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Labeling Strategy and Signal Broadening Mechanism of Protein NMR Spectroscopy in Xenopus laevis Oocytes
Author(s) -
Ye Yansheng,
Liu Xiaoli,
Chen Yanhua,
Xu Guohua,
Wu Qiong,
Zhang Zeting,
Yao Chendie,
Liu Maili,
Li Conggang
Publication year - 2015
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201500279
Subject(s) - xenopus , nuclear magnetic resonance spectroscopy , spectroscopy , biophysics , chemistry , cytoplasm , nuclear magnetic resonance , biology , biochemistry , physics , gene , quantum mechanics
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.