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Nearly a decade ago the Molecular Interactions Research Group (MIRG) of the ABRF sought to develop a relatively simple model system for the quantitative analysis of biomolecular interactions of proteins and their ligands. Ideally, the components would be sufficiently discrete to provide an instructional system for the quantitative analysis of association reactions, but also robust enough to permit the estimation of binding constants over a wide range of affinity for more demanding analyses. For several reasons MIRG selected the Bacillus amyloliquefaciens extracellular ribonuclease barnase and its intracellular inhibitor barstar as a model. Both barnase and barstar are small, monomeric, highly soluble proteins that have been extensively studied as models for protein folding and protein interactions. The binding of wild-type barstar with wild-type barnase is extremely tight, with a dissociation constant estimated to be approximately 6 × 10-14 M, yet there many variants of barnase and barstar with substantially reduced affinity, with observed binding constants as low as the mM range. MIRG investigators found good agreement for the strength and stoichiometry of the barnase-barstar interactions using several biophysical approaches with a number of variants, lending support to their use as reference reagents for molecular interactions. In an effort to develop a more complex, challenging system, a bivalent variant of barnase was constructed that incorporated amino acid substitutions that rendered two nonequivalent barstar binding sites. The construction and utility of this system for biomolecular interactions will be presented.