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Studying excited states of proteins by NMR spectroscopy

Nature Structural Biology volume 8, pages 932–935 (2001)Cite this article

Abstract

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 (ΔH = 7.1 kcal mol−1) and entropic (TΔS = 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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Figure 1: Dispersion profiles of T4 lysozyme L99A.
Figure 2: Values of the forward (kGE) and backward (kEG) rate constants and the equilibrium constant (KG/E = kEG / kGE) obtained from global fits of kex and pE to 15N (open circle) and 13C (open square) relaxation dispersion measurements for all residues as a function of 1 / T, including best-fit lines (see Methods).
Figure 3: Slow dynamics cluster about the cavity.

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Acknowledgements

This work was support by grants from the Medical Research Council of Canada (L.E.K.) and the National Institutes of Health (F.W.D.). L.E.K. is a foreign investigator of the Howard Hughes Medical Research Institute.

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Author notes

  1. Frans A.A. Mulder, Anthony Mittermaier, Bin Hon and Frederick W. Dahlquist: These authors contributed equally to this work.

Authors and Affiliations

  1. Protein Engineering Network Centers of Excellence and Departments of Medical Genetics, Biochemistry and Chemistry, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    Frans A.A. Mulder, Anthony Mittermaier & Lewis E. Kay

  2. Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, 97403, Oregon, USA

    Bin Hon & Frederick W. Dahlquist

Authors
  1. Frans A.A. Mulder
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  2. Anthony Mittermaier
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  5. Lewis E. Kay
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Correspondence to Lewis E. Kay.

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Mulder, F., Mittermaier, A., Hon, B. et al. Studying excited states of proteins by NMR spectroscopy. Nat Struct Mol Biol 8, 932–935 (2001). https://doi.org/10.1038/nsb1101-932

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