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 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 degrees 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 (Delta H = 7.1 kcal mol(-1)) and entropic (T Delta S = 5.1 kcal mol(-1), 25 degrees C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.
Mulder
F A
FA
Protein Engineering Network Centers of Excellence and Department of Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Mittermaier
A
A
Hon
B
B
Dahlquist
F W
FW
Kay
L E
LE
D019943:Amino Acid Substitution / Q000235:genetics; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D019277:Entropy; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009038:Motion; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013696:Temperature
D008024:Ligands; D009113:Muramidase