19F NMR spectroscopy of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate reductase: equilibrium folding and ligand binding studies.

Escherichia coli dihydrofolate reductase contains five tryptophan residues distributed throughout its structure. In order to examine the regions of the protein surrounding these tryptophan residues, we have incorporated 6-fluorotryptophan into the protein. To assign the five resonances observed in the 19F NMR spectrum, five site-directed mutants of the enzyme were made, each with one tryptophan replaced by a phenylalanine. The 19F NMR spectra of the apoprotein, two binary complexes (with NADPH or methotrexate), and one ternary complex (with NADPH and methotrexate) were obtained. The chemical shifts of two of the tryptophan resonances (at positions 22 and 74) are particularly sensitive to ligand binding, while the remaining three (at positions 30, 47, and 133) change, but by less. Since several of the tryptophans are distant from the binding site, these results suggest that 19F NMR can detect ligand-induced changes that are propagated throughout the structure. In the apoprotein, the resonances of the tryptophans at positions 22 and 30 are broadened. In the binary complex with NADPH, the resonances of tryptophans 30 and 74 are broadened while that of tryptophan 22 almost disappears. The line broadening of the tryptophan 22 resonance may reflect motion in that part of the protein, since it is near a region that is disordered in the crystal structure of the apoprotein and its NADP+ complex. In contrast, in the ternary complex this region has a defined structure, and all resonances are of equal intensity and line width. The 19F NMR spectra of the apoprotein and the three ligand complexes were also examined as a function of urea concentration.(ABSTRACT TRUNCATED AT 250 WORDS)