BPS2026 – Unveiling the thermodynamics of multivalent protein complexes: Insights into intra-domain interactions

Multivalent interactions mediated by multidomain proteins arise from a complex interplay of intermolecular and intramolecular contacts, making their thermodynamic interpretation inherently challenging. While intermolecular contributions can often be inferred from conventional binding analysis, the thermodynamics of intra-domain interactions have remained particularly elusive. In this study, we measured temperature-dependent kinetics of protein-peptide systems with valences spanning monomer to tetramer using surface plasmon resonance (SPR) and screened them with discrete multivalent scaffolds designed to minimize nonspecific binding. We then applied a refined error-guided fitting strategy and incorporated the resulting kinetic constants into a theoretical framework to disentangle inter- and intramolecular contributions. This integration enabled quantitative extraction of intra-domain thermodynamic parameters—including free energies and degeneracy coefficients—that had not been experimentally accessible. Our results demonstrate an integrated experimental-theoretical approach to uncover hidden thermodynamic features relevant to protein design and engineering.