Structure, dynamics and causality in protein interaction networks

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Problem and Hypothesis: The importance of protein-protein interactions (PPIs) to biology is reflected in the enormous efforts to study them on a large scale, including our own efforts, which resulted in a first description of a PPI network in vivo (Tarassov, et al. Science, 2008). In the past funding period we have developed and validated a novel approach to study dynamics of biochemical networks and to determine protein functions, discovery of pathways and a novel mechanism of allostery. The method is based on measuring dynamics of homomeric protein complexes (HPC) on a large scale.***Objectives: Our specific aims are: Aim 1., to construct high-quality, mechanism-based sentinels of signal transduction and metabolic pathways based on HPC and analysis of underlying mechanisms of HPC dynamics. Aim 2. To use HPC analysis and mechanism-based HPC sentinels to predict signaling and metabolic network topology under specific nutrient limitation. Aim 3. To extend HPC dynamics to multiple perturbations and prediction of organization of biochemical networks in general. ***Methodologies: In addition to the DHFR PCA strain array described above, we will dissect the physical basis of changes in HPCs using mass spectroscopy, GFP fusions to the proteins to determine localization and abundance and a novel infrared fluorescent protein PCA to determine changes in subcellular localization of HPC and confocal microscopy. This will result in an array of HPCs with known underlying spatiotemporal changes in proteins complexes or abundance that we call HPC "sentinels" We will further apply a pipeline of analysis tools, largely based on existing strategies, to interpret HPC dynamics on specific signaling and metabolic pathways. ***This study will contribute to advancement of knowledge by: ***1- Providing a strategy to directly probe the dynamics of biochemical networks in vivo. ***2- Providing a complement to existing genetic and mass-spectroscopic strategies for determining gene function. ***3 - Revealing new principles and mechanisms of dynamic biochemical network organization. ***4 - Revealing novel functions of genes for which function is presently unknown.***5 - Contributing towards solving the mystery of the principles of formation and evolution of the cellular machinery, on which we have already made some progress. **