REFOLDING OF [6-F-19]TRYPTOPHAN-LABELED ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE IN THE PRESENCE OF LIGAND - A STOPPED-FLOW NMR-SPECTROSCOPY STUDY

Escherichia coli dihydrofolate reductase contains five tryptophan resi dues that are spatially distributed throughout the protein and located in different secondary structural elements. When these tryptophans ar e replaced with [6-F-19]tryptophan, distinct native and unfolded reson ances can be resolved in the 1-D F-19 NMR spectra. Using site-directed mutagenesis, these resonances have been assigned to individual trypto phans [Hoeltzli, S. D., and Frieden, C. (1994) Biochemistry 33, 5502-5 504], allowing both the native and unfolded environments of each trypt ophan to be monitored during the refolding process. We have previously used these assignments and stopped-flow NMR to investigate the behavi or of specific regions of the protein during refolding of apo dihydrof olate reductase from urea in real time. These studies now have been ex tended to investigate the real time behavior of specific regions of th e protein during refolding of dihydrofolate reductase in the presence of either NADPt or dihydrofolate. As observed for the apoprotein, in t he presence of either ligand, unfolded resonance intensities present a t the first observed time point (1.5 s) disappear in two phases simila r to those monitored by either stopped-flow fluorescence or circular d ichroism spectroscopy. The existence of unfolded resonances which disa ppear slowly indicates that an equilibrium exists between the unfolded side chain environment and one or more intermediates, and that format ion of at least one intermediate is cooperative, The results of this s tudy are consistent with previous fluorescence studies demonstrating t hat dihydrofolate binds at an earlier step in the folding process than does NADP(+) [Frieden, C, (1990) Proc, Natl. Acad. Sci. U.S.A. 87, 44 13-4416] and provide a structural interpretation of the previous resul ts. :in the apoprotein as well as in the presence of either ligand, th e protein folds via at least one cooperatively formed, solvent-protect ed intermediate which contains secondary structure, In the presence of NADP(+), a stable native-like side chain environment forms in the reg ions around tryptophans 30, 133, and 37 in an intermediate which canno t bind NADP(+) tightly. Native side chain environment forms in the reg ions around tryptophans 22 and 74 only in the structure which is able to bind NADP(+) tightly. In the presence of dihydrofolate, stable nati ve-like side chain environment forms cooperatively in the regions arou nd each tryptophan in a non-native intermediate which must undergo a c onformational change prior to binding NADP(+). The presence of ligands influences the processes which occur during the folding of dihydrofol ate reductase, and the ligand may in effect serve as part of the hydro phobic core.