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Valerie Daggett - One of the best experts on this subject based on the ideXlab platform.
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multimolecule test tube simulations of Protein Unfolding and aggregation
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Michelle E Mccully, David A C Beck, Valerie DaggettAbstract:Molecular dynamics simulations of Protein folding or Unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the Unfolding/folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -Unfolding Protein for which the folding/Unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/Unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring Proteins, which were mostly hydrophobic in nature and ultimately caused the Proteins to aggregate. Protein–water hydrogen bonds were also replaced with Protein–Protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in Protein–Protein interactions, the Protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study Protein Unfolding and also provide insight into the types of interactions that occur as Proteins aggregate at high temperature at an atomic level.
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Understanding Protein Unfolding from molecular simulations
Wiley Interdisciplinary Reviews: Computational Molecular Science, 2012Co-Authors: Rudesh D. Toofanny, Valerie DaggettAbstract:Experimental biophysical techniques can probe the native, transition, intermediate, and denatured states of Proteins. The characterization of these states and the conversion between states provide us with information about folding, dynamics, misfolding, and the origin of mechanical strength in response to force. Molecular dynamics (MD) simulations are a complementary theoretical technique that provides atomic detail to the experimental measurements particularly through careful benchmarking and validation against experiment. Furthermore, MD simulations often correctly predict the outcome of experiments and provide new and interesting avenues of investigation. Our understanding of Protein Unfolding is being pushed forward by the symbiosis of experimental and theoretical methods. Here, we review investigations of several Protein systems and highlight the close interplay between experiment and simulation in providing an atomic resolution view of Protein Unfolding, which provide us with the general principles for folding and the origin of mechanical strength. © 2012 John Wiley & Sons, Ltd.
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Ensemble versus single-molecule Protein Unfolding.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Ryan Day, Valerie DaggettAbstract:Molecular dynamics (MD) simulations are the classic single-molecule “experiments,” providing atomic-resolution structural and dynamic information. However, the single-molecule nature of the technique has also been its shortcoming, with frequent criticisms of sampling inadequacies and questions regarding the ensemble behavior of large numbers of molecules. Given the increase in computer power, we now address this issue by performing a large number of simulations and comparing individual and ensemble properties. One hundred independent MD simulations of the Protein chymotrypsin inhibitor 2 were carried out for 20 ns each at 498 K in water to more fully describe the potentially diverse routes of Protein Unfolding and investigate how representative a single trajectory can be. Rapid Unfolding was observed in all cases with the trajectories distributed about an average “ensemble” path in which secondary and tertiary structure was lost concomitantly, with tertiary structure loss occurring slightly faster. Individual trajectories did, however, sample conformations far from the average path with very heterogeneous time-dependent properties. Nevertheless, all of the simulations but one followed the average ensemble pathway, such that a small number of simulations (5-10) are sufficient to capture the average properties of these states and the Unfolding pathway.
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Increasing temperature accelerates Protein Unfolding without changing the pathway of Unfolding.
Journal of molecular biology, 2002Co-Authors: Ryan Day, Brian J. Bennion, Sihyun Ham, Valerie DaggettAbstract:Abstract We have traditionally relied on extremely elevated temperatures (498 K, 225 °C) to investigate the Unfolding process of Proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298 K to 498 K. The simulation lengths vary from 94 ns to 20 ns, for a total simulation time of 344 ns, or 0.34 μs. At 298 K, the Protein is very stable over the full 50 ns simulation. At 348 K, corresponding to the experimentally observed melting temperature of CI2, the Protein unfolds over the first 25 ns, explores partially unfolded conformations for 20 ns, and then refolds over the last 35 ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early Unfolding is characterized by sliding or breathing motions in the Protein core, leading to an Unfolding transition state with a weakened core and some loss of secondary structure. After the Unfolding transition, the core contacts are rapidly lost as the Protein passes on to the fully denatured ensemble. While the overall character and order of events in the Unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498 K simulations are suitable for elucidating the details of Protein Unfolding at a minimum of computational expense.
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molecular dynamics simulations of the Protein Unfolding folding reaction
Accounts of Chemical Research, 2002Co-Authors: Valerie DaggettAbstract:All-atom molecular dynamics simulations of Proteins in solvent are now able to realistically map the Protein-Unfolding pathway. The agreement with experiments probing both folding and Unfolding suggests that these simulated Unfolding events also shed light on folding. The simulations have produced detailed models of Protein folding transition, intermediate, and denatured states that are in both qualitative and quantitative agreement with experiment. The various studies presented here highlight how such simulations both complement and extend experiment.
Chiwook Park - One of the best experts on this subject based on the ideXlab platform.
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Probing Membrane Protein Unfolding with Pulse Proteolysis
Journal of Molecular Biology, 2010Co-Authors: Jonathan P. Schlebach, James U. Bowie, Chiwook ParkAbstract:Abstract Technical challenges have greatly impeded the investigation of membrane Protein folding and Unfolding. To develop a new tool that facilitates the study of membrane Proteins, we tested pulse proteolysis as a probe for membrane Protein Unfolding. Pulse proteolysis is a method to monitor Protein folding and Unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded Proteins. This method requires only a small amount of Protein and, in many cases, may be used with unpurified Proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane Protein Unfolding, we chose Halobacterium halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A560). In this work, we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact Protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (Cm) shows excellent agreement with that determined by A560. The Cm values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A560. Our results suggest that pulse proteolysis is a quantitative tool to probe membrane Protein Unfolding. Combining pulse proteolysis with Western blotting may allow the investigation of membrane Protein Unfolding in situ without overexpression or purification.
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Revisiting absorbance at 230 nm as a Protein Unfolding probe
Analytical biochemistry, 2009Co-Authors: Pei-fen Liu, Larisa Avramova, Chiwook ParkAbstract:Thermodynamic stability and Unfolding kinetics of Proteins are typically determined by monitoring Protein Unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230nm (A(230)) is also known to be sensitive to Protein conformation. However, its feasibility for quantitative analysis of Protein energetics has not been assessed. Here we evaluate A(230) as a structural probe to determine thermodynamic stability and Unfolding kinetics of Proteins. By using Escherichia coli maltose binding Protein (MBP) and E. coli ribonuclease H (RNase H) as our model Proteins, we monitored their Unfolding in urea and guanidinium chloride with A(230). Significant changes in A(230) were observed with both Proteins on Unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A(230) in varying concentrations of denaturants. Also, Unfolding kinetics was investigated by monitoring the change in A(230) under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A(230) allowed us to monitor Protein Unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A(230) is a valid and convenient structural probe to determine thermodynamic stability and Unfolding kinetics of Proteins with many potential applications.
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Investigating Protein Unfolding kinetics by pulse proteolysis
Protein Science, 2009Co-Authors: Yu-ran Na, Chiwook ParkAbstract:Investigation of Protein Unfolding kinetics of Proteins in crude samples may provide many exciting opportunities to study Protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed “pulse proteolysis” to investigate Protein Unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of Proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded Proteins. Electrophoretic separation after proteolysis allows monitoring Protein Unfolding without Protein purification. We employed pulse proteolysis to determine Unfolding kinetics of E. coli maltose binding Protein (MBP) and E. coli ribonuclease H (RNase H). The Unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an Unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate Unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on Unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for Unfolding of MBP•maltose complex, we have determined the dissociation equilibrium constant (Kd) of the complex from Unfolding kinetic constants, which is also in good agreement with known Kd values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate Protein Unfolding kinetics.
Patrick Senet - One of the best experts on this subject based on the ideXlab platform.
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Curvature and Torsion of Protein Main Chain as Local Order Parameters of Protein Unfolding
Journal of Physical Chemistry B, 2020Co-Authors: Paul Grassein, Patrice Delarue, Adrien Nicolaï, Fabrice Neiers, Harold A. Scheraga, Gia G. Maisuradze, Patrick SenetAbstract:Thermal Protein Unfolding resembles a global (two-state) phase transition. At the local scale, Protein Unfolding is, however, heterogeneous and probe dependent. Here, we consider local order parameters defined by the local curvature and torsion of the Protein main chain. Because chemical shift (CS) measured by NMR spectroscopy is extremely sensitive to the local atomic environment, CS has served as a local probe of thermal Unfolding of Proteins by varying the position of the atomic isotope along the amino-acid sequence. The variation of the CS of each C(alpha) atom along the sequence as a function of the temperature defines a local heat-induced denaturation curve. We demonstrate that these local heat-induced denaturation curves mirror the local Protein nativeness defined by the free-energy landscape of the local curvature and torsion of the Protein main chain described by the C(alpha)-C(alpha) virtual bonds. Comparison between molecular dynamics simulations and CS data of the gpW Protein demonstrates that some local native states defined by the local curvature and torsion of the main chain, mainly located in secondary structures, are coupled to each other whereas others, mainly located in flexible Protein segments, are not. Consequently, CS of some residues are faithful reporters of global Protein Unfolding, with heat-induced denaturation curves similar to the average global one, whereas other residues remain silent about the Protein unfolded state. For these latter, the local deformation of the Protein main chain, characterized by its local curvature and torsion, is not cooperatively coupled to global Unfolding.
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Curvature and Torsion of Protein Main Chain as Local Order Parameters of Protein Unfolding.
The journal of physical chemistry. B, 2020Co-Authors: Paul Grassein, Patrice Delarue, Adrien Nicolaï, Fabrice Neiers, Harold A. Scheraga, Gia G. Maisuradze, Patrick SenetAbstract:Thermal Protein Unfolding resembles a global (two-state) phase transition. At the local scale, Protein Unfolding is, however, heterogeneous and probe dependent. Here, we consider local order parame...
Paul Grassein - One of the best experts on this subject based on the ideXlab platform.
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Curvature and Torsion of Protein Main Chain as Local Order Parameters of Protein Unfolding
Journal of Physical Chemistry B, 2020Co-Authors: Paul Grassein, Patrice Delarue, Adrien Nicolaï, Fabrice Neiers, Harold A. Scheraga, Gia G. Maisuradze, Patrick SenetAbstract:Thermal Protein Unfolding resembles a global (two-state) phase transition. At the local scale, Protein Unfolding is, however, heterogeneous and probe dependent. Here, we consider local order parameters defined by the local curvature and torsion of the Protein main chain. Because chemical shift (CS) measured by NMR spectroscopy is extremely sensitive to the local atomic environment, CS has served as a local probe of thermal Unfolding of Proteins by varying the position of the atomic isotope along the amino-acid sequence. The variation of the CS of each C(alpha) atom along the sequence as a function of the temperature defines a local heat-induced denaturation curve. We demonstrate that these local heat-induced denaturation curves mirror the local Protein nativeness defined by the free-energy landscape of the local curvature and torsion of the Protein main chain described by the C(alpha)-C(alpha) virtual bonds. Comparison between molecular dynamics simulations and CS data of the gpW Protein demonstrates that some local native states defined by the local curvature and torsion of the main chain, mainly located in secondary structures, are coupled to each other whereas others, mainly located in flexible Protein segments, are not. Consequently, CS of some residues are faithful reporters of global Protein Unfolding, with heat-induced denaturation curves similar to the average global one, whereas other residues remain silent about the Protein unfolded state. For these latter, the local deformation of the Protein main chain, characterized by its local curvature and torsion, is not cooperatively coupled to global Unfolding.
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Curvature and Torsion of Protein Main Chain as Local Order Parameters of Protein Unfolding.
The journal of physical chemistry. B, 2020Co-Authors: Paul Grassein, Patrice Delarue, Adrien Nicolaï, Fabrice Neiers, Harold A. Scheraga, Gia G. Maisuradze, Patrick SenetAbstract:Thermal Protein Unfolding resembles a global (two-state) phase transition. At the local scale, Protein Unfolding is, however, heterogeneous and probe dependent. Here, we consider local order parame...
Ryan Day - One of the best experts on this subject based on the ideXlab platform.
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Ensemble versus single-molecule Protein Unfolding.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Ryan Day, Valerie DaggettAbstract:Molecular dynamics (MD) simulations are the classic single-molecule “experiments,” providing atomic-resolution structural and dynamic information. However, the single-molecule nature of the technique has also been its shortcoming, with frequent criticisms of sampling inadequacies and questions regarding the ensemble behavior of large numbers of molecules. Given the increase in computer power, we now address this issue by performing a large number of simulations and comparing individual and ensemble properties. One hundred independent MD simulations of the Protein chymotrypsin inhibitor 2 were carried out for 20 ns each at 498 K in water to more fully describe the potentially diverse routes of Protein Unfolding and investigate how representative a single trajectory can be. Rapid Unfolding was observed in all cases with the trajectories distributed about an average “ensemble” path in which secondary and tertiary structure was lost concomitantly, with tertiary structure loss occurring slightly faster. Individual trajectories did, however, sample conformations far from the average path with very heterogeneous time-dependent properties. Nevertheless, all of the simulations but one followed the average ensemble pathway, such that a small number of simulations (5-10) are sufficient to capture the average properties of these states and the Unfolding pathway.
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Increasing temperature accelerates Protein Unfolding without changing the pathway of Unfolding.
Journal of molecular biology, 2002Co-Authors: Ryan Day, Brian J. Bennion, Sihyun Ham, Valerie DaggettAbstract:Abstract We have traditionally relied on extremely elevated temperatures (498 K, 225 °C) to investigate the Unfolding process of Proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298 K to 498 K. The simulation lengths vary from 94 ns to 20 ns, for a total simulation time of 344 ns, or 0.34 μs. At 298 K, the Protein is very stable over the full 50 ns simulation. At 348 K, corresponding to the experimentally observed melting temperature of CI2, the Protein unfolds over the first 25 ns, explores partially unfolded conformations for 20 ns, and then refolds over the last 35 ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early Unfolding is characterized by sliding or breathing motions in the Protein core, leading to an Unfolding transition state with a weakened core and some loss of secondary structure. After the Unfolding transition, the core contacts are rapidly lost as the Protein passes on to the fully denatured ensemble. While the overall character and order of events in the Unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498 K simulations are suitable for elucidating the details of Protein Unfolding at a minimum of computational expense.
