Folding of dihydrofolate reductase from Escherichia coli

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Abstract
The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15.degree. C is 6.13 .+-. 0.36 kcal mol-1 by difference UV, 5.32 .+-. 0.67 kcal mol-1 by CD, and 5.42 .+-. 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated .tau.1 and .tau.2 that correspond to the slow phases in unfolding and three faster reactions designated .tau.3, .tau.4, and .tau.5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm. For refolding in the presence of saturating methotrexate, the four slower reactions, .tau.1-.tau.4, lead to the binding of the inhibitor; the .tau.5 phase does not. The increase in fluorescence intensity observed for the .tau.5 phase is opposite to the changes observed for the .tau.1-.tau.4 phases and is contrary to what would be expected for a progressive folding reaction. This result suggests that the early stages of folding may involved a hydrophobic cluster that protects tryptophan residues from solvent-associated quenching mechanisms. The urea dependence of the .tau.1-.tau.5 reactions is characteristic of folding, and the activation energies range from 13 to 37 kcal mol-1. A folding model which incorporates all of these results is proposed that involves a multiplicity of native, intermediate, and unfolded forms.