Simultaneous measurement of ~1H-~(15)N and Methyl ~1H_m-~(13)C_m residual dipolar couplings in large proteins

摘要

A two-dimensional TROSY-based SIM-~(13)C_m- ~1H_m/~1H-~(15)N NMR experiment for simultaneous measurements of methyl ~1 D _(CH) and backbone amide ~1 D _(NH) residual dipolar couplings (RDC) in {U-[ ~(15)N,~2H]; Ileδ1-[~(13)CH_3]; Leu,Val-[~(13)CH_3/~(12)CD_3]}-labeled samples of large proteins is described. Significant variation in the alignment tensor of the 82-kDa enzyme Malate synthase G is observed as a function of only slight changes in experimental conditions. The SIM-~(13)C _m-~1H_m/~1H-~(15)N data sets provide convenient means of establishing the alignment tensor characteristics via the measurement of ~1 D _(NH) RDCs in the same protein sample.