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Protein Science (2007), 16:30-42. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: Application to adaptor protein complexes in cell signaling

Jon C.D. Houtman1, Patrick H. Brown2, Brent Bowden3, Hiroshi Yamaguchi4, Ettore Appella4, Lawrence E. Samelson3, and Peter Schuck2

1 Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa 52242, USA
2 National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA
3 Laboratory of Cellular and Molecular Biology, National Institutes of Health, Bethesda, Maryland 20892, USA
4 Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

(RECEIVED September 13, 2006; FINAL REVISION October 16, 2006; ACCEPTED October 16, 2006)

Multisite interactions and the formation of ternary or higher-order protein complexes are ubiquitous features of protein interactions. Cooperativity between different ligands is a hallmark for information transfer, and is frequently critical for the biological function. We describe a new computational platform for the global analysis of isothermal titration calorimetry (ITC) data for the study of binary and ternary multisite interactions, implemented as part of the public domain multimethod analysis software SEDPHAT. The global analysis of titrations performed in different orientations was explored, and the potential for unraveling cooperativity parameters in multisite interactions was assessed in theory and experiment. To demonstrate the practical potential and limitations of global analyses of ITC titrations for the study of cooperative multiprotein interactions, we have examined the interactions of three proteins that are critical for signal transduction after T-cell activation, LAT, Grb2, and Sos1. We have shown previously that multivalent interactions between these three molecules promote the assembly of large multiprotein complexes important for T-cell receptor activation. By global analysis of the heats of binding observed in sets of ITC injections in different orientations, which allowed us to follow the formation of binary and ternary complexes, we observed negative and positive cooperativity that may be important to control the pathway of assembly and disassembly of adaptor protein particles.

Keywords: ITC; multiprotein complexes; cooperativity; signal transduction; adaptor protein; Sos1; LAT; Grb2; protein interactions; reversible associations


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