Introduction: Proteins that can bind several ligands at the same time are widespread in nature. We have developed an approach to study such interactions. The proposed method is based on the attachment of several binding domains to a single oligomeric backbone thus creating proteins with a specific number of binding sites.

Methods: To obtain proteins with different numbers of binding sites, we denatured and then renatured a mixture of the Sm-like protein from Sulfolobus acidocaldarius (SacSm) and the same protein but with the apical domain of the GroEL protein attached (ADGroEL_SacSm). By varying the ratio of the components we can change the number of apical domains on each resulting oligomer. After producing proteins with 1 to 7 binding domains, we determined the strength and stoichiometry of their interaction with the model non-native protein lactalbumin (LA). The interaction constants were assessed quantitatively by analyzing the results of SDS gel electrophoresis of the formed complexes.

Results: The data obtained suggest that the interaction between a single apical domain (AD) and non-native proteins is relatively weak and is not well-defined by our methods. However, when several ADs are closely located on a single protein backbone, non-native proteins may interact with several domains simultaneously, and the stability of this complex increases exponentially. LA cannot interact with more than two ADs at the same time, which is manifested by the lack of change in the dissociation constant as the number of binding sites increases. At the same time, its small size allows the binding of up to 4 molecules on the complete heptameric ADGroEL_SacSm ring.

Conclusion: We have developed a workable method for studying multivalent protein-protein interactions with a range of binding sites, from 1 to 7, which is essential for understanding the complexity of these interactions in biological systems.