Stopped-flow NMR spectroscopy: Real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase

Sydney D. Hoeltzli, Carl Frieden

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Escherichia coli dihydrofolate reductase (DHFR; EC 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 stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced 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%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k ≈ 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-f low circular dichroism (representing 80% of total amplitude). These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate. Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above. The NMR data show that ≈20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein.

Original languageEnglish
Pages (from-to)9318-9322
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number20
StatePublished - Sep 26 1995


  • Circular dichroism
  • Fluorescence
  • Unfolding intermediate


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