Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes
- Departments of *Biological Chemistry and Molecular Pharmacology and
- §Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115; and
- ‡Department of Molecular Pharmacology, Stanford University Medical School, 269 West Campus Drive, Stanford, CA 94305
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Contributed by Joan Ruderman, June 11, 2006
Abstract
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.
Footnotes
- †To whom correspondence may be addressed. E-mail: philipp_selenko{at}hms.harvard.edu, gerhard_wagner{at}hms.harvard.edu, or joan_ruderman{at}hms.harvard.edu
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↵ ¶Present address: Cancer Biology and Genetics Program, Memorial Sloan–Kettering Cancer Center, New York, NY 10021.
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Author contributions: P.S. and Z.S. designed research; P.S., Z.S., and B.G. performed research; P.S. contributed new reagents/analytic tools; P.S. and Z.S. analyzed data; and P.S., J.R., and G.W. wrote the paper.
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Conflict of interest statement: No conflicts declared.
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See Commentary on page 11817.
- Abbreviation:
- HSQC,
- heteronuclear single quantum coherence.
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Freely available online through the PNAS open access option.
- © 2006 by The National Academy of Sciences of the USA
