F-19 NMR-SPECTROSCOPY OF [6-F-19]TRYPTOPHAN-LABELED ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE - EQUILIBRIUM FOLDING AND LIGAND-BINDING STUDIES

Escherichia coli dihydrofolate reductase contains five tryptophan resi dues distributed throughout its structure. In order to examine the reg ions of the protein surrounding these tryptophan residues, we have inc orporated 6-fluorotryptophan into the protein. To assign the five reso nances observed in the F-19 NMR spectrum, five site-directed mutants o f the enzyme were made, each with one tryptophan replaced by a phenyla lanine. The F-19 NMR spectra of the apoprotein, two binary complexes ( with NADPH or methotrexate), and one ternary complex (with NADPH and m ethotrexate) were obtained. The chemical shifts of two of the tryptoph an resonances (at positions 22 and 74) are particularly sensitive to l igand binding, while the remaining three (at positions 30, 47, and 133 ) change, but by less. Since several of the tryptophans are distant fr om the binding site, these results suggest that F-19 NMR can detect li gand-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 re flect motion in that part of the protein, since it is near a region th at is disordered in the crystal structure of the apoprotein and its NA DP(+) complex. In contrast, in the ternary complex this region has a d efined structure, and all resonances are of equal intensity and line w idth. The F-19 NMR spectra of the apoprotein and the three ligand comp lexes were also examined as a function of urea concentration. At urea concentrations well below the denaturation midpoint, the resonance ass igned to tryptophan 22 in the apoprotein narrows and moves toward its denatured chemical shift. This behavior is predicted by a model that s uggests that this region is undergoing rapid conformational changes be tween native-like and unfolded-like forms. The disappearance of the br oadened tryptophan 22 resonance from its native chemical shift in the binary NADPH complex at very low urea concentrations may have a simila r explanation. In the binary complex with methotrexate or the ternary complex with NADPH and methotrexate, all peaks decrease equally with i ncreasing urea concentration, and all regions of the protein appear to be in slow exchange between the folded and unfolded forms.