title: Labeling strategy and signal broadening mechanism of Protein NMR spectroscopy in Xenopus laevis oocytes.
abstract: We used Xenopus laevis oocytes, a paradigm for a variety of biological studies, as a eukaryotic model system for in-cell protein NMR spectroscopy. The small globular protein GB1 was one of the first studied in Xenopus oocytes, but there have been few reports since then of high-resolution spectra in oocytes. The scarcity of data is at least partly due to the lack of good labeling strategies and the paucity of information on resonance broadening mechanisms. Here, we systematically evaluate isotope enrichment and labeling methods in oocytes injected with five different proteins with molecular masses of 6 to 54 kDa. (19) F labeling is more promising than (15) N, (13) C, and (2) H enrichment. We also used (19) F NMR spectroscopy to quantify the contribution of viscosity, weak interactions, and sample inhomogeneity to resonance broadening in cells. We found that the viscosity in oocytes is only about 1.2 times that of water, and that inhomogeneous broadening is a major factor in determining line width in these cells.
authors.affiliation: Key Laboratory of Magnetic Resonance in Biological Systems State, Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance Department, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071 (P.R. China).|Graduate University of Chinese Academy of Sciences, Beijing, 100049 (P.R. China).
authors.forename: Yansheng
authors.initials: Y
authors.lastname: Ye
authors.affiliation: Key Laboratory of Magnetic Resonance in Biological Systems State, Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance Department, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071 (P.R. China).
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Liu
authors.affiliation: Key Laboratory of Magnetic Resonance in Biological Systems State, Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance Department, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071 (P.R. China).|Graduate University of Chinese Academy of Sciences, Beijing, 100049 (P.R. China).
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Key Laboratory of Magnetic Resonance in Biological Systems State, Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance Department, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071 (P.R. China).
authors.forename: Guohua
authors.initials: G
authors.lastname: Xu
authors.affiliation: Key Laboratory of Magnetic Resonance in Biological Systems State, Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance Department, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071 (P.R. China).
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Key Laboratory of Magnetic Resonance in Biological Systems State, Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance Department, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071 (P.R. China).
authors.forename: Zeting
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Magnetic Resonance in Biological Systems State, Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance Department, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071 (P.R. China).|Graduate University of Chinese Academy of Sciences, Beijing, 100049 (P.R. China).
authors.forename: Chendie
authors.initials: C
authors.lastname: Yao
authors.affiliation: Key Laboratory of Magnetic Resonance in Biological Systems State, Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance Department, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071 (P.R. China).
authors.forename: Maili
authors.initials: M
authors.lastname: Liu
authors.affiliation: Key Laboratory of Magnetic Resonance in Biological Systems State, Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance Department, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071 (P.R. China). Conggangli@wipm.ac.cn.
authors.forename: Conggang
authors.initials: C
authors.lastname: Li
mesh_terms: D000818:Animals; D005260:Female; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009865:Oocytes / Q000378:metabolism*; D011487:Protein Conformation; D014982:Xenopus laevis / Q000235:genetics*
keywords: NMR spectroscopy; Xenopus laevis oocytes; in-cell NMR spectroscopy; proteins