Houtman Jon C D, Brown Patrick H, Bowden Brent, Yamaguchi Hiroshi, Appella Ettore, Samelson Lawrence E, Schuck Peter
Department of Microbiology, University of Iowa, Iowa 52242, USA.
Protein Sci. 2007 Jan;16(1):30-42. doi: 10.1110/ps.062558507.
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.
多位点相互作用以及三元或更高级别蛋白质复合物的形成是蛋白质相互作用中普遍存在的特征。不同配体之间的协同作用是信息传递的标志，并且通常对生物学功能至关重要。我们描述了一个新的计算平台，用于对等温滴定量热法（ITC）数据进行全局分析，以研究二元和三元多位点相互作用，该平台作为公共领域多方法分析软件SEDPHAT的一部分得以实现。我们探索了对以不同方向进行的滴定进行全局分析，并在理论和实验上评估了在多位点相互作用中解析协同参数的潜力。为了证明ITC滴定全局分析在研究协同多蛋白相互作用方面的实际潜力和局限性，我们研究了三种对T细胞活化后信号转导至关重要的蛋白质LAT、Grb2和Sos1之间的相互作用。我们之前已经表明，这三种分子之间的多价相互作用促进了对T细胞受体活化重要的大型多蛋白复合物的组装。通过对在不同方向的ITC注射组中观察到的结合热进行全局分析，这使我们能够追踪二元和三元复合物的形成，我们观察到了可能对控制衔接蛋白颗粒组装和解聚途径很重要的负协同作用和正协同作用。
