Senior Lecturer in Chemical Systems Biology
Mechanism of Membrane Transporters
Our work aims to provide a molecular description of the transport mechanism(s) of sugar transporters from the Major Facilitator Superfamily (MFS) using a novel combination of mass-spectrometry based methods and modelling. According to the widely acknowledged alternating-access model, MFS transporters have to adopt at least two conformations open to opposite sides of the membrane to allow vectorial translocation of their substrate. However, a molecular description of the mechanism underlying such transition is not well established and the actual models fail to integrate the role of the lipid environment. By combining reconstitution in different lipid environments, mutagenesis and modelling, we hope to derive a detailed conformational landscape of the transporters XylE and LacY in lipid bilayers. We use native mass spectrometry to identify with high-resolution the binding of lipid species, ion-mobility mass spectrometry to assess the global conformational changes and the variation in stability of the protein, and hydrogen-deuterium exchange to follow the structural dynamics.
Nucleic Acid Binding Proteins
Within living cells, proteins, nucleic acids and other substances physically interact to form complex macromolecular assemblies that control all cellular processes. Using hybrid mass spectrometry-based approaches, we aim to probe the conformational states and dynamics and understand how the cellular DNA and RNA binding protein machineries interact. We base our research on the DNA-repair complex HerA-NurA and La-related proteins (LARPs).
Mechanism of multi-drug efflux pump using hybrid-MS methodology
Drug resistance is a pertinent medical problem associated with drug effectiveness and antibiotic treatment. Multidrug efflux transporters pump toxic compounds out of the cell conferring resistance to antibiotics and other substances. The structure and dynamics of many of these transporters remain largely unknown using traditional structural approaches. Despite the realization that the lipids play a critical role for the structure and function of membrane proteins, the complexity of the lipid-protein interactions makes them challenging to characterize by conventional tools. Here, we use state-of-the-art hybrid-mass spectrometry to uncover the molecular level interactions of drugs with multidrug efflux pumps, enabling the exploration of their synergistic binding with lipids and to identify stable complex intermediates.
Antibody structure and function
Antibodies play a vital role in the regulation of the human immune system and are often considered as targets for treatment of severe diseases such as cancers, allergies and inflammatory diseases. As well as targets, antibodies are also a popular type of biotherapeutic for treatment of these diseases. Understanding antibody structure and function is imperative for the advancement of medical intervention. For these reasons, methods to better probe their conformational dynamics is of great interest.
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Firstly, our work aims to improve our understanding of how antibodies behave in the gas phase of a mass spectrometer. Secondly, to advance current mass spectrometry methods for probing the structure and function of antibodies. Ultimately, to apply these methods to improve our understanding of the structure and function of immunoglobulin E, a therapeutic target to combat asthma and allergies.
Integrative modelling
Although there is a wealth of structural information available for soluble proteins, a significant gap remains in the understanding of how protein structure relates to its function. The conformational dynamics of proteins are often poorly represented by traditional structure-deriving biophysical techniques, such as NMR (with size limitations) and X-ray crystallography (which may be time consuming and provides only a static snapshot of the most stable conformation).
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Within our group, we utilise a combination of mass spectrometry (MS) techniques, such as native MS, ion-mobility MS (IM-MS), chemical cross-linking MS (XL-MS), covalent labelling MS and hydrogen-deuterium exchange MS (HDX-MS). Structural information derived from these techniques, in combination with computational modelling, can be implemented in a restraint-guided modelling process.
Modelling of Membrane Proteins
Using the novel combination of both super resolution imaging (SRI) and hybrid mass spectrometry (MS) techniques, the structure, dynamics and transient interactions of membrane proteins can be elucidated. We aim to develop computational tools to integrate structural information from both SRI and MS-based sources into a computational workflow and produce models of membrane protein complexes which can describe the elusive conformational dynamics of these species. These data will provide the link between structural information yielded from MS, with the functional states of the complex found in vivo. So far, we base our work on a well characterized membrane protein dimer, UapA, a purine/H+ symporter, to provide proof of concept for this novel methodology. This approach has high potential in the analysis of highly dynamic, heterologous, and broadly uncharacterized membrane protein complexes, such as G-protein coupled receptors (GPCRs). Our approach may help clarify the often complex behavior observed in the activation and function of such complexes.
Modelling of Soluble Macromolecular Assemblies
In order to describe the protein dynamics which bridge between structure and function, we combine our integrative modelling workflow with molecular dynamics and network modelling techniques to further describe their motions and explore their conformational space. The modular nature of our integrative modelling workflow means that additional data procured from techniques such as cryo-electron microscopy, ion mobility MS, hydrogen-deuterium exchange MS and chemical cross linking MS can later be implemented to further improve the model selection process.