Protocol abstract
Nature Protocols 4, - 1641 - 1648 (2009)
Published online: 22 October 2009 | doi:10.1038/nprot.2009.118
Subject Categories: Biochemistry and protein analysis | Spectroscopy and structural analysis
Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy
Patrik Lundström1, Pramodh Vallurupalli2, D Flemming Hansen2 & Lewis E Kay2
Abstract
The utility of nuclear magnetic resonance (NMR) spectroscopy as a tool for the study of biomolecular structure and dynamics has benefited from the development of facile labeling methods that incorporate NMR active probes at key positions in the molecule. Here we describe a protocol for the labeling of proteins that facilitates their study using a technique that is sensitive to millisecond conformational exchange processes. The samples necessary for an analysis of exchange dynamics are discussed, using the Abp1p SH3 domain from Saccharomyces cerevisiae as an example. For this system, the time frame for production of each sample, including in vitro refolding, is about 80 h. The samples so produced facilitate the measurement of accurate chemical shifts of low populated, invisible conformers that are part of the exchange pathway. The accuracy of the methodology has been established experimentally and the chemical shifts that are obtained provide important restraints in structure calculations of the excited state.
- Division of Molecular Biotechnology, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
- Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, Ontario, Canada.
Correspondence to: Lewis E Kay2 e-mail: kay@pound.med.utoronto.ca.
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