Biophysical characterization of the C-terminal region of FliG, an essential rotor component of the Na+-driven flagellar motor

Abstract

The bacterial flagellar motor generates a rotational force by the flow of ions through the membrane. The rotational force is generated by the interaction between the cytoplasmic regions of the rotor and the stator. FliG is directly involved in the torque generation of the rotor protein by its interaction. FliG is composed of three domains: the N-terminal, Middle and C-terminal domains, based on its structure. The C-terminal domain of FliG is assumed to be important for the interaction with the stator that generates torque. In this study, using CD spectra, gel filtration chromatography and DSC (differential scanning calorimetry), we characterized the physical properties of the C-terminal domain (G214-Stop) of wild-type (WT) FliG and its non-motile phenotype mutant derivatives (L259Q, L270R and L271P), which were derived from the sodium-driven motor of Vibrio. The CD spectra and gel filtration chromatography revealed a slight difference between the WT and the mutant FliG proteins, but the DSC results suggested a large difference in their stabilities. That structural difference was confirmed by differences in protease sensitivity. Based on these results, we conclude that mutations which confer the non-motile phenotype destabilize the C-terminal domain of FliG.

Domain-based biophysical characterization of the structural and thermal stability of FliG, an essential rotor component of the Na⁺-driven flagellar motor

“…Fitting this peak by a two state model, the T m value is ca. 59°C and the corrected Van’t Hoff enthalpy, ΔH, is 208 kJ/mol, which are similar to our previous report [28]. In contrast, the DSC curves of FliG MC and FliG Full can be fitted with two and three peaks, respectively.…”

“…Recently we reported on the biophysical characteristics of the FliG C-terminal domain, FliG C , from V. alginolyticus [28]. Using the same analytical techniques, we characterized two other constructs, FliG full length (FliG Full ), and FliG middle and C-terminal domain (FliG MC ), in addition to FliG C (Fig.…”

“…Cells were grown and induced as described previously [28]. Cells were harvested by centrifugation, suspended in TN buffer [50 mM Tris-HCl (pH 8.0), 0.5 M NaCl] containing 20 mM imidazole and protease inhibitors, and were disrupted by sonication (LV 5.0, duty cycle 50%, 1 min×5).…”

“…Circular dichroism spectroscopy was measured using a J-720W instrument (Jasco) as described previously [28]. All sample concentrations were 0.1 mg/ml and buffer conditions were 50 mM sodium phosphate (pH 6.5 or pH 8.0).…”

“…In addition to this mutant, three other Mot − mutants, L259Q, L270R, and L271P of FliG, were reported in V. alginolyticus [27]. We have characterized the physical properties of the C-terminal domain (G214–L351) of wild-type FliG and its non-motile phenotype mutant derivatives [28]. The CD spectra and size exclusion chromatography did not show a significant difference between the wild-type and mutant FliG proteins, however, the DSC data were very different between the mutants.…”