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Key Protein Interactions Show Promise for Difficult Biomolecular Systems

Published: Tuesday, December 10, 2013
Last Updated: Tuesday, December 10, 2013
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Wyatt Technology publishes a new 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. It offers a new perspective over traditional methods to understand complex protein-protein interactions. To obtain a copy of the application note, please click here.

The application note demonstrates how the use of the Calypso® II composition-gradient system combined with a DAWN® HELEOS® from Wyatt Technology provides insights into complicated protein-protein interactions, which are not measurable by nuclear magnetic resonance (NMR) or other traditional techniques.  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 (FliGM and FliGC) were measured individually. The results demonstrate a 100-fold difference in binding affinity between FliGM (KD = 6.6 μM) and FliGC (KD = 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 FliGc 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. 

With this application note, Wyatt Technology demonstrates how CG-MALS provided invaluable information about the complex interactions between the proteins involved in the bacterial flagellar motor switching mechanism.  Sophia Kenrick from Wyatt Technology explains, “CG-MALS provides new data explaining the binding of FliG to FliM, which could not have been obtained by other methods. 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 Calypso II combined with a DAWN HELEOS or miniDAWN® TREOS® MALS detector encompass a complete composition-gradient multi-angle light scattering (CG-MALS) system, capable of characterizing a wide range of interactions.  These instruments prepare solutions of different molecular composition or concentration and measure the change in molar mass as complexes form or dissociate. No special modifications, e.g., sample tagging or immobilization procedures, are necessary: samples are unlabeled and entirely in solution. Calypso's automation enhances productivity while the CALYPSO software provides an unparalleled selection of interaction models to provide the affinity and absolute stoichiometry of the complexes formed in solution. Several application notes have been drafted on this product, detailing the success and ease Calypso II can bring to your studies. 

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