Studying excited states of proteins by NMR spectroscopy
Frans A.A. Mulder1, 2, Anthony Mittermaier1, 2, Bin Hon3, Frederick W. Dahlquist3
& Lewis E. Kay1
1
Protein Engineering Network Centers of Excellence and Departments of Medical Genetics, Biochemistry and Chemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
2
These authors contributed equally to this work.
3
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403, USA.
Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. 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 techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 °C) ground state conformation and an excited state that is 2.0 kcal mol-1 higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (H = 7.1 kcal mol-1) and entropic (TS = 5.1 kcal mol-1, 25 °C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.
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H = 7.1 kcal mol-1) and entropic (T
