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Updated: Mar 30, 2026
Identifying Protein-protein Interaction Sites Using Peptide Arrays
Published on: November 18, 2014
Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site
Aaron P Yamniuk1, John A Newitt1, Michael L Doyle1
11 Bristol-Myers Squibb, Princeton, New Jersey 08540, USA; 2 Tokyo Institute of Technology, Yokohama 226-8503, Japan; 3 Google[x], Google Life Sciences, Mountain View, California 94043, USA; 4 SystaMedic, Incorporated, Groton, Connecticut 06340, USA; 5 Biosensor Tools LLC, Salt Lake City, Utah 84103, USA; 6 National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA; 7 Polaris Pharmaceuticals, Incorporated, San Diego, California 92121, USA; and 8 Institute for Bioscience and Biotechnology Research, Fischell Department of Bioengineering, University of Maryland, Rockville, Maryland 20850, USA.
Abstract:
A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.
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