Towards a complete description of the structural and dynamic properties ofthe denatured state of barnase and the role of residual structure in folding

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 th e resulting data are too sparse to be transformed into molecular models usi ng conventional techniques. Molecular dynamics simulations can complement N MR by providing detailed structural information for components of the denat ured ensemble. Here, we describe three independent 4 ns high-temperature mo lecular dynamics simulations of barnase in water. The simulated denatured s tate was conformationally heterogeneous with respect to the conformations p opulated both within a single simulation and between simulations. Nonethele ss, there were some persistent interactions that occurred to varying degree s in all simulations and primarily involved the formation of fluid hydropho bic clusters with participating residues changing over time. The region of the beta(3-4) hair pin contained a particularly high degree of such side-ch ain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal a lpha-helices (alpha 1 and alpha 2) adopted dynamic helical structure. In ad dition, there were persistent contacts that pinched off core 2 from the bod y of the protein. The rest of the protein was unstructured, aside from tran sient and mostly local sidechain interactions. Overall, the simulated denat ured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha 1 and alpha 2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha 1 and beta(3-4), in ag reement with previous NMR studies. Here, we also show that these regions co ntaining residual structure display impaired mobility by both molecular dyn amics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repai ring disrupted regions. For example, tertiary contacts between beta(3-4) an d alpha 1 assisted in the refolding of alpha 1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha 1 a lone and complexed, and, as such, alpha 1 and beta(3-4) appear to be foldin g initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that ea rlier time-points from the simulations provide models of the major intermed iate and transition states in quantitative agreement with data from both un folding and refolding experiments. Both beta(3-4) and alpha 1 are dynamic i n the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. T he beta-structure condenses about beta(3-4), while alpha 1 aids in stabiliz ing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transit ion state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensa tion of the structure then occurs to yield the native state. (C) 2000 Acade mic Press.