SUNY at Albany
June 19-23, 2001
Elucidation of the Folding Mechanism of Fis, A dimeric DNA-Binding Protein
Equilibrium and kinetic analyses have been carried out to define the folding mechanism of factor for inversion stimulation (Fis), a small homodimeric DNA-binding protein that is involved in a number of regulatory processes, including the Hin- and Gin-catalyzed site-specific DNA inversion reactions in E. coli. The urea-induced equilibrium denaturation pathway of Fis was studied using multiple techniques, including fluorescence and circular dichroism (CD) spectroscopy. The equilibrium denaturation of Fis as monitored by near and far-UV CD occurs in a concerted two state process. CD and fluorescence experiments using Fis variants in which the native tyrosines residues were replaced, one at a time, by a tryptophan, the two state unfolding of Fis was confirmed and the DNA binding region was determined to have an increased mobility under mild denaturant conditions (0.2-2M urea).
The urea-induced refolding and unfolding of Fis and the tryptophan variants were studied using stopped-flow CD. Stopped-flow fluorescence experiments could only be performed with the tryptophan variants, which have a much stronger fluorescence signal than the wild type. This provides an indirect method of obtaining fluorescence kinetic data on the wild type. Despite Fis being such a small protein, it seems to display complicated kinetics both in folding and unfolding as seen by the presence of refolding burst phase and a leveling off of the unfolding rate at high urea concentrations. The preliminary data seems to indicate a refolding mechanism that includes a dimeric intermediate as well as an intermediate in unfolding.
Sarah A. Hobart(1), Sergey Ilin(1), Robert Osuna(2), and Wilfredo Colon(1)
Rensselaer Polytechnic Institute(1), Department of Chemistry, 110 8th Street, Troy, NY 12180, and
Department of Biological Sciences(2), State University of New York at Albany, Albany, NY 12180