A Novel Crosslinking Strategy for MS Structural Biology

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.

Integrative structural biology seeks to compute models of multicomponent protein assemblies using proximity constraints derived from a variety of data sets including chemical cross-linking mass spectrometry [XL-MS].
MS, reaction protocols, and XL-MS spectral analysis algorithms have advanced, such that XL-MS is now a key technique, particularly in situations that have proved challenging by other methods. Despite such developmental strides, XL-MS has struggled to come of age and current application is often limited to specialist labs. This is chiefly because XL-MS poses several technical challenges. This project aims to tackle the current limiting issues:
1. Enrichment of low abundance cross-linked peptide species from highly complex protease digests of cross-linked protein-protein interaction partners.
2. Circumvention of the analytical challenges currently associated with chromatographic analysis of high charge-state, high-mass cross-linked peptide species.
3. Provision of a means to discriminate between desired and undesired cross-linked peptide species.
4. Enablement of interpretation of complex MS/MS fragmentation spectra of cross-linked species using universal proteomic identification tools.
These issues will be addressed herein through development of a novel class of cross-linking reagents which will enable, post-digestion enrichment, detection of the correct end-products and automated LC/MS analysis. This will result in a new, easy to use, scientifically powerful XL-MS approach.

Our aim here is to demonstrate a significant improvement in the performance of the cross-linking mass spectrometry technique, with the ultimate aim of new tool provision. This research project however is aimed not only at improving the methodology of cross-linking mass spectrometry in the short-term, but at enabling significant advancement in our scientific understanding of protein-protein interactions in the longer-term, through the application of our gained knowledge and dissemination of our developed tools and protocols.

As such, the immediate impact of this work will be of benefit to the following different groups:
1. First is the impact on the wider scientific community of this new strategy, improving the quality and availability for scientists of key information about important proteome interactions. The ability to access this information, will both enhance our understanding and inform new experiments; as well as giving the UK research base a competitive edge and enhancing its international reputation.
2. Secondly the development of a significantly improved technology platform for interaction analysis will benefit not just UK researchers, but will also be widely adopted by many groups in the international biological sciences community where there is a need to understand one of the many cellular processes that are mediated by stable protein complexes.
3. With regard to UK business, this technology will appeal to many researchers in the pharmaceutical, agrochemical and biologics industries where the analysis of protein interactions is required for the discovery of druggable protein targets. This is important in the development of biologically active small molecules with novel mode of action, such as drugs, pesticides and herbicides, critical to the economic success of these companies. The consequence of having access to tools accelerating scientific discovery and maximising the information attainable from studying protein interactions will improve the competitiveness of UK research based companies.
4. This proposal will give rise to primary IP with the potential for technology exploitation through licensing agreements, thereby providing a regional/national economic impact through engagement and/or collaboration with industrial partners, e.g. commercialisation of tools, reagents and techniques. [See attached letters of interest].

Finally, this technology tool has the potential to offer significant insight into basic biological interaction mechanisms relevant to understanding the aetiology of, or the development of treatments for many common serious diseases or conditions, with the consequent potential impact to enhance the quality of health/life for everyone.