Application note shows CG-MALS can quantify complex proteins

Wyatt Technology has published an application note highlighting a study on the complex interactions between proteins and how they modulate the rotational direction of bacteria flagella. The application note highlights the benefit of using composition-gradient multi-angle static light scattering (CG-MALS) to confirm the specific binding sites of the protein, thus offering a new perspective over traditional methods to understand complex protein-protein interactions.

The application note demonstrates how a combination of the company’s Calypso II and Dawn Heleos instruments provides insights into complicated protein-protein interactions, which are not measurable by nuclear magnetic resonance (NMR) or other traditional techniques. The products encompass a complete CG-MALS system, preparing solutions of different molecular composition or concentration and measuring the change in molar mass as complexes form or dissociate. No special modifications, eg, sample tagging or immobilisation procedures, are necessary.

The study focuses on the bacteria flagella, an electrical motor which aids the movement of the bacteria, and the proteins which affect its function. In particular, the different domains of one flagellar protein (FliG) bind two different sites on its binding partner (FliM) with different affinity, as part of the flagellar motor switching mechanism.

In the first part of the analysis, the binding affinity between FliM and the two FliG domains (FliG_M and FliG_C) were measured individually. The results demonstrate a 100-fold difference in binding affinity between FliG_M (K_D = 6.6 μM) and FliG_C (K_D = 580 μM) for FliM. This large difference in affinity supports the current hypothesis for switching the rotational direction of the flagella: the tighter-binding FliGM domain remains bound while the weaker-binding FliG_c domain can be displaced by other regulatory proteins to change the direction of rotation.

When the binding between FliM and full-length FliG was tested, a slow, time-dependent association into large complexes was observed. This large association was hinted at by previous NMR studies but could not be quantified by this technique. Measuring the molar mass as a function of time by CG-MALS provides direction for future studies and may help determine the mechanism of flagellar motor switching.

“CG-MALS provides new data explaining the binding of FliG to FliM, which could not have been obtained by other methods,” said Sophia Kenrick from Wyatt Technology. “The same CG-MALS technique used to investigate the structure-function relationship of the flagellar proteins can be extended to other complex protein assemblies, such as the large microchannels bacteria and other pathogens use to inject toxins into a host. Understanding how the proteins work together may help with research into how to disrupt these interactions and control the spread of these pathogens.”

The application note thus demonstrates how CG-MALS provided invaluable information about the complex interactions between the proteins involved in the bacterial flagellar motor switching mechanism. It can be viewed here.