| 19448976 |
Lundstrom P, Lin H, Kay LE: Measuring 13Cbeta chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy. J Biomol NMR. 2009 Jul;44(3):139-55. Epub 2009 May 16.
A labeling scheme is introduced that facilitates the measurement of accurate (13) C (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 (13) C enrichment (30-40%) at C (beta) side-chain carbon positions for 15 of the amino acids with little (13) C label at positions one bond removed (approximately 5%). A pair of samples are produced using [1-(13) C]-glucose/NaH (12) CO (3) or [2-(13) C]-glucose as carbon sources with isolated and enriched (> 30%) (13) C (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 (13) C (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. |
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