A POSSIBLE INITIAL FOLDING INTERMEDIATE - THE C-TERMINAL PROTEOLYTIC DOMAIN OF TRYPTOPHAN SYNTHASE-BETA CHAINS FOLDS IN LESS THAN 4 MILLISECONDS INTO A CONDENSED STATE WITH NON-NATIVE-LIKE SECONDARY STRUCTURE

The isolated F2-V8 peptide corresponding to the 101 C-terminal residues of Escherichia coli tryptophan synthase beta-chains folds into a heat-stable, yet fluctuating, condensed state that contains a lot of secondary structure. However, this state has non-native-like secondary and supersecondary structures [Chaffotte, A., Guillou, Y., Delepierre, M., Hinz, H.-J., & Goldberg, M. E. (1991) Biochemistry 30, 8067-8074]. To characterize the rate of appearance of this state, stopped-flow studies on the far-ultraviolet circular dichroism (CD) and on the binding of 1-anilino-8-naphthalenesulfonate (ANS) have been conducted during the folding of guanidine-unfolded F2-V8. It was shown that both the CD signal at 222 nm and the ANS binding properties of folded isolated F2-V8 were regained, at 20-degrees-C, within the dead time of the stopped-flow apparatus, which was 4 ms. At 12-degrees-C, the binding of ANS was also completed within this dead time, but the ellipticity showed some minor later changes. After a rapid overshoot of the CD signal that occurred during the 4-ms dead time, a small readjustment of the ellipticity to the final value occurred more slowly and was completed after about 25 ms. Thus, even at 12-degrees-C, the hydrophobic core and most of the secondary structure of folded F2-V8 were formed in less than 4 ms. These observations strongly suggest that the previously described condensed non-native-like state of F2-V8 results from a very rapid, nonspecific, hydrophobic collapse. It is proposed that such a state may be a general early intermediate in protein folding.