Evaluation of TOCSY mixing for sensitivity-enhancement in solid-state NMR and application of 4D experiments for side-chain assignments of the full-length 30 kDa membrane protein GlpG

Chemical shift assignments of large membrane proteins by solid-state NMR experiments are challenging. Recent advancements in sensitivity-enhanced pulse sequences, have made it feasible to acquire ¹H-detected 4D spectra of these challenging protein samples within reasonable timeframes. However, obtaining unambiguous assignments remains difficult without access to side-chain chemical shifts. Drawing inspiration from sensitivity-enhanced TOCSY experiments in solution NMR, we have explored the potential of ¹³C- ¹³C TOCSY mixing as a viable option for triple sensitivity-enhanced 4D experiments aimed at side-chain assignments in solid-state NMR. Through simulations and experimental trials, we have identified optimal conditions to achieve uniform transfer efficiency for both transverse components and to minimize undesired cross-transfers. Our experiments, conducted on the 30 kDa membrane protein GlpG embedded in E. coli liposomes, have demonstrated enhanced sensitivity compared to the most effective dipolar and J-coupling-based ¹³C- ¹³C mixing sequences. Notably, a non-uniformly sampled 4D hCXCANH spectrum with exceptionally high sensitivity was obtained in just a few days using a 600 MHz spectrometer equipped with a 1.3 mm probe operating at a magic angle spinning rate of 55 kHz.