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 13C chemical shift tensor, the transverse relaxation of one component of the 13C doublet in aromatic 13C−1H 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 13C 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 1H and 13C steady-state magnetizations.With selective observation of the slowly relaxing component of the 13C doublets in experiments recorded without 1H decoupling during the 13C chemical shift evolution period, a 4−10-fold sensitivity gain for individual aromatic 13C−1H correlation peaks was achieved for the uniformly 13C-labeled 18 kDa protein cyclophilin A. A new 3D ct-TROSY-HCCH-COSY experiment is presented, which correlates the resonances of 13C nuclei with those of covalently bound 13C−1H groups and can be applied for complete identification of aromatic spin systems. In this scheme the chemical shift evolution of neighboring aromatic 13C spins are recorded in two indirectly detected spectral dimensions, so that the additional third dimension is obtained without increase of the number of delays.