Protein-RNA and protein-protein interaction in the immune defense system of prokaryotes

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Mass spectrometry (MS) and its current applications address various questions of high relevance for cellular and structural biology. These applications include not only proteomic approaches, but also investigations of protein¿protein and protein¿ligand interactions, and protein structures. Here we will apply MS to various projects revolving around the immune defence system of prokaryotes, including protein-protein, protein-RNA, protein-DNA crosslinking of various CRISPR-Cas complexes, as well as to studies that monitor quantitative changes of prokaryotic proteomes upon phage invasion. The main goals of this project in a second funding period are to expand our MS-based investigations from the single protein-RNA complexes that we targeted during the first funding period to entire protein-protein, protein-RNA and protein-DNA complexes, which will enable a realistic structural modelling of these molecular machines. Mass spectrometry-based crosslinking approaches will supply us with sets of spatial constraints to allow for molecular modelling of the complexes, especially where structural information acquired by e.g. X ray crystallography is not, or only partially available. Furthermore, the methods that we have developed during the first funding period will allow us to apply our protein-RNA crosslinking approach to in vivo samples, i.e. to entire prokaryotic cells in order to identify protein-RNA contact sites in living cells. By relating the crosslinking results to quantitative proteome studies of prokaryotes under phage invasion, we aim to develop a detailed understanding of how protein-RNA(DNA) interactions in the CRISPR-Cas system contribute to the integration of the phage genome into the host genome.