Thermal adaptation of mesophilic and thermophilic FtsZ assembly by modulation of the critical concentration.

Cytokinesis is the last stage in the cell cycle. In prokaryotes, the protein FtsZ guides cell constriction by assembling into a contractile ring-shaped structure termed the Z-ring. Constriction of the Z-ring is driven by the GTPase activity of FtsZ that overcomes the energetic barrier between two protein conformations having different propensities to assemble into polymers. FtsZ is found in psychrophilic, mesophilic and thermophilic organisms thereby functioning at temperatures ranging from subzero to >100°C. To gain insight into the functional adaptations enabling assembly of FtsZ in distinct environmental conditions, we analyzed the energetics of FtsZ function from mesophilic Escherichia coli in comparison with FtsZ from thermophilic Methanocaldococcus jannaschii. Presumably, the assembly may be similarly modulated by temperature for both FtsZ orthologs. The temperature dependence of the first-order rates of nucleotide hydrolysis and of polymer disassembly, indicated an entropy-driven destabilization of the FtsZ-GTP intermediate. This destabilization was true for both mesophilic and thermophilic FtsZ, reflecting a conserved mechanism of disassembly. From the temperature dependence of the critical concentrations for polymerization, we detected a change of opposite sign in the heat capacity, that was partially explained by the specific changes in the solvent-accessible surface area between the free and polymerized states of FtsZ. At the physiological temperature, the assembly of both FtsZ orthologs was found to be driven by a small positive entropy. In contrast, the assembly occurred with a negative enthalpy for mesophilic FtsZ and with a positive enthalpy for thermophilic FtsZ. Notably, the assembly of both FtsZ orthologs is characterized by a critical concentration of similar value (1-2 µM) at the environmental temperatures of their host organisms. These findings suggest a simple but robust mechanism of adaptation of FtsZ, previously shown for eukaryotic tubulin, by adjustment of the critical concentration for polymerization.

Studies on the dissociation and urea-induced unfolding of FtsZ support the dimer nucleus polymerization mechanism.

FtsZ is a major protein in bacterial cytokinesis that polymerizes into single filaments. A dimer has been proposed to be the nucleating species in FtsZ polymerization. To investigate the influence of the self-assembly of FtsZ on its unfolding pathway, we characterized its oligomerization and unfolding thermodynamics. We studied the assembly using size-exclusion chromatography and fluorescence spectroscopy, and the unfolding using circular dichroism and two-photon fluorescence correlation spectroscopy. The chromatographic analysis demonstrated the presence of monomers, dimers, and tetramers with populations dependent on protein concentration. Dilution experiments using fluorescent conjugates revealed dimer-to-monomer and tetramer-to-dimer dissociation constants in the micromolar range. Measurements of fluorescence lifetimes and rotational correlation times of the conjugates supported the presence of tetramers at high protein concentrations and monomers at low protein concentrations. The unfolding study demonstrated that the three-state unfolding of FtsZ was due to the mainly dimeric state of the protein, and that the monomer unfolds through a two-state mechanism. The monomer-to-dimer equilibrium characterized here (K(d) = 9 µM) indicates a significant fraction (~10%) of stable dimers at the critical concentration for polymerization, supporting a role of the dimeric species in the first steps of FtsZ polymerization.