PhD defence: Structural characteristics of mitochondrial protein assemblies probed by mass spectrometry

Most biological processes that maintain cellular life depend on interacting proteins, which are forming complexes that correlate with the type of biological processes they are involved in. To understand these processes, X-ray crystallography, NMR spectroscopy, and cryogenic electron microscopy are commonly used to structurally characterize such protein assemblies. However, a common drawback of these methods is that they require a highly purified sample, hampering the characterization of protein complexes in-vivo.

In this thesis the versatility of cross-linking mass-spectrometry (XL-MS), as complementary method for structural biology is explored, with the aim to characterize protein assemblies in-vivo. We demonstrate that XL-MS enables the characterization of protein complexes in their naive cellular environment.

However, conventional XL-MS workflows present drawbacks that significantly hamper a full structural characterization. We therefore explored how coupling XL-MS with other technologies can improve the structural characterization of protein complexes. By combining XL-MS and complexome profiling we were able to uncover the macromolecular organization of the mitochondrial interactome and to characterize several multi-protein assemblies in detail. Moreover, we demonstrate that XL-MS especially in combination with cryoEM sub-tomographic averaging and computer-aided structural modeling provides improved methodologies to explore the structure-function relationships of macromolecular complexes in vivo.