Studying excited states of proteins by NMR spectroscopy

Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray stud ies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is steric ally inaccessible to ligand, yet the protein is able to bind substituted be nzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highl y populated (97%, 25 degreesC) ground state conformation and an excited sta te 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 all ows the separation of the free energy change into enthalpic (DeltaH = 7.1 k cal mol(-1)) and entropic (T DeltaS = 5.1 kcal mol(-1), 25 degreesC) compon ents. The residues involved cluster about the cavity providing evidence tha t the excited state facilitates ligand entry.