title: Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.
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.
authors.affiliation: 1 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.
authors.forename: Aaron P
authors.initials: AP
authors.lastname: Yamniuk
authors.affiliation: 1 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.
authors.forename: John A
authors.initials: JA
authors.lastname: Newitt
authors.affiliation: 1 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.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Doyle
authors.affiliation: 1 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.
authors.forename: Fumio
authors.initials: F
authors.lastname: Arisaka
authors.affiliation: 1 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.
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Giannetti
authors.affiliation: 1 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.
authors.forename: Preston
authors.initials: P
authors.lastname: Hensley
authors.affiliation: 1 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.
authors.forename: David G
authors.initials: DG
authors.lastname: Myszka
authors.affiliation: 1 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.
authors.forename: Fred P
authors.initials: FP
authors.lastname: Schwarz
authors.affiliation: 1 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.
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
authors.affiliation: 1 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.
authors.forename: Edward
authors.initials: E
authors.lastname: Eisenstein
mesh_terms: D000595:Amino Acid Sequence; D019540:Area Under Curve; D001426:Bacterial Proteins / Q000737:chemistry; D004722:Endoribonucleases / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; C057438:barstar protein, Bacillus amyloliquefaciens; D004722:Endoribonucleases; C022543:ribonuclease T(2)
keywords: analytical ultracentrifugation; isothermal titration calorimetry; surface plasmon resonance