The research in the Allison Lab is primarily interested in how proteins do their jobs, the myriad of structural forms and interactions that affect their functioning, and by using structure-function relationships as a scaffold, we are interested in how this function can be tuned to yield desirable functional features. This involves studying both soluble and membrane proteins, with a particular emphasis on studying enzymes. Alongside the proteinaceous research, by studying the fundamentals of the experimental techniques we commonly use, and developing supporting software to expedite and enable new analyses, we try to uncover new and enabling methodologies.
Proteins in their simplest sense are composed of folded chains of basic building blocks called amino acids. However, these amino acids can be covalently modified, consequently generating the potential for huge diversity in protein structure. Post-translational modification occurs after protein biosynthesis, and most commonly involves the enzymatic addition of different functional groups. These may include hydrophobic groups for membrane localization, cofactors for enhancement of enzymatic activity, or the addition of a variety of different smaller chemical groups. These modifications can commonly cause differences in protein behaviour, for example they can affect stability, cellular lifetime, and of course function, but these effects are poorly understood both generally, and for specific proteins.
Protein glycosylation is a form of co-translational and post-translational modification whereby enzymes attach a carbohydrate to a glycosyl acceptor on a protein. Glycolsylations are classified depending on the site of attachment, with attachment to the nitrogen of the amino acid asparagine (or arginine side-chains) referred to as N-linked glycosylation. In this case, the glycan that is attached almost exclusively derives from a precursor in which the glycan is attached to a special lipid molecule. The glycans that are attached to protein are species and strain specific, and in part, for this reason protein glycosylation is fundamentally important for cellular recognition, such as those involved in host-pathogen interactions and immune responses.
The genomic DNA of an organism indirectly encodes glycan structure and under this notion we are interested in furthering our understanding of how the glycans are built by glycosyltransferases on their lipid-linked precursor molecules. As a glycan is being built, how are the two substrates (the growing glycan attached to a lipid, and the carbohydrate to be added) recognised? How is processitivity controlled? Are different strategies employed at different stages of glycan biosynthesis? Can we predict from sequence or structure the substrate specificity? How readily can glycan assembly be engineered to produce glycans with specific and desirable structure? How have different organisms been able to generate such diversity in their glycans? We study these ideas using a plethora of different biochemical tools and analyses, and have particular expertise in the application of mass spectrometry and crystallography approaches.
Native mass spectrometry is a form of structural proteomics interested in the characterisation of protein structure and interactions. It is a type of mass spectrometry where non-covalent interactions of the analyte are maintained during analysis, which is distinct from all other methodologies using the same instrumentation. This enables us to measure, for example, the oligomeric state of proteins, protein-ligand binding, and other similar interactions. It is almost unique in that rather than being an ensemble technique, it can detect the populations within a sample.
We use native mass spectrometry to optimise protein purifications, study oligomeric state and protein-protein interactions, and the interaction of substrates and ligands with the proteins we study. But we are also interested in method development, and furthering our understanding of how macromolecules behave in the gas phase. We also develop software tools, that are used for the analysis of ion mobility-mass spectrometry data.
The lab houses a Waters Synapt XS ion mobility-mass spectrometer, equipped with ETD and a 32k quadrupole, configured for offline nano-electrospray ionisation, for which we fabricate custom glass capillary emitters. We also have a Waters ACQUITY UPLC M-Class that enables LC-MS workflows.
See the Resources page for some of the software we develop.
Protein crystal structures
The protein models we create through protein crystallography (neat videos explaining the technique (Part 1, Part 2) from Elspeth Garman, University of Oxford) are all deposited in the Protein Data Bank. View all our structures in the Protein Data Bank or follow the individually linked PDB codes below to view each structure.
- Sahin, C., Österlund, N., Leppert, A., Johansson, J., Marklund, E. G., Benesch, J. L. P., Ilag, L. L., Allison, T. M., Landreh, M. (2021) Ion mobility-mass spectrometry shows stepwise protein unfolding under alkaline conditions. Chem. Commun. 57 1450–1453.
- Allison, T. M., Barran, P., Cianferani, S., Degiacomi, M. T., Gabelica, V., Grandori, R., Marklund, E. G., Menneteau, T., Migas, L. G., Politis, A., Sharon, M., Sobott, F., Thalassinos, K., Benesch, J. L. P. (2020) Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology. Anal. Chem. 92 10872–10880.
- Landreh, M., Shahin, C., Gault, J., Sadeghi, S., Drum, C. L., Uzdavinys, P., Drew, D., Allison, T. M., Degiacomi, M. T., Marklund, E. G. (2020) Predicting the Shapes of Protein Complexes through Collision Cross Section Measurements and Database Searches. Anal. Chem. 92 12297–12303.
- Allison, T. M., Barran, P., Benesch, J. L. P., Cianferani, S., Degiacomi, M. T., Gabelica, V., Grandori, R., Marklund, E. G., Menneteau, T., Migas, L. G., Politis, A., Sharon, M., Sobott, F., Thalassinos, K. (2020) Software Requirements for the Analysis and Interpretation of Native Ion Mobility Mass Spectrometry Data. Anal. Chem. 92 10881–10890.
- Bolla, J. R., Corey, R. A., Sahin, C., Gault, J., Hummer, A., Hopper, J. T. S., Lane, D. P., Drew, D., Allison, T. M., Stansfeld, P. J., Robinson, C. V., Landreh, M. (2020) A mass spectrometry-based approach to distinguish annular and specific lipid binding to membrane proteins. Angew. Chem. Int. Ed. Engl. 59 3523–3528.
- Allison, T. M., Agasid, M. T. (2020) Native Protein Mass Spectrometry. Protein Nanotechnology. Methods in Molecular Biology v.2073.
- Kaldmäe, M., Österlund, N., Lianoudaki, D., Sahin, C., Bergman, P., Nyman, T., Kronqvist, N., Ilag, L. L., Allison, T. M., Marklund, E. G., Landreh, M. (2019) Gas-phase collisions with trimethylamine-N-oxide enable activation-controlled protein ion charge reduction. J. Am. Soc. Mass Spectrom. 30 1385–1388.
- Collier, M. P., Alderson, T. R., de Villiers, C. P., Nicholls, D., Gastall, H. Y., Allison, T. M., Degiacomi, M. T., Jiang, H., Mlynek, G., Fürst, D. O., van der Ven, P. F. M., Djinovic-Carugo, K., Baldwin, A. J., Watkins, H., Gehmlich, K., Benesch, J. L. P. (2019) HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C. Sci. Adv. 5, eaav8421.
- Allison, T. M., Bechara, C. (2019) Structural mass spectrometry comes of age: new insight into protein structure, function and interactions. Biochem. Soc. Trans. 47, 317–327.
- Allison, T. M., Landreh, M. (2019) Ion Mobility in Structural Biology. Comprehensive Analytical Chemistry 83, 161–195.
- Gault, J., Lianoudaki, D., Kaldmäe, M., Kronqvist, N., Rising, A., Johansson, J., Lohkamp, B., Laín, S., Allison, T. M., Lane, D. P., Marklund, E. G., Landreh, M. (2018) Mass spectrometry reveals the direct action of a chemical chaperone. J. Phys. Chem. Lett. 9 4082–4086.
- Yewdall, N. A., Allison, T. M., Pearce, F. G., Robinson, C. V., Gerrard, J. A. (2018) Self-assembly of toroidal proteins explored using native mass spectrometry. Chem. Sci. 9 6099–6106.
- Liko, I., Degiacomi, M. T., Lee, S., Newport, T. D., Gault, J., Reading, E., Hopper, J. T. S., Housden, N. G., White, P., Colledge, M., Sula, A., Wallace, B. A., Kleanthous, C., Stansfeld, P. J., Bayley, H., Benesch, J. L. P., Allison, T. M., Robinson, C. V. (2018) Lipid binding attenuates channel closure of the outer membrane protein OmpF. Proc. Natl. Acad. Sci. U.S.A. 115 6691–6696.
- Hochberg, G. K. A., Shepherd, D. A., Marklund, E. G., Santhanagoplan, I., Degiacomi, M. T., Laganowksy, A., Allison, T. M., Basha, E., Marty, M. T., Galpin, M. R., Struwe, W. B., Baldwin, A. J., Vierling, E., Benesch, J. L. P. (2018) Structural principles that enable oligomeric small heat-shock protein paralogs to evolve distinct functions. Science 359 930–935.
- Bolla, J. R., Sauer, J. B., Wu, D., Mehmood, S., Allison, T. M., Robinson, C. V. (2018) Direct observation of the influence of cardiolipin and antibiotics on lipid II binding to MurJ. Nat. Chem. 10 363–371.
- Yen, H., Hopper, J. T. S., Liko, I., Allison, T. M., Zhu, Y., Wang, D., Stegmann, M., Mohammed, S., Wu, B., Robinson, C. V. (2017) Ligand binding to a G protein-coupled receptor captured in a mass spectrometer. Sci. Adv. 3 e1701016.
- Hopper, J. T. S., Ambrose, S., Grant, O. C., Krumm, S., Allison, T. M., Degiacomi, M. T., Tully, M. D., Pritchard, L. K., Ozorowski, G., Ward, A. B., Crispin, M., Doores, K. J., Woods, R. J., Benesch, J. L. P., Robinson, C. V., Struwe, W. B. (2017) The tetrameric plant lectin BanLec neutralises HIV through bidentate binding to specific viral glycans. Structure 25 773–782.
- Liko, I., Allison, T. M., Hopper, J. T. S., Robinson, C. V. (2016) Mass spectrometry guided structural biology. Curr. Opin. Struct. Biol. 40 136–144.
- Nazmi, A. R., Lang, E. J. M., Bai, Y, Allison, T. M., Othman, M. H., Panjikar, S., Arcus, V. L., Parker, E. J. (2016) Interdomain conformational changes provide allosteric regulation en route to chorismate. J. Biol. Chem. 291 21836–21847.
- Liko, I., Hopper, J. T. S., Allison, T. M., Benesch, J. L. P., Robinson, C. V. (2016) Negative ions enhance survival of membrane protein complexes. J. Am. Soc. Mass Spectrom. 27 1099–1104.
- *Allison T. M., *Landreh, M., Benesch, J. L. P., Robinson, C. V. (2016) Low charge and reduced mobility of membrane protein complexes has implications for calibration of collision cross section measurements. Anal. Chem. 88 5879–5884.
- Podobnik, M., Savory, P., Rojko, N., Kisovec, M., Wood, N., Hambley, R., Pugh, J., Wallace, J., McNeill, L., Bruce, M., Liko, I., Allison, T. M., Mehmood, S., Yilmaz, N., Kobayashi, T., Gilbert, R. J. C., Robinson, C. V., Jayasinghe, L., Anderluh, G. (2016) Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly. Nat. Commun. 7, 11598.
- Allison T. M., Reading, E., Liko, I., Baldwin, A. J., Laganowsky, A., Robinson C. V. (2015) Quantifying the stabilizing effects of protein-ligand interactions in the gas phase. Nat. Commun. 6, 8551.
- *Reading, E., *Liko, I., Allison T. M., Benesch, J. L. P., Laganowsky, A., Robinson, C. V. (2015) The role of the detergent micelle in preserving the structure of membrane proteins in the gas phase. Angew. Chem. Int. Ed. Engl. 54, 4577–4581.
- *Mehmood, S., *Allison T. M., Robinson C. V. (2015) Mass spectrometry of protein complexes: from origins to applications. Annu. Rev. Phys. Chem. 66, 453–474.
- Mehmood, S., Marcoux, J., Hopper, J. T, Allison, T. M., Liko, I., Borysik, A. J., Robinson, C. V. (2014) Charge reduction stabilizes intact membrane protein complexes for mass spectrometry. J. Am. Chem. Soc. 136, 17010–17012.
- *Laganowsky, A., *Reading, E., Allison, T. M., Ulmschneider, M. B., Degiacomi, M. T., Baldwin A. J., Robinson, C. V. (2014) Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510, 172–175.
- Cross, P. J., Pietersma, A. L., Allison, T. M., and Parker, E. J. (2013) Neisseria meningitidis expresses a single 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase that is inhibited primarily by phenylalanine. Protein Science 22, 1087–1099.
- Allison, T. M., Cochrane, F. C., Jameson, G. B., and Parker, E. J. (2013) Examining the role of intersubunit contacts in catalysis by 3-deoxy-d-manno-octulosonate 8-phosphate synthase. Biochemistry 52, 4676–4686.
- Cross, P. J., Allison, T. M., Dobson, R. C. J., Jameson, G. B., and Parker, E. J. (2013) Engineering allosteric control to an unregulated enzyme by transfer of a regulatory domain. Proc. Natl. Acad. Sci. U.S.A. 110 2111–2116.
- Allison, T. M., Hutton, R. D., Wanting, J., Gloyne, B. J., Nimmo, E. B., Jameson, G. B., and Parker, E. J. (2011) An extended b7a7 substrate-binding loop is essential for efficient catalysis by 3-deoxy-d- manno-octulosonate 8-phosphate synthase. Biochemistry 50, 9318–9327.
- Allison, T. M., Hutton, R. D., Cochrane, F. C., Yeoman, J. A., Jameson, G. B., and Parker, E.J. (2011) Targeting the role of a key conserved motif for substrate selection and catalysis by 3- deoxy-d-manno-octulosonate 8-phosphate synthase. Biochemistry 50, 3686–3695.
- Allison, T. M., Yeoman, J. A., Hutton, R. D., Cochrane, F. C., Jameson, G. B., and Parker, E. J. (2010) Specificity and mutational analysis of the metal-dependent 3-deoxy-d-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans. Biochim. Biophys. Acta, Proteins Proteomics 1804, 1526–1536.
* These authors contributed equally