Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes
- 8 August 2006
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 103 (32) , 11904-11909
- https://doi.org/10.1073/pnas.0604667103
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.Keywords
This publication has 28 references indexed in Scilit:
- Macromolecular Crowding in the Escherichia coli Periplasm Maintains α-Synuclein DisorderJournal of Molecular Biology, 2006
- Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR)Nature Methods, 2006
- Multidimensional NMR Spectroscopy for Protein Characterization and Assignment inside CellsJournal of the American Chemical Society, 2005
- Protein Dynamics in Living CellsBiochemistry, 2005
- Methyl Groups as Probes for Proteins and Complexes in In-Cell NMR ExperimentsJournal of the American Chemical Society, 2004
- The Roboocyte: Automated cDNA/mRNA Injection and Subsequent TEVC Recording on Xenopus Oocytes in 96-Well Microtiter PlatesReceptors and Channels, 2003
- Apparent diffusion of water, ions, and small molecules in the Xenopus oocyte is consistent with Brownian displacementMagnetic Resonance in Medicine, 2002
- In-Cell NMR SpectroscopyBiochemistry, 2001
- NMRPipe: A multidimensional spectral processing system based on UNIX pipesJournal of Biomolecular NMR, 1995
- Investigation of the backbone dynamics of the igg‐binding domain of streptococcal protein g by heteronuclear two‐dimensional 1H‐15N nuclear magnetic resonance spectroscopyProtein Science, 1994