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William A Eaton - One of the best experts on this subject based on the ideXlab platform.
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Native contacts determine protein Folding Mechanisms in atomistic simulations
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Robert B. Best, Gerhard Hummer, William A EatonAbstract:The recent availability of long equilibrium simulations of protein Folding in atomistic detail for more than 10 proteins allows us to identify the key interactions driving Folding. We find that the collective fraction of native amino acid contacts, Q, captures remarkably well the transition states for all the proteins with a Folding free energy barrier. Going beyond this global picture, we devise two different measures to quantify the importance of individual interresidue contacts in the Folding Mechanism: (i) the log-ratio of lifetimes of contacts during Folding transition paths and in the unfolded state and (ii) a Bayesian measure of how predictive the formation of each contact is for being on a transition path. Both of these measures indicate that native, or near-native, contacts are important for determining Mechanism, as might be expected. More remarkably, however, we found that for almost all the proteins, with the designed protein α3D being a notable exception, nonnative contacts play no significant part in determining Folding Mechanisms.
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Protein Folding studied by single-molecule FRET
Current Opinion in Structural Biology, 2008Co-Authors: Benjamin Schuler, William A EatonAbstract:A complete understanding of a protein-Folding Mechanism requires description of the distribution of microscopic pathways that connect the folded and unfolded states. This distribution can, in principle, be described by computer simulations and theoretical models of protein Folding, but is hidden in conventional experiments on large ensembles of molecules because only average properties are measured. A long-term goal of single-molecule fluorescence studies is to time-resolve the structural events as individual molecules make transitions between folded and unfolded states. Although such studies are still in their infancy, the work till now shows great promise and has already produced novel and important information on current issues in protein Folding that has been impossible or difficult to obtain from ensemble measurements.
Sheena E. Radford - One of the best experts on this subject based on the ideXlab platform.
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Malleability of the Folding Mechanism of the outer membrane protein PagP: parallel pathways and the effect of membrane elasticity.
Journal of Molecular Biology, 2012Co-Authors: Gerard H. M. Huysmans, Sheena E. Radford, Stephen A. Baldwin, David J. BrockwellAbstract:Understanding the interactions between membrane proteins and the lipid bilayer is key to increasing our ability to predict and tailor the Folding Mechanism, structure and stability of membrane proteins. Here, we have investigated the effects of changing the membrane composition and the relative concentrations of protein and lipid on the Folding Mechanism of the bacterial outer membrane protein PagP. The Folding pathway, monitored by tryptophan fluorescence, was found to be characterized by a burst phase, representing PagP adsorption to the liposome surface, followed by a time course that reflects the Folding and insertion of the protein into the membrane. In 1,2-dilauroyl-sn-glycero-3-phosphocholine (diC12:0PC) liposomes, the post-adsorption time course fits well to a single exponential at high lipid-to-protein ratios (LPRs), but at low LPRs, a second exponential phase with a slower Folding rate constant is observed. Interrupted reFolding assays demonstrated that the two exponential phases reflect the presence of parallel Folding pathways. Partitioning between these pathways was found to be modulated by the elastic properties of the membrane. Folding into mixed 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine:diC12:0PC liposomes resulted in a decrease in PagP adsorption to the liposomes and a switch to the slower Folding pathway. By contrast, inclusion of 1,2-dilauroyl-sn-glycero-3-phosphoserine into diC12:0PC liposomes resulted in a decrease in the Folding rate of the fast pathway. The results highlight the effect of lipid composition in tailoring the Folding Mechanism of a membrane protein, revealing that membrane proteins have access to multiple, competing Folding routes to a unique native structure.
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Semisynthesis of a glycosylated Im7 analogue for protein Folding studies
Journal of the American Chemical Society, 2005Co-Authors: Christian P.r. Hackenberger, Claire T. Friel, Sheena E. Radford, Barbara ImperialiAbstract:To establish a system to address questions concerning the influence of glycosylation on protein Folding pathways, we have developed a semisynthetic route toward the immunity protein Im7. This fourhelix protein has been used extensively as model protein for Folding studies. Native chemical ligation (NCL) affords an N-linked chitobiose glycoprotein analogue of Im7 with an Ala29Cys mutation. The semisynthetic approach relies on the solid-phase peptide synthesis (SPPS) of N-terminal thioesters (including helix I), in glycosylated or unglycosylated form, in combination with the expression of the C-terminal fragment of Im7 (containing helices II-IV). Detailed kinetic and thermodynamic analysis of the protein Folding behavior reveals that semisynthetic Im7 analogues are well suited for protein Folding studies and that the Folding Mechanism of the glycoprotein of this Im7 variant is not significantly altered over the unglycosylated analogue.
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im7 Folding Mechanism misFolding on a path to the native state
Nature Structural & Molecular Biology, 2002Co-Authors: Andrew P Capaldi, Colin Kleanthous, Sheena E. RadfordAbstract:Many proteins populate collapsed intermediate states during Folding. In order to elucidate the nature and importance of these species, we have mapped the structure of the on-pathway intermediate of the four-helix protein, Im7, together with the conformational changes it undergoes as it folds to the native state. Kinetic data for 29 Im7 point mutants show that the intermediate contains three of the four helices found in the native structure, packed around a specific hydrophobic core. However, the intermediate contains many non-native interactions; as a result, hydrophobic interactions become disrupted in the rate-limiting transition state before the final helix docks onto the developing structure. The results of this study support a hierarchical Mechanism of protein Folding and explain why the misFolding of Im7 occurs. The data also demonstrate that non-native interactions can play a significant role in Folding, even for small proteins with simple topologies.
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GroEL accelerates the reFolding of hen lysozyme without changing its Folding Mechanism
Nature Structural & Molecular Biology, 1999Co-Authors: Joseph E. Coyle, Frieda L. Texter, Alison E. Ashcroft, Dimitris Masselos, Carol V. Robinson, Sheena E. RadfordAbstract:The chaperonin GroEL binds Folding intermediates of four-disulfidehen lysozyme transiently within its central cavity. Using stopped flow fluorescence we show that GroEL binds early intermediates in Folding and accelerates the slow kinetic phase that reflects the reversal of non-native interactions involving tryptophan residues and the formation of the native state. Pulsed hydrogen exchange monitored by electrospray ionization mass spectrometry demonstrates that GroEL does not alter the Folding Mechanism, nor are protected species unfolded by the chaperonin. The data suggest a Mechanism for GroEL-assisted Folding in which the reorganization of non-native tertiary interactions is facilitated but domain Folding is unperturbed.
Jan Kubelka - One of the best experts on this subject based on the ideXlab platform.
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Refined Folding Mechanism of a Helix-turn-helix Motif
Biophysical Journal, 2014Co-Authors: Ginka S. Kubelka, Jan KubelkaAbstract:Helix-turn-helix motifs are valuable models for elucidating the protein Folding Mechanism. The de novo designed helix-turn-helix motif αtα has been studied thoroughly in our group by infrared spectroscopy and circular dichroism, applying site-specific isotopic labels and mutations. This preliminary investigation of αtα has provided detailed site-specific information on its Folding Mechanism, revealing increased stability in the middle of the helices and unFolding starting from the loose turn and the ends of the helices. The hydrophobic core contributes significantly to the stability of this little protein. To get further information on the Folding Mechanism of αtα, provided by additional independent probes, Forster resonance energy transfer (FRET) and differential scanning calorimetry (DSC) were employed to monitor thermal unFolding. FRET allows for measuring distances within a protein during its denaturation. In αtα, the intrinsic Trp residue in position 2 at the N-terminus and EDANS attached to the C-terminus served as FRET donor and acceptor, respectively, so end-to-end distances could be obtained. Thermodynamic parameters of a protein can be directly measured using DSC. Therefore, these directly obtained data were used to verify preliminary parameters from spectroscopic measurements. Overall, the results from FRET and DSC measurements were compared to the preliminary data on αtα to refine the proposed picture of its Folding Mechanism.
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Folding Mechanism of a Helix-Turn-Helix Protein from Combined 13C-Edited IR and Mutational Studies
Biophysical Journal, 2011Co-Authors: Ginka S. Buchner, Krista E. Amunson, Jan KubelkaAbstract:Helix-turn-helix motifs are important super-secondary protein structural elements and excellent models for studying the Mechanism of protein Folding. We have been investigating Folding of a de novo designed 38-residue helix-turn-helix motif alpha-t-alpha using IR spectroscopy with site-specific 13C isotopic editing. Our preliminary site-specific thermal unFolding data revealed that alpha-t-alpha is most stable near the centers of both alpha-helices, and likely unfolds from the helical termini and the loose turn region. To obtain more detail about the distribution of thermodynamic stabilities along the structure, additional six isotopically labeled proteins were synthesized and investigated using isotopically-edited IR. The new site-specific thermal unFolding data support the proposed Folding Mechanism, but reveal additional detail about the Folding Mechanism. The roles of the individual residue-residue stabilizing interactions were further studied by mutations, designed to destabilize the hydrophobic core near the helical centers and to stabilize the helical termini. Additional mutations both stabilized and destabilized the turn/loop sequence. The effect of the mutations on the overall thermodynamic stability was measured using CD and IR spectroscopies. Five isotopically labeled variants of a selected stabilized and destabilized mutant were synthesized to further study the influence of the mutations on the structural stability of the labeled segments. The effects of the mutations on the global and, especially, local unFolding provide important clues about the stabilization of the helix-turn-helix motif by specific interactions. Our results support and refine the proposed Folding Mechanism and point out the significance of tertiary interactions for the stability of alpha-t-alpha.
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Effects of Mutations on Side-Specific Folding Mechanism of a Helix-Turn-Helix Protein
Biophysical Journal, 2010Co-Authors: Ginka S. Buchner, Krista E. Amunson, Jan KubelkaAbstract:Helix-turn-helix motifs are important super-secondary protein structural elements and excellent models for studying the Mechanism of protein Folding. We have been investigating Folding of a de novo designed 38-residue helix-turn-helix motif α-t-α using IR spectroscopy with site-specific 13C isotopic editing. Our preliminary site-specific thermal unFolding data revealed that α-t-α is most stable near the centers of both α-helices, and likely unfolds from the helical termini and the loose turn region. To obtain further insights into the Folding Mechanism, and to investigate the roles of the individual residue-residue stabilizing interactions, we have begun mutational studies of the α-t-α protein. The mutations were designed to both destabilize and further stabilize the hydrophobic core near the helical centers. Additional mutations were designed to stabilize the helical termini and the turn/loop sequence. The overall thermodynamic stability of the α-t-α was measured using CD and IR spectroscopies. The core mutations appreciably decreased or increased the overall Folding stability as intended, however, stabilizing the turn and helical termini proved to be a rather challenging task. Site-specific thermal unFolding of the mutated α-t-α were probed with IR on multiple 13C isotopically labeled variants of each mutant. The effects of the mutations on both the global and, in particular, local site-specific unFolding provide important clues about the stabilization of the helix-turn-helix motif by specific interactions. Although additional mutational studies are underway, thus far all the data are consistent with the proposed Folding Mechanism.
Benjamin Schuler - One of the best experts on this subject based on the ideXlab platform.
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Protein Folding studied by single-molecule FRET
Current Opinion in Structural Biology, 2008Co-Authors: Benjamin Schuler, William A EatonAbstract:A complete understanding of a protein-Folding Mechanism requires description of the distribution of microscopic pathways that connect the folded and unfolded states. This distribution can, in principle, be described by computer simulations and theoretical models of protein Folding, but is hidden in conventional experiments on large ensembles of molecules because only average properties are measured. A long-term goal of single-molecule fluorescence studies is to time-resolve the structural events as individual molecules make transitions between folded and unfolded states. Although such studies are still in their infancy, the work till now shows great promise and has already produced novel and important information on current issues in protein Folding that has been impossible or difficult to obtain from ensemble measurements.
Tadanori Goto - One of the best experts on this subject based on the ideXlab platform.
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a small ocean bottom electromagnetometer and ocean bottom electrometer system with an arm Folding Mechanism technical report
Exploration Geophysics, 2009Co-Authors: Takafumi Kasaya, Tadanori GotoAbstract:Natural magnetic fields are attenuated by electrically conductive water. For that reason, marine magnetotelluric surveys have collected data at long periods (1000–100 000 s). The mantle structure has been the main target of seafloor magnetotelluric measurements. To ascertain crustal structure, however, electromagnetic data at shorter periods are important, e.g. in investigations of megathrust earthquake zones, or in natural resource surveys. To investigate of the former, for example, electromagnetic data for periods of less than 1000 s are necessary. Because no suitable ocean bottom electromagnetometer (OBEM) has been available, we have developed a small OBEM and ocean bottom electrometer (OBE) system with a high sample rate, which has an arm-Folding Mechanism to facilitate assembly and recovering operations. For magnetic observation, we used a fluxgate sensor. Field observations were undertaken to evaluate the field performance of our instruments. All instruments were recovered and their electromagnetic ...