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

质谱学(MS)及其目前的应用解决了与细胞和结构生物学高度相关的各种问题。这些应用不仅包括蛋白质组学方法，还包括蛋白质和蛋白质配体相互作用以及蛋白质结构的研究。在这里，我们将把MS应用于围绕原核生物免疫防御系统的各种项目，包括各种CRISPR-Cas复合体的蛋白质-蛋白质、蛋白质-RNA、蛋白质-DNA交联，以及监测原核蛋白质组在噬菌体入侵时的数量变化的研究。这个项目在第二个资助期的主要目标是将我们基于MS的研究从我们在第一个资助期瞄准的单一蛋白质-RNA复合体扩展到整个蛋白质-蛋白质、蛋白质-RNA和蛋白质-DNA复合体，这将使这些分子机器的现实结构建模成为可能。基于质谱学的交联方法将为我们提供一组空间约束，以允许对络合物进行分子建模，特别是在无法获得或仅部分获得X射线结晶学结构信息的情况下。此外，我们在第一个资助期开发的方法将使我们能够将我们的蛋白质-RNA交联法应用于活体样本，即整个原核细胞，以便确定活细胞中的蛋白质-RNA接触位点。通过将交联结果与噬菌体入侵下原核生物的定量蛋白质组研究联系起来，我们旨在详细了解CRISPR-Cas系统中的蛋白质-RNA(DNA)相互作用如何有助于噬菌体基因组整合到宿主基因组中。

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.