Refolding of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate reductase in the presence of ligand: a stopped-flow NMR spectroscopy study.
Related Articles Refolding of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate reductase in the presence of ligand: a stopped-flow NMR spectroscopy study.
Biochemistry. 1998 Jan 6;37(1):387-98
Authors: Hoeltzli SD, Frieden C
Escherichia coli dihydrofolate reductase contains five tryptophan residues that are spatially distributed throughout the protein and located in different secondary structural elements. When these tryptophans are replaced with [6-19F]tryptophan, distinct native and unfolded resonances can be resolved in the 1-D 19F NMR spectra. Using site-directed mutagenesis, these resonances have been assigned to individual tryptophans [Hoeltzli, S. D., and Frieden, C. (1994) Biochemistry 33, 5502-5509], allowing both the native and unfolded environments of each tryptophan to be monitored during the refolding process. We have previously used these assignments and stopped-flow NMR to investigate the behavior of specific regions of the protein during refolding of apo dihydrofolate reductase from urea in real time. These studies now have been extended to investigate the real time behavior of specific regions of the protein during refolding of dihydrofolate reductase in the presence of either NADP+ or dihydrofolate. As observed for the apoprotein, in the presence of either ligand, unfolded resonance intensities present at the first observed time point (1.5 s) disappear in two phases similar to those monitored by either stopped-flow fluorescence or circular dichroism spectroscopy. The existence of unfolded resonances which disappear slowly indicates that an equilibrium exists between the unfolded side chain environment and one or more intermediates, and that formation of at least one intermediate is cooperative. The results of this study are consistent with previous fluorescence studies demonstrating that dihydrofolate binds at an earlier step in the folding process than does NADP+ [Frieden, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4413-4416] and provide a structural interpretation of the previous results. In the apoprotein as well as in the presence of either ligand, the protein folds via at least one cooperatively formed, solvent-protected intermediate which contains secondary structure. In the presence of NADP+, a stable native-like side chain environment forms in the regions around tryptophans 30, 133, and 47 in an intermediate which cannot bind NADP+ tightly. Native side chain environment forms in the regions around tryptophans 22 and 74 only in the structure which is able to bind NADP+ tightly. In the presence of dihydrofolate, stable native-like side chain environment forms cooperatively in the regions around each tryptophan in a non-native intermediate which must undergo a conformational change prior to binding NADP+. The presence of ligands influences the processes which occur during the folding of dihydrofolate reductase, and the ligand may in effect serve as part of the hydrophobic core.
PMID: 9425060 [PubMed - indexed for MEDLINE]
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PubMed