Seeing the Unseen: Sampling the Excited State of T4 Lysozyme L99A with Simulations on the Anton Supercomputer

Abstract

Proteins are the workhorses of the cell, converting small molecules into energy, harnessing energy for macromolecular synthesis, and interacting with one another to relay important cellular messages. All of these tasks are performed through the coordinated movement of a protein's atoms, which guide a protein from one conformational state to another. While experimental techniques provide insight into protein atomic motions, experiments cannot track all atomic-level motions across the nanosecond (ns) to millisecond (ms) time regimes. Even for proteins like T4 lysozyme, which have been extensively characterized (1-3), mysteries remain about the mechanism of converting between different protein states. The Leu99⇒Ala (L99A) mutant of T4 lysozyme is a model system for studying rarely seen, hidden excited states, even though the structure of its excited state has never been solved (4-9). Here we have sampled the excited state of L99A at atomic resolution with computational simulation on the Anton supercomputer (10). In this simulation, phenylalanine 114 (F114) and helices F and G undergo conformational changes that are indicative of a transition to the excited state. This MD-generated excited state also reproduces known excited state chemical shifts (R2 = 0.95) and agrees with multiple lines of experiment (4, 7, 11, 12). Our results of the L99A excited state cohere decades of data on this invisible conformation. We anticipate that this structure will provide experimentalists studying this protein with the tools to understand the atomic level details underlying L99A dynamics.