An essential intermediate in the folding of dihydrofolate reductase

The folding of Escherichia coli dihydrofolate reductase was examined at pH 7.8 and 15 degrees C by using stopped-flow fluorescence and absorbance spec troscopies. The formation of a highly fluorescent intermediate occurs with relaxation times ranging between 142 and 343 msec, whereas stopped-flow abs orbance 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 forma tion of this ensemble of intermediates is an obligatory step in the folding reaction.