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A focus is made on molecular dynamics (MD) simulations of proteins in solution to investigate the detailed processes of protein folding and unfolding. Proteins can be expected to fairly reliably depict protein folding/unfolding transition states, intermediate states and denatured states, provided explicit solvent and good simulation techniques are employed.

Molecular dynamics simulations was used to reveal the detailed conformational behaviors of peptides and proteins and by comparing fragment and full length protein simulations, the role of each peptide segment in the folding process was investigated. The segments with different helical propensities play an important role during protein folding.

Homology models are constructed for both human catechol O-methyltransferase (COMT) polymorphs and molecular dynamics simulations of these models are performed at 25, 37 and 50 degree C to explore the structural consequences of the 108V/M polymorphism. The addition of cosubstrate S-adenosylmethionine (SAM) tightens up the cosubstrate pocket in both proteins and prevents the altered packing at the polymorphic site in 108M COMT.

The self-assembly of beta(sub 2)-microglobulin into fibrils leading to dialysis-related amyloidosis is described. It is proposed that the forming of alpha sheet facilitates self assembly into partially unfolded prefibrillar amyloidogenic intermediates.

An assessment of three spherical atom-based cutoff approaches such as abrupt truncation, a CHARMM-style electrostatic shift truncation and a force-shift truncation for use with all-atom explicit solvent molecular dynamics (MD) is described. This work demonstrates that by using proper and rigorous techniques, it is possible to correctly model polypeptide dynamics in solution with a spherical cutoff.

High-temperature molecular dynamics simulations were used to gain insight into the early steps in the unfolding pathway of azurin, a blue copper protein with a beta-barrel structure formed by two sheets arranged in a Greek key folding topology. The results reveal that unfolding of the beta-barrel in azurin is associated with dislocation of its unique alpha-helix with respect to the protein scaffold.

The molecular dynamics (MD) simulations are employed to determine the effects of trimethylamine N-oxide (TMAO) on the dynamics and structure of the prion protein at the atomic level. It is shown that the misfolded, PrP(super Sc) -like prion protein obtained through MD simulation under the acidic conditions can be restored to a more globular structure upon addition of 1M TMAO.

Transthyretin, [[beta].sub.2]-microglobulin, lysozyme, and the prion protein are four of the best-characterized proteins implicated in amyloid disease. Upon partial acid denaturation, these proteins undergo conformational change into an amyloidogenic intermediate that can self-assemble into amyloid fibrils. Many experiments have shown that pH-mediated changes in structure are required for the formation of the amyloidogeneic intermediate, but it has proved impossible to characterize these conformational changes at high resolution using experimental means. To probe these conformational changes at atomic resolution, we have performed molecular dynamics simulations of these proteins at neutral and low pH. In low-pH simulations of all four proteins, we observe the formation of [alpha]-pleated sheet secondary structure, which was first proposed by L. Pauling and R. B. Corey [(1951) Proc. Natl. Acad. Sci. USA 37, 251-256]. In all [beta]-sheet proteins, transthyretin and [[beta].sub.2]-microglobulin, [alpha]-pleated sheet structure formed over the strands that are highly protected in hydrogen-exchange experiments probing amyloidogenic conditions. In lysozyme and the prion protein, [alpha]-sheets formed in the specific regions of the protein implicated in the amyloidogenic conversion. We propose that the formation of [alpha]-pleated sheet structure may be a common conformational transition in amyloidosis.

Here, we describe the folding/unfolding kinetics of [[alpha].sub.3]D, a small designed three-helix bundle. Both IR temperature jump and ultrafast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 [+ or -] 1.2 [micro]s (at [approximately equal to] 50[degrees]C), indicating that a protein can fold on the 1- to 5-[micro]s time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably [greater than or equal to] 1 [([micro]s).sup.-1] for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. [[alpha].sub.3]D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways.

Author(s): Ugo Mayor [1]; Nicholas R. Guydosh [1]; Christopher M. Johnson [1]; J. Günter Grossmann [2]; Satoshi Sato [1]; Gouri S. Jas [3, 4]; Stefan M. V. Freund [1]; Darwin [...]

Proteins appear to fold by diverse pathways, but variations of a simple mechanism--nucleation-condensation--describe the overall features of folding of most domains. In general, secondary structure is inherently unstable and its stability is enhanced by tertiary interactions. Consequently, an extensive interplay of secondary and tertiary interactions determines the transition-state for folding, which is structurally similar to the native state, being formed in a general collapse (condensation) around a diffuse nucleus. As the propensity for stable secondary structure increases, folding becomes more hierarchical and eventually follows a framework mechanism where the transition state is assembled from pre-formed secondary structural elements.

Research has been conducted on the proteins' all-atom molecular dynamics simulations in solvent. The analysis of these simulations which have produced the models of protein folding transition, intermediate and denatured states is presented.

The authors review the publications on protein folding events. The topic of interest include the application of the experimental and theoretical methods of protein folding at atomic level resolution and their results.

Chemical synthesis allows the incorporation of nonnatural amino acids into proteins that may provide previously untried probes of their folding pathway and thermodynamic stability. We have used a flexible thioether linker as a loop mimetic in the human yes kinase-associated protein (YAP 65) WW domain, a three-stranded, 44-residue, [beta]-sheet protein. This linkage avoids problems of incorporating sequences that constrain loops to the extent that they significantly change the nature of the denatured state with concomitant effects on the folding kinetics. An NMR solution structure shows that the thioether linker had little effect on the global fold of the domain, although the loop is apparently more dynamic. The thioether variants are destabilized by up to 1.4 kcal/mol (1 cal = 4.18 J). Preliminary [PHI]-value analysis showed that the first loop is highly structured in the folding transition state, and the second loop is essentially unstructured. These data are consistent with results from simulated unfolding and detailed protein-engineering studies of structurally homologous WW domains. Previously, [PHI]-value analysis was limited to studying side-chain interactions. The linkers used here extend the protein engineering method directly to secondary-structure interactions.

Research has been conducted on the hydrophobic deletion mutants of chymotrypsin inhibitor 2. The free energy perturbation of these mutants has been calculated via transition- and denatured-state structures from various denaturation molecular dynamics simulations.

The binding interface between cytochrome b(sub)5 and cytochrome c is investigated to understand the molecular mechanism involved in the electron-transport reactions between the cytochromes. Data show that 51% of cytochrome b(sub)5 residues exhibit chemical shift changes and these residues are localized to the binding interface.

The dynamic characteristics of the ribonuclease inhibitor barstar was studied through molecular dynamics simulations in explicit water. Results indicated that the entropy of the native state can be important and should not be neglected in thermodynamic studies of protein folding. The side chain motion shows the main source of the residual entropy of the native state and entropic studies based only on the backbone dynamics are insufficient.