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Quantitative NMR Analysis of the Protein G B1 Domain in Xenopus laevis Egg Extracts and Intact Oocytes
Philipp Selenko, Zach Serber, Bedrick Gadea, Joan Ruderman and Gerhard Wagner
Proceedings of the National Academy of Sciences of the United States of America
Vol. 103, No. 32 (Aug. 8, 2006), pp. 11904-11909
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/30051638
Page Count: 6
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We introduce a eukaryotic cellular system, the Xenopus laevis oocyte, for in-cell NMR analyses of biomolecules at high resolution and delineate the experimental reference conditions for successful implementations of in vivo NMR measurements in this cell type. This approach enables quantitative NMR experiments at defined intracellular concentrations of exogenous proteins, which is exemplified by the description of in-cell NMR properties of the protein G B1 domain (GB1). Additional experiments in Xenopus egg extracts and artificially crowded in vitro solutions suggest that for this biologically inert protein domain, intracellular viscosity and macromolecular crowding dictate its in vivo behavior. These contributions appear particularly pronounced for protein regions with high degrees of internal mobility in the pure state. We also evaluate the experimental limitations of this method and discuss potential applications toward the in situ structural characterization of eukaryotic cellular activities.
Proceedings of the National Academy of Sciences of the United States of America © 2006 National Academy of Sciences