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Sampling Protein cOmplex Conformational Space with native top down Mass Spectrometry

Periodic Reporting for period 4 - SPOCkS MS (Sampling Protein cOmplex Conformational Space with native top down Mass Spectrometry)

Reporting period: 2022-07-01 to 2023-12-31

The main question to be addressed by SPOCk’S MS is how protein complex conformation adapts to local changes, such as processing of polyproteins, protein phosphorylation or conversion of substrates. While labelling strategies combined with mass spectrometry (MS), such as hydrogen deuterium exchange and hydroxyl footprinting, are very versatile in studying protein structure, these techniques are employed on bulk samples averaging over all species present. SPOCk’S MS will remedy these by studying the footprinting and therefore exposed surface area on conformation and mass selected species. Labelling still happens in solution avoiding gas phase associated artefacts. The labelling positions are then read out using newly developed top-down MS technology. Ultra-violet and free-electron lasers will be employed to fragment the protein complexes in the gas phase. In order to achieve the highest possible sequence and thus structural coverage, lasers will be complemented by additional dissociation and separation stages to allow MS^N. SPOCk’S MS will allow sampling conformational space of proteins and protein complexes and especially report about the transient nature of protein interfaces. Constraints derived in MS will be fed into a dedicated software pipeline to derive atomistic models. SPOCk’S MS will be used to study intracellular viral protein complexes, especially coronaviral replication/transcription complexes, which are highly flexible and often resist crystallisation and are barely accessible by conventional structural biology techniques.
- Integrate labelling with complex species selective native MS for time-resolved structural studies
- Combine fragmentation techniques to maximise information content from MS
- Develop software suite to analyse data and model protein complex structures based on MS constraints
- Apply SPOCk’S MS to protein complexes of human pathogenic viruses
Components for the instrumental developments have been obtained and separate benchmarking especially of the main mass spectrometer and the ion mobility device are ongoing. Moreover, the shape of the spray tips is systematically investigated to facilitate automation and online labelling.
Main efforts in the reporting period have focussed on assessing soft X-rays at the FLASH free-electron laser and PETRA III synchrotron for efficient fragmentation and dissociation of standard proteins and protein complexes. Data evaluation from four measurement campaigns has been completed showing potential for the methodology and suggesting that an optimized setup as foreseen in the project will likely be beneficial. The results are being prepared for publication.
The main biological question of SPOCk’S MS is how coronaviral replication/transcription complexes assemble and function and thereby facilitate replication of SARS-CoV. In a first publication (Krichel et al 2020), polyprotein processing of the regulatory region nsp7-10 through the viral protease was monitored by native mass spectrometry assessing processing order and efficiency of the three cleavage sides. Crucially, complex formation of two released proteins nsp7 and nsp8 could be observed simultaneously showing a heterotetramer, which topology was deduced. The study has been extended to other coronaviruses revealing two distinct assembly pathways for nsp7+8 complexes resulting in alternate stoichiometry and topology (
Importantly, in light of the SARS-CoV-2 pandemic, efforts on the biological aspects of the project were intensified. Instead of SARS-CoV, we switched to production and analysis of SARS-CoV-2 proteins. Several proteins are available now for more in depth analysis of the replication/transcription complex. Moreover, we assisted in screening antiviral targeting the main viral protease ( which identified several lead compounds, and further mass spectrometric investigations of this emergent virus.
X-ray induced fragmentation has a potential for top-down mass spectrometry that warrants further investigations and setup optimization. However, our data suggests that its combination with other fragmentation techniques will be most successful to achieve the project’s object and unearth the full potential of native top-down mass spectrometry. Although the instrumental developments are a bit delayed, it is still expected that the proposed new methodology can be developed.
With the current progress, we expect that functional coronaviral replication/transcription complexes comprising at least the soluble parts will be at reach before the end of the project. It will important to not only focus on SARS-CoV-2 and compare to other coronaviruses that cause severe disease in animals and deduce whether similar strategies can be employed to inhibit their replication.