Monitoring and Visualizing Protein Societal Behavior in the Cell; an Integrative Approach

Cellular processes underlying health and disease are regulated by the cell through a diverse set of mechanisms, including protein-protein interactions and protein post translation modifications (PTMs), like protein phosphorylation or ubiquitination. Protein-complex assembly is one of the key drivers of the highly dynamic and complex social behavior between all molecules within the cell. Many unique protein complexes have been discovered through elaborate and detailed biochemical investigations, which have yielded surprising biological insights, but also valuable biochemical tools such as the PCR reaction and CRISPR/Cas gene editing (with a pioneering role of co-applicant van der Oost). Their study in an unbiased fashion through a genuinely integrative approach, which is at the core of this proposal, is still challenging, but now timely as each of the technologies has sufficiently progressed to make it all possible. The main goal of this proposal is to develop an integrated approach to characterize proteins and their assemblies by a combination of analytical and biophysical techniques with integrative structural modelling strategies. Distinctively, our novel workflow will combine data from the rapidly developing fields of cryo-electron tomography (cryo-ET), native MS, top-down proteomics and crosslinking mass spectrometry (XL-MS) to uncover protein complexes in an unbiased fashion, ideally in their native state and ultimately in situ. For the first time, all such diverse data streams will be combined through integrative structural modelling approaches that will allow to generate improved and novel 3D models of protein assemblies. The resulting integrated pipeline will be applied to the study of protein complexes in the thermophiles Pyrococcus furiosus and Thermus thermophilus. This proposal has the following main aims: (1) Developments of new workflows to analyze proteoforms and protein complexes in their native state by mass spectrometry, led by the group of Heck (UU). (2) Development of cryo-electron tomography (cryo-ET) and analytical workflows optimized to facilitate cartography of the protein organization in situ, led by the group of Förster (UU). (3) The development of XL-MS, to analyze protein complex formation in situ to derive distance restraints useful for structural modeling, led by the group of Scheltema (UU). (4) The development of software tools for the integration of data generated from all above approaches to guide the modelling of protein assemblies, led by the group of Bonvin (UU). Our integrative approach will be applied to study protein networks in the thermophiles P. furiosus and T. thermophilus, which will be led by the group van der Oost (WUR). The reasons for selecting these model systems are manifold: 1) improved understanding of the structural organization of these 3-billion-year-old bacteria and archaea will assist us in understanding the origin of eukaryotic life, 2) their limited size of their genome/proteome will make our studies more comprehensive, 3) the robustness of their enzymes under harsh conditions may find broad application in biotechnology, 4) and their culture conditions and coccoid morphology allow us to monitor and visualize the societal behavior of all proteins within the cell. Through secondments and training sessions we will share the successful outcomes of the work and train PhDs, PD, and Master students from UU and WUR, and encourage interactions with the industrial partners TFS and PM. A summer school will be organized to disseminate results to a broader group of scientists. The project will speed up developments in MS and/or EM, data analysis and structural modelling software. The resulting integrated workflow will allow us to ultimately tackle the complex environment of the human organelles and even the whole cell.