STRUCTURE PREDICTION OF SUBTILISIN BPN' MUTANTS USING MOLECULAR-DYNAMICS METHODS

In this paper we describe the achievements and pitfalls encountered in doing structure predictions of protein mutants using molecular dynamics simulation techniques in which properties of atoms are slowly changed as a function of time. Basically the method consists of a thermodynamic integration (slow growth) calculation used for free energy determination, but aimed at structure prediction; this allows for a fast determination of the mutant structure. We compared the calculated structure of the mutants Met222Ala, Met222Phe and Met222Gln of subtilisin BPN' with the respective X-ray structures and found good agreement between predicted and X-ray structure. The conformation of the residue subject to the mutation is relatively easy to predict and is mainly determined by packing criteria. When the side chain has polar groups its exact orientation may pose problems; long-range Coulomb interactions may generate a polarization feedback involving system relaxation times beyond the simulation time. Changes induced in the environment are harder to predict using this method. In particular, rearrangement of the hydration structure was difficult to predict correctly, probably because of the long relaxation times. In all conversions made the changes observed in the environment were found to be history-dependent and in particular the hydrogen bonding patterns provided evidence for metastable substates. In all cases the structure predicted was compared with available kinetic data and the reduced activity could be explained in terms of changes in the configuration of the active site.