Real-time refolding studies of 6-19F-tryptophan labeled Escherichia coli dihydrofolate reductase using stopped-flow NMR spectroscopy.
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].
Hoeltzli
S D
SD
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Frieden
C
C
D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D005461:Fluorine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
D005461:Fluorine; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase