Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stoppedflow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during ureainduced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (-:,20%) The mechanism by which a protein unfolds from or folds to its correct tertiary structure remains one of the key unanswered questions in biochemistry. Common experimental approaches measure rates of unfolding or refolding by monitoring changes in intrinsic fluorescence, absorbance, or circular dichroism.Unfolding that occurs in a single phase (1, 2) has been interpreted as indicating that the unfolding of a protein represents a single process and that observable unfolding intermediates do not exist. When multiple phases are observed, they may represent either separate pathways or the formation of intermediates on a single pathway. Fluorescence, absorbance, or circular dichroism measurements do not readily distinguish between these mechanisms; if intermediates exist, these techniques cannot provide specific information about the structure of such intermediates.A number of studies have identified specific regions of secondary structure formed during the folding process by using hydrogen-deuterium exchange combined with multidimensional proton NMR spectroscopy (reviewed in refs. [3][4][5]. This technique, by monitoring rates of amide-proton exchange, provides detailed and specific information about the behavior of the backbone of the protein and the formation of secondary structure but does not, in general, monitor side-chain environment. Thus, structural information about the formation of specific regions of tertiary structure during the folding process is still limited. In addition, few studies have addressed the structural changes which occur during unfolding.Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) is a monomer of 159 amino acids and molecular weight 17,680 which catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate. Its small size, well-characterized enzyme mechanism (6, 7), and well-refined structure (8-10), as well as the reversibility of its folding reaction in the presence of chemical denaturants (11,12), make this protein a good model for protein-folding studies. E. coli DHFR contains five tryptophan residues distributed throughout its structure. We have previously prepared 6-19F-tryptophan labeled E. coli DHFR, assigned the resonances observed in the 19F spectrum of this protein to individual tryptophans, and studied its behavior at equilibrium in the presence of chemical denaturant (13).In this paper, we monitor the real-time changes in the NMR spectrum of 6-19F-tryptophan labeled E. coli DHFR to study t...

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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)

NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen-deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use "F-substituted amino acids to follow changes in side-chain environment upon folding. Current techniques of molecular biology allow assignments of I9F resonances to specific amino acids by site-directed mutagenesis. It is possible to follow changes and to analyze results from I9F spectra in real time using a stoppedflow device incorporated into the NMR spectrometer.

Escherichia coli dihydrofolate reductase (ecDHFR, EC1.5.1.3) contains 5 tryptophan residues that have been replaced with 6-19F-tryptophan. Five native and four of the five unfolded tryptophan resonances can be resolved in the 1D 19F NMR spectra and have been assigned [Hoeltzli, S. D., & Frieden, C. (1994) Biochemistry 33, 5502-5509]. This resolution allows the behavior of the native and the unfolded resonances assigned to each individual tryptophan to be monitored during the unfolding or refolding process. We now use these assignments and stopped-flow NMR to investigate the real-time behavior of specific regions of the protein during refolding of DHFR after dilution from 4.6 to 2.3 M urea (midpoint of the transition = 3.8 M) at 5 degrees C. Approximately half of the intensity of each of the four unfolded resonances is present at the first measurable time point (1.5 s). Little native resonance intensity is detectable at this time. The remaining unfolded resonance intensities present then disappear in two phases, with rates similar to the two slowest phases observed by either stopped-flow fluorescence or circular dichroism spectroscopy upon refolding under the same conditions. Substantial total resonance intensity is missing during the first 20 s of the refolding process. The appearance of the majority of native resonance intensity (as assessed by the height of each of the five native tryptophan resonances) is slow and similar for all five tryptophans. In contrast, the largest amplitude changes observed by either stopped-flow far-UV circular dichroism spectroscopy or fluorescence spectroscopy, and the greatest loss of unfolded resonance intensity, occur much more rapidly. We conclude from these studies: (1) that, under these conditions, the unfolded state remains substantially populated after initiation of refolding; (2) that the early steps in refolding involve a solvent protected intermediate containing substantial secondary structure, but (3) that the stable native side chain interactions form slowly and are associated with the final rate-limiting phase of the refolding process. Preliminary analysis of the area of broadened native resonances suggests that these resonances may appear at different rates, indicating that some regions of the protein begin to sample a native-like side chain environment while side chain environment in other regions of the protein remains less ordered. The results of this study are consistent with the earlier studies demonstrating that mobility of side chains is an early step in unfolding [Hoeltzli, S. D., & Frieden, C. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318-9322] and that recovery of enzymatic activity occurs as a late step in the folding process [Frieden, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4413-4416].