文献类型：期刊文献

中文题名：A random fractional ^(13)C labeling strategy for P.pastoris expressed eukaryotic membrane proteins for solid-state NMR studies

作者：Meihui Sang[1];Zejun Fan[1];Qiongqiong Ren[1];Jing Liu[2];Shaokai Zhao[2];Shenlin Wang[1]

机构：[1]State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai,200237,China;[2]College of Chemistry and Chemical Engineering,Peking University,Beijing,100087,China

年份：2026

卷号：6

期号：2

起止页码：97

中文期刊名：Magnetic Resonance Letters

外文期刊名：磁共振快报(英文)

基金：supported by the National Key R&D Program of China(2024YFA0917100);the National Natural Science Foundation of China(22274050);the Shanghai Science and Technology Commission(contract number:23J21900300,24HC2810700);the Fundamental Research Funds for the Central Universities.

语种：英文

中文关键词：Solid-state NMR;Sparse^(13)C labeling;Eukaryotic membrane proteins;P.pastoris expression system

摘要：Solid-state NMR(SSNMR)has emerged as an important technique for characterizing membrane protein structures.Using sparse ^(13)C labeling of membrane proteins can significantly enhance spectral resolution.In this study,we present an investigation of a random fractional ^(13)C 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 ^(13)C-methanol in the expression medium enables random fractional ^(13)C labeling of the expressed proteins.The labeling strategy was assessed using a eukaryotic rhodopsin from Leptosphaeria maculans(LR).A comparison of 1D-^(13)C and 2D^(15)N–^(13)C spectra of LR expressed in media with different ratios of ^(13)C-methanol to NA-methanol revealed a notable decrease in the ^(13)C linewidth of LR as the proportion of ^(13)C-methanol decreased.A^(13)C enrichment level of 25%was determined for balance between spectral resolution and sensitivity,resulting in an average ^(13)C line-width for LR that is half of uniform ^(13)C labeling.This reduction in linewidths led to a 50%increase in the number of well-resolved cross-peaks on ^(15)N-^(13)Cαspectra.The random fractional ^(13)C labeling method only uses economic ^(13)C-labeled methanol,providing a cost-effective approach for sparse ^(13)C 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.