Robust HeteronuclearCorrelations for Sub-milligramProtein 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 developed a robust method for selective polarization between insensitive nuclei (SPINE). This approach significantly enhances the heteronuclear ¹³C–¹⁵N correlation efficiency over CP, with gain factors of 1.75 for ¹³CA–¹⁵N and 1.9 and ¹³CO–¹⁵N transfers. SPINE’s efficacy was validated 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 multidimensional experiments to about one-third of that using a single ¹³C–¹⁵N CP and to about one-tenth with dual 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.