文献类型：期刊文献

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

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

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Peking Univ, Coll Chem & Chem Engn, Beijing 100087, Peoples R China

年份：2026

卷号：6

期号：2

外文期刊名：MAGNETIC RESONANCE LETTERS

收录：WOS:【ESCI(收录号:WOS:001803601300001)】；

基金：All of the NMR experiments were conducted at the Beijing NMR Center, the NMR Facility at the National Center for Protein Sciences at Peking University, the National Center for Protein Science (Shanghai), and the 800 MHz spectrometer at East China University of Science and Technology. The work was 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) and the Fundamental Research Funds for the Central Universities. We are grateful for the assistance of Dr. Zhi-jun Liu of the National Center for Protein Science, Shanghai, on NMR data collection. We also thank the staff members of the Nuclear Magnetic Resonance System at the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, China for providing technical support and assistance in data collection and analysis.r (2024YFA0917100) , the National Natural Science Foundation of China (22274050) , the Shanghai Science and Technology Commission (contract number: 23J21900300, 24HC2810700) and the Fundamental Research Funds for the Central Universities. We are grateful for the assistance of Dr. Zhi-jun Liu of the National Center for Protein Science, Shanghai, on NMR data collection. We also thank the staff members of the Nuclear Magnetic Resonance System at the National Facility for Protein Science in Shanghai (NFPS) , Shanghai Advanced Research Institute, Chinese Academy of Sciences, China for providing technical support and assistance in data collection and analysis.

语种：英文

外文关键词：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 2D15N-13C 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. A13C 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 line-widths led to a 50% increase in the number of well-resolved cross-peaks on 15N-13C alpha spectra. 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. (c) 2025 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).