While spectroscopy readily samples the average conformation of well-populated protein states, protein excited states are often too sparsely populated to allow for direct observation. This presents an issue for defining the relationship between the dynamics and function of a protein, as these transient excited states can dictate how that protein performs a vast array of functions. Even for the well characterized excited state of the Leu99 to Ala (L99A) mutant of T4 lysozyme (1-3), the structure and role in ligand binding of the rare excited state remain unknown. Understanding the relationship between the structure and function of the excited state of L99A has vast implications for understanding the dynamics of residues near buried pockets of proteins in general. Here we sample the transition to and structure of the L99A high-energy state with all-atom simulation on the Anton supercomputer (4). Previously prescribed conformational changes in phenylalanine 114 (F114) and in the F/G helices occur in the molecular dynamics (MD) simulation to the excited state. The theoretical chemical shifts of this excited state conformation agree with previously solved chemical shifts for the excited state (R2 = 0.945). Additionally, these conformational changes are similar to those found in crystal structures of L99A bound to a congeneric series of ligands (22). Our results cohere decades of data on this invisible conformation, providing atomic-level characterization for the first time on the excited state of this historically studied protein.

Long-timescale simulation on the Anton supercomputer reveals the "invisible" excited state of the L99A mutant of T4 lysozyme

251st National Meeting (2016)

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