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

We describe new correlation experiments suitable for determining long-range ¹H-¹H distances in ²H,¹⁵N-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 ¹H-¹H dipolar couplings that broaden ¹H magic angle spinning (MAS) spectra of solids. In the approach described here, we retain 100% of the ¹H 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 ¹H detection provides a gain in sensitivity of >5 compared to the ¹⁵N detected version of the experiment (at a MAS frequency of 13.5kHz). The pulse schemes make use of the favorable dispersion of the amide ¹⁵Ns resonances in the protein backbone. The experiments are demonstrated on a sample of the uniformly ²H,¹⁵N-labeled dipeptide N-Ac-Val-Leu-OH and are analogous to the solution-state suite of HSQC-NOESY experiments. In this compound the ¹H amide linewidths at 750 MHz vary from ∼0.67 ppm at ω_r/2π ∼ 5 kHz to ∼0.20 ppm at ω_r/2π ∼ 30 kHz, indicating that useful resolution is available in the ¹H spectrum via this approach. Since the experiments circumvent the problem of dipolar truncation in the ¹H-¹H 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.