Periodic Reporting for period 5 - SPOCkS MS (Sampling Protein cOmplex Conformational Space with native top down Mass Spectrometry)
Reporting period: 2024-01-01 to 2024-09-30
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.
Objectives:
- 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
Objectives:
- 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
Over the entire project period, work focussed mainly on objectives two and four. Due to the COVID-19 pandemic, the focus shifted further to investigating especially coronaviral protein complexes using mainly the basic, unmodified mass spectrometric setup.
Different components to modify an orbitrap UHMR have been obtained and tested. Multi-stage fragmentation and MS^N is now enabled by an omnitrap interfaced with the commercial system. This allows for slow CID, ECD and UVPD fragmentation. The setup will be further explored in follow-up projects and represents a unique resource for native top-down MS.
Main efforts in the project have focussed on assessing soft X-rays at FLASH and FLASH II free-electron lasers and PETRA III synchrotron for efficient fragmentation and dissociation of standard proteins and protein complexes. The results show potential for the methodology especially in combination with conformational separation in ion mobility.
The main biological question of SPOCk’S MS is how coronaviral replication/transcription complexes assemble and function and thereby facilitate replication of coronaviruses (CoVs). In a first publication (Krichel et al 2020), polyprotein processing of the regulatory region nsp7-10 through the viral protease in SARS-CoV-1 was monitored by native MS assessing processing order and efficiency of the three cleavage sites. Crucially, complex formation of released nsp7 and nsp8 could be observed simultaneously showing a heterotetramer, of which the topology was deduced. The complex was examined further in a follow-up (Krichel et al. 2021). The study revealed two distinct assembly pathways for nsp7+8 complexes resulting in alternate stoichiometry and topology in different CoVs. Processing was then also compared for nsp7-11 (the full intermediate observed in cells) in four different CoVs, which were also included in the complexation study. While processing order is largely conserved, subtle differences exist and are also reflected upon assembly of released subunits to nsp16. Crucially, we could deduce rate constants for all four cleavage sites in parallel (Schamoni-Kast et al. 2024 bioRxiv).
Importantly, in light of the SARS-CoV-2 pandemic, efforts on the biological aspects of the project were intensified. Instead of SARS-CoV-1, we switched to production and analysis of SARS-CoV-2 proteins and then also compared to various human CoVs to deduce general principles. Moreover, we assisted in screening antiviral targeting the main viral protease and further mass spectrometric investigations of this emergent virus (Günther et al 2021). Additionally, we performed investigations on Lassa virus, which revealed insights into viral assembly (Sänger et al 2023).
Different components to modify an orbitrap UHMR have been obtained and tested. Multi-stage fragmentation and MS^N is now enabled by an omnitrap interfaced with the commercial system. This allows for slow CID, ECD and UVPD fragmentation. The setup will be further explored in follow-up projects and represents a unique resource for native top-down MS.
Main efforts in the project have focussed on assessing soft X-rays at FLASH and FLASH II free-electron lasers and PETRA III synchrotron for efficient fragmentation and dissociation of standard proteins and protein complexes. The results show potential for the methodology especially in combination with conformational separation in ion mobility.
The main biological question of SPOCk’S MS is how coronaviral replication/transcription complexes assemble and function and thereby facilitate replication of coronaviruses (CoVs). In a first publication (Krichel et al 2020), polyprotein processing of the regulatory region nsp7-10 through the viral protease in SARS-CoV-1 was monitored by native MS assessing processing order and efficiency of the three cleavage sites. Crucially, complex formation of released nsp7 and nsp8 could be observed simultaneously showing a heterotetramer, of which the topology was deduced. The complex was examined further in a follow-up (Krichel et al. 2021). The study revealed two distinct assembly pathways for nsp7+8 complexes resulting in alternate stoichiometry and topology in different CoVs. Processing was then also compared for nsp7-11 (the full intermediate observed in cells) in four different CoVs, which were also included in the complexation study. While processing order is largely conserved, subtle differences exist and are also reflected upon assembly of released subunits to nsp16. Crucially, we could deduce rate constants for all four cleavage sites in parallel (Schamoni-Kast et al. 2024 bioRxiv).
Importantly, in light of the SARS-CoV-2 pandemic, efforts on the biological aspects of the project were intensified. Instead of SARS-CoV-1, we switched to production and analysis of SARS-CoV-2 proteins and then also compared to various human CoVs to deduce general principles. Moreover, we assisted in screening antiviral targeting the main viral protease and further mass spectrometric investigations of this emergent virus (Günther et al 2021). Additionally, we performed investigations on Lassa virus, which revealed insights into viral assembly (Sänger et al 2023).
X-ray induced fragmentation has potential for native top-down MS that warrants further investigations and setup optimization. However, our data suggests that its combination with other fragmentation techniques will be most successful to achieve desired analytical depth and unearth the full potential of native top-down MS. Such combined fragmentation will be facilitated by our setup including the omnitrap.
Obtaining rate constant for multi-reaction kinetics as in polyprotein processing is extremely challenging and to our knowledge has not been achieved at this precision before. Moreover, we monitor in real time intact substrates, intermediates, products and subsequently formed assemblies. Thereby a complete picture emerges as opposed to using peptide surrogates covering individual cleavage sites. Comparing processing and complexation in different CoVs and assessing formation of chimeric species, instead of just looking at pandemic strains, will aid understanding evolutionary and fitness aspects.
Obtaining rate constant for multi-reaction kinetics as in polyprotein processing is extremely challenging and to our knowledge has not been achieved at this precision before. Moreover, we monitor in real time intact substrates, intermediates, products and subsequently formed assemblies. Thereby a complete picture emerges as opposed to using peptide surrogates covering individual cleavage sites. Comparing processing and complexation in different CoVs and assessing formation of chimeric species, instead of just looking at pandemic strains, will aid understanding evolutionary and fitness aspects.