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Joshua A Wand - One of the best experts on this subject based on the ideXlab platform.
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membrane proteins have distinct fast internal motion and residual Conformational Entropy
Angewandte Chemie, 2020Co-Authors: Evan S Obrien, Matthew A Stetz, Bryan S Marques, Brian Fuglestad, Henry J Lessen, Kushol Gupta, Karen G Fleming, Joshua A WandAbstract:For a variety of reasons, the internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side chain dynamics of the 7-transmembrane helix protein sensory rhodopsin II and the beta-barrel bacterial outer membrane channel protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Though of quite different topologies, both proteins are found to have a similar and striking distribution of methyl-bearing amino acid side chain motion that is independent of membrane mimetic. The methyl-bearing side chains of both proteins are, on average, more dynamic in the ps-ns time regime than any soluble protein characterized to date. Approximately one third of methyl-bearing side chains in both proteins exhibit extreme rotameric averaging on this timescale. Accordingly, both proteins retain an extraordinary residual Conformational Entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect that normally stabilizes the folded state of water-soluble proteins. Furthermore, the large reservoir of Conformational Entropy that is observed provides the potential to greatly influence the thermodynamics underlying a plethora of membrane protein functions including ligand binding, allostery and signaling.
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membrane proteins have distinct fast internal motion and residual Conformational Entropy
bioRxiv, 2020Co-Authors: Evan S Obrien, Joshua A Wand, Matthew A Stetz, Bryan S Marques, Brian Fuglestad, Henry J Lessen, Kushol Gupta, Karen G FlemingAbstract:For a variety of reasons, the internal motions of integral membrane proteins have largely eluded comprehensive experiential characterization. Here, the fast side chain dynamics of the 7-transmembrane helix protein sensory rhodopsin II and the beta-barrel bacterial outer membrane channel protein W have been characterized in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Though of quite different topologies, both proteins are found to have a similar and striking distribution of methyl-bearing amino acid side chain motion that is independent of membrane mimetic. The methyl-bearing side chains of both proteins, on average, are more dynamic in the ps-ns time regime than any soluble protein characterized to date. Approximately one third of methyl-bearing side chains exhibit extreme rotameric averaging on this timescale. Accordingly, both proteins retain an extraordinary residual Conformational Entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect that normally stabilizes the folded state of water-soluble proteins. Furthermore, the large reservoir of Conformational Entropy that is observed provides the potential to greatly influence the thermodynamics underlying a plethora of membrane protein functions including ligand binding, allostery and signaling.
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characterization of internal protein dynamics and Conformational Entropy by nmr relaxation
2019Co-Authors: Matthew A Stetz, Kathleen G Valentine, J A Caro, Sravya Kotaru, Bryan S Marques, Joshua A WandAbstract:Abstract Recent studies suggest that the fast timescale motion of methyl-bearing side chains may play an important role in mediating protein activity. These motions have been shown to encapsulate the residual Conformational Entropy of the folded state that can potentially contribute to the energetics of protein function. Here, we provide an overview of how to characterize these motions using nuclear magnetic resonance (NMR) spin relaxation methods. The strengths and limitations of several techniques are highlighted in order to assist with experimental design. Particular emphasis is placed on the practical aspects of sample preparation, data collection, data fitting, and statistical analysis. Additionally, discussion of the recently refined “Entropy meter” is presented and its use in converting NMR observables to Conformational Entropy is illustrated. Taken together, these methods should yield new insights into the complex interplay between structure and dynamics in protein function.
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on the relationship between nmr derived amide order parameters and protein backbone Entropy changes
Proteins, 2015Co-Authors: Kim A Sharp, Vignesh Kasinath, Evan S Obrien, Joshua A WandAbstract:Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone Entropy. Amide order parameters (O(2) NH ) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O(2) NH < 0.8) shows that these report both on the local flexibility of the protein and on a different component of the Conformational Entropy than that reported by the side chain methyl axis order parameters, O(2) axis . A calibration curve for backbone Entropy vs. O(2) NH is developed, which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O(2) NH changes obtained by NMR relaxation measurements to extract backbone Entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain Entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein Conformational Entropy changes from NMR relaxation measurements.
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coupled motion in proteins revealed by pressure perturbation
Journal of the American Chemical Society, 2012Co-Authors: Vignesh Kasinath, Veronica R Moorman, Nathaniel V Nucci, Vincent J Hilser, Joshua A WandAbstract:The cooperative nature of protein substructure and internal motion is a critical aspect of their functional competence about which little is known experimentally. NMR relaxation is used here to monitor the effects of high pressure on fast internal motion in the protein ubiquitin. In contrast to the main chain, the motions of the methyl-bearing side chains have a large and variable pressure dependence. Within the core, this pressure sensitivity correlates with the magnitude of motion at ambient pressure. Spatial clustering of the dynamic response to applied hydrostatic pressure is also seen, indicating localized cooperativity of motion on the sub-nanosecond time scale and suggesting regions of variable compressibility. These and other features indicate that the native ensemble contains a significant fraction of members with characteristics ascribed to the recently postulated “dry molten globule”. The accompanying variable side-chain Conformational Entropy helps complete our view of the thermodynamic archit...
Kathleen G Valentine - One of the best experts on this subject based on the ideXlab platform.
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characterization of internal protein dynamics and Conformational Entropy by nmr relaxation
2019Co-Authors: Matthew A Stetz, Kathleen G Valentine, J A Caro, Sravya Kotaru, Bryan S Marques, Joshua A WandAbstract:Abstract Recent studies suggest that the fast timescale motion of methyl-bearing side chains may play an important role in mediating protein activity. These motions have been shown to encapsulate the residual Conformational Entropy of the folded state that can potentially contribute to the energetics of protein function. Here, we provide an overview of how to characterize these motions using nuclear magnetic resonance (NMR) spin relaxation methods. The strengths and limitations of several techniques are highlighted in order to assist with experimental design. Particular emphasis is placed on the practical aspects of sample preparation, data collection, data fitting, and statistical analysis. Additionally, discussion of the recently refined “Entropy meter” is presented and its use in converting NMR observables to Conformational Entropy is illustrated. Taken together, these methods should yield new insights into the complex interplay between structure and dynamics in protein function.
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motion and Conformational Entropy in protein function creation of an nmr based Entropy meter
Biophysical Journal, 2015Co-Authors: Vignesh Kasinath, Veronica R Moorman, Kyle W Harpole, Kathleen G Valentine, Kendra K Frederick, Kim A Sharp, Joshua WandAbstract:Conformational Entropy is a potentially important thermodynamic parameter contributing to protein function. Quantitative measures of Conformational Entropy are necessary for an understanding of its role but have been difficult to obtain. We have recently introduced empirical method that utilizes changes in Conformational dynamics as a proxy for changes in Conformational Entropy. We have now used molecular dynamics simulations to probe the microscopic origins of the link between Conformational dynamics and Conformational Entropy. Simulation of seven proteins gave an excellent correlation with measures of side-chain motion derived from NMR relaxation. The simulations show that the motion of methyl-bearing side-chains are sufficiently coupled to that of other side chains to serve as excellent reporters of the overall side-chain Conformational Entropy. These results tend to validate the use of experimentally accessible measures of methyl motion - the NMR-derived generalized order parameters - as a proxy from which to derive changes in protein Conformational Entropy due to a perturbation such as the binding of a ligand. A slightly modified weighting scheme to project the change in dynamics of experimental methyl dynamics into Conformational Entropy is presented. Originally based on data from the calmodulin system, we will describe experimental results from other systems that indicate that the “Entropy meter” approach is both robust and general and that the involved Conformational entropies are often large and cannot be ignored. Supported by the NIH and the Mathers Foundation.
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the role of Conformational Entropy in molecular recognition by calmodulin
Nature Chemical Biology, 2010Co-Authors: Michael S Marlow, Jakob Dogan, Kathleen G Valentine, Kendra K Frederick, Joshua A WandAbstract:NMR-measured order parameters of methyl groups can be used to quantitate the Entropy of protein Conformational change associated with calmodulin-peptide ligand interactions. This Conformational Entropy is a major contributor to the affinity of calmodulin interactions.
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Conformational Entropy in molecular recognition by proteins
Nature, 2007Co-Authors: Kendra K Frederick, Kathleen G Valentine, Michael S Marlow, Joshua A WandAbstract:Molecular recognition by proteins is fundamental to almost every biological process, particularly the protein associations underlying cellular signal transduction. Understanding the basis for protein-protein interactions requires the full characterization of the thermodynamics of their association. Historically it has been virtually impossible to experimentally estimate changes in protein Conformational Entropy, a potentially important component of the free energy of protein association. However, nuclear magnetic resonance spectroscopy has emerged as a powerful tool for characterizing the dynamics of proteins. Here we employ changes in Conformational dynamics as a proxy for corresponding changes in Conformational Entropy. We find that the change in internal dynamics of the protein calmodulin varies significantly on binding a variety of target domains. Surprisingly, the apparent change in the corresponding Conformational Entropy is linearly related to the change in the overall binding Entropy. This indicates that changes in protein Conformational Entropy can contribute significantly to the free energy of protein-ligand association.
Kendra K Frederick - One of the best experts on this subject based on the ideXlab platform.
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motion and Conformational Entropy in protein function creation of an nmr based Entropy meter
Biophysical Journal, 2015Co-Authors: Vignesh Kasinath, Veronica R Moorman, Kyle W Harpole, Kathleen G Valentine, Kendra K Frederick, Kim A Sharp, Joshua WandAbstract:Conformational Entropy is a potentially important thermodynamic parameter contributing to protein function. Quantitative measures of Conformational Entropy are necessary for an understanding of its role but have been difficult to obtain. We have recently introduced empirical method that utilizes changes in Conformational dynamics as a proxy for changes in Conformational Entropy. We have now used molecular dynamics simulations to probe the microscopic origins of the link between Conformational dynamics and Conformational Entropy. Simulation of seven proteins gave an excellent correlation with measures of side-chain motion derived from NMR relaxation. The simulations show that the motion of methyl-bearing side-chains are sufficiently coupled to that of other side chains to serve as excellent reporters of the overall side-chain Conformational Entropy. These results tend to validate the use of experimentally accessible measures of methyl motion - the NMR-derived generalized order parameters - as a proxy from which to derive changes in protein Conformational Entropy due to a perturbation such as the binding of a ligand. A slightly modified weighting scheme to project the change in dynamics of experimental methyl dynamics into Conformational Entropy is presented. Originally based on data from the calmodulin system, we will describe experimental results from other systems that indicate that the “Entropy meter” approach is both robust and general and that the involved Conformational entropies are often large and cannot be ignored. Supported by the NIH and the Mathers Foundation.
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the role of Conformational Entropy in molecular recognition by calmodulin
Nature Chemical Biology, 2010Co-Authors: Michael S Marlow, Jakob Dogan, Kathleen G Valentine, Kendra K Frederick, Joshua A WandAbstract:NMR-measured order parameters of methyl groups can be used to quantitate the Entropy of protein Conformational change associated with calmodulin-peptide ligand interactions. This Conformational Entropy is a major contributor to the affinity of calmodulin interactions.
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Conformational Entropy in molecular recognition by proteins
Nature, 2007Co-Authors: Kendra K Frederick, Kathleen G Valentine, Michael S Marlow, Joshua A WandAbstract:Molecular recognition by proteins is fundamental to almost every biological process, particularly the protein associations underlying cellular signal transduction. Understanding the basis for protein-protein interactions requires the full characterization of the thermodynamics of their association. Historically it has been virtually impossible to experimentally estimate changes in protein Conformational Entropy, a potentially important component of the free energy of protein association. However, nuclear magnetic resonance spectroscopy has emerged as a powerful tool for characterizing the dynamics of proteins. Here we employ changes in Conformational dynamics as a proxy for corresponding changes in Conformational Entropy. We find that the change in internal dynamics of the protein calmodulin varies significantly on binding a variety of target domains. Surprisingly, the apparent change in the corresponding Conformational Entropy is linearly related to the change in the overall binding Entropy. This indicates that changes in protein Conformational Entropy can contribute significantly to the free energy of protein-ligand association.
David D. Boehr - One of the best experts on this subject based on the ideXlab platform.
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Different Solvent and Conformational Entropy Contributions to the Allosteric Activation and Inhibition Mechanisms of Yeast Chorismate Mutase.
Biochemistry, 2020Co-Authors: Scott D. Gorman, Dennis S. Winston, Debashish Sahu, David D. BoehrAbstract:Allosteric regulation is important in many biological processes, including cell signaling, gene regulation and metabolism. Saccharomyces cerevisiae chorismate mutase (ScCM) is a key homodimeric enz...
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different solvent and Conformational Entropy contributions to the allosteric activation and inhibition mechanisms of yeast chorismate mutase
Biochemistry, 2020Co-Authors: Scott D. Gorman, Dennis S. Winston, Debashish Sahu, David D. BoehrAbstract:Allosteric regulation is important in many biological processes, including cell signaling, gene regulation, and metabolism. Saccharomyces cerevisiae chorismate mutase (ScCM) is a key homodimeric enzyme in the shikimate pathway responsible for the generation of aromatic amino acids, where it is allosterically inhibited and activated by Tyr and Trp, respectively. Our previous studies indicated that binding of both allosteric effectors is negatively cooperative, that is binding at one allosteric binding site discourages binding at the other, due to the entropic penalty of binding the second allosteric effector. We utilized variable temperature isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) experiments to better understand the entropic contributions to allosteric effector binding, including changes to solvent Entropy and protein Conformational Entropy. Upon binding either Tyr or Trp, ScCM experiences a quenching of motions on the picosecond-to-nanosecond time scale, which we could relate to a loss of protein Conformational Entropy. Further ITC and NMR studies were consistent with the Tyr-bound form of ScCM being associated with more water molecules compared to the Trp-bound form and Tyr binding being associated with a less positive solvent Entropy change. These studies provide insight into the role of structural dynamics in ScCM function and highlight the importance of solvent Entropy changes in allosteric regulation, a historically underappreciated concept.
Vignesh Kasinath - One of the best experts on this subject based on the ideXlab platform.
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on the relationship between nmr derived amide order parameters and protein backbone Entropy changes
Proteins, 2015Co-Authors: Kim A Sharp, Vignesh Kasinath, Evan S Obrien, Joshua A WandAbstract:Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone Entropy. Amide order parameters (O(2) NH ) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O(2) NH < 0.8) shows that these report both on the local flexibility of the protein and on a different component of the Conformational Entropy than that reported by the side chain methyl axis order parameters, O(2) axis . A calibration curve for backbone Entropy vs. O(2) NH is developed, which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O(2) NH changes obtained by NMR relaxation measurements to extract backbone Entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain Entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein Conformational Entropy changes from NMR relaxation measurements.
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motion and Conformational Entropy in protein function creation of an nmr based Entropy meter
Biophysical Journal, 2015Co-Authors: Vignesh Kasinath, Veronica R Moorman, Kyle W Harpole, Kathleen G Valentine, Kendra K Frederick, Kim A Sharp, Joshua WandAbstract:Conformational Entropy is a potentially important thermodynamic parameter contributing to protein function. Quantitative measures of Conformational Entropy are necessary for an understanding of its role but have been difficult to obtain. We have recently introduced empirical method that utilizes changes in Conformational dynamics as a proxy for changes in Conformational Entropy. We have now used molecular dynamics simulations to probe the microscopic origins of the link between Conformational dynamics and Conformational Entropy. Simulation of seven proteins gave an excellent correlation with measures of side-chain motion derived from NMR relaxation. The simulations show that the motion of methyl-bearing side-chains are sufficiently coupled to that of other side chains to serve as excellent reporters of the overall side-chain Conformational Entropy. These results tend to validate the use of experimentally accessible measures of methyl motion - the NMR-derived generalized order parameters - as a proxy from which to derive changes in protein Conformational Entropy due to a perturbation such as the binding of a ligand. A slightly modified weighting scheme to project the change in dynamics of experimental methyl dynamics into Conformational Entropy is presented. Originally based on data from the calmodulin system, we will describe experimental results from other systems that indicate that the “Entropy meter” approach is both robust and general and that the involved Conformational entropies are often large and cannot be ignored. Supported by the NIH and the Mathers Foundation.
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coupled motion in proteins revealed by pressure perturbation
Journal of the American Chemical Society, 2012Co-Authors: Vignesh Kasinath, Veronica R Moorman, Nathaniel V Nucci, Vincent J Hilser, Joshua A WandAbstract:The cooperative nature of protein substructure and internal motion is a critical aspect of their functional competence about which little is known experimentally. NMR relaxation is used here to monitor the effects of high pressure on fast internal motion in the protein ubiquitin. In contrast to the main chain, the motions of the methyl-bearing side chains have a large and variable pressure dependence. Within the core, this pressure sensitivity correlates with the magnitude of motion at ambient pressure. Spatial clustering of the dynamic response to applied hydrostatic pressure is also seen, indicating localized cooperativity of motion on the sub-nanosecond time scale and suggesting regions of variable compressibility. These and other features indicate that the native ensemble contains a significant fraction of members with characteristics ascribed to the recently postulated “dry molten globule”. The accompanying variable side-chain Conformational Entropy helps complete our view of the thermodynamic archit...