Sampling long time scale protein motions: OSRW simulation of active site loop conformational free energies in...

X-ray crystallographic snapshots have shown that conformational changes of a tetraglycine loop in the active site of formyl-CoA:oxalate CoA transferase (FRC) play an important role in the catalytic cycle of the enzyme. Orthogonal space random walk (OSRW) simulations have been applied to obtain quantitative computational estimates of the relative free energy of the "open" and "closed" conformations of this loop together with the energetic barrier for interconversion of these states in wild type FRC. These OSRW calculations not only show that the two conformations have similar free energies but also predict a barrier that is consistent with the observed turnover number of the enzyme. In an effort to quantitate the importance of specific residues in the tetraglycine loop, OSRW simulations have also been performed on the G258A, G259A, G260A, and G261A FRC variants both to examine the energetic effects of replacing each glycine residue and to correlate the computed energies with kinetic and structural observations. In enzymes with substantially reduced catalytic efficiency (k(cat)/K(M)), the OSRW simulations reveal the adoption of additional low energy loop conformations. In the case of the G260A FRC variant, the new conformation identified by simulation is similar to that observed in the X-ray crystal structure of the protein. These results provide further evidence for the power of the OSRW method in sampling conformational space and, hence, in providing quantitative free energy estimates for the conformations adopted by functionally important active site loops. In addition, these simulations model the motions of side chains that are correlated with changes in loop conformation thereby permitting access to long time-scale motions through the use of nanosecond simulations.