Transverse Relaxation-Optimized Spectroscopy (TROSY) for NMR Studies of Aromatic Spin Systems in 13C-Labeled Proteins

Abstract

Transverse relaxation-optimized spectroscopy (TROSY) yields greatly improved sensitivity for multidimensional NMR experiments with aromatic spin systems in proteins. TROSY makes use of the fact that due to the large anisotropy of the 13 C chemical shift tensor, the transverse relaxation of one component of the 13 C doublet in aromatic 13 C− 1 H moieties is reduced by interference of dipole−dipole (DD) coupling and chemical shift anisotropy (CSA) relaxation. The full advantage of TROSY for studies of aromatic spin systems is obtained at presently available resonance frequencies from 500 to 800 MHz. Since the 13 C chemical shifts are recorded using a constant-time evolution period, the TROSY improvement in signal-to-noise relative to corresponding conventional NMR experiments increases with increasing molecular size and can be further significantly enhanced by combined use of the 1 H and 13 C steady-state magnetizations.With selective observation of the slowly relaxing component of the 13 C doublets in experiments recorded without 1 H decoupling during the 13 C chemical shift evolution period, a 4−10-fold sensitivity gain for individual aromatic 13 C− 1 H correlation peaks was achieved for the uniformly 13 C-labeled 18 kDa protein cyclophilin A. A new 3D ct-TROSY-HCCH-COSY experiment is presented, which correlates the resonances of 13 C nuclei with those of covalently bound 13 C− 1 H groups and can be applied for complete identification of aromatic spin systems. In this scheme the chemical shift evolution of neighboring aromatic 13 C spins are recorded in two indirectly detected spectral dimensions, so that the additional third dimension is obtained without increase of the number of delays.