Studying Protein-Excited States by Nmr

. Much of structural biology has focused on obtaining static three-dimensional representations of molecules and molecular complexes. However, a complete description of a protein requires not only a three-dimensional picture of the average structure but knowledge of how that structure changes with time. Proteins are inherently dynamic and the dynamics are critical for function. For example, it is often the case that static three-dimensional structures alone do not completely explain results from functional biological assays, nor do they necessarily illuminate the path for protein engineering or rational drug design. This is, of course, not surprising. A three-dimensional static structure provides a description of the ground state of a molecule. Macromolecular function is, in many cases, highly dependent on excursions to excited molecular states and hence intimately coupled to flexibility. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR methods to characterize the transition between a ligand-inaccessible closed form of the protein and an excited state that binds ligands that is 2.0 kcal mol higher in free energy.