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. 2000 May 23;97(11):5866-70.
doi: 10.1073/pnas.100547697.

An essential intermediate in the folding of dihydrofolate reductase

D K Heidary  1 , J C O'Neill Jr, M Roy, P A Jennings
Affiliations

An essential intermediate in the folding of dihydrofolate reductase

D K Heidary et al. Proc Natl Acad Sci U S A. .

Abstract

The folding of Escherichia coli dihydrofolate reductase was examined at pH 7.8 and 15 degrees C by using stopped-flow fluorescence and absorbance spectroscopies. The formation of a highly fluorescent intermediate occurs with relaxation times ranging between 142 and 343 msec, whereas stopped-flow absorbance spectroscopy using methotrexate binding assays shows a distinct lag phase during these time frames for the native state. The lag in absorbance kinetics and the lack of fast-track folding events indicate that the formation of this ensemble of intermediates is an obligatory step in the folding reaction.

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Figures

Figure 1
Figure 1
Kinetic simulations of on- (Scheme 1) and off- (Scheme 2) pathway simple folding models. The starting point for all simulations is 100% U, where the U to I transition is designated as k1, whereas the I to U transition is k−1 and the ratio of k1/k−1 is kept constant. (a) Plot of the kinetic simulations for a simple on-pathway folding mechanism by using kinsim with k1 = 3.33 s−1, k−1 = 0.833 s−1 and k2 = 1 s−1 with the population of native species shown in red. (b) Simulation for an off-pathway folding model with k1 = 3.33 s−1, k−1 = 0.833 s−1 and k2 = 1 s−1. (c) Plot of the simulation depicted in a, but with k1 = 333 s−1, k−1 = 83.3 s−1 and k2 = 1 s−1. (d) Depicts the off-pathway folding mechanism in b, with k1 = 333 s−1, k−1 = 83.3 s−1 and k2 = 1 s−1.
Figure 2
Figure 2
Plot of the change in the relative fluorescence intensity as a function of time for a refolding jump from 6 M to 0.54 M urea at pH 7.80 and 15°C, measured on the Applied Photophysics SX.17MV stopped-flow unit. Tryptophan fluorescence was followed by using an excitation wavelength of 293 nm and by monitoring the emission for wavelengths >320 nm.
Figure 3
Figure 3
(a) Plot of the difference in absorbance on methotrexate binding as a function of time for a refolding jump to 0.54 M urea. (b) Plot of the absorbance difference for a refolding jump to 2 M urea. Spectra were acquired on the Applied Photophysics SX.17MV stopped-flow unit. The change in signal was observed at 380 nm. The solid lines correspond to the simulation of the proposed four-channel folding model. Insets show the first 2 sec of the reaction. The data were collected at pH 7.80 and 15°C.
Figure 4
Figure 4
(a) Plot of the change in relative fluorescence intensity as a function of time for a refolding jump from 6 M to 0.54 M urea at pH 7.80 and 15°C, measured on the Applied Photophysics SX.17MV stopped-flow unit. Tryptophan fluorescence was monitored by an excitation wavelength of 293 nm and emission observed for wavelengths >320 nm. (b) Plot of the difference in absorbance on methotrexate binding as a function of time for a refolding jump from 6 M to 0.54 M urea, pH 7.80 and 15°C.
Figure 5
Figure 5
Ribbon diagram of E. coli dihydrofolate reductase by using the x-ray coordinates from the Bolin et al. structure (28). The molecule is oriented to show the two subdomains with the interconnecting loops. The two tryptophans forming the exciton coupling (47 and 74) are shown in purple and blue, respectively, whereas the substrate-binding pocket is shown with the bound methotrexate in red. The program molscript was used to prepare this diagram (29).
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