title: Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy.
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.
authors.affiliation: Division of Molecular Biotechnology, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.
authors.forename: Patrik
authors.initials: P
authors.lastname: Lundström
authors.forename: Pramodh
authors.initials: P
authors.lastname: Vallurupalli
authors.forename: D Flemming
authors.initials: DF
authors.lastname: Hansen
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D002247:Carbon Isotopes; D005591:Chemical Fractionation; D003903:Deuterium; D007553:Isotope Labeling / Q000379:methods*; D008840:Microfilament Proteins / Q000737:chemistry*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
chemical_list: C062612:ABP1 protein, S cerevisiae; D002247:Carbon Isotopes; D008840:Microfilament Proteins; D009587:Nitrogen Isotopes; D029701:Saccharomyces cerevisiae Proteins; D003903:Deuterium