Simulations of the structural and dynamical properties of denatured proteins: the “molten coil” state of bovine pancreatic trypsin inhibitor
ABSTRACT The dynamic nature of denatured, unfolded proteins makes it difficult to characterize their structures experimentally. To complement experiment and to obtain more detailed information about the structure and dynamic behavior of the denatured state, we have performed eleven 2.5 ns molecular dynamics simulations of reduced bovine pancreatic trypsin inhibitor (BPTI) at high temperature in water and a control simulation at 298 K, for a total of 30 ns of simulation time. In a neutral pH environment (acidic residues ionized), the unfolded protein structures were compact with an average radius of gyration 9% greater than the native state. The compact conformations resulted from the transient formation of non-native hydrophobic clusters, turns and salt bridges. However, when the acidic residues were protonated, the protein periodically expanded to a radius of gyration of 18 to 20 Å. The early steps in unfolding were similar in the different simulations until passing through the major transition state of unfolding. Afterwards, unfolding proceeded through one of two general pathways with respect to secondary structure: loss of the C-terminal helix followed by loss of β-structure or the opposite. To determine whether the protein preferentially sampled particular conformational substates in the denatured state, pairwise Cα root-mean-square deviations were measured between all structures, but similar structures were found between only two trajectories. Yet, similar composite properties (secondary structure content, side-chain and water contacts, solvent accessible surface area, etc.) were observed for the structures that unfolded through different pathways. Somewhat surprisingly, the unfolded structures are in agreement with both past experiments suggesting that reduced BPTI is a random coil and more recent experiments providing evidence for non-random structure, demonstrating how ensembles of fluctuating structures can give rise to experimental observables that are seemingly at odds.
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ABSTRACT: The 'new view' of protein folding is based on a statistical analysis of the landscape for protein folding. This perspective leads to the investigation of the statistical distributions of protein conformations as the folding protein approaches the native state from unfolded states. Molecular dynamics simulations of both the thermodynamics of protein folding and the kinetics of unfolding are beginning to explore the statistical nature of these distributions. They also provide connections between the theory of protein folding landscapes and the experimental observations of the properties of key ensembles of the conformations populated as folding progresses.Current Opinion in Structural Biology 05/1998; 8(2):222-6. DOI:10.1016/S0959-440X(98)80043-2 · 8.75 Impact Factor
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ABSTRACT: Experimental data from protein engineering studies and NMR spectroscopy have been used by theoreticians to develop algorithms for helix propensity and to benchmark computer simulations of folding pathways and energy landscapes. Molecular dynamic simulations of the unfolding of chymotrypsin inhibitor 2 (CI2) have provided detailed structural models of the transition state ensemble for unfolding/folding of the protein. We now have used the simulated transition state structures to design faster folding mutants of CI2. The models pinpoint a number of unfavorable local interactions at the carboxyl terminus of the single alpha-helix and in the protease-binding loop region of CI2. By removing these interactions or replacing them with stabilizing ones, we have increased the rate of folding of the protein up to 40-fold (tau = 0.4 ms). This correspondence, and other examples of agreement between experiment and theory in general, Phi-values and molecular dynamics simulations, in particular, suggest that significant progress has been made toward describing complete folding pathways at atomic resolution by combining experiment and simulation.Proceedings of the National Academy of Sciences 08/1998; 95(15):8473-8. DOI:10.1073/pnas.95.15.8473 · 9.81 Impact Factor
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ABSTRACT: The folding/unfolding pathway of barnase has been analyzed using a method similar to the classical Brønsted-βapproach: Φ-value analysis. Kinetic and equilibrium measurements on the folding/unfolding of over 100 designed mutants have led to a residue-by-residue description of the transition state. The transition state responds to mutation and changes in solvent in a manner analogous to both classical Hammond and anti-Hammond behavior as the energy landscape is perturbed. Here, we compare the Φ-value analysis with an explicit structural analysis of the transition state by molecular dynamics simulations of thermal denaturation of wild-type and two mutant forms of barnase. We look for similarities in the results of experiment and simulation to provide a detailed and reliable description of the folding reaction and for differences that could point to deficiencies in the methods. In general, there is excellent agreement between simulation and experiment, with a correlation coefficient of 0.93 between observed and simulated Φ-values for the transition state for unfolding, with the exception of the second helix, α2. In the simulations, wild-type barnase unfolded by disruption of the hydrophobic cores and β-structure, followed by unraveling of the principal α-helix, α1. The Ile 88 →Val mutant unfolded by the same mechanism as the wild-type protein, albeit more rapidly. Tyr 17 is one of the residues that, when mutated, leads to anti-Hammond effects; the helix unfolds earlier, relative to gross unfolding of the rest of the protein. In the simulation of the unfolding of the Tyr 17 →Gly mutant, the main helix unraveled before substantial loss of β-structure, showing more precisely the structural change in the transition state. The major difference between simulation and experiment is that α2 is present in the simulated transition state, but Φ-values suggest that it is unstructured. Although this could result from simulation overestimating the helical content, there is an alternative explanation that reconciles simulation and experiment. The segment of protein containing α2 is autonomous and makes few interactions with the body of the protein in the simulated transition state. If the folding of a segment of the protein is not coupled to the rest of the molecule, then the mutations may not be felt until significant interactions are made between these portions of the protein. Such an effect could occur for any multimodular protein.Journal of the American Chemical Society 12/1998; 120(49):12740-12754. DOI:10.1021/ja981558y · 11.44 Impact Factor