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Studying excited states of proteins by NMR spectroscopy

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  • F A Mulder
  • A Mittermaier, University of Toronto, Canada
  • B Hon, University of Oregon, United States
  • F W Dahlquist, University of Oregon, United States
  • L E Kay, University of Toronto, Canada

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.

Original languageEnglish
JournalNature Structural and Molecular Biology
Pages (from-to)932-935
Number of pages4
Publication statusPublished - Nov 2001
Externally publishedYes

    Research areas

  • Amino Acid Substitution, Bacteriophage T4, Binding Sites, Entropy, Kinetics, Ligands, Magnetic Resonance Spectroscopy, Models, Molecular, Motion, Muramidase, Protein Structure, Secondary, Temperature

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