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.

Lock and Key Binding of the HOX YPWM Peptide to the PBX Homeodomain

“…The effect was most pronounced for the aromatic and C1Ј-H1Ј cross-peaks, for which only the slowly relaxing component of each 13 C doublet was observed. The large chemical shift anisotropy of aromatic carbons (Ͼ100 ppm) results in favorable conditions for implementation of TROSY-type experiments in the assignment of nucleic acids and aromatic residues of proteins (31)(32)(33). The transverse relaxation rates of multiple quantum coherences are longer than those of the corresponding single quantum coherences.…”

Resolution-optimized NMR measurement of 1DCH, 1DCC and 2DCH residual dipolar couplings in nucleic acid bases

“…The pulse scheme of Figure 1 starts with magnetization transfer by the regular INEPT scheme from 1 H to 13 C. No phase alternation of the 90 • y ( 1 H) pulse is used, such that for the downfield TROSY doublet component the 13 C Boltzmann magnetization co-adds to the INEPT component (Pervushin et al, 1998). Depending on the frequencies at which the shaped pulses, a − e, are applied, the same pulse scheme can be used for magnetization that starts on H 5 or H 6 , and different nuclei are selected for decoupling to yield optimal resolution and sensitivity of the resonances corresponding to the interactions of interest.…”

“…Here, we focus on the types of couplings that provide the highest normalized accuracy. Optimal results are obtained with TROSY-based pulse schemes (Brutscher et al, 1998;Pervushin et al, 1998;Fiala et al, 2000). Not only do these methods yield RDCs which provide excellent cross-validation (rms errors less than 3% of their potential range), they also yield very narrow bands for the isotropic values of the various J couplings.…”

Solution NMR-derived global fold of a monomeric 82-kDa enzyme

“…Large gains in both the sensitivity and the resolution of NMR spectra of large molecules can be achieved by using the so-called transverse relaxation-optimized spectroscopy (TROSY) approach (3), in which only the slowly decaying components of nuclear magnetization contribute to the final signal. Since the original pioneering developments involving studies of amide (3) and aromatic moieties (4), more recent applications with methyl (5) and methylene groups (6) have appeared. A second major advance has involved the ''reintroduction'' of magnetic interactions that would normally average to zero in isotropic solution by means of the use of media leading to a weak alignment of the macromolecule of interest (7).…”