9L]LHSPUN[OL(MÄUP[`VM Individual and Combined FliG Domains for FliM in the Bacterial Flagellar Motor Complex

Significance of the Topic

Bacterial flagella change rotational direction through a complex of switch proteins. Quantitative insight into how FliG domains bind FliM is essential for understanding the molecular mechanism of motor switching and for applications in microbiology, synthetic nanomotors, and targeted antimicrobial strategies.

Objectives and Study Overview

This work applies composition-gradient multi-angle static light scattering (CG-MALS) to characterize the binding interactions between FliM and three constructs of FliG: the middle domain (FliGM), the C-terminal domain (FliGC), and the combined multidomain construct (FliGMC). The goals were to determine dissociation constants, stoichiometries, and assembly kinetics of the resulting complexes.

Methodology and Instrumentation

• Sample Preparation: FliM and FliG constructs were prepared in 10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.5, filtered to 0.02 µm, and quantified by UV absorbance.
• CG-MALS Setup: Automated composition gradients were generated by a Calypso II system interfaced with a DAWN HELEOS MALS detector. Inline filtration (0.1 µm and 0.02 µm) ensured sample clarity.
• Data Analysis: Calypso software fit light scattering data to hetero‐association models, yielding equilibrium dissociation constants (KD) and complex stoichiometries.

Main Results and Discussion

• FliGM–FliM Interaction: A strong 1:1 complex formed with KD = 6.6 µM, consistent with previous SEC‐MALS estimates.
• FliGC–FliM Interaction: A weaker 1:1 association was observed with KD = 580 µM, in line with prior NMR data, indicating that FliGC can be readily displaced by regulatory proteins.
• Multidomain FliGMC–FliM Assembly: Slow, concentration‐dependent association yielded higher‐order complexes (e.g., 2:1 and 3:2 FliM:FliG ratios). Equilibrium was not reached within one hour, revealing self‐assembly kinetics inaccessible to conventional techniques.

Benefits and Practical Applications of the Method

• CG-MALS provides real-time, solution‐phase analysis of complex biomolecular assemblies without surface immobilization, capturing kinetic and stoichiometric details.
• The quantitative affinity and stoichiometry data inform mechanistic models of the flagellar switch and support design of synthetic motor systems or inhibitors targeting bacterial motility.

Future Trends and Opportunities

Integration of CG-MALS with complementary techniques (ITC, SPR, analytical ultracentrifugation) and advances in kinetic modeling will expand its use for dynamic, multi‐component systems. Correlating in vitro assembly kinetics with in vivo conditions may elucidate chaperone‐mediated assembly pathways and regulatory mechanisms.

Conclusion

Employing CG-MALS has revealed a 100-fold difference in binding affinity between FliGM and FliGC for FliM, and uncovered slow formation of higher‐order assemblies by the multidomain construct. These findings enhance our understanding of the molecular basis for bacterial flagellar switching and demonstrate the unique capability of CG-MALS to dissect complex protein interactions.

Reference

1. Dyer CM et al. Revealing the Affinity of Individual and Combined FliG Domains for FliM in the Bacterial Flagellar Motor Complex. J. Mol. Biol. 388, 71–82 (2009).
2. Blair DF. Flagellar motor of bacteria. J. Bacteriol. 188, 7033–7040 (2006).
3. Protein Data Bank: FliG structure, PDB ID 1LKV; FliM structure, PDB ID 2HP7.
4. Park S et al. Binding of CheY-P to FliM induces molecular changes controlling flagellar motor switching. Proc. Natl. Acad. Sci. U.S.A. 103, 11886–11891 (2006).