Multiple-Site Exchange in Proteins Studied with a Suite of Six NMR Relaxation Dispersion Experiments:  An Application to the Folding of a Fyn SH3 Domain Mutant

Dmitry M. Korzhnev,† Philipp Neudecker,† Anthony Mittermaier,† Vladislav Yu. Orekhov,‡ and Lewis E. Kay*†
Contribution from the Departments of Medical Genetics, Biochemistry, and Chemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada, and Swedish NMR Center at Gteborg University, Box 465, 405 30 Gteborg, Sweden
J. Am. Chem. Soc., 2005, 127 (44), pp 15602–15611
DOI: 10.1021/ja054550e
Publication Date (Web): October 12, 2005
Copyright © 2005 American Chemical Society
†

 University of Toronto.

,
‡

 Swedish NMR Center at Göteborg University.

,
*

In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about the paper should be addressed.

, kay@pound.med.utoronto.ca

Abstract

Abstract Image

The three-site exchange folding reaction of an 15N-labeled, highly deuterated Gly48Met mutant of the Fyn SH3 domain has been characterized at 25 °C using a suite of six CPMG-type relaxation dispersion experiments that measure exchange contributions to backbone 1H and 15N transverse relaxation rates in proteins. It is shown that this suite of experiments allows the extraction of all the parameters of this multisite exchange process in a robust manner, including chemical shift differences between exchanging states, from a data set recorded at only a single temperature. The populations of the exchanging folded, intermediate, and unfolded states that are fit are 94, 0.7, and 5%, respectively. Despite the small fraction of the intermediate, structural information is obtained for this state that is consistent with the picture of SH3 domain folding that has emerged from other studies. Taken together, the six dispersion experiments facilitate the complete reconstruction of 1H−15N correlation spectra for the unfolded and intermediate states that are “invisible” in even the most sensitive of NMR experiments.

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  • Published In Issue November 09, 2005
  • Received July 8, 2005

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