A reexamination of the folding mechanism of dihydrofolate reductase from Escherichia coli: Verification and refinement of a four-channel model

Abstract

The mechanism of folding of dihydrofolate reductase from Escherichia coli was reinvestigated by studying the unfolding and refolding kinetics using absorbance and fluorescence spectroscopies. The original kinetic model proposed that folding involved a series of native, intermediate, and unfolded forms which interconverted through four independent channels linked by slow cis/trans isomerization reactions at Xaa-Pro peptide bonds [Touchette, N. A., Perry, K. M., & Matthews, C. R. (1986) Biochemistry 25, 5445]. Recently, alternative sequential models have been proposed [Frieden, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4413; Kuwajima et al. (1991) Biochemistry 30, 7693] which challenge the original proposal. Stopped-flow studies of the intrinsic tryptophan fluorescence demonstrated the presence of three (and tentatively four) kinetic phases in unfolding which correlated well with four phases previously observed in refolding experiments. By monitoring the binding of the inhibitor methotrexate during folding at varying relative concentrations of inhibitor to protein, it was found that the selective loss of the slow-folding phases at substoichiometric levels could only be explained by a four-channel folding model. Double-jump experiments (native-->unfolded-->native) showed that the four refolding channels are populated within 20 s at 15 degrees C and are not likely to be due to proline isomerization. Reverse double-jump experiments (unfolded-->native-->unfolded) demonstrated that interconversions between native conformers are more rapid than originally proposed. Interestingly, the majority of the protein folds through a channel to a native conformer that is minimally populated at equilibrium. This implies that although the folding of dihydrofolate reductase is ultimately under thermodynamic control, kinetic factors contribute to the transient populations of native species during folding.

Folding pathway of a multidomain protein depends on its topology of domain connectivity

“…However, in its circular permutant, for which the topological complexity of wild-type DHFR is resolved, two pathways coexist, resulting in a complex folding behavior. [24][25][26][27]. It is plausible that the slow process (several hundred seconds) during the τ 1 − τ 4 phases is due to intense "internal friction" (30)(31)(32) in the glassy dynamics of conformation (33), including formation/disruption of nonnative contacts, the effects of proline isomerization, and the cis-trans isomerization of Gly95 and Gly96.…”

“…Because DLD does not include a single contiguous region of the chain, but rather includes separate N-and C-terminal parts, the structural ordering of DLD can be correlated with the structural ordering of ABD. As a model protein, DHFR has been intensively investigated (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), which has resulted in a picture that DHFR folds along the following pathway:…”

Significant hydrogen exchange protection in GroEL‐bound DHFR is maintained during iterative rounds of substrate cycling

“…These sites are located throughout the eight-stranded P-sheet in DHFR, demonstrating the formation of a native-like topology early in folding (Jennings et al, 1993). The close similarity of the hydrogen exchange properties of the kinetic folding intermediate with those of GroEL-bound DHFR suggests the intriguing possibility that a similar intermediate could be the substrate for GroEL.…”

“…It is interesting in this regard that an intermediate formed within 141 ms of the initiation of folding of E. coli DHFR (unassisted by GroEL) protects a similar number of amides from hydrogen exchange, their protection factors exceeding 100 after this refolding time (Jennings et al, 1993). These sites are located throughout the eight-stranded P-sheet in DHFR, demonstrating the formation of a native-like topology early in folding (Jennings et al, 1993).…”

“…1). DHFR is an ideal protein for these studies because the native protein has been studied in great detail by NMR (Stockman et al, 1991(Stockman et al, , 1992 and X-ray (Davies et al, 1990) methods, and much is known about the spontaneous folding pathway of the protein from Escherichia coli (Jennings et al, 1993;Jones & Matthews, 1995). By contrast with E. coli DHFR (Clark et al, 1996;Rospert et al, 1996), however, several mammalian DHFR species, although they can be refolded reversibly, form a well-defined stable complex with GroEL in the absence of nucleotides (Martin et al, 1991;Viitanen et al, 1991;Mayhew et al, 1996).…”