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

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.