Thermodynamic studies of the Escherichia coli factor for inversion stimulation and the eukaryotic nucleosome core particle

The stability and kinetics of the Factor for Inversion Stimulation (FIS) were determined and compared with other intertwined segment swapped alpha-helical DNA binding dimers to aid in understanding oligomeric protein folding. The equilibrium stability of recombinant FIS had been determined by both urea and guanidinium-induced denaturation, using circular dichroism (CD). Stopped-flow CD was used to determine the folding mechanism of the FIS using both urea and guanidinium as the denaturants. Similar to the folding mechanisms of the eukaryotic histones determined previously, the kinetic mechanism of FIS is a sequential process: 1) unfolded monomers associate in a burst phase reaction to form a dimeric intermediate; 2) this intermediate further folds in a first-order reaction to yield the native dimer in the rate-limiting step of the folding reaction. The sequential formation of an on-pathway dimeric intermediate was confirmed using urea double jump experiments. To investigate the role of protein stability in higher order protein-DNA complexes, a Fo?rster resonance energy transfer (FRET) system was created to monitor the salt-induced unfolding of nucleosome core particles (NCPs) containing H2A.1 and H2A.Z. The donor (D)-acceptor (A) pair chosen was tryptophan and Cys-AEDANS, respectively, with a Fo?rster distance of 20 A?. The FRET system was able to monitor the dissociation of the H2A-H2B dimers from nucleosome core particles reconstituted with the 601 artificial positioning sequence. The free energy of dissociation, DG, for the dimers was determined from FRET equilibrium studies in which two transitions were observed. These transitions correlate to the dissociation of each dimer with positive cooperativity. Although the stability of the FRET NCPs incorporated with H2A.Z were not significantly altered, an increase in the cooperativity of the H2A.Z dimers was observed