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.
Subject(s)
Bacterial Proteins/chemistry , Tetrahydrofolate Dehydrogenase/chemistry , Bacterial Proteins/metabolism , Escherichia coli/enzymology , Methotrexate/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Protein Folding , Spectrometry, Fluorescence , Tetrahydrofolate Dehydrogenase/metabolismABSTRACT
The folding of the beta-sheet protein, interleukin-1 beta, was examined at pH 5.0 and 25 degrees C using pulse-labelling hydrogen exchange and electrospray ionization mass spectrometric analysis, as well as stopped-flow circular dichroism and fluorescence spectroscopies. The first detectable event is the formation of a partially folded intermediate in a kinetic step with a relaxation time of 126 +/- 26 ms. There is a lag in native protein production of at least 400 ms. Optical studies indicate that the intermediate is converted to the native species in a reaction with a relaxation time of 43 +/- 5 s. The kinetic rates determined from stopped-flow fluorescence, circular dichroism and pulse-labelling experiments are similar and consistent with a simple sequential model for the folding pathway of interleukin-1 beta at pH 5.0 and 25 degrees C. Taken together, our data provide kinetic evidence that formation of the native state of interleukin-1 beta proceeds through an obligatory intermediate. We explain our results in terms of the classical and new views of protein folding.
