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C. Robert Matthews - One of the best experts on this subject based on the ideXlab platform.
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Frustration and folding of a TIM Barrel protein
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Kevin T. Halloran, Yanming Wang, Karunesh Arora, Srinivas Chakravarthy, Thomas C. Irving, Osman Bilsel, Charles L. Brooks, C. Robert MatthewsAbstract:Triosephosphate isomerase (TIM) Barrel proteins have not only a conserved architecture that supports a myriad of enzymatic functions, but also a conserved folding mechanism that involves on- and off-pathway intermediates. Although experiments have proven to be invaluable in defining the folding free-energy surface, they provide only a limited understanding of the structures of the partially folded states that appear during folding. Coarse-grained simulations employing native centric models are capable of sampling the entire energy landscape of TIM Barrels and offer the possibility of a molecular-level understanding of the readout from sequence to structure. We have combined sequence-sensitive native centric simulations with small-angle X-ray scattering and TIMe-resolved Förster resonance energy transfer to monitor the formation of structure in an intermediate in the Sulfolobus solfataricus indole-3-glycerol phosphate synthase TIM Barrel that appears within 50 μs and must at least partially unfold to achieve productive folding. Simulations reveal the presence of a major and 2 minor folding channels not detected in experiments. Frustration in folding, i.e., backtracking in native contacts, is observed in the major channel at the initial stage of folding, as well as late in folding in a minor channel before the appearance of the native conformation. Similarities in global and pairwise dimensions of the early intermediate, the formation of structure in the central region that spreads progressively toward each terminus, and a similar rate-limiting step in the closing of the β-Barrel underscore the value of combining simulation and experiment to unravel complex folding mechanisms at the molecular level.
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Correlation of fitness landscapes from three orthologous TIM Barrels originates from sequence and structure constraints
Nature communications, 2017Co-Authors: Yvonne H. Chan, Konstantin B. Zeldovich, Sergey V. Venev, C. Robert MatthewsAbstract:Sequence divergence of orthologous proteins enables adaptation to environmental stresses and promotes evolution of novel functions. Limits on evolution imposed by constraints on sequence and structure were explored using a model TIM Barrel protein, indole-3-glycerol phosphate synthase (IGPS). Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Analysis of beneficial mutations pointed to an unexpected, long-range allosteric pathway towards the active site of the protein. Significant correlations between the fitness landscapes of distant orthologues implicate both sequence and structure as primary forces in defining the TIM Barrel fitness landscape and suggest that fitness landscapes can be translocated in sequence space. Exploration of fitness landscapes in the context of a protein fold provides a strategy for elucidating the sequence-structure-fitness relationships in other common motifs.
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Interplay between drying and stability of a TIM Barrel protein: a combined simulation-experimental study.
Journal of the American Chemical Society, 2013Co-Authors: Payel Das, Kevin T. Halloran, Divya Kapoor, Ruhong Zhou, C. Robert MatthewsAbstract:Recent molecular dynamics simulations have suggested important roles for nanoscale dewetting in the stability, function, and folding dynamics of proteins. Using a synergistic simulation–experimental approach on the αTS TIM Barrel protein, we validated this hypothesis by revealing the occurrence of drying inside hydrophobic amino acid clusters and its manifestation in experimental measures of protein stability and structure. Cavities created within three clusters of branched aliphatic amino acids [isoleucine, leucine, and valine (ILV) clusters] were found to experience strong water density fluctuations or intermittent dewetting transitions in simulations. Individually substituting 10 residues in the large ILV cluster at the N-terminus with less hydrophobic alanines showed a weakening or diminishing effect on dewetting that depended on the site of the mutation. Our simulations also demonstrated that replacement of buried leucines with isosteric, polar asparagines enhanced the wetting of the N- and C-termina...
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Clusters of Branched Aliphatic Side Chains Serve As Cores of Stability in the Native State of the HisF TIM Barrel Protein
Journal of molecular biology, 2013Co-Authors: Basavanapura N. Gangadhara, Sagar V. Kathuria, Jennifer M. Laine, Francesca Massi, C. Robert MatthewsAbstract:Imidazole-3-glycerol phosphate synthase is a heterodimeric allosteric enzyme that catalyzes consecutive reactions in imidazole biosynthesis through its HisF and HisH subunits. The unusually slow unfolding reaction of the isolated HisF TIM Barrel domain from the thermophilic bacteria, Thermotoga mariTIMa, enabled an NMR-based site-specific analysis of the main-chain hydrogen bonds that stabilize its native conformation. Very strong protection against exchange with solvent deuterium in the native state was found in a subset of buried positions in α-helices and pervasively in the underlying β-strands associated with a pair of large clusters of isoleucine, leucine and valine (ILV) side chains located in the α7(βα)8(βα)1–2 and α2(βα)3–6β7 segments of the (βα)8 Barrel. The most densely packed region of the large cluster, α3(βα)4–6β7, correlates closely with the core of stability previously observed in computational, protein engineering and NMR dynamics studies, demonstrating a key role for this cluster in determining the thermodynamic and structural properties of the native state of HisF. When considered with the results of previous studies where ILV clusters were found to stabilize the hydrogen-bonded networks in folding intermediates for other TIM Barrel proteins, it appears that clusters of branched aliphatic side chains can serve as cores of stability across the entire folding reaction coordinate of one of the most common motifs in biology.
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βα-Hairpin Clamps Brace βαβ Modules and Can Make Substantive Contributions to the Stability of TIM Barrel Proteins
PloS one, 2009Co-Authors: Xiaoyan Yang, Ramakrishna Vadrevu, Sagar V. Kathuria, C. Robert MatthewsAbstract:Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive betaalpha or alphabeta elements of secondary structure in alphaTS from E. coli, a TIM Barrel protein, have previously been found to contribute 4-6 kcal mol(-1) to the stability of the native conformation. Experimental analysis of similar betaalpha-hairpin clamps in a homologous pair of TIM Barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to alphaTS. A survey of 71 TIM Barrel proteins demonstrates a 4-fold symmetry for the placement of betaalpha-hairpin clamps, bracing the fundamental betaalphabeta building block and defining its register in the (betaalpha)(8) motif. The preferred sequences and locations of betaalpha-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM Barrel proteins.
Ramakrishna Vadrevu - One of the best experts on this subject based on the ideXlab platform.
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diversity in αβ and βα loop connections in TIM Barrel proteins implications for stability and design of the fold
Interdisciplinary Sciences: Computational Life Sciences, 2018Co-Authors: Rajashekar Varma Kadumuri, Ramakrishna VadrevuAbstract:The (βα)8/TIM Barrel is one of the most common folds of known protein structures facilitating diverse catalytic functions. The fold is formed by the repetition of the basic βαβ building block in which the β-strands are followed by α-helices eight TIMes alternating in sequence and structure. αβ and βα loops connecting α-helices to the β-strands and the β-strands to the α-helices contribute to stability and function, respectively, an inherent imposition by the TIM Barrel architecture itself. In this study, αβ and βα loops from a data set of 430 non-redundant, high-resolution triosephosphate isomerase (TIM) Barrels bearing sequence homology of <30% were analyzed for their amino acid propensities, sequence profiles, and positional preferences of amino acids. While the distribution of short connections is significantly higher in αβ loops, there appears to be no such preference in βα loops. Glycine, proline, lysine, and arginine tend to show greater preference to occur in αβ loops, whereas serine, threonine, cysteine, tryptophan, and histidine occur more frequently in βα loops. In addition, striking dissimilarities in sequence and positional preferences of amino acids, especially, in short, αβ and βα loops are observed. Together, the analysis suggests the role for short loops and charged residues in promoting both non-polar and polar interactions and in β strand registry. The observed diversity, perhaps, dictates the distinct role of αβ and βα loops in stability and function, respectively. In summary, the overall observations and reasoning, in addition to steering protein engineering efforts on TIM Barrel design and stabilization can provide the basis for incorporating consensus loop sequences for designing independently folding βαβ modules.
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Diversity in αβ and βα Loop Connections in TIM Barrel Proteins: Implications for Stability and Design of the Fold.
Interdisciplinary sciences computational life sciences, 2017Co-Authors: Rajashekar Varma Kadumuri, Ramakrishna VadrevuAbstract:The (βα)8/TIM Barrel is one of the most common folds of known protein structures facilitating diverse catalytic functions. The fold is formed by the repetition of the basic βαβ building block in which the β-strands are followed by α-helices eight TIMes alternating in sequence and structure. αβ and βα loops connecting α-helices to the β-strands and the β-strands to the α-helices contribute to stability and function, respectively, an inherent imposition by the TIM Barrel architecture itself. In this study, αβ and βα loops from a data set of 430 non-redundant, high-resolution triosephosphate isomerase (TIM) Barrels bearing sequence homology of
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βα-Hairpin Clamps Brace βαβ Modules and Can Make Substantive Contributions to the Stability of TIM Barrel Proteins
PloS one, 2009Co-Authors: Xiaoyan Yang, Ramakrishna Vadrevu, Sagar V. Kathuria, C. Robert MatthewsAbstract:Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive betaalpha or alphabeta elements of secondary structure in alphaTS from E. coli, a TIM Barrel protein, have previously been found to contribute 4-6 kcal mol(-1) to the stability of the native conformation. Experimental analysis of similar betaalpha-hairpin clamps in a homologous pair of TIM Barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to alphaTS. A survey of 71 TIM Barrel proteins demonstrates a 4-fold symmetry for the placement of betaalpha-hairpin clamps, bracing the fundamental betaalphabeta building block and defining its register in the (betaalpha)(8) motif. The preferred sequences and locations of betaalpha-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM Barrel proteins.
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βα hairpin clamps brace βαβ modules and can make substantive contributions to the stability of TIM Barrel proteins
PLOS ONE, 2009Co-Authors: Xiaoyan Yang, Ramakrishna Vadrevu, Sagar V. Kathuria, Robert C MatthewsAbstract:Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive βα or αβ elements of secondary structure in αTS from E. coli, a TIM Barrel protein, have previously been found to contribute 4–6 kcal mol−1 to the stability of the native conformation. Experimental analysis of similar βα-hairpin clamps in a homologous pair of TIM Barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to αTS. A survey of 71 TIM Barrel proteins demonstrates a 4-fold symmetry for the placement of βα-hairpin clamps, bracing the fundamental βαβ building block and defining its register in the (βα)8 motif. The preferred sequences and locations of βα-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM Barrel proteins.
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NMR Analysis of Partially-Folded States and Persistent Structure in the Alpha Subunit of Tryptophan Synthase: Implications for the Equilibrium Folding Mechanism of a 29 kDa TIM Barrel Protein
Journal of molecular biology, 2007Co-Authors: Ramakrishna Vadrevu, C. Robert MatthewsAbstract:Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (alpha TS) from Escherichia coli, a (beta alpha)(8) TIM Barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several (15)N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of alpha TS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several beta-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact beta-Barrel and a frayed alpha-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of alpha TS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.
Robert C Matthews - One of the best experts on this subject based on the ideXlab platform.
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sequence and misfolding in the ancient TIM Barrel motif
The FASEB Journal, 2015Co-Authors: Kevin T. Halloran, Srinivas Chakravarthy, Thomas C. Irving, Osman Bilsel, Robert C MatthewsAbstract:Protein folding energy landscapes have been driven by evolution to minimize the energetic and topological frustration experienced during the folding reaction. Small 2 state folders, whose folding r...
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βα hairpin clamps brace βαβ modules and can make substantive contributions to the stability of TIM Barrel proteins
PLOS ONE, 2009Co-Authors: Xiaoyan Yang, Ramakrishna Vadrevu, Sagar V. Kathuria, Robert C MatthewsAbstract:Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive βα or αβ elements of secondary structure in αTS from E. coli, a TIM Barrel protein, have previously been found to contribute 4–6 kcal mol−1 to the stability of the native conformation. Experimental analysis of similar βα-hairpin clamps in a homologous pair of TIM Barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to αTS. A survey of 71 TIM Barrel proteins demonstrates a 4-fold symmetry for the placement of βα-hairpin clamps, bracing the fundamental βαβ building block and defining its register in the (βα)8 motif. The preferred sequences and locations of βα-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM Barrel proteins.
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structural analysis of kinetic folding intermediates for a TIM Barrel protein indole 3 glycerol phosphate synthase by hydrogen exchange mass spectrometry and gō model simulation
Journal of Molecular Biology, 2007Co-Authors: Maithreyi K. Rao, John M. Finke, William R. Forsyth, Robert C MatthewsAbstract:Abstract The structures of partially folded states appearing during the folding of a (βα) 8 TIM Barrel protein, the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (βα) 4 region, modest protection in the neighboring (βα) 1–3 and (βα) 5 β 6 segments and no significant protection in the remaining N and C-terminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (βα) 2–5 β 6 region after 5 s of folding demonstrates that these species represent kinetically distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate, while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a C α Gō model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of the structure offering protection against exchange in the on-pathway intermediate(s). Because the native-centric Gō model simulations do not explicitly include sequence-specific information, the simulation results support the hypothesis that the topology of TIM Barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Gō model simulation.
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specific structure appears at the n terminus in the sub millisecond folding intermediate of the alpha subunit of tryptophan synthase a TIM Barrel protein
Journal of Molecular Biology, 2005Co-Authors: Ramakrishna Vadrevu, Xiaoyan Yang, Robert C MatthewsAbstract:Competing views of the products of sub-millisecond folding reactions observed in many globular proteins have been ascribed either to the formation of discrete, partially folded states or to the random collapse of the unfolded chain under native-favoring conditions. To test the validity of these alternative interpretations for the stopped-flow burst-phase reaction in the (βα)8, TIM Barrel motif, a series of alanine replacements were made at five different leucine or isoleucine residues in the alpha subunit of tryptophan synthase (αTS) from Escherichia coli. This protein has been proposed to fold, in the sub-millisecond TIMe range, to an off-pathway intermediate with significant stability and ∼50% of the far-UV circular dichroism (CD) signal of the native conformation. Individual alanine replacements at any of three isoleucine or leucine residues in either α1, β2 or β3 completely eliminate the off-pathway species. These variants, within 5 ms, access an intermediate whose properties closely resemble those of an on-pathway equilibrium intermediate that is highly populated at moderate urea concentrations in wild-type αTS. By contrast, alanine replacements for leucine residues in either β4 or β6 destabilize but preserve the off-pathway, burst-phase species. When considered with complementary thermodynamic and kinetic data, this mutational analysis demonstrates that the sub-millisecond appearance of CD signal for αTS reflects the acquisition of secondary structure in a distinct thermodynamic state, not the random collapse of an unfolded chain. The contrasting results for replacements in the contiguous α1/β2/β3 domain and the C-terminal β4 and β6 strands imply a heterogeneous structure for the burst-phase species. The α1/β2/β3 domain appears to be tightly packed, and the C terminus appears to behave as a molten-globule-like structure whose folding is tightly coupled to that of the α1/β2/β3 domain.
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multi state unfolding of the alpha subunit of tryptophan synthase a TIM Barrel protein insights into the secondary structure of the stable equilibrium intermediates by hydrogen exchange mass spectrometry
Journal of Molecular Biology, 2004Co-Authors: Teerapat Rojsajjakul, Patrick L Wintrode, Ramakrishna Vadrevu, Robert C Matthews, David L SmithAbstract:The urea-induced unfolding of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded (beta/alpha)(8) TIM Barrel protein, has been shown to involve two stable equilibrium intermediates, I1 and I2, well populated at approximately 3 M and 5 M urea, respectively. The characterization of the I1 intermediate by circular dichroism (CD) spectroscopy has shown that I1 retains a significant fraction of the native ellipticity; the far-UV CD signal for the I2 species closely resembles that of the fully unfolded form. To obtain detailed insight into the disruption of secondary structure in the urea-induced unfolding process, a hydrogen exchange-mass spectrometry study was performed on alphaTS. The full-length protein was destabilized in increasing concentration of urea, the amide hydrogen atoms were pulse-labeled with deuterium, the labeled samples were quenched in acid and the products were analyzed by electrospray ionization mass spectrometry. Consistent with the CD results, the I1 intermediate protects up to approximately 129 amide hydrogen atoms against exchange while the I2 intermediate offers no protection. Electrospray ionization mass spectrometry analysis of the peptic fragments derived from alphaTS labeled at 3 M urea indicates that most of the region between residues 12-130, which constitutes the first four beta strands and three alpha helices, (beta/alpha)(1-3)beta(4), is structured. The (beta/alpha)(1-3)beta(4) module appears to represent the minimum sub-core of stability of the I1 intermediate. A 4+2+2 folding model is proposed as a likely alternative to the earlier 6+2 folding mechanism for alphaTS.
D Alejandro Fernandez Velasco - One of the best experts on this subject based on the ideXlab platform.
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Correction: Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM Barrel proteins.
Physical chemistry chemical physics : PCCP, 2016Co-Authors: Sergio Romero-romero, Miguel Costas, Adela Rodríguez-romero, D Alejandro Fernandez VelascoAbstract:Correction for ‘Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM Barrel proteins’ by Sergio Romero-Romero et al., Phys. Chem. Chem. Phys., 2015, 17, 20699–20714.
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De novo design of a four-fold symmetric TIM-Barrel protein with atomic-level accuracy
Nature Chemical Biology, 2016Co-Authors: Po-ssu Huang, Kaspar Feldmeier, Birte Höcker, Fabio Parmeggiani, D Alejandro Fernandez Velasco, David BakerAbstract:Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-Barrel fold. Here we describe the computational design of four-fold symmetrical (β/α)_8 Barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of side chain–backbone hydrogen bonds for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical to that of the designed TIM-Barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-Barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-Barrel superfamilies, suggesting that Nature has sampled only a subset of the sequence space available to the TIM-Barrel fold. The ability to design TIM Barrels de novo opens new possibilities for custom-made enzymes. Despite substantial effort, the de novo design of a stable TIM-Barrel protein fold has remained elusive. A Rosetta-based computational strategy identifies a unique 184-residue sequence that adopts a TIM-Barrel structure, as revealed by X-ray crystallography.
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Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM Barrel proteins.
Physical chemistry chemical physics : PCCP, 2015Co-Authors: Sergio Romero-romero, Miguel Costas, Adela Rodríguez-romero, D Alejandro Fernandez VelascoAbstract:Temperature is one of the main variables that modulate protein function and stability. Thermodynamic studies of oligomeric proteins, the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. There are no reports on the reversible equilibrium thermal unfolding of proteins composed of (β/α)8 Barrel subunits, albeit this "TIM Barrel" topology is one of the most abundant and versatile in nature. We studied the eponymous TIM Barrel, triosephosphate isomerase (TIM), belonging to five species of different bacterial taxa. All of them were found to be catalytically efficient dimers. The three-dimensional structure of four enzymes was solved at high/medium resolution. Irreversibility and kinetic control were observed in the thermal unfolding of two TIMs, while for the other three the thermal unfolding was found to follow a two-state equilibrium reversible process. Shifts in the global stability curves of these three proteins are related to the organismal temperature range of opTIMal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. Reversibility appears to correlate with the low isoelectric point, the absence of a residual structure in the unfolded state, small cavity volume in the native state, low conformational stability and a low melting temperature. Furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce aggregation and favour reversibility. It is therefore very thought-provoking to find that a common topological ensemble, such as the TIM Barrel, can unfold/refold in the Anfinsen way, i.e. without the help of the cellular machinery.
Xiaoyan Yang - One of the best experts on this subject based on the ideXlab platform.
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βα-Hairpin Clamps Brace βαβ Modules and Can Make Substantive Contributions to the Stability of TIM Barrel Proteins
PloS one, 2009Co-Authors: Xiaoyan Yang, Ramakrishna Vadrevu, Sagar V. Kathuria, C. Robert MatthewsAbstract:Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive betaalpha or alphabeta elements of secondary structure in alphaTS from E. coli, a TIM Barrel protein, have previously been found to contribute 4-6 kcal mol(-1) to the stability of the native conformation. Experimental analysis of similar betaalpha-hairpin clamps in a homologous pair of TIM Barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to alphaTS. A survey of 71 TIM Barrel proteins demonstrates a 4-fold symmetry for the placement of betaalpha-hairpin clamps, bracing the fundamental betaalphabeta building block and defining its register in the (betaalpha)(8) motif. The preferred sequences and locations of betaalpha-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM Barrel proteins.
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βα hairpin clamps brace βαβ modules and can make substantive contributions to the stability of TIM Barrel proteins
PLOS ONE, 2009Co-Authors: Xiaoyan Yang, Ramakrishna Vadrevu, Sagar V. Kathuria, Robert C MatthewsAbstract:Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive βα or αβ elements of secondary structure in αTS from E. coli, a TIM Barrel protein, have previously been found to contribute 4–6 kcal mol−1 to the stability of the native conformation. Experimental analysis of similar βα-hairpin clamps in a homologous pair of TIM Barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to αTS. A survey of 71 TIM Barrel proteins demonstrates a 4-fold symmetry for the placement of βα-hairpin clamps, bracing the fundamental βαβ building block and defining its register in the (βα)8 motif. The preferred sequences and locations of βα-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM Barrel proteins.
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long range side chain main chain hydrogen bonds a molecular signature of the TIM Barrel architecture a dissertation
2009Co-Authors: Xiaoyan YangAbstract:The hydrophobic effect and hydrogen bonding interactions have long been considered to be the dominant forces in protein folding. However, the contribution of hydrogen bonds to stabilizing proteins has been difficult to clarify. As the intramolecular hydrogen bonds are formed in place of hydrogen bonds with solvent during folding, measures of stability fail to give a significant change in free energy. Previous studies on hydrogen bonding interactions have shown that they are only marginally important. Three long-range side chain-main chain hydrogen bonds have been found in the alpha subunit of tryptophan synthase (αTS), a (βα) 8 TIM Barrel protein. These long-range noncovalent interactions connect either the N-terminus of one β-strand with the C-terminus of the succeeding and anti-parallel α-helix (F19-D46 and I97-D124) or the N-terminus of an α-helix with the C-terminus of the succeeding β-strand (A103-D130). By analogy, these interactions are designated as βα- or αβ-hairpin clamps. Surprisingly, the removal of any one of these clamp interactions, by replacement of the aspartic acid with alanine, results in significantly decreased thermodynamic stability for the native state and a substantial loss of secondary structure. When compared to several other side chain-side chain and short-range side chain-main chain interactions in αTS, these hairpin clamps clearly play a unique role in the structure and stability of αTS. The generality of these observations for βα-hairpin clamps in TIM Barrel proteins was tested by experimental analysis of the clamps in a pair of homologous indole-3-glycerol phosphate synthase (IGPS) TIM Barrels of low sequence identity. The results suggest that only the subset of conserved βα-hairpin clamps with hydrogen bond length less than 2.80 A make substantive contributions to stability and/or structure. Those clamps with longer hydrogen bonds make modest contributions to stability and structure, similar to other types of side chain-main chain or side chain-side chain hydrogen bonds. The role of these clamps in defining the structures of the super-family of TIM Barrel proteins was examined by a survey of 71 TIM Barrel proteins from the structural database. Conserved features of βα-hairpin clamps are consistent with a 4-fold symmetry, with a predominance of main chain amide hydrogen bond donors near the N-terminus of the odd-number β-strands and side chain hydrogen bond acceptors in the loops between the subsequent α-helices and even-numbered β-strands. In this configuration, the clamps provide an N-terminal cap to odd-number β- strands in the β-Barrel. Taken together, these findings suggest that βα-hairpin clamps are a vestigial signature of the fundamental βαβ building block for the (βα)8 motif and an integral part of the basic TIM Barrel architecture. The relative paucity of βα-hairpin clamps remaining in TIM Barrel structures and their variable contributions to stability imply that other determinants for structure and stability of the Barrel have evolved to…
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A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the alpha subunit of tryptophan synthase, a TIM Barrel protein.
Journal of molecular biology, 2006Co-Authors: Ramakrishna Vadrevu, Xiaoyan Yang, Sagar V. Kathuria, C. Robert MatthewsAbstract:Protein misfolding is now recognized as playing a crucial role in both normal and pathogenic folding reactions. An interesting example of misfolding at the earliest state of a natural folding reaction is provided by the alpha-subunit of tryptophan synthase, a (beta/alpha)(8) TIM Barrel protein. The molecular basis for the formation of this off-pathway misfolded intermediate, I(BP), and a subsequent on-pathway intermediate, I1, was probed by mutational analysis of 20 branched aliphatic side-chains distributed throughout the sequence. The elimination of I(BP) and the substantial destabilization of I1 by replacement of a selective set of the isoleucine, leucine or valine residues (ILV) with alanine in a large ILV cluster external-to-the-Barrel and spanning the N and C termini (cluster 2) implies tight-packing at most sites in both intermediates. Differential effects on I(BP) and I1 for replacements in alpha3, beta4 and alpha8 at the boundaries of cluster 2 suggest that their incorporation into I1 but not I(BP) reflects non-native folds at the edges of the crucial (beta/alpha)(1-2)beta(3) core in I(BP). The retention of I(BP) and the smaller and consistent destabilization of both I(BP) and I1 by similar replacements in an internal-to-the-Barrel ILV cluster (cluster 1) and a second external-to-the-Barrel ILV cluster (cluster 3) imply molten globule-like packing. The tight packing inferred, in part, for I(BP) or for all of I1 in cluster 2, but not in clusters 1 and 3, may reflect the larger size of cluster 2 and/or the enhanced number of isoleucine, leucine and valine self-contacts in and between contiguous elements of secondary structure. Tightly packed ILV-dominated hydrophobic clusters could serve as an important driving force for the earliest events in the folding and misfolding of the TIM Barrel and other members of the (beta/alpha)(n) class of proteins.
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Specific Structure Appears at the N terminus in the Sub-millisecond Folding Intermediate of the Alpha Subunit of Tryptophan Synthase, a TIM Barrel Protein
Journal of molecular biology, 2005Co-Authors: Ramakrishna Vadrevu, Xiaoyan Yang, C. Robert MatthewsAbstract:Competing views of the products of sub-millisecond folding reactions observed in many globular proteins have been ascribed either to the formation of discrete, partially folded states or to the random collapse of the unfolded chain under native-favoring conditions. To test the validity of these alternative interpretations for the stopped-flow burst-phase reaction in the (betaalpha)8, TIM Barrel motif, a series of alanine replacements were made at five different leucine or isoleucine residues in the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. This protein has been proposed to fold, in the sub-millisecond TIMe range, to an off-pathway intermediate with significant stability and approximately 50% of the far-UV circular dichroism (CD) signal of the native conformation. Individual alanine replacements at any of three isoleucine or leucine residues in either alpha1, beta2 or beta3 completely eliminate the off-pathway species. These variants, within 5 ms, access an intermediate whose properties closely resemble those of an on-pathway equilibrium intermediate that is highly populated at moderate urea concentrations in wild-type alphaTS. By contrast, alanine replacements for leucine residues in either beta4 or beta6 destabilize but preserve the off-pathway, burst-phase species. When considered with complementary thermodynamic and kinetic data, this mutational analysis demonstrates that the sub-millisecond appearance of CD signal for alphaTS reflects the acquisition of secondary structure in a distinct thermodynamic state, not the random collapse of an unfolded chain. The contrasting results for replacements in the contiguous alpha1/beta2/beta3 domain and the C-terminal beta4 and beta6 strands imply a heterogeneous structure for the burst-phase species. The alpha1/beta2/beta3 domain appears to be tightly packed, and the C terminus appears to behave as a molten-globule-like structure whose folding is tightly coupled to that of the alpha1/beta2/beta3 domain.
