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V Daggett

Publications and source records attributed to V Daggett.

54 records · Page 3Linked to original sources
PubMed1996

Structure of the transition state for folding of a protein derived from experiment and simulation.

Independent experimental and theoretical studies of the unfolding of barley chymotrypsin inhibitor 2 (CI2) are compared in an attempt to derive plausible three-dimensional structural models of the transition states. A very simple structure index is calculated along the sequence for the molecular dynamics-generated transition state models to facilitate comparison with the phi F values. The two are in good agreement overall (correlation coefficient = 0.87), which suggests that the theoretical models should provide a structural framework for interpretation of the phi F values. Both experiment and simulation indicate that the transition state is a distorted form of the native state in which the alpha-helix is weakened but partially intact and the beta-sheet is quite disrupted. As inferred from the phi f values and observed directly in the simulations, the unfolding of CI2 is cooperative and there is a "folding core" comprising a patch on the alpha-helix and a portion of the beta-sheet, nucleated by interactions between Ala16, Ile49 and other neighbouring residues. The protein becomes less structured radiating away from this core. Overall the data indicate that CI2 folds by a nucleation-collapse mechanism. In the absence of experimental information, we have little confidence that the molecular dynamics simulations are correct, especially when only one or a few simulations are performed. On the other hand, even though the experimentally derived phi values may reflect the extent of overall structure formation, they do not provide an actual atomic-resolution three dimensional structure of the transition state. By combining the two approaches, however, we have a framework for interpreting phi F values and can hopefully arrive at a more trustworthy model of the transition state. The process is in some ways similar to the combination of molecular dynamics and NMR data to solve the tertiary structure of proteins.

Amino Acid Sequence↗
PubMed1996

Molecular dynamics simulations of apocytochrome b562--the highly ordered limit of molten globules.

BACKGROUND: Cytochrome B562 is a heme-containing, four-helix bundle. It has been proposed that the apo form of the protein is a molten globule. We present a molecular dynamics study of apocytochrome b562 to investigate its structural and dynamic properties. RESULTS: Our simulations suggest that all four helices are essentially intact and confirm that the experimental difficulties of assigning helical NOEs in the C-terminal helix are not due to structural disorder. The increased 'moltenness' of the apoprotein is due to an increased mobility of the sidechains. The small observed increase in compressibility for the apoprotein is proposed to be the result of an increase in the intrinsic protein compressibility, which is opposed by the increase in the size of the protein hydration shell. CONCLUSIONS: Apocytochrome b562 is postulated to be near the highly ordered limit of the molten globule state, a structure whose molten character is due primarily to increased sidechain mobility with concurrent loss in tertiary contacts between the helices, rather than changes in the folding topology or substantially increased disorder of the secondary structure.

Animals↗
PubMed1995

Molecular dynamics simulation of cytochrome b5: implications for protein-protein recognition.

Cytochrome b5 participates in electron-transfer reactions with a variety of different proteins. To explore how this protein might discern between structurally varied proteins, we have performed a molecular dynamics simulation focusing on its structural stability and dynamic behavior in solution. The protein was simulated in water at 298 K and pH 6.9 for 2.5 ns. The protein deviated significantly from the crystal structure midway through the simulation, but ultimately the crystalline conformation was regained. The simulation was at all times well behaved as judged by comparison to structural NMR data obtained in solution. One region of the protein backbone that deviated from the crystal conformation contains acidic residues implicated in electrostatic-based protein-protein recognition. The mobility in this region caused the protein to display different patterns of residues at the surface with time, as well as the formation of a large cleft partially exposing the hydrophobic core lining the heme pocket. Furthermore, the position and cyclical formation of this cleft suggest that hydrophobic interactions may be important in protein-protein recognition events and possibly even electron transfer, as the cleft allows for easy access to the heme group. These results indicate that thermal motion could provide a low-energy mechanism for controlling recognition events. Thus, the dynamical behavior observed through the varying solution conformations sampled may be important in influencing the diverse range of protein-protein interactions in which cytochrome b5 participates.

Computer Simulation↗
PubMed1995

pH-dependent conformations of the amyloid beta(1-28) peptide fragment explored using molecular dynamics.

Molecular dynamics simulations were used to successfully reproduce the experimentally observed pH-dependent conformational behavior of a monomeric peptide in aqueous solution. Simulations were conducted at 298 K on a peptide corresponding to residues 1-28 of the amyloid beta-peptide [referred to as beta(1-28)], which is the primary component of the plaques associated with Alzheimer's disease. beta(1-28) was found to be entirely alpha-helical at low pH. Upon deprotonation of acidic residues at medium pH, helical structure was lost in the N-terminal region. At high pH, some secondary structure was recovered to yield two helices joined by a kink. These results are in good agreement with the NMR solution structure at low pH [Zagorski and Barrow (1992) Biochemistry 31, 5621-5631; Talafous et al. (1994) Biochemistry 33, 7788-7796] and CD and NMR evidence of an alpha-helix to beta-sheet transition at mid-range pH [Barrow et al. (1992) J. Mol. Biol. 225, 1075-1093]. Additional simulations were also able to regenerate folded species from partially unfolded conformers. A mechanism for the pH-dependent structural rearrangements is proposed that involves the creation of a hydrogen-bonded pair between Ser 8 and Glu 11. The evidence for the existence of a multiconformational equilibrium of folded and unfolded species of the peptide is discussed.

Amino Acid Sequence↗
PubMed1995

Sequence effects on the conformational properties of the amyloid beta (1-28) peptide: testing a proposed mechanism for the alpha-->beta transition.

Molecular dynamics simulations have been used to successfully reproduce the observed pH-dependent conformational properties of the amyloid beta(1-28) peptide [Kirshenbaum and Daggett (1995) Biochemistry, 34, 7629-7639]. On the basis of these simulations a mechanism was proposed for the unfolding of the N-terminal portion of the peptide at neutral pH when beginning from the helical conformation. It was proposed that interactions between the side chains of Ser 8 and Glu 11 are important in determining the pH dependence of the helix content. Here we further investigate this proposed mechanism and the residues involved in the conformational transition by performing computational "mutagenesis" studies. On the basis of simulations of the mutant peptides, the importance of the Ser 8-Glu 11 interaction is substantiated, and further details of the conformational transition are elucidated.

Alanine↗
PubMed1995

Molecular dynamics simulations of protein unfolding and limited refolding: characterization of partially unfolded states of ubiquitin in 60% methanol and in water.

Extensive experimental data are available on the native, partially and fully unfolded states of ubiquitin. Two and three-dimensional NMR experiments of a partially unfolded form of the protein in 60% methanol indicate that approximately one-half of the molecule contains disrupted but native-like structure while the other half is unstructured and/or contains non-native structure. In contrast, the interpretation of hydrogen-exchange data have led to the conclusion that this state is native-like. Thus, there are discrepancies between the experimental studies, or interpretations based on the data. We compare the results of molecular dynamics simulations, under varying conditions, with the experimental results. The simulations extend past 0.5 ns and include explicit solvent molecules: either pure water or 60% methanol. To begin with, ubiquitin was thermally denatured in water (at 498 K). Two particular structures, or "aliquots", during the unfolding process were selected for further study (60 and 198 ps). These structures were then simulated separately in water and 60% methanol at a lower and experimentally meaningful temperature (335 K). The conformations generated from the structure extracted later in the simulation contained significant amounts of non-native structure in the presence of methanol while satisfying both the NMR and hydrogen exchange data. In fact, clearly non-native regions of the structure yielded the desired protection from hydrogen exchange. In contrast, an earlier, more native-like, intermediate did not do as well at predicting the hydrogen-exchange behavior and was inconsistent with the NMR data. These data suggest that the results and interpretations using the different experimental techniques can be reconciled by a single state. This finding also brings into question the practice of interpreting protection to hydrogen exchange in terms of native secondary and tertiary structure, especially when one has weak patterns and low protection factors. When the partially unfolded states were placed in pure water, the protein collapsed and began to refold. Therefore, the desired solvent-dependent properties were observed: the partially unfolded conformations with increased exposure of hydrophobic residues remained expanded in methanol but collapsed in water as the non-polar groups minimized their exposure to solvent.

Amino Acid Sequence↗
PubMed1995

Theoretical studies of sequence effects on the conformational properties of a fragment of the prion protein: implications for scrapie formation.

BACKGROUND: Prion diseases are neurodegenerative disorders that appear to be due to a conformational change, involving the conversion of alpha-helices in the normal, cellular isoform of the prion protein (PrPC) to beta-structure in the infectious scrapie form (PrPSc). One form of Gerstmann-Sträussler-Scheinker syndrome (GSS), an inherited prion disease, is caused by mutation of Ala117 of PrPC to Val. We therefore set out to evaluate the effects of this mutation on the stability of the PrPC form. RESULTS: We have performed molecular dynamics simulations of a portion of the PrPC sequence (residues 109-122, termed H1) that is proposed to figure prominently in the conversion of PrPC to PrPSc. In particular, beginning with H1 in the alpha-helical state, the conformational consequences of sequence changes at position 117 were investigated for six hydrophobic mutations. Of these, only the Val mutation was helix-destabilizing. Portions of this mutant peptide adopted and retained an extended conformation during a 2 ns simulation of the peptide in water. CONCLUSIONS: The conformational transitions and structures observed in the simulation of the mutant peptide with Val at position 117 provide insight into the possible early steps in the conversion of PrPC to PrPSc.

Animals↗
PubMed1995

Investigation of the solution structure of chymotrypsin inhibitor 2 using molecular dynamics: comparison to x-ray crystallographic and NMR data.

The native solution structure and dynamics of chymotrypsin inhibitor 2 (CI2) have been studied using a long (5.3 ns) molecular dynamics (MD) simulation without any imposed restraints. The majority of the experimentally observed spin-spin coupling constants, short- and long-range nuclear Overhauser effect (NOE) cross peaks and the amide hydrogen exchange behavior were reproduced by the MD simulation. This good correspondence suggests that the major structural features of the protein during the simulation are representative of the true protein structure in solution. Two water molecules formed hydrogen bond bridges between beta2 and beta3, in agreement with X-ray crystallographic data and a recent reassessment of the solution structure using time-averaged NMR restraints during MD refinement. The active-site loop of the protein displayed the greatest structural changes and the highest mobility. When this loop region was excluded, the average Calpha r.m.s. deviation of the simulated solution structures from the crystal structure was approximately 1.5 Angstrom from 0.5 to 5.3 ns. There is structural heterogeneity in particular regions of the NMR-derived solution structures, which could be a result of imprecision or true internal motion. A study of the distribution of mobility through the protein allows us to distinguish between these two alternatives. In particular, deviations in the active-site loop appear to be a result of heightened mobility, which is also supported by good correspondence between calculated and experimental S2 N-H order parameters. On the other hand, other ill-defined regions of the NMR-derived structures are well defined in the simulation and are probably the result of a lack of structural restraints (i.e. NOEs), as opposed to reflecting the true mobility.

Computer Simulation↗
PubMed1994

Characterization of the transition state of protein unfolding by use of molecular dynamics: chymotrypsin inhibitor 2.

Temperature-induced unfolding of chymotrypsin inhibitor 2 in water was investigated by molecular dynamics simulations. The major transition state of unfolding was identified on the basis of structural and conformational changes in the protein during the unfolding reaction. The native tertiary contacts in the hydrophobic core were considerably disrupted in the transition state, whereas the secondary structure was partially intact. The extent of structural change of the protein around a particular residue was represented quantitatively by the ratio of the number of contacts the residue makes in the transition state relative to the native state, phi MD, which allows quantitative comparison with the experimentally determined phi F values. For the region of the unfolding trajectory that is identified as the transition state, the phi MD and phi F values are in good agreement, suggesting that the transition state identified in the unfolding simulation corresponds to that probed with protein engineering methods. Although speculative, the transition state identified in the simulation is consistent with available experimental data and provides an in-depth view of what the transition state of unfolding may look like.

Amino Acid Sequence↗
PubMed1993

Protein unfolding pathways explored through molecular dynamics simulations.

Herein we describe the results of molecular dynamics simulations of the bovine pancreatic trypsin inhibitor (BPTI) in solution at a variety of temperatures both with and without disulfide bonds. The reduced form of the protein unfolded at high temperature to an ensemble of conformations with all the properties of the molten globule state. In this account we outline the structural details of the actual unfolding process between the native and molten globule states. The first steps of unfolding involved expansion of the protein, which disrupted packing interactions. The solvent-accessible surface area also quickly increased. The unfolding was localized mostly to the turn and loop regions of the molecule, while leaving the secondary structure intact. Then, there was more gradual unfolding of the secondary structure and non-native turns became prevalent. This same trajectory was continued and more drastic unfolding occurred that resulted in a relatively compact state devoid of stable secondary structure.

Aprotinin↗
PubMed1992

A model of the molten globule state from molecular dynamics simulations.

It is generally accepted that a protein's primary sequence determines its three-dimensional structure. It has proved difficult, however, to obtain detailed structural information about the actual protein folding process and intermediate states. We present the results of molecular dynamics simulations of the unfolding of reduced bovine pancreatic trypsin inhibitor. The resulting partially "denatured" state was compact but expanded relative to the native state (11-25%); the expansion was not caused by an influx of water molecules. The structures were mobile, with overall secondary structure contents comparable to those of the native protein. The protein experienced relatively local unfolding, with the largest changes in the structure occurring in the loop regions. A hydrophobic core was maintained although packing of the side chains was compromised. The properties displayed in the simulation are consistent with unfolding to a molten globule state. Our simulations provide an in-depth view of this state and details of water-protein interactions that cannot yet be obtained experimentally.

Animals↗
PubMed1992

Molecular dynamics simulations of helix denaturation.

An understanding of the structural transitions that an alpha-helix undergoes will help to elucidate such motions in proteins and their role in protein folding. We present the results of molecular dynamics simulations to investigate these transitions in a short polyalanine peptide (13 residues) both in vacuo and in the presence of solvent. The denaturation of this peptide was monitored as a function of temperature (ranging from 5 to 200 degrees C). In vacuo, the helical state predominated at all temperatures, whereas in solution the helix melted with increasing temperature. The peptide was predominantly helical at low temperature in solution, while at intermediate temperatures the peptide spent the bulk of the time fluctuating between different conformations with intermediate amounts of helix, e.g. not completely helical nor entirely non-helical. Many of these conformations consisted of short helical segments with intervening non-helical residues. At high temperature the peptide unfolded and adopted various collapsed unstructured states. The intrahelical hydrogen bonds that break at high temperature were not fully compensated by hydrogen bonds with water molecules in the partially unfolded forms of the peptide. Increases in temperature disrupted both the helical structure and the peptide-water interactions. Water played a major but indirect role in facilitating unfolding, as opposed to specifically competing for the intrapeptide hydrogen bonds. The implications of our results to protein folding are discussed.

Alanine↗
PubMed1991

Molecular dynamics simulations of small peptides: dependence on dielectric model and pH.

There has been much interest recently in the structure of small peptides in solution. A recent study by Bradley and co-workers [(1989) in Techniques of Protein Chemistry, Hugli, T.E., Ed., Academic Press, Orlando, FL, pp. 531-546; (1990) Journal of Molecular Biology, 215, pp. 607-622] describes a 17-residue peptide that is stable as a monomeric helix in aqueous solution at low pH, as determined by two-dimensional nmr and CD spectroscopy. They also have determined the helix content of the peptide as a function of pH using CD. We performed molecular dynamics simulations, with an empirical force field, of this peptide at low pH, with three different dielectric models: a linear distance-dependent dielectric function (epsilon = R); a modified form [J. Ramstein and R. Lavery (1988) Proceedings of the National Academy of Science, USA, Vol. 85, pp. 7231-7235] of the sigmoidal distance-dependent dielectric function of Hingerty and co-workers [(1985) Biopolymers, Vol. 24, pp. 427-439]; and epsilon = 1 with the peptide immersed in a bath of water molecules. We found that simulations with the sigmoidal dielectric function and the model with explicit water molecules resulted in average distances for particular interactions that were consistent with the experimental nmr results, with the sigmoidal function best representing the data. However, these models exhibited very different helix-stabilizing interactions. We also performed simulations using the sigmoidal function at moderate and high pH to compare to experimental determinations of the pH dependence of helix content. Helix content did not decrease with increases in pH, as shown experimentally. We did, however, observe changes in a specific side chain-helix dipole interaction that was implicated in determining the pH-dependent behavior of this peptide. Overall, the sigmoidal dielectric function was a reasonable alternative to adding explicit water molecules. In comparing 100 ps molecular dynamics simulations, the sigmoidal function was much less computer intensive and sampled more of conformational space than the treatment using explicit water molecules. Sampling is especially important for this system since the peptide has been shown experimentally to populate both helical and nonhelical conformations.

Amino Acid Sequence↗
PubMed1991

The application of computational methods to the study of enzyme catalysis by triose-phosphate isomerase and stabilities of variants of bacteriophage T4 lysozyme.

We review our research on triose-phosphate isomerase and bacteriophage T4 lysozyme. In our studies over the last ten years we have used electrostatic potentials, computer graphics, quantum mechanics, molecular mechanics, molecular dynamics and free energy calculations to try to understand why triose-phosphate isomerase is such an efficient enzyme and why its efficiency is dramatically decreased by several site-specific mutations. For T4 lysozyme we have used free energy methods to analyse and try to understand why Thr-157----Val and Thr-157----Ala mutations decrease protein stability by about 1-2 kcal/mol.

Amino Acid Sequence↗
PubMed1991

A molecular dynamics simulation of polyalanine: an analysis of equilibrium motions and helix-coil transitions.

An understanding of helix dynamics can aid in interpreting the motions of proteins. The conformational transitions that occur also appear to play a role in protein folding. Structural studies of isolated peptides in solution are just becoming available. However, detailed analysis of the helix-coil transition is still not available and will be difficult to obtain experimentally. For these reasons, we performed a long molecular dynamics simulation of polyalanine at high temperature. Using this approach, we obtain a description of the overall structure and inherent flexibility of the chain as well as a structural picture of the conformational changes that occur. In this way, we can address both equilibrium properties of the peptide and the dynamics and mechanisms of the structural transitions. Our results correlate fairly well with the available experimental data and previous simulations aimed at addressing alpha-helix dynamics. The peptide spends the bulk of its time fluctuating between different conformations with intermediate helix contents. Transitions between highly ordered and highly disordered structures were rare, but they occurred rapidly. Our distribution of conformations favored collapsed states. Hence, our transitions to structures with high helical content were from fluctuating compact structures. The conversion between helix and coil occurred sequentially on a residue-by-residue basis. However, there was local cooperativity; the transition of a residue to the coil state was facilitated after a neighboring group became nonhelical. The relevance of our results to protein folding is also discussed.

Models, Chemical↗
PubMed1990

Molecular dynamics simulations of active site mutants of triosephosphate isomerase.

Molecular dynamics simulations of triosephosphate isomerase (TIM) and of some active site TIM mutants were performed in an attempt to elucidate possible interactions important for catalytic activity and binding. A variety of active site residues in TIM have been altered, resulting in all cases in decreases in catalytic activity. Second-site suppressor mutants were characterized for two of these active site mutants. The pseudorevertants have increased activity compared to the single mutant from which they were derived and, surprisingly, in both cases the increase in activity is a result of the replacement of an active site serine for proline. We performed simulations of wild-type TIM and the active site mutants with the substrate dihydroxyacetone phosphate bound both noncovalently and covalently. The noncovalent complexes were used to examine interactions important to binding while the covalent complexes are models of the transition state structure for enolization, which is the rate-determining step for the mutants. The difference between these two states, then, is related to the catalytic activity. We found various protein-substrate interactions that improved in the noncovalent mutant complexes, which correlates with the experimentally observed increase in binding affinity upon mutation. In the covalent complexes we observed improved electrostatic stabilization of the transition state upon introduction of Pro, which is also consistent with the experimental data. Our simulations reproduce the highly co-operative nature of the interactions in the active site and suggest that this approach may be useful for identifying particularly promising sites for mutation.

Binding Sites↗
PubMed1987

Protein degradation: the role of mixed-function oxidases.

The mechanisms by which protein oxidation is mediated in the cell are of both biological and pharmacological importance. Oxidases responsible for the metabolism of xenobiotics catalyze the oxidative inactivation of select enzymes. Oxidation mediated by mixed-function oxidase (MFO) systems renders proteins more susceptible to proteolysis and, consequently, appears to be a signal for protein degradation. The mode of action of MFO systems is discussed in detail for a specific, well-characterized system--the MFO-catalyzed oxidation of glutamine synthetase (GS). Findings for this system are then generalized to help explain how other metabolic enzymes are oxidized by MFO systems. The broader consequences of oxidative mechanisms are discussed. For example, the accumulation of modified proteins during aging and in some premature aging diseases may be due in part to shifts in the relative rates of oxidation and degradation for these proteins. Further, the oxidation of key metabolic enzymes appears to be responsible for the bacteriocidal action of neutrophils. There is also some evidence that the degradation of endogenous proteins increases following ingestion of, or exposure to, agents that induce MFO activity.

Animals↗