Characterizing Discrete Binding Conformations of T4 L99A via Markov State Modeling

As a model system, the binding pocket of the L99A mutant of T4 lysozyme has been the subject of numerous computational free energy studies. However, some previous studies have failed to fully sample and account for the observed changes in the binding pocket of T4 L99A upon binding of a congeneric ligand series, limiting the accuracy of results. In this work, we establish definitions for the conformational states of the T4 L99A binding pocket based on the dynamics of the system rather than from experimental crystal structures. Using these definitions, we estimate the timescales for the transitions between states in this protein, which will have important implications for future binding studies in this system. We also discuss the need to develop enhanced sampling methods to generally account for significant changes in protein conformation due to relatively small ligand perturbations.