JE-TROSY: combined J- and TROSY-spectroscopy for the measurement of one-bond couplings in macromolecules.

With the application of RDCs in high-resolution NMR studies of macromolecules, there has been an interest in the development of accurate, sensitive methods for measuring 15N-1H and 13C-1H one-bond coupling constants. Most methods for determining these couplings are based on the measurement of the displacement between cross-peak components in J-coupled spectra. However, for large macromolecules and macromolecular complexes, these methods are often unreliable since differential relaxation can significantly broaden one of the multiplet components (i.e., the anti-TROSY component) and thereby make accurate determination of its position difficult. To overcome this problem, a J-evolved transverse relaxation optimized (JE-TROSY) method is presented for the determination of one-bond couplings that involves J-evolution of the sharpest cross-peak multiplet component selected in a TROSY experiment. Couplings are measured from the displacement of the TROSY component in the additional J-evolution dimension relative to a zero frequency origin. The JE-TROSY method is demonstrated on uniformly labeled 15N, 13C-labeled RNA and peptide samples, as well as with an RNA-protein complex, in which the protein is uniformly 15N, 13C-labeled. In all cases, resolved, sensitive spectra are obtained from which heteronuclear one-bond J-couplings could be accurately and easily measured.