Exp 13 C NMR Studies of Bacterial Dihydrofolate Reductase Containing (Methyl- exp 13 C)Methionine and (Guanido- exp 13 C)Arginine

摘要

(Methyl- exp 13 C)methionine and (guanido- exp 13 C)arginine have been incorporated with high efficiency by Streptococcus faecium var. Durans strain A into dihydrofolate reductase isoenzyme 2 and exp 13 C NMR spectra have been obtained for the labeled enzymes and their complexes with substrates, co-factors, and inhibitors. The exp 13 C NMR spectra exhibit a high degree of discrimination--up to six guanido- exp 13 C resonances spanning a 1.2 ppM range have been resolved for the eight arginine residues and, under certain conditions, seven methyl- exp 13 C resonances spanning a 3 ppM chemical shift range have been resolved for the seven methionine residues of the enzyme. The exp 13 C chemical shifts and spin lattice relaxation times of these distinct, relatively narrow resonances can be interpreted in terms of the conformational states of the enzyme and the interactions of the exp 13 C-labeled residues with bound ligands. In a larger context, the results reported here provide experimental data which bear on a central question in the use of exp 13 C NMR spectroscopy to probe the structure of labeled macromolecules, vis.: Where should the exp 13 C label be incorporated to ensure a relatively narrow resonance whose chemical shift is nonetheless sensitive to perturbations of the macromolecule. Contrary to one accepted view, this study demonstrates that a significant degree of internal motion for a class of amino acid residues is not necessarily incompatible with a large chemical shift dispersion within the class. (ERA citation 03:053552)