title: Fractional enrichment of proteins using [2-(13)C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity.
abstract: A selective isotope labeling scheme based on the utilization of [2-(13)C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state (13)Cα chemical shifts using Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-(13)C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state (13)Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s(-1), despite the small fraction of (13)Cα-(13)Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using (13)Cα spin probes.
authors.affiliation: Division of Chemistry, Department of Physics, Chemistry and Biology, Linköping University, 58183, Linköping, Sweden.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Ahlner
authors.forename: Cecilia
authors.initials: C
authors.lastname: Andresen
authors.forename: Shahid N
authors.initials: SN
authors.lastname: Khan
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
authors.forename: Patrik
authors.initials: P
authors.lastname: Lundström
mesh_terms: D002247:Carbon Isotopes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity
chemical_list: D002247:Carbon Isotopes; D011506:Proteins; D005990:Glycerol