A Novel Crosslinking Strategy for MS Structural Biology

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Proteins may be thought of as the essential 'factories' within living cells. They are intricate and complex in construction, and carry out a variety of functions essential for the existence of life. Understanding the inner workings of these factories requires knowledge of how they are assembled, both at the atomic level and at the level of higher-order multi-complex protein entities that perform essential functions, such as respiration and cell replication. Protein factories are dynamic structures, responding to input signals through interactions with other molecules, most frequently other proteins. Knowing what happens during the processing of signals and how output responses are generated relies on knowledge of both the protein 3D structure and any dynamic changes that take place to it at the molecular-level during such events.A deeper understanding of how one protein recognises and binds to another protein in order to regulate its function underpins most of the biological activity in living cells and is therefore crucial to how we may go about designing new molecules to prevent or enhance such interactions. Any advancement in our understanding of protein-protein interactions has the potential to widen the number of targets for modifying biological function by the use of drugs. Such knowledge may help to accelerate the development of new molecular medicines to treat most of our major serious diseases, as these often manifest themselves through protein interaction pathways. Structural biology is concerned with the study of protein 'shape' and how alterations in shape affect function. Structures ranging from individual proteins to large multicomponent cellular assemblies are studied. This challenging problem requires the integration of many different biophysical and biochemical protein analysis techniques. One comparatively recent technique to be applied to the problem of determining the spatial relationships between proteins in a complex or in close proximity through a binding event is the use of chemical cross-linking followed mass spectrometry analysis (XL-MS).Chemical cross-linking seeks to 'freeze' the 3D arrangement of protein chains in a complex by tethering them together using a reagent that forms a strong covalent link between adjacent regions, provided they fall within the distance of the span of the cross-linker. The covalently-linked regions of the proximal protein chains can then be excised from the protein backbone using an enzyme and the cross-linked complex carrying residues of both parent proteins analysed using mass spectrometry (MS). Modern MS can break these linked fragments into smaller pieces and determine their constituent amino acid residues. Using clever software algorithms we can decipher not only the identity of the two linked proteins that were in close proximity, but also ID the actual sites of linkage. This increases the resolution of this method for structure determination from the level of large protein domains down to even smaller sections, dubbed ''peptide-level resolution''. Better resolution in turn permits us to build better, more accurate models of the structures of multi-protein complexes.Unfortunately XL-MS in its present format exhibits a number of weaknesses. Current cross-linkers in general use are non-cleavable. This gives rise to large linked complexes upon excision that are tricky to analyse by MS due to their size and complex fragmentation patterns. Also XL-MS reagents tend not to react efficiently, making the linked peptides difficult to detect in protein digests.To tackle these current limiting issues we propose the introduction of an entirely new innovative cross-linking and MS analysis strategy that involves developing a novel class of cross-linking reagents which will allow detection of the linked peptides combined with simple automated MS data analysis.