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

Under ultrafast magic-angle spinning (MAS), proton-detected solid-state nuclear magnetic resonance (ssNMR) has emerged as a powerful technique for elucidating structures from sub-milligram protein, where establishing 13C-15N correlations is essential. However, traditional 13C-15N cross-polarization (CP), which performs well at lower MAS frequencies, suffers diminished efficiency under ultrafast MAS conditions. To address this challenge, we have developed a robust method for selective polarization between insensitive nuclei (SPINE). This approach significantly enhances the efficiency of heteronuclear 13C-15N correlation compared to CP, achieving gain factors of 1.75 for 13CA-15N and 1.9 and 13CO-15N transfers. These enhancements can reduce the duration of current multi-dimensional experiments to approximately one-third of that required by 13C-15N CP and to approximately one-tenth when involving two 13C-15N transfers. The effectiveness of SPINE has been validated through experiments on four distinct 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). These findings highlight the practical utility and versatility of SPINE in ssNMR spectroscopy, making it a valuable approach for structural biology applications.