Spectral editing at ultra-fast magic-angle-spinning in solid-state NMR: facilitating protein sequential signal assignment by HIGHLIGHT approach
Abstract
This study demonstrates a novel spectral editing technique for protein solid-state NMR (SSNMR) to simplify the spectrum drastically and to reduce the ambiguity for protein main-chain signal assignments in fast magic-angle-spinning (MAS) conditions at a wide frequency range of 40â??80Â*kHz. The approach termed HIGHLIGHT (Wang et al., in Chem Comm 51:15055â??15058, 2015) combines the reverse 13C, 15N-isotope labeling strategy and selective signal quenching using the frequency-selective REDOR pulse sequence under fast MAS. The scheme allows one to selectively observe the signals of â??highlightedâ?? labeled amino-acid residues that precede or follow unlabeled residues through selectively quenching 13CO or 15N signals for a pair of consecutively labeled residues by recoupling 13COâ??15N dipolar couplings. Our numerical simulation results showed that the scheme yielded only ~15Â*% loss of signals for the highlighted residues while quenching as much as ~90Â*% of signals for non-highlighted residues. For lysine-reverse-labeled micro-crystalline GB1 protein, the 2D 15N/13Cα correlation and 2D 13Cα/13CO correlation SSNMR spectra by the HIGHLIGHT approach yielded signals only for six residues following and preceding the unlabeled lysine residues, respectively. The experimental dephasing curves agreed reasonably well with the corresponding simulation results for highlighted and quenched residues at spinning speeds of 40 and 60Â*kHz. The compatibility of the HIGHLIGHT approach with fast MAS allows for sensitivity enhancement by paramagnetic assisted data collection (PACC) and 1H detection. We also discuss how the HIGHLIGHT approach facilitates signal assignments using 13C-detected 3D SSNMR by demonstrating full sequential assignments of lysine-reverse-labeled micro-crystalline GB1 protein (~300Â*nmol), for which data collection required only 11Â*h. The HIGHLIGHT approach offers valuable means of signal assignments especially for larger proteins through reducing the number of resonance and clarifying multiple starting points in sequential assignment with enhanced sensitivity.
Source: Journal of Biomolecular NMR