While RBFE calculations in principle capture protein conformational changes if the underlying simulations are run long enough, these conformational changes have often not been thoroughly assessed in prior studies on this system. Thus, prior attempts at calculating binding free energies for a congeneric series of alkyl benzene ligands bound to T4 L99A have likely been inaccurate. In a study by Lim et al., the authors found that the accuracy of RBFE calculations could be significantly influenced by choice of protein starting conformation. The study demonstrated that improving RBFE calculations may require knowledge of the conformational changes of a system and their relative timescales, which is often difficult to define and sufficiently sample in the course of MD timescales typically used for these calculations. Interconversion between binding pocket states is slow, requiring significant simulation time to capture even a single event. However, insufficient sampling of different protein conformational states leads to significant errors in RBFE calculations. In this study, we seek to capture transitions among the experimentally observed conformational states of the T4 L99A F-helix and make order of magnitude estimates for the timescales of these transitions. We begin by defining the discrete conformational states of the T4 L99A F-helix based on the slow dynamics of the system. Using Markov state models (MSMs), we estimate the timescales for transitions between discrete states in MD. Furthermore, we show how defining metastable states of a system from the slowest motions of the system is preferable to defining states based on the RMSD to experimental crystal structures. Our approach demonstrates how changes in protein conformation for the T4 L99A system are not observed on MD timescales typical for binding studies, supporting the need to apply Markov state modeling to short, parallel MD simulations of systems to identify potential changes in protein conformation. We conclude by discussing the need to further develop and employ enhanced sampling methods to account for significant changes in protein conformation in response to small ligand perturbations.