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Article
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl–methyl nuclear overhauser enhancement spectroscopy
Journal of Biomolecular NMR (2011)
  • Vincenzo Venditti, National Institutes of Health
  • Nicolas L. Fawzi, National Institutes of Health
  • G. Marius Clore, National Institutes of Health
Abstract

Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ~1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl–methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.

Keywords
  • paramagnetic relaxation enhancement,
  • methyl-TROSY,
  • 1h-13hmqc,
  • paramagnetic spin-labeling,
  • methyl group labeling
Publication Date
2011
Publisher Statement
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.
Citation Information
Vincenzo Venditti, Nicolas L. Fawzi and G. Marius Clore. "Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl–methyl nuclear overhauser enhancement spectroscopy" Journal of Biomolecular NMR Vol. 51 Iss. 3 (2011)
Available at: http://0-works.bepress.com.library.simmons.edu/vincenzo_venditti/9/