Low‐Power Solid‐State NMR Experiments for Resonance Assignment under Fast Magic‐Angle Spinning

Abstract

Solid-state NMR has evolved in the past decade into a powerful technique for the characterization of biomolecular structure and dynamics. Micro-crystalline globular proteins, amyloid fibrils, and membrane proteins can now be routinely studied using solid-state NMR techniques. This was made possible in part due to the development of 2D and 3D homonuclear and heteronuclear experiments that correlate C and N spins for resonance assignment as well as for obtaining longrange distance restraints in structure elucidation. Remarkable developments in magic-angle spinning (MAS) probe technology also contributed to this success. Now, a new generation of commercially available 1.3 mm probes can reach above 60 kHz of MAS. This allows for more efficient averaging of strong dipolar couplings, hence providing better resolution in highly crowded protein spectra. On the other hand, fast spinning reduces the effectiveness of many of the routinely used NMR experiments for obtaining resonance assignments. For example, at low MAS ( 15 kHz), C C correlations are often measured by proton-driven spin diffusion (PDSD). Under very fast MAS, efficient averaging of dipolar couplings renders PDSD experiments ineffective. Instead, selective dipolar recoupling of spins becomes necessary to allow for efficient polarization transfer. Herein, we introduce a complete set of low-power solidstate NMR experiments sufficient for protein resonance assignment under fast MAS (>60 kHz), including sequential N C correlation experiments. The low rf (radio frequency)-field requirements of our experiments prevent considerable heating of the sample, thus avoiding protein degradation and making this approach well-suited for the investigation of temperaturesensitive biomolecules. As an application, NCA, N(CO)CA, and C C correlation spectra were recorded at 60 kHz MAS on less than 1 mg of [C, N] isotope-labeled sample. We also demonstrate that our approach can be readily performed on protein samples in which the H T1 relaxation times are shortened by means of paramagnetic doping. Here, the reduced recycle delay enhances the sensitivity but requires the use of NMR sequences with low-power deposition, as described herein. Figure 1 presents the different pulse schemes that were used to obtain N C and C C correlations. At low MAS, the initial cross-polarization (CP) transfer from protons to low-g nuclei generally requires high power irradiation on both channels. In contrast, under fast MAS, efficient CP transfer is also possible at low rf fields. Our pulse schemes use second-order cross-polarization (SOCP) for the initial magnetization transfer. SOCP at the n=0 Hartman–Hahn condition relies on second-order crossterms between homonuclear and heteronuclear couplings. SOCP works efficiently at low rf fields if sufficient care is taken to avoid detrimental dipolar and/or CSA recoupling conditions at the used rf-field amplitudes. We employed rf fields of 9 kHz—well below all resonance conditions. SOCP is intrinsically band-selective as only weak rf fields are applied. The rf fields employed here are sufficient to excite all N protein backbone