LARGEMS · The Dynamic Composition of Protein Complexes: A New Perspective in Structural Biology

80% of the proteome exists in complexes or large macromolecular assemblies. It is accepted that revealing the structure of these protein complexes is a key towards mechanistic understanding of cellular processes. Yet, this might not be sufficient; a higher level of complexity probably exists and protein complexes may not be static and uniform in form and function as thought. A protein complex may actually represent an ensemble of compositionally distinct entities with functional versatility. My main aim is to provide evidence for this conceptual change and to reveal the dynamic architecture of a protein assembly. As a model system, I will investigate the COP9 signalosome (CSN), an evolutionary conserved multisubunit complex, which is involved in a variety of essential functions ranging from cell-cycle progression, DNA-repair and apoptosis. My strategy is based on a comprehensive approach, made up of four main steps; i) Revealing the structural organization of the native complex. ii) Establishing whether the complex has co-existing independent modules that function separately of, or coordinately with the holocomplex. iii) Monitoring in real-time the biogenesis and activation pathway of the complex and developing an approach for shifting its oligomerization equilibrium. iv) Determining the correlation between modularity of the complex and cell cycle progression and comparing its composition in healthy versus cancerous cells. I will integrate genetic, biochemical and structural biology approaches. In particular, I will apply a state of the art mass spectrometry technique, that will enable us to define the stoichiometry, subunit composition, dynamic interactions and structural organization of protein complexes isolated directly from the cellular environment.