1. Transverse relaxation and the molecular size limit in liquid state NMR 161

2. TROSY: how does it work? 163

2.1 Transverse relaxation in coupled spin systems 163

2.2 The TROSY effect, relaxation due to remote protons and 2H isotope labeling 165

3. Direct heteronuclear chemical shift correlations 168

3.1 Single-Quantum [15N,1H]-TROSY 168

3.2 Zero-Quantum [15N,1H]-TROSY 171

3.3 Single-Quantum TROSY with aromatic 13C–1H moieties 176

4. Resonance assignment and NOE spectroscopy of large biomolecules 180

4.1 TROSY-based triple resonance experiments for 13C, 15N and 1HN backbone resonance assignment in uniformly 2H, 13C, 15N labeled proteins 180

4.2 TROSY-type NOE spectroscopy 186

5. Scalar coupling across hydrogen bonds observed by TROSY 187

6. The use of TROSY for measurements of residual dipolar coupling constants 190

7. Conclusions 191

8. Acknowledgements 191

9. References 191

The application of nuclear magnetic resonance (NMR) spectroscopy for structure determination of proteins and nucleic acids (Wüthrich, 1986) with molecular mass exceeding 30 kDa is largely constrained by two factors, fast transverse relaxation of spins of interest and complexity of NMR spectra, both of which increase with increasing molecular size (Wagner, 1993b; Clore & Gronenborn, 1997, 1998b; Kay & Gardner, 1997). The good news is that neither of these factors represent a fundamental limit for the application of NMR techniques to protein structure determination in solution (Clore & Gronenborn, 1998a; Wüthrich, 1998; Wider & Wüthrich, 1999). In fact, in the past few years the size limitations imposed by these factors have been pushed up to 50–70 kDa by the use of 13C, 15N and 2H isotope labeling combined with selective reprotonation of individual chemical groups in conjunction with the use of triple-resonance experiments (Bax, 1994; Gardner et al. 1997; Gardner & Kay, 1998) and heteronuclear-resolved NMR (Fesik & Zuiderweg, 1988; Marion et al. 1989a; Otting & Wüthrich, 1990). Among the largest biomolecules whose 3D structure was solved by NMR are the 44 kDa trimeric ectodomain of simian immunodeficiency virus (SIV) gp41 (Caffrey et al. 1998) and 40–60 kDa particles of the elongation initiation factor 4E solubilized in CHAPS micelles (Matsuo et al. 1997; McGuire et al. 1998).