Robust heteronuclear correlations for proteins in ultrafast magic-angle spinning solid-state NMR

Proton-detected solid-state nuclear magnetic resonance (ssNMR) under ultrafast magic-angle spinning (MAS) has become a powerful tool for elucidating the structures of proteins with sub-milligram quantities, where establishing 13C-15N correlations is essential. However, traditional 13C-15N cross-polarization (CP), effective at lower MAS frequencies, suffers diminished efficiency under ultrafast MAS conditions. To overcome this limitation, we have developed a robust method for selective polarization between insensitive nuclei (SPINE). This approach significantly enhances the efficiency of heteronuclear 13C-15N correlation relative to CP, achieving gain factors of 1.75 for 13CA-15N and 1.9 and 13CO-15N transfers. The efficacy of SPINE has been validated through experiments on four diverse proteins: the microcrystalline β1 immunoglobulin binding domain of protein G (GB1), the large-conductance mechanosensitive ion channel from Methanosarcina acetivorans (MaMscL), fibrillar septum-forming protein (SepF), and the vertex protein of the β-carboxysome shell (CcmL). This enhancement can reduce the duration of current multi-dimensional experiments to approximately one-third of that using 13C-15N CP and to about one-tenth when using two 13C-15N transfers. Our findings underscore the practical utility and versatility of SPINE in ssNMR spectroscopy, making it a valuable approach for advancing structural biology studies of sub-milligram protein.