Kinetics-Based\nState Definitions for Discrete Binding\nConformations of T4 L99A in MD via Markov State Modeling

As\na model system, the binding pocket of the L99A mutant of T4\nlysozyme has been the subject of numerous computational free energy\nstudies. However, previous studies have failed to fully sample and\naccount for the observed changes in the binding pocket of T4 L99A\nupon binding of a congeneric ligand series, limiting the accuracy\nof results. In this work, we resolve the closed, intermediate, and\nopen states for T4 L99A previously reported in experiment in MD and\nestablish definitions for these states based on the dynamics of the\nsystem. From this analysis, we arrive at two primary conclusions.\nFirst, assignment of simulation trajectories into discrete states\nshould not be done simply based on RMSD to crystal structures as this\ncan result in misassignment of states. Second, the different metastable\nconformations studied here need to be carefully treated, as we estimate\nthe time scales for conformational interconversion to be on the order\nof 10<sup>2</sup> to 10<sup>3</sup> nsfar longer than time\nscales for typical binding calculations. We conclude with a discussion\non the need to develop enhanced sampling methods to generally account\nfor significant changes in protein conformation due to relatively\nsmall ligand perturbations.