Evaluation binding of individual and combined domains in the bacterial flagellar motor complex by CG-MALS

Importance of the topic

Complex protein interactions govern bacterial flagellar motor switching, which is essential for bacterial motility, chemotaxis, and adaptation. Probing the binding dynamics of these motor components at high resolution advances our understanding of microbial behavior and informs potential antimicrobial strategies.

Objectives and study overview

This study employs composition-gradient multi-angle static light scattering (CG-MALS) to characterize interactions between the middle (FliG M), C-terminal (FliG C), and multidomain (FliG MC) constructs of the FliG protein with its binding partner FliM. Key goals include quantifying binding affinities, determining complex stoichiometries, and capturing real-time assembly kinetics.

Used instrumentation

• Calypso composition-gradient system for automated gradient generation
• DAWN MALS detector for multi-angle static light scattering measurements
• Anotop 0.02 µm and polycarbonate 0.1 µm filter membranes for sample preparation
• UV-Visible spectroscopy for concentration determination

Applied methodology

CG-MALS experiments generated dual-component composition gradients, followed by stop-flow equilibration within the MALS flow cell. Light scattering and concentration signals were analyzed using Calypso software to fit binding models, extract equilibrium dissociation constants (K_d), and derive molecular weight distributions under both fast (seconds) and slow (minutes to hours) timescales.

Main results and discussion

• FliG M–FliM binding exhibited strong affinity with K_d ≈ 6.6 µM, consistent with prior estimates of 1–10 µM.
• FliG C–FliM interaction was weaker (K_d ≈ 580 µM), confirming an order-of-magnitude lower affinity compared to FliG M.
• Multidomain FliG MC and FliM formed larger assemblies (>1:1 stoichiometry) with slow association kinetics (τ ≈ 30–60 min), approaching an apparent molecular weight of ~230 kDa, indicative of 2:1 and 3:2 FliM:FliG complexes.

Benefits and practical applications of the method

CG-MALS provides a powerful solution-phase approach to resolve complex protein–protein interactions without surface immobilization, delivering simultaneous insights into binding affinity, stoichiometry, and assembly kinetics. This capability is particularly valuable for systems prone to oligomerization or slow self-assembly.

Future trends and possible applications

Extending CG-MALS to other dynamic biomolecular assemblies could uncover kinetic pathways in chaperone-mediated folding, ribonucleoprotein complex formation, or virus capsid self-assembly. Integration with complementary techniques (e.g., cryo-EM, NMR) may yield comprehensive mechanistic models.

Conclusion

This study demonstrates that CG-MALS uniquely captures the binding hierarchy and self-assembly behavior of flagellar motor proteins. The marked affinity difference between FliG domains and the slow, higher-order complex formation by multidomain constructs provide new mechanistic insights into flagellar switch assembly.

Reference

• Dyer CM et al. J. Mol. Biol. 388, 71 (2009).
• Blair DF. J. Bacteriol. 188, 7033 (2006).
• Wyatt Technology Corporation. PDB IDs: FliG 1LKV; FliM 2HP7.
• Park S et al. Proc. Natl. Acad. Sci. USA 103, 11886 (2006).