Transient Access to the Protein Interior: Simulation versus NMR

Many proteins rely on rare structural fluctuations for their function, whereby solvent and other small molecules gain transient access to internal cavities. In magnetic relaxation dispersion (MRD) experiments, water molecules buried in such cavities are used as intrinsic probes of the intermittent protein motions that govern their exchange with external solvent. While this has allowed a detailed characterization of exchange kinetics for several proteins, little is known about the exchange mechanism. Here, we use a millisecond all-atom MD trajectory produced by Shaw et al. (Science 2010, 330, 341) to characterize water exchange from the four internal hydration sites in the protein bovine pancreatic trypsin inhibitor. Using a recently developed stochastic point process approach, we compute the survival correlation function probed by MRD experiments as well as other quantities designed to validate the exchange-mediated orientational randomization (EMOR) model used to interpret the MRD data. The EMOR model is found to be quantitatively accurate, and the simulation reproduces the experimental mean survival times for all four sites with activation energy discrepancies in the range 0-3 k(B)T. On the other hand, the simulated hydration sites are somewhat too flexible, and the water flip barrier is underestimated by up to 6 k(B)T. The simulation reveals that water molecules gain access to the internal sites by a transient aqueduct mechanism, migrating as single-file water chains through transient (<5 ns) tunnels or pores. The present study illustrates the power of state-of-the-art molecular dynamics simulations in validating and extending experimental results.