H-1 detected H-1, N-15 correlation spectroscopy in rotating solids

We describe new correlation experiments suitable for determining long-range H-1-H-1 distances in H-2,N-15-labeled peptides and proteins. The approach uses perdeuteration together with back substitution of exchangeable protons during sample preparation as a means of attenuating the strong H-1-H-1 dipolar couplings that broaden H-1 magic angle spinning (MAS) spectra of solids. In the approach described here, we retain 100% of the H-1 sensitivity by labeling and detecting all exchangeable sites. This is in contrast to homonuclear multiple pulse decoupling sequences that are applied during detection and that compromise sensitivity because of the requirement of sampling between pulses. As a result H-1 detection provides a gain in sensitivity of >5 compared to the N-15 detected version of the experiment (at a MAS frequency of 13.5 kHz). The pulse schemes make use of the favorable dispersion of the amide (15)Ns resonances in the protein backbone. The experiments are demonstrated on a sample of the uniformly H-2,N-15-labeled dipeptide N-Ac-Val-Leu-OH and are analogous to the solution-state suite of HSQC-NOESY experiments. In this compound the H-1 amide linewidths at 750 MHz vary from similar to0.67 ppm at omega(r)/2pi similar to 5 kHz to similar to0.20 ppm at omega(r)/2pi similar to 30 kHz, indicating that useful resolution is available in the H-1 spectrum via this approach. Since the experiments circumvent the problem of dipolar truncation in the H-1-H-1 spin system, they should make it possible to measure long-range distances in a uniformly labeled environment. Thus, we expect the experiments to be useful in constraining the global fold of a protein. (C) 2002 Elsevier Science (USA). All rights reserved.