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

A simple labeling approach is presented based on protein expression in [1-C-13]- or [2-C-13]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C-alpha sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta 1) are produced with isolated C-13 spins (i.e., no C-13-C-13 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-alpha sites are labeled without concomitant labeling at C-beta positions for 17 of the common 20 amino acids and there are no cases for which C-13(alpha)-(CO)-C-13 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 N-15 studies. The utility of the labeling is established by recording C-13 R-1 rho and CPMG-based experiments on a number of different protein systems.