Annu. Rev. Biophys. Biomol. Struct. 1998. 27:357–406 Copyright c  1998 by Annual Reviews. All rights reserved THE USE OF 2 H, 13 C, 15 N MULTIDIMENSIONAL NMR TO STUDY THE STRUCTURE AND DYNAMICS OF PROTEINS Kevin H. Gardner and Lewis E. Kay Protein Engineering Network Centres of Excellence and Departments of Medical Genetics and Microbiology, Biochemistry, and Chemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8; e-mail: gardner@bloch.med.utoronto.ca; kay@bloch.med.utoronto.ca KEY WORDS: protein deuteration, triple resonance NMR, amino acid-specific isotopic labeling, structure determination, sidechain dynamics ABSTRACT During the past thirty years, deuterium labeling has been used to improve the resolution and sensitivity of protein NMR spectra used in a wide variety of appli- cations. Most recently, the combination of triple resonance experiments and 2 H, 13 C, 15 N labeled samples has been critical to the solution structure determination of several proteins with molecular weights on the order of 30 kDa. Here we review the developments in isotopic labeling strategies, NMR pulse sequences, and structure-determination protocols that have facilitated this advance and hold promise for future NMR-based structural studies of even larger systems. As well, we detail recent progress in the use of solution 2 H NMR methods to probe the dynamics of protein sidechains. CONTENTS INTRODUCTION ........................................................... 358 Background: Deuteration prior to 1993 ....................................... 358 Scope of This Review: Deuteration since 1993 ................................. 360 DEUTERIUM LABELING METHODS .......................................... 362 Uniform Deuteration ...................................................... 363 Site-Specific Protonation in a Highly Deuterated Environment ..................... 364 Practical Aspects of Producing Deuterated Proteins in Escherichia coli .............. 370 357 1056-8700/98/0610-0357$08.00 Annu. Rev. Biophys. Biomol. Struct. 1998.27:357-406. Downloaded from arjournals.annualreviews.org by KAROLINSKA INSTITUTET UNIVERSITY LIBRARY on 03/20/06. For personal use only.