Metacomplex Formation & Binding Affinity of Multivalent Binding Partners

Importance of the topic

Multivalent interactions underpin critical biological processes such as cell signaling, immune recognition and supramolecular assembly. Accurately characterizing these interactions in solution is essential to avoid artifacts from surface immobilization and to understand binding stoichiometry and affinity in complex systems.

Objectives and overview of the study

This study employs composition-gradient multi-angle light scattering (CG-MALS) to quantify the metacomplex formation between tetrameric streptavidin and a bivalent anti-streptavidin antibody. The goal is to determine absolute binding affinities, stoichiometries and presence of self- or hetero-association in solution.

Methodology and instrumentation

A Calypso automated composition-gradient system delivered controlled concentration gradients to an inline UV/Vis detector and a DAWN MALS instrument. Single-component gradients assessed self-association; dual-component “crossover” gradients probed hetero-association. Equilibrium was reached under stop-flow conditions prior to data collection. Data modeling and parameter fitting were performed in CALYPSO software, testing three interaction models: infinite self-association (ISA), ISA plus defined 1:2 and 2:1 terms, and a full “2×2” multivalent model.

Main results and discussion

Measured light scattering signals exceeded those expected for simple 1:1 or 1:2 binding, indicating formation of higher-order complexes. No self-association of either protein was detected. Crossover gradient data peaked near a 1:1 molar ratio, with weight-average molar mass (~350 kDa) consistent with n:n assemblies. Model fitting revealed:

• ISA model fit (χ2=0.485) suggested a base 1:1 unit with Kd≈5 nM and weaker chain assembly (Kd≈44 nM).
• Refined ISA+1:2+2:1 model (χ2=0.26) yielded convergent Kd values (~23±7 nM) for single-site interactions across complexes.
• The 2×2 model, incorporating all possible n:n and adjacent stoichiometries, provided the best fit (χ2=0.19) with Kd≈34 nM per binding site.

Benefits and practical applications

CG-MALS offers unique advantages for studying multivalent-multivalent interactions entirely in solution. It quantifies absolute stoichiometry and affinity without immobilization artifacts, making it valuable for analyzing complex protein assemblies, antibody design, aptamer development and biopharmaceutical characterization.

Future trends and applications

Anticipated developments include integration with complementary detection methods (e.g., light scattering fluorescence), advanced modeling of cooperative effects, and broader application to nucleic acid complexes, cell-surface receptors and polymeric systems. High-throughput CG-MALS could streamline screening of multivalent therapeutics and nanomaterials.

Conclusion

This study demonstrates that CG-MALS is a powerful tool for elucidating metacomplex formation between multivalent proteins. By measuring interactions in solution and applying comprehensive models, CG-MALS determines binding affinity and stoichiometry accurately, guiding the design and characterization of complex biomolecular assemblies.

Reference

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