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
2007
Lundström, Patrik | Teilum, Kaare | Carstensen, Tommy | Bezsonova, Irina | Wiesner, Silke | Hansen, D Flemming | Religa, Tomasz L | Akke, Mikael | Kay, Lewis E
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.
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