Carbon detected protein resonance assignment at 55 kHz magic-angle spinning

The acquisition of dipolar-based multidimensional spectra is the foundation for amino-acid specific assignment protocols for solid proteins using magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. The development of faster MAS has opened new possibilities for proton-detected assignment strategies, and at the same time reduced the sample amount as compared with their carbon-detected counterparts. Here we demonstrate the surprising utility of carbon-detected experiments at the fast MAS rate of 55 kHz. In particular, two pulse sequences are introduced, namely, (H)CAN(CO)CA, and (H)CON(CA)CO, which link sequential CA-CA and CO-CO resonances, respectively, with N providing the 3rd dimension. Sensitivity is maintained, despite the small sample volume, by incorporation of low power decoupling, enabling fast repetition rates, and magnetization transfer elements developed for the fast MAS regime. The sequences were tested for a microcrystalline protein, a membrane protein, and a fibril, and required less than one week of instrument time. Additionally, a quantitative comparison with the comparable proton-detected 3D spectrum, (H)N(CACO)NH, reveals competitive sensitivity for the (H)CAN(CO)CA spectrum for a fibril sample. Notably, the carbon-detected spectrum did not reveal the undesired diagonal peaks arising from transfer back to the initial amino-acid residues, which was observed in (H)N(CACO)NH spectrum. The sequence is particularly utile for assignment of proline, which does not contain an amide proton, and therefore is missing from many proton-detected spectra.