Dr. Valerie Daggett
Department of Medicinal Chemistry
University of Washington
Seattle, WA

Monday, March 22, 1999
3:00 pm (CT)
3269 Beckman Institute

Abstract

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models. Molecular dynamics (MD) simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here we describe 3 independent 4 ns high-temperature MD simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the b(3-4) hairpin contained a particularly high degree of such side chain interactions but it lacked b-structure in two of the three denatured ensembles. The two principle a-helices (a1 and a2) adopted dynamic helical structure. The rest of the protein was unstructured aside from transient and mostly local side-chain interactions. The residual structure is important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between b(3-4) and a1 assisted in the refolding of a1. The contact-assisted helix formation was confirmed in fragment simulations of b(3-4) and a1 alone and complexed, and, as such, a1 and b(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulaion in reverse, noting that earlier time points from the simulations provide models of the major intermediate and transition states in quantitative agreement with experiments.

Tea and coffee will be served in R3151 Beckman Institute at 2:15pm.