles folding of the 40 amino acid, three-helix headpiece of the HIV accessory protein in a recently developed all-atom free-energy forcefield. Six of twenty simulations using an adapted basin-hopping method converged to better than 3 \\AA backbone RMS deviation to the experimental structure. Using over 60,000 low-energy conformations of this protein, we constructed a decoy tree that completely characterizes its folding funnel.

  11. A light-harvesting antenna protein retains its folded conformation in the absence of protein-lipid and protein-pigment interactions.

    Science.gov (United States)

    Kikuchi, J; Asakura, T; Loach, P A; Parkes-Loach, P S; Shimada, K; Hunter, C N; Conroy, M J; Williamson, M P

    1999-04-15

    The first study by nmr of the integral membrane protein, the bacterial light-harvesting (LH) antenna protein LH1 beta, is reported. The photosynthetic apparatus of purple bacteria contains two different kinds of antenna complexes (LH1 and LH2), which consist of two small integral membrane proteins alpha and beta, each of approximately 6 kDa, and bacteriochlorophyll and carotenoid pigments. We have purified the antenna polypeptide LH1 beta from Rhodobacter sphaeroides, and have recorded CD spectra and a series of two-dimensional nmr spectra. A comparison of CD spectra of LH1 beta observed in organic solvents and detergent micelles shows that the helical character of the peptide does not change appreciably between the two milieus. A significantly high-field shifted methyl signal was observed both in organic solvents and in detergent micelles, implying that a similar three-dimensional structure is present in each case. However, the 1H-nmr signals observed in organic solvents had a narrower line width and better resolution, and it is shown that in this case organic solvents provide a better medium for nmr studies than detergent micelles. A sequential assignment has been carried out on the C-terminal transmembrane region, which is the region in which the pigment is bound. The region is shown to have a helical structure by the chemical shift values of the alpha-CH protons and the presence of nuclear Overhauser effects characteristic of helices. An analysis of the amide proton chemical shifts of the residues surrounding the histidine chlorophyll ligand suggests that the local structure is well ordered even in the absence of protein-lipid and protein-pigment interactions. Its structure was determined from 348 nmr-derived constraints by using distance geometry calculations. The polypeptide contains an alpha-helix extending from Leu19 (position of cytoplasmic surface) to Trp44 (position of periplasmic surface). The helix is bent, as expected from the amide proton chemical

  12. Pressure-assisted protein extraction: a novel method for recovering proteins from archival tissue for proteomic analysis.

    Science.gov (United States)

    Fowler, Carol B; Waybright, Timothy J; Veenstra, Timothy D; O'Leary, Timothy J; Mason, Jeffrey T

    2012-04-06

    Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.

  13. A galaxy of folds.

    Science.gov (United States)

    Alva, Vikram; Remmert, Michael; Biegert, Andreas; Lupas, Andrei N; Söding, Johannes

    2010-01-01

    Many protein classification systems capture homologous relationships by grouping domains into families and superfamilies on the basis of sequence similarity. Superfamilies with similar 3D structures are further grouped into folds. In the absence of discernable sequence similarity, these structural similarities were long thought to have originated independently, by convergent evolution. However, the growth of databases and advances in sequence comparison methods have led to the discovery of many distant evolutionary relationships that transcend the boundaries of superfamilies and folds. To investigate the contributions of convergent versus divergent evolution in the origin of protein folds, we clustered representative domains of known structure by their sequence similarity, treating them as point masses in a virtual 2D space which attract or repel each other depending on their pairwise sequence similarities. As expected, families in the same superfamily form tight clusters. But often, superfamilies of the same fold are linked with each other, suggesting that the entire fold evolved from an ancient prototype. Strikingly, some links connect superfamilies with different folds. They arise from modular peptide fragments of between 20 and 40 residues that co-occur in the connected folds in disparate structural contexts. These may be descendants of an ancestral pool of peptide modules that evolved as cofactors in the RNA world and from which the first folded proteins arose by amplification and recombination. Our galaxy of folds summarizes, in a single image, most known and many yet undescribed homologous relationships between protein superfamilies, providing new insights into the evolution of protein domains.

  14. VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.

    Science.gov (United States)

    Ren, Hong Yu; Grove, Diane E; De La Rosa, Oxana; Houck, Scott A; Sopha, Pattarawut; Van Goor, Fredrick; Hoffman, Beth J; Cyr, Douglas M

    2013-10-01

    Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.

  15. Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells.

    Directory of Open Access Journals (Sweden)

    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.

  16. Relevance of structural segregation and chain compaction for the thermodynamics of folding of a hydrophobic protein model.

    Science.gov (United States)

    Barbosa, Marco Aurélio A; de Araújo, Antônio F Pereira

    2003-05-01

    The relevance of inside-outside segregation and chain compaction for the thermodynamics of folding of a hydrophobic protein model is probed by complete enumeration of two-dimensional chains of up to 18 monomers in the square lattice. The exact computation of Z scores for uniquely designed sequences confirms that Z tends to decrease linearly with sigma square root of N, as previously suggested by theoretical analysis and Monte Carlo simulations, where sigma, the standard deviation of the number of contacts made by different monomers in the target structure, is a measure of structural segregation and N is the chain length. The probability that the target conformation is indeed the unique global energy minimum of the designed sequence is found to increase dramatically with sigma, approaching unity at maximal segregation. However, due to the huge number of conformations with sub-maximal values of sigma, which correspond to intermediate, only mildly discriminative, values of Z, in addition to significant oscillations of Z around its estimated value, the probability that a correctly designed sequence corresponds to a maximally segregated conformation is small. This behavior of Z also explains the observed relation between sigma and different measures of folding cooperativity of correctly designed sequences.

  17. A Hamiltonian Replica Exchange Molecular Dynamics (MD Method for the Study of Folding, Based on the Analysis of the Stabilization Determinants of Proteins

    Directory of Open Access Journals (Sweden)

    Massimiliano Meli

    2013-06-01

    Full Text Available Herein, we present a novel Hamiltonian replica exchange protocol for classical molecular dynamics simulations of protein folding/unfolding. The scheme starts from the analysis of the energy-networks responsible for the stabilization of the folded conformation, by means of the energy-decomposition approach. In this framework, the compact energetic map of the native state is generated by a preliminary short molecular dynamics (MD simulation of the protein in explicit solvent. This map is simplified by means of an eigenvalue decomposition. The highest components of the eigenvector associated with the lowest eigenvalue indicate which sites, named “hot spots”, are likely to be responsible for the stability and correct folding of the protein. In the Hamiltonian replica exchange protocol, we use modified force-field parameters to treat the interparticle non-bonded potentials of the hot spots within the protein and between protein and solvent atoms, leaving unperturbed those relative to all other residues, as well as solvent-solvent interactions. We show that it is possible to reversibly simulate the folding/unfolding behavior of two test proteins, namely Villin HeadPiece HP35 (35 residues and Protein A (62 residues, using a limited number of replicas. We next discuss possible implications for the study of folding mechanisms via all atom simulations.

  18. A Hamiltonian replica exchange molecular dynamics (MD) method for the study of folding, based on the analysis of the stabilization determinants of proteins.

    Science.gov (United States)

    Meli, Massimiliano; Colombo, Giorgio

    2013-06-06

    Herein, we present a novel Hamiltonian replica exchange protocol for classical molecular dynamics simulations of protein folding/unfolding. The scheme starts from the analysis of the energy-networks responsible for the stabilization of the folded conformation, by means of the energy-decomposition approach. In this framework, the compact energetic map of the native state is generated by a preliminary short molecular dynamics (MD) simulation of the protein in explicit solvent. This map is simplified by means of an eigenvalue decomposition. The highest components of the eigenvector associated with the lowest eigenvalue indicate which sites, named "hot spots", are likely to be responsible for the stability and correct folding of the protein. In the Hamiltonian replica exchange protocol, we use modified force-field parameters to treat the interparticle non-bonded potentials of the hot spots within the protein and between protein and solvent atoms, leaving unperturbed those relative to all other residues, as well as solvent-solvent interactions. We show that it is possible to reversibly simulate the folding/unfolding behavior of two test proteins, namely Villin HeadPiece HP35 (35 residues) and Protein A (62 residues), using a limited number of replicas. We next discuss possible implications for the study of folding mechanisms via all atom simulations.

  19. Essential roles of protein-solvent many-body correlation in solvent-entropy effect on protein folding and denaturation: comparison between hard-sphere solvent and water.

    Science.gov (United States)

    Oshima, Hiraku; Kinoshita, Masahiro

    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

  20. Genes Related to Mitochondrial Functions, Protein Degradation, and Chromatin Folding Are Differentially Expressed in Lymphomonocytes of Rett Syndrome Patients

    Science.gov (United States)

    Leoni, Guido; Cervellati, Franco; Canali, Raffaella; Cortelazzo, Alessio; De Felice, Claudio; Ciccoli, Lucia; Hayek, Joussef

    2013-01-01

    Rett syndrome (RTT) is mainly caused by mutations in the X-linked methyl-CpG binding protein (MeCP2) gene. By binding to methylated promoters on CpG islands, MeCP2 protein is able to modulate several genes and important cellular pathways. Therefore, mutations in MeCP2 can seriously affect the cellular phenotype. Today, the pathways that MeCP2 mutations are able to affect in RTT are not clear yet. The aim of our study was to investigate the gene expression profiles in peripheral blood lymphomonocytes (PBMC) isolated from RTT patients to try to evidence new genes and new pathways that are involved in RTT pathophysiology. LIMMA (Linear Models for MicroArray) and SAM (Significance Analysis of Microarrays) analyses on microarray data from 12 RTT patients and 7 control subjects identified 482 genes modulated in RTT, of which 430 were upregulated and 52 were downregulated. Functional clustering of a total of 146 genes in RTT identified key biological pathways related to mitochondrial function and organization, cellular ubiquitination and proteosome degradation, RNA processing, and chromatin folding. Our microarray data reveal an overexpression of genes involved in ATP synthesis suggesting altered energy requirement that parallels with increased activities of protein degradation. In conclusion, these findings suggest that mitochondrial-ATP-proteasome functions are likely to be involved in RTT clinical features. PMID:24453408

  1. Genes Related to Mitochondrial Functions, Protein Degradation, and Chromatin Folding Are Differentially Expressed in Lymphomonocytes of Rett Syndrome Patients

    Directory of Open Access Journals (Sweden)

    Alessandra Pecorelli

    2013-01-01

    Full Text Available Rett syndrome (RTT is mainly caused by mutations in the X-linked methyl-CpG binding protein (MeCP2 gene. By binding to methylated promoters on CpG islands, MeCP2 protein is able to modulate several genes and important cellular pathways. Therefore, mutations in MeCP2 can seriously affect the cellular phenotype. Today, the pathways that MeCP2 mutations are able to affect in RTT are not clear yet. The aim of our study was to investigate the gene expression profiles in peripheral blood lymphomonocytes (PBMC isolated from RTT patients to try to evidence new genes and new pathways that are involved in RTT pathophysiology. LIMMA (Linear Models for MicroArray and SAM (Significance Analysis of Microarrays analyses on microarray data from 12 RTT patients and 7 control subjects identified 482 genes modulated in RTT, of which 430 were upregulated and 52 were downregulated. Functional clustering of a total of 146 genes in RTT identified key biological pathways related to mitochondrial function and organization, cellular ubiquitination and proteosome degradation, RNA processing, and chromatin folding. Our microarray data reveal an overexpression of genes involved in ATP synthesis suggesting altered energy requirement that parallels with increased activities of protein degradation. In conclusion, these findings suggest that mitochondrial-ATP-proteasome functions are likely to be involved in RTT clinical features.

  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.

  3. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

    Energy Technology Data Exchange (ETDEWEB)

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M.F.; Downes, C. Peter; Batty, Ian H. (Toronto); (Dundee)

    2012-10-16

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

  4. Alternative splice variants in TIM barrel proteins from human genome correlate with the structural and evolutionary modularity of this versatile protein fold.

    Science.gov (United States)

    Ochoa-Leyva, Adrián; Montero-Morán, Gabriela; Saab-Rincón, Gloria; Brieba, Luis G; Soberón, Xavier

    2013-01-01

    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 (βα)8 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 (βα)2, (βα)4, (βα)5, (βα)6, and (βα)7 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 diversify the

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

    Science.gov (United States)

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

    2014-06-01

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

  6. Quantitative conformational analysis of partially folded proteins from residual dipolar couplings: application to the molecular recognition element of Sendai virus nucleoprotein.

    Science.gov (United States)

    Jensen, Malene Ringkjøbing; Houben, Klaartje; Lescop, Ewen; Blanchard, Laurence; Ruigrok, Rob W H; Blackledge, Martin

    2008-06-25

    A significant fraction of proteins coded in the human proteome do not fold into stable three-dimensional structures but are either partially or completely unfolded. A key feature of this family of proteins is their proposed capacity to undergo a disorder-to-order transition upon interaction with a physiological partner. The mechanisms governing protein folding upon interaction, in particular the extent to which recognition elements are preconfigured prior to formation of molecular complexes, can prove difficult to resolve in highly flexible systems. Here, we develop a conformational model of this type of protein, using an explicit description of the unfolded state, specifically modified to allow for the presence of transient secondary structure, and combining this with extensive measurement of residual dipolar couplings throughout the chain. This combination of techniques allows us to quantitatively analyze the level and nature of helical sampling present in the interaction site of the partially folded C-terminal domain of Sendai virus nucleoprotein (N(TAIL)). Rather than fraying randomly, the molecular recognition element of N(TAIL) preferentially populates three specific overlapping helical conformers, each stabilized by an N-capping interaction. The unfolded strands adjacent to the helix are thereby projected in the direction of the partner protein, identifying a mechanism by which they could achieve nonspecific encounter interactions prior to binding. This study provides experimental evidence for the molecular basis of helix formation in partially folded peptide chains, carrying clear implications for understanding early steps of protein folding.

  7. Impact of P-Site tRNA and antibiotics on ribosome mediated protein folding: studies using the Escherichia coli ribosome.

    Directory of Open Access Journals (Sweden)

    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.

  8. Aggregation of a slow-folding mutant of a beta-clam protein proceeds through a monomeric nucleus.

    Science.gov (United States)

    Ignatova, Zoya; Gierasch, Lila M

    2005-05-17

    Mechanistic understanding of protein aggregation, leading either to structured amyloid fibrils or to amorphous inclusion body-like deposits, should facilitate the identification of potential therapeutic intervention strategies for the devastating amyloid-based diseases. Here we focus on the in vitro aggregation of a slow-folding mutant of the beta-clam protein, cellular retinoic acid-binding protein I (P39A CRABP I), which forms inclusion bodies when expressed in Escherichia coli. Aggregation was monitored by observing the fluorescence of a fluorescein-based biarsenical dye (FlAsH) that ligates to a tetra-Cys motif, here incorporated into a flexible Omega-loop. The fluorescence signal of FlAsH on the tetra-Cys-containing P39A CRABP I is sensitive to whether this protein is native or unfolded, and was used in combination with other techniques to follow aggregate formation. The aggregation time course is compatible with a nucleation-dependent polymerization model, and detailed kinetic analysis showed that the energetically unfavorable nucleus is monomeric. A similar conclusion was reached previously for poly(Gln) species [Chen, S., Ferrone, F. A., and Wetzel, R. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11884-11889] and points to an unfavorable equilibrium between the misfolded intermediate and the bulk pool of monomers as causative in aggregation. The P39A mutation, which removes a helix-stop signal, may slow closure of the beta-barrel in P39A CRABP I relative to the wild type, leaving it vulnerable to aggregation. Wide-angle X-ray scattering showed that the amorphous aggregates formed by the aggregation-prone intermediates of P39A CRABP I contain predominantly beta-strands structured in a lamellar fashion with 10.03 A spacing between adjacent beta-sheets.

  9. 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

    The glucose-sensitive insulin-secretion (GSIS) phenotype is relatively unstable in long-term culture of beta cells. The purpose of this study was to investigate relative changes in the proteome between glucose-responsive (low passage) and glucose non-responsive (high passage) murine MIN-6...... pancreatic beta cells. The 2D-DIGE and subsequent DeCyder analysis detected 3351 protein spots in the pH range of 4-7. Comparing MIN-6(H) to MIN-6(L) and using a threshold of 1.2-fold, the number of proteins with a decrease in expression level was 152 (4.5%), similar was 3140 (93.7%) and increased 59 (1.......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...

  10. The adsorption and unfolding kinetics determines the folding state of proteins at the air-water interface and thereby the equation of state

    NARCIS (Netherlands)

    Wierenga, P.A.; Egmond, M.R.; Voragen, A.G.J.; Jongh, H.H.J.de

    2006-01-01

    Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends o

  11. 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

    2010-01-01

    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 indispe

  12. Characterization of single-tryptophan mutants of histidine-containing phosphocarrier protein : Evidence for local rearrangements during folding from high concentrations of denaturan

    NARCIS (Netherlands)

    Azuaga, AI; Canet, D; Smeenk, G; Berends, R; Titgemeijer, F; Duurkens, R; Mateo, PL; Scheek, RM; Robillard, GT; Dobson, CM; van Nuland, NAJ; Azuaga, Ana I.; Mateo, Pedro L.; Dobson, Christopher M.

    2003-01-01

    We have used site-directed mutagenesis in combination with a battery of biophysical techniques to probe the stability and folding behavior of a small globular protein, the histidine-containing phosphocarrier protein (HPr). Specifically, the four phenylalanine residues (2, 22, 29, and 48) of the wild

  13. 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.

  14. RNA-Seq reveals expression signatures of genes involved in oxygen transport, protein synthesis, folding, and degradation in response to heat stress in catfish.

    Science.gov (United States)

    Liu, Shikai; Wang, Xiuli; Sun, Fanyue; Zhang, Jiaren; Feng, Jianbin; Liu, Hong; Rajendran, K V; Sun, Luyang; Zhang, Yu; Jiang, Yanliang; Peatman, Eric; Kaltenboeck, Ludmilla; Kucuktas, Huseyin; Liu, Zhanjiang

    2013-06-17

    Temperature is one of the most prominent abiotic factors affecting ectotherms. Most fish species, as ectotherms, have extraordinary ability to deal with a wide range of temperature changes. While the molecular mechanism underlying temperature adaptation has long been of interest, it is still largely unexplored with fish. Understanding of the fundamental mechanisms conferring tolerance to temperature fluctuations is a topic of increasing interest as temperature may continue to rise as a result of global climate change. Catfish have a wide natural habitat and possess great plasticity in dealing with environmental variations in temperature. However, no studies have been conducted at the transcriptomic level to determine heat stress-induced gene expression. In the present study, we conducted an RNA-Seq analysis to identify heat stress-induced genes in catfish at the transcriptome level. Expression analysis identified a total of 2,260 differentially expressed genes with a cutoff of twofold change. qRT-PCR validation suggested the high reliability of the RNA-Seq results. Gene ontology, enrichment, and pathway analyses were conducted to gain insight into physiological and gene pathways. Specifically, genes involved in oxygen transport, protein folding and degradation, and metabolic process were highly induced, while general protein synthesis was dramatically repressed in response to the lethal temperature stress. This is the first RNA-Seq-based expression study in catfish in response to heat stress. The candidate genes identified should be valuable for further targeted studies on heat tolerance, thereby assisting the development of heat-tolerant catfish lines for aquaculture.

  15. 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.

  16. N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

    Directory of Open Access Journals (Sweden)

    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.

  17. 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

    2016-01-01

    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. Availability: the webserver can be accessed via http://compbio.clemson.edu/SAAFEC/. PMID:27070572

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. Recovering kinetics from a simplified protein folding model using replica exchange simulations: a kinetic network and effective stochastic dynamics.

    Science.gov (United States)

    Zheng, Weihua; Andrec, Michael; Gallicchio, Emilio; Levy, Ronald M

    2009-08-27

    We present an approach to recover kinetics from a simplified protein folding model at different temperatures using the combined power of replica exchange (RE), a kinetic network, and effective stochastic dynamics. While RE simulations generate a large set of discrete states with the correct thermodynamics, kinetic information is lost due to the random exchange of temperatures. We show how we can recover the kinetics of a 2D continuous potential with an entropic barrier by using RE-generated discrete states as nodes of a kinetic network. By choosing the neighbors and the microscopic rates between the neighbors appropriately, the correct kinetics of the system can be recovered by running a kinetic simulation on the network. We fine-tune the parameters of the network by comparison with the effective drift velocities and diffusion coefficients of the system determined from short-time stochastic trajectories. One of the advantages of the kinetic network model is that the network can be built on a high-dimensional discretized state space, which can consist of multiple paths not consistent with a single reaction coordinate.

  3. A universal molecular clock of protein folds and its power in tracing the early history of aerobic metabolism and planet oxygenation.

    Science.gov (United States)

    Wang, Minglei; Jiang, Ying-Ying; Kim, Kyung Mo; Qu, Ge; Ji, Hong-Fang; Mittenthal, Jay E; Zhang, Hong-Yu; Caetano-Anollés, Gustavo

    2011-01-01

    The standard molecular clock describes a constant rate of molecular evolution and provides a powerful framework for evolutionary timescales. Here, we describe the existence and implications of a molecular clock of folds, a universal recurrence in the discovery of new structures in the world of proteins. Using a phylogenomic structural census in hundreds of proteomes, we build phylogenies and time lines of domains at fold and fold superfamily levels of structural complexity. These time lines correlate approximately linearly with geological timescales and were here used to date two crucial events in life history, planet oxygenation and organism diversification. We first dissected the structures and functions of enzymes in simulated metabolic networks. The placement of anaerobic and aerobic enzymes in the time line revealed that aerobic metabolism emerged about 2.9 billion years (giga-annum; Ga) ago and expanded during a period of about 400 My, reaching what is known as the Great Oxidation Event. During this period, enzymes recruited old and new folds for oxygen-mediated enzymatic activities. Remarkably, the first fold lost by a superkingdom disappeared in Archaea 2.6 Ga ago, within the span of oxygen rise, suggesting that oxygen also triggered diversification of life. The implications of a molecular clock of folds are many and important for the neutral theory of molecular evolution and for understanding the growth and diversity of the protein world. The clock also extends the standard concept that was specific to molecules and their timescales and turns it into a universal timescale-generating tool.

  4. 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%)…

  5. Tissue engineered skin substitutes created by laser-assisted bioprinting form skin-like structures in the dorsal skin fold chamber in mice.

    Science.gov (United States)

    Michael, Stefanie; Sorg, Heiko; Peck, Claas-Tido; Koch, Lothar; Deiwick, Andrea; Chichkov, Boris; Vogt, Peter M; Reimers, Kerstin

    2013-01-01

    Tissue engineering plays an important role in the production of skin equivalents for the therapy of chronic and especially burn wounds. Actually, there exists no (cellularized) skin equivalent which might be able to satisfactorily mimic native skin. Here, we utilized a laser-assisted bioprinting (LaBP) technique to create a fully cellularized skin substitute. The unique feature of LaBP is the possibility to position different cell types in an exact three-dimensional (3D) spatial pattern. For the creation of the skin substitutes, we positioned fibroblasts and keratinocytes on top of a stabilizing matrix (Matriderm®). These skin constructs were subsequently tested in vivo, employing the dorsal skin fold chamber in nude mice. The transplants were placed into full-thickness skin wounds and were fully connected to the surrounding tissue when explanted after 11 days. The printed keratinocytes formed a multi-layered epidermis with beginning differentiation and stratum corneum. Proliferation of the keratinocytes was mainly detected in the suprabasal layers. In vitro controls, which were cultivated at the air-liquid-interface, also exhibited proliferative cells, but they were rather located in the whole epidermis. E-cadherin as a hint for adherens junctions and therefore tissue formation could be found in the epidermis in vivo as well as in vitro. In both conditions, the printed fibroblasts partly stayed on top of the underlying Matriderm® where they produced collagen, while part of them migrated into the Matriderm®. In the mice, some blood vessels could be found to grow from the wound bed and the wound edges in direction of the printed cells. In conclusion, we could show the successful 3D printing of a cell construct via LaBP and the subsequent tissue formation in vivo. These findings represent the prerequisite for the creation of a complex tissue like skin, consisting of different cell types in an intricate 3D pattern.

  6. Tissue engineered skin substitutes created by laser-assisted bioprinting form skin-like structures in the dorsal skin fold chamber in mice.

    Directory of Open Access Journals (Sweden)

    Stefanie Michael

    Full Text Available Tissue engineering plays an important role in the production of skin equivalents for the therapy of chronic and especially burn wounds. Actually, there exists no (cellularized skin equivalent which might be able to satisfactorily mimic native skin. Here, we utilized a laser-assisted bioprinting (LaBP technique to create a fully cellularized skin substitute. The unique feature of LaBP is the possibility to position different cell types in an exact three-dimensional (3D spatial pattern. For the creation of the skin substitutes, we positioned fibroblasts and keratinocytes on top of a stabilizing matrix (Matriderm®. These skin constructs were subsequently tested in vivo, employing the dorsal skin fold chamber in nude mice. The transplants were placed into full-thickness skin wounds and were fully connected to the surrounding tissue when explanted after 11 days. The printed keratinocytes formed a multi-layered epidermis with beginning differentiation and stratum corneum. Proliferation of the keratinocytes was mainly detected in the suprabasal layers. In vitro controls, which were cultivated at the air-liquid-interface, also exhibited proliferative cells, but they were rather located in the whole epidermis. E-cadherin as a hint for adherens junctions and therefore tissue formation could be found in the epidermis in vivo as well as in vitro. In both conditions, the printed fibroblasts partly stayed on top of the underlying Matriderm® where they produced collagen, while part of them migrated into the Matriderm®. In the mice, some blood vessels could be found to grow from the wound bed and the wound edges in direction of the printed cells. In conclusion, we could show the successful 3D printing of a cell construct via LaBP and the subsequent tissue formation in vivo. These findings represent the prerequisite for the creation of a complex tissue like skin, consisting of different cell types in an intricate 3D pattern.

  7. Further Development of the FFT-based Method for Atomistic Modeling of Protein Folding and Binding under Crowding: Optimization of Accuracy and Speed.

    Science.gov (United States)

    Qin, Sanbo; Zhou, Huan-Xiang

    2014-07-01

    Recently, we (Qin, S.; Zhou, H. X. J. Chem. Theory Comput.2013, 9, 4633-4643) developed the FFT-based method for Modeling Atomistic Proteins-crowder interactions, henceforth FMAP. Given its potential wide use for calculating effects of crowding on protein folding and binding free energies, here we aimed to optimize the accuracy and speed of FMAP. FMAP is based on expressing protein-crowder interactions as correlation functions and evaluating the latter via fast Fourier transform (FFT). The numerical accuracy of FFT improves as the grid spacing for discretizing space is reduced, but at increasing computational cost. We sought to speed up FMAP calculations by using a relatively coarse grid spacing of 0.6 Å and then correcting for discretization errors. This strategy was tested for different types of interactions (hard-core repulsion, nonpolar attraction, and electrostatic interaction) and over a wide range of protein-crowder systems. We were able to correct for the numerical errors on hard-core repulsion and nonpolar attraction by an 8% inflation of atomic hard-core radii and on electrostatic interaction by a 5% inflation of the magnitudes of protein atomic charges. The corrected results have higher accuracy and enjoy a speedup of more than 100-fold over those obtained using a fine grid spacing of 0.15 Å. With this optimization of accuracy and speed, FMAP may become a practical tool for realistic modeling of protein folding and binding in cell-like environments.

  8. Disulfide bond formation and folding of plant peroxidases expressed as inclusion body protein in Escherichia coli thioredoxin reductase negative strains

    DEFF Research Database (Denmark)

    Teilum, K; Ostergaard, L; Welinder, K G

    1999-01-01

    , two Ca2+ ions, and a heme group. We have studied the expression yield and folding efficiency of (i) a novel Arabidopsis thaliana peroxidase, ATP N; and (ii) barley grain peroxidase, BP 1. The expression yield ranges from 0 to 60 microgram/ml of cell culture depending on the peroxidase gene...... and the vector/host combination. The choice of E. coli strain in particular affects the yield of active peroxidase obtained in the folding step. Thus, the yield of active ATP N peroxidase can be increased 50-fold by using thioredoxin reductase negative strains, which facilitate the formation of disulfide bonds...

  9. Nanosecond-time-resolved infrared spectroscopic study of fast relaxation kinetics of protein folding by means of laser-induced temperature-jump

    Institute of Scientific and Technical Information of China (English)

    Zhang Qing-Li; Wang Li; Weng Yu-Xiang; Qiu Xiang-Gang; Wang Wei-Chi; Yan Ji-Xiang

    2005-01-01

    Elucidating the initial kinetics of folding pathways is critical to the understanding of the protein folding mechanism. Transient infrared spectroscopy has proved a powerful tool to probe the folding kinetics. Herein we report the construction of a nanosecond laser-induced temperature-jump (T-jump) technique coupled to a nanosecond timeresolved transient mid-infrared (mid-IR) spectrometer system capable of investigating the protein folding kinetics with a temporal resolution of 50 ns after deconvolution of the instrumental response function. The mid-IR source is a liquid N2 cooled CO laser covering a spectral range of 5.0μm (2000 cm-1) ~ 6.5μm (1540 cm-1). The heating pulse was generated by a high pressure H2 Raman shifter at wavelength of 1.9μm. The maximum temperature-jump could reach as high as 26±1℃. The fast folding/unfolding dynamics of cytochrome C was investigated by the constructed system,providing an example.

  10. Stress proteins in CNS inflammation

    NARCIS (Netherlands)

    Noort, J.M. van

    2008-01-01

    Stress proteins or heat shock proteins (HSPs) are ubiquitous cellular components that have long been known to act as molecular chaperones. By assisting proper folding and transport of proteins, and by assisting in the degradation of aberrant proteins, they play key roles in cellular metabolism. The

  11. The critical role of N- and C-terminal contact in protein stability and folding of a family 10 xylanase under extreme conditions.

    Directory of Open Access Journals (Sweden)

    Amit Bhardwaj

    Full Text Available BACKGROUND: Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive. METHODOLOGY: In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX with a TIM-barrel structure that shows stability under high temperature, alkali pH, and protease and SDS treatment. Based on crystal structure, an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the N- and C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stability under poly-extreme conditions. CONCLUSION: A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly, substitution of Phe4 with Trp increased stability in SDS treatment. Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as DeltaF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y cluster destabilizes the N- and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.

  12. Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

    Science.gov (United States)

    Reiz, Bela; Li, Liang

    2010-09-01

    Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein.

  13. Ambient pH stress inhibits spore germination of Penicillium expansum by impairing protein synthesis and folding: a proteomic-based study.

    Science.gov (United States)

    Li, Boqiang; Lai, Tongfei; Qin, Guozheng; Tian, Shiping

    2010-01-01

    Spore germination is the first step for fungal pathogens to infect host plants. The pH value, as one of the most important environmental parameters, has critical influence on spore germination. In this study, effects of ambient pH on spore germination were determined by culturing spores of Penicillium expansum in medium with pH values at 2.0, 5.0 and 8.0, and involved mechanisms were further investigated through methods of comparative proteomics. The results demonstrated that spore germination of P. expansum was obviously inhibited at pH 2.0 and 8.0. Using quadrupole time-of-flight tandem mass spectrometer, 34 proteins with significant changes in abundance were identified. Among them, 17 proteins were related to protein synthesis and folding, and most of them were down-regulated at pH 2.0 and 8.0. Accordingly, lower content of total soluble proteins and higher ratio of aggregated proteins were observed in spores at pH 2.0 and 8.0. In addition, it was found that ambient pH could affect intracellular pH and ATP level of P. expansum spores. These findings indicated that ambient pH might affect spore germination of P. expansum by changing intracellular pH and regulating protein expression. Further, impairing synthesis and folding of proteins might be one of the main reasons.

  14. Structure of TatA paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that transports folded proteins of differing diameter.

    Science.gov (United States)

    Baglieri, Jacopo; Beck, Daniel; Vasisht, Nishi; Smith, Corinne J; Robinson, Colin

    2012-03-01

    The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ∼50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70-90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6-8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed.

  15. A comparison of the pH, urea, and temperature-denatured states of barnase by heteronuclear NMR: implications for the initiation of protein folding.

    Science.gov (United States)

    Arcus, V L; Vuilleumier, S; Freund, S M; Bycroft, M; Fersht, A R

    1995-11-24

    The denatured states of barnase that are induced by urea, acid, and high temperature and acid have been assigned and characterised by high resolution heteronuclear NMR. The assignment was completed using a combination of triple-resonance and magnetisation-transfer methods. The latter was facilitated by selecting a suitable mutant of barnase (Ile-->Val51) which has an appropriate rate of interconversion between native and denatured states in urea. 3J NH-C alpha H coupling constants were determined for pH and urea-denatured barnase and intrinsic "random coil" coupling constants are shown to be different for different residue types. All the denatured states are highly unfolded. But, a consistent series of weak correlations in chemical shift, NOESY and coupling constant data provides evidence that the acid-denatured state has some residual structure in regions that form the first and second helices and the central strands of beta-sheet in the native protein. The acid/temperature-denatured states has less structure in these regions, and the urea-denatured state, less still. These observations may be combined with detailed analyses of the folding pathway of barnase from kinetic studies to illuminate the relevance of residual structure in the denatured states of proteins to the mechanism of protein folding. First, the folding of barnase is known to proceed in its later stages through structures in which the first helix and centre of the beta-sheet are extensively formed. Thus, embryonic initiation sites for these do exist in the denatured states and so could well develop into true nuclei. Second, it has been clearly established that the second helix is unfolded in these later states, and so residual structure in this region of the protein is non-productive. These data fit a model of protein folding in which local nucleation sites are latent in the denatured state and develop only when they make interactions elsewhere in the protein that stabilise them during the folding

  16. 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.

    2014-01-01

    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. Re

  17. Integron-associated mobile gene cassettes code for folded proteins: the structure of Bal32a, a new member of the adaptable alpha+beta barrel family.

    Science.gov (United States)

    Robinson, Andrew; Wu, Peter S-C; Harrop, Stephen J; Schaeffer, Patrick M; Dosztányi, Zsuzsanna; Gillings, Michael R; Holmes, Andrew J; Nevalainen, K M Helena; Stokes, H W; Otting, Gottfried; Dixon, Nicholas E; Curmi, Paul M G; Mabbutt, Bridget C

    2005-03-11

    The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent