Fractional 13C enrichment of isolated carbons using [1-13C]- or [2-13C]-glucose facilitates the accurate measurement of dynamics at backbone Cα and side-chain methyl positions in proteins
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A simple labeling approach is presented based on protein expression in [1-13C]- or [2-13C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone Cα sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated 13C spins (i.e., no 13C–13C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling at Cβ positions for 17 of the common 20 amino acids and there are no cases for which 13Cα−13CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone 15N studies. The utility of the labeling is established by recording 13C R 1ρ and CPMG-based experiments on a number of different protein systems.
KeywordsSelective 13C labeling Protein expression [1-13C]-glucose [2-13C]-glucose 13C relaxation measurements CPMG relaxation dispersion T1ρ
P.L., K.T. and D.F.H. are supported by fellowships from the Hellmuth Hertz foundation, EMBO and The Danish Agency for Science, Technology and Innovation (J.no. 272-05-0232), respectively. Useful discussions with Prof. Mike Rosen (University of Texas Southwestern Medical School) are acknowledged. This research was supported by grants from the Canadian Institutes of Health Research (L.E.K.) and the Swedish Research Council (M.A.). L.E.K. holds a Canada Research Chair in Biochemistry.
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