EQUILIBRIUM UNFOLDING OF FTSZ FROM ESCHERICHIA COLI BY FCS

University Of California-Irvine, Irvine CA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The use of multiple spectroscopic techniques to trace the structural changes that occur during protein unfolding provide support for a particular folding model. We have studied the function and structure of FtsZ from Escherichia coli. The FtsZ protein displays GTPase activity and polymerization in vitro. The unfolding induced by guanidinium chloride was analyzed with two spectroscopic techniques, circular dichroism and intrinsic fluorescence. The unfolding reaction showed a biphasic behavior with the presence of an intermediary. This information led to the proposal of a model in which the monomer of FtsZ unfolds in two steps (with three states) and the intermediary is the apo-protein. In the same line of investigation, in this thesis, by means of analytic size-exclusion chromatography, it was found that a solution of FtsZ contains monomers, dimers and trimers. For this reason, the equilibrium unfolding of FtsZ was analyzed again by incorporating a phase of dissociation of the monomers from the dimers, accounting for the intermediate state observed on earlier thermodynamic studies, this implies the presence of a dissociation constant. The dissociation of oligomers diminishes the molecular weight and increases the number of molecules, this leads to a change in diffusion of the fluorophore, an aspect which could be monitored directly. Observing the number of molecules and the diffusion coefficients of the fluorophore during the unfolding of FtsZ could shed lights about the oligomerization state that contributes to the stability of the native protein. Fluorescence correlation spectroscopy allows the calculation of the number and the diffusion coefficient of molecules moving in and out of a small volume. The technique provides high sensibility because it requires very small amounts of material, in the range of sub-micromolar concentrations. The same methodology was used to analyze the dissociation of tubulin heterodimer induced by denaturant agents.