Breaking the ¹³C-¹³C Polarization Transfer Barrier for High-Dimensional Protein Solid-State NMR with Ultra-Fast MAS

Recent advances in ¹H-detected solid-state NMR spectroscopy (SSNMR) using ultra-fast magic angle spinning (UFMAS) at frequencies above 60 kHz potentially facilitate high-dimensional SSNMR (HD-SSNMR) for protein analysis. A major limitation of HD-SSNMR is the exponential signal loss that occurs during successive polarization transfers. To overcome this bottleneck in HD-SSNMR for the most problematic ¹³C-¹³C transfers, we introduce a simple yet exceptionally efficient homonuclear cross-polarization (HCP) scheme called SeMi-selective Adiabatic Recoupling Transfer with HCP (SMART-HCP). We demonstrate that SMART-HCP with UFMAS at 90 kHz achieved nearly complete transfer with an efficiency of 76% from ¹³CO to ¹³C_α and 70% from ¹³C_α to ¹³CO for uniformly ¹³C,¹⁵N-labeled l-alanine. Semiselective HCP was achieved by optimizing radio frequency (RF)-offset frequency and amplitude modulations via a simple graphical method. For uniformly ¹³C,¹⁵N-labeled immunoglobulin-binding protein G (GB1) proteins, compared with the conventional dipolar recoupling enhancement through amplitude modulation (DREAM) scheme, SMART-HCP enhanced ¹³CO-¹³C_α transfers up to ∼3-fold (average 1.7-fold) for nonglycine residues, thereby accelerating various protein SSNMR experiments, including HD-SSNMR, by up to ∼9-fold. Our 3D ¹H-detected (H)-CACO-(N)H SSNMR spectra of the GB1 sample suggest that with the SMART-HCP method a usually time-consuming 3D protein SSNMR experiment can be achieved within 3.5 h for a trace amount of the protein sample (∼200 μg). Besides biological applications, this method is likely applicable to ¹³C SSNMR analysis of a wide range of samples, such as polymers, peptide-based pharmacological agents, and other solid organic materials.