Time-resolved fluorescence anisotropy study of the refolding reaction of the alpha-subunit of tryptophan synthase reveals nonmonotonic behavior of the rotational correlation time

Time-resolved fluorescence anisotropy of a bound extrinsic probe was studie d in an effort to characterize dynamic properties of the transient partiall y folded forms that appear during the folding of the a-subunit of tryptopha n synthase (alpha TS) from Escherichia coli. Previous studies have shown th at aTS, a single structural domain, can be cleaved into autonomously foldin g amino- and carboxy-folding units comprising residues 1-188 and 189-268, r espectively [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistr y 18, 4827-4835]. By use of a double-kinetic approach [Jones, B. E., Beeche m, J. M., and Matthews, C. R. (1995) Bioclzemistry 34, 1867-1877], the rota tional correlation time of 1-anilino-8-naphthalene sulfonate bound to nonpo lar surfaces of folding intermediates was measured by time-correlated singl e photon counting at varying time delays following initiation of folding fr om the urea-denatured form by stopped-flow techniques. Comparison of the ro tational correlation times for the full-length alpha TS and the amino-termi nal fragment suggests that folding of the amino-terminal fragment and carbo xy-terminal fragment is coordinated, not autonomous, on the milliseconds to seconds time scale. If a spherical shape is assumed, the apparent hydrodyn amic radius of alpha TS after 5 ms is 26.8 Angstrom. The radius increases t o 28.5 Angstrom by 1 s before decreasing to the radius for native alpha TS, 24.7 Angstrom, on a longer time scale (>25 s). Viewed within the context o f the kinetic folding model of alpha TS [Bilsel, O., Zitzewitz, J. A., Bowe rs, K. E., and Matthews, C, R. (1999) Biochemistry 38, 1018-1029], the init ial collapse reflects the formation of an off-pathway burst-phase intermedi ate in which at least part of the carboxy folding unit interacts with the a mino folding unit. The subsequent increase in rotational correlation time c orresponds to the formation of an on-pathway intermediate that leads to the native conformation. The apparent increase in the radius for the on-pathwa y intermediate may reflect a change in the interaction of the two-folding u nits, thereby forming a direct precursor for the alpha/beta barrel structur e.