Measuring 13Cβ chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy

A labeling scheme is introduced that facilitates the measurement of accurate C-13(beta) chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of C-13 enrichment (30-40%) at C-beta side-chain carbon positions for 15 of the amino acids with little C-13 label at positions one bond removed (a parts per thousand 5%). A pair of samples are produced using [1-C-13]-glucose/(NaHCO3)-C-12 or [2-C-13]-glucose as carbon sources with isolated and enriched (> 30%) C-13(beta) positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of C-13(beta) chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein-ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.