A random fractional 13C labeling strategy for P. pastoris expressed eukaryotic membrane proteins for solid-state NMR studies.

Solid-state NMR (SSNMR) has emerged as an important technique for characterizing membrane protein structures. Using sparse 13C labeling of membrane proteins can significantly enhance spectral resolution. In this study, we present an investigation of a random fractional 13C labeling approach for large eukaryotic membrane proteins produced in P. pastoris for SSNMR research. This method takes advantage of the fact that P. pastoris can use the C1 compound methanol as the sole carbon source. Thus, incorporating a mixture of natural abundance (NA) methanol and 13C-methanol in the expression medium enables random fractional 13C labeling of the expressed proteins. The labeling strategy was assessed using a eukaryotic rhodopsin from Leptosphaeria maculans (LR). A comparison of 1D-13C and 2D15N-13C spectra of LR expressed in media with different ratios of 13C-methanol to NA-methanol revealed a notable decrease in the 13C linewidth of LR as the proportion of 13C-methanol decreased. A13C enrichment level of 25% was determined for balance between spectral resolution and sensitivity, resulting in an average 13C line-width for LR that is half of uniform 13C labeling. This reduction in linewidths led to a 50% increase in the number of well-resolved cross-peaks on 15N-13Cα spectra. The random fractional 13C labeling method only uses economic 13C-labeled methanol, providing a cost-effective approach for sparse 13C labeling to improve the SSNMR spectral resolution of membrane proteins. It will be beneficial to site-specific characterization on membrane protein structure, dynamics and interactions.