Molecular dynamics simulation of the unfolding of barnase: characterization of the major intermediate.

The folding/unfolding pathway of barnase has been studied extensively using the protein engineering method, which has provided indirect structural information for the transition state and the major folding intermediate. To further characterize the structural properties of the intermediate, we have simulated the thermal denaturation of barnase beginning from the average NMR structure. Our results indicate that there are at least two intermediates on the unfolding pathway. The three hydrophobic cores are partially formed in the major intermediate (I1), with core1 and core3 being slightly stronger than core2. Helix alpha 1 is substantially formed, with the center being stronger than the termini. The first turn of alpha 2 is lost and alpha 3 is unfolded. The center of the beta-sheet is substantially formed, but the edges are disrupted. These structural characteristics are in good qualitative agreement with the experimental data. For semi-quantitative comparison with experimental data, the extent of native structure of individual residues is characterized by a structure index, S, that reflects both secondary and tertiary structure. There is good agreement between S and the experimentally measured phi values, which are based on energetics, except for three residues. These residues are polar and non-conservative mutations were made to obtain phi values, which can complicate structural interpretations. These residues make strong side-chain interactions in I1, but the backbone structure is disrupted, leading to low S values. Thus, this discordance highlights possible limitations in both the phi value and S value analyses: strong polar interactions in the intermediate may give rise to high phi values that are not reflective of structure per se; however, due to sampling limitations, any one simulation is not expected to capture all of the features of the true conformational ensemble. In any case, these simulations provide an experimentally testable, atomic-level structural model for the major folding intermediate of barnase, as well as the detailed pathway from the native to the intermediate state.