An Exchange-Free Measure of 15N Transverse Relaxation:  An NMR Spectroscopy Application to the Study of a Folding Intermediate with Pervasive Chemical Exchange

D. Flemming Hansen,† Daiwen Yang,‡ Haniqiao Feng,§ Zheng Zhou,§ Silke Wiesner, Yawen Bai,§ and Lewis E. Kay*†
Contribution from the Departments of Medical Genetics, Biochemisty and Chemistry, The University of Toronto, Toronto, Ontario, Canada, M5S 1A8, Department of Biological Sciences, National University of Singapore, Singapore 117543, Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland 20892, and Structural Biology and Biochemistry, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8
J. Am. Chem. Soc., 2007, 129 (37), pp 11468–11479
DOI: 10.1021/ja072717t
Publication Date (Web): August 28, 2007
Copyright © 2007 American Chemical Society
†

 The University of Toronto.

,
‡

 National University of Singapore.

,
§

 NCI, NIH.

,

 The Hospital for Sick Children.

,
*

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

A series of experiments are presented that provide an exchange-free measure of dipole−dipole 15N transverse relaxation, Rdd, that can then be substituted for 15N R1ρ or R2 rates in the study of internal protein dynamics. The method is predicated on the measurement of a series of relaxation rates involving 1H−15N longitudinal order, anti-phase 1H and 15N single-quantum coherences, and 1H−15N multiple quantum coherences; the relaxation rates of all coherences are measured under conditions of spin-locking. Results from detailed simulations and experiments on a number of protein systems establish that Rdd values are independent of exchange and systematic errors from dipolar interactions with proximal protons are calculated to be less than 1−2%, on average, for applications to perdeuterated proteins. Simulations further indicate that the methodology is rather insensitive to the exact level of deuteration so long as proteins are reasonably highly deuterated (>50%). The utility of the methodology is demonstrated with applications involving protein L, ubiquitin, and a stabilized folding intermediate of apocytochrome b562 that shows large contributions to 15N R1ρ relaxation from chemical exchange.

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History

  • Published In Issue September 19, 2007
  • Received April 18, 2007

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