A5042031499- Mass Spectrometry Techniques and Applications
- Protein Structure and Dynamics
- Analytical Chemistry and Chromatography
- Lipid Membrane Structure and Behavior
- RNA and protein synthesis mechanisms
- Advanced Proteomics Techniques and Applications
- Bacterial Genetics and Biotechnology
- Metabolomics and Mass Spectrometry Studies
- Receptor Mechanisms and Signaling
- Ion-surface interactions and analysis
- Enzyme Structure and Function
- Bacteriophages and microbial interactions
- Pharmacological Effects and Assays
- Molecular Junctions and Nanostructures
- Adenosine and Purinergic Signaling
- RNA Research and Splicing
- Analytical chemistry methods development
- Growth Hormone and Insulin-like Growth Factors
- Carbohydrate Chemistry and Synthesis
- Toxin Mechanisms and Immunotoxins
- Hypothalamic control of reproductive hormones
- Estrogen and related hormone effects
- Cell death mechanisms and regulation
- Transgenic Plants and Applications
- Bacterial biofilms and quorum sensing
University of Oxford
2012-2021
Oxford Technologies (United Kingdom)
2017-2018
Laboratoire de Chimie
2015
Oxfam
2015
University of Nottingham
2009-2012
Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in presence absence bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated collision argon gas induce unfolding their structures. The collisional cross sections (Ω) folded unfolded measured T-Wave cell a Waters Synapt HDMS...
Porins are β-barrel outer-membrane proteins through which small solutes and metabolites diffuse that also exploited during cell death. We have studied how the bacteriocin colicin E9 (ColE9) assembles a cytotoxic translocon at surface of Escherichia coli incorporates trimeric porin OmpF. Formation involved ColE9's unstructured N-terminal domain threading in opposite directions two OmpF subunits, capturing its target TolB on other side membrane fixed orientation triggers import. Thus, an...
Abstract G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation β 1 -adrenergic receptor in response various ligands. By simultaneously capturing effects ligand binding coupling different proteins, probed relative specific...
The study of intact soluble protein assemblies by means mass spectrometry is providing invaluable contributions to structural biology and biochemistry. A recent breakthrough has enabled similar membrane complexes, following their release from detergent micelles in the gas phase. Careful optimization conditions, particularly with respect energy regimes, essential for maintaining compact folded states as removed. However, many saccharide detergents widely employed can cause unfolding proteins...
Abstract The prolactin receptor is an archetype member of the class I cytokine family, comprising receptors with fundamental functions in biology as well key drug targets. Structurally, each these represent intriguing diversity, providing exceptionally challenging target for structural biology. Here, we access molecular architecture monomeric human by combining experimental and computational efforts. We solve NMR structure its transmembrane domain micelles collect data on overlapping...
The benefits of lowering protein ion charge states in electrospray ionization (ESI) have attracted recent interest. We describe a simple approach to decrease by exposure droplets neutral solvent vapor such as acetonitrile. technique allows detection weak noncovalent complexes, provides preferred for tandem mass spectrometry (MS/MS) dissociation and has the added benefit reducing common adducts, alkali metals, without addition solution additives or requirement secondary spray.
G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors belong to the largest family of membrane-embedded cell surface proteins and are involved in a diverse array physiological processes. Despite progress mass spectrometry membrane complexes, protein-coupled have remained intractable because their low yield instability after extraction from membranes. We established conditions spectrometer that preserve noncovalent ligand binding human purinergic receptor P2Y
Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES (DESI) MS has however transformed the study of tissue surfaces through release characterisation small molecules. Motivated by desire to screen ligand binding we report development a native DESI platform. By establishing conditions that preserve non-covalent interactions exploit surface capture rapid...
Extracellular signal-regulated kinase 2 (ERK2) is an S/T with more than 200 known substrates, and critical roles in regulation of cell growth differentiation currently no membrane proteins have been linked to ERK2 scaffolding. Here, we identify the human Na+/H+ exchanger 1 (hNHE1) as a scaffold protein for show direct hNHE1-ERK1/2 interaction cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy immunofluorescence analysis demonstrate that scaffolding by hNHE1 occurs one...
TolR is a 15-kDa inner membrane protein subunit of the Tol-Pal complex in Gram-negative bacteria, and its function poorly understood. recruited to cell division sites where it involved maintaining integrity outer membrane. related MotB, peptidoglycan (PG)-binding stator from flagellum, suggesting might serve similar role Tol-Pal. The only structure thus far reported for periplasmic domain Haemophilus influenzae which N- C-terminal residues had been deleted (TolR(62-133), Escherichia coli...
Strong interactions between lipids and proteins occur primarily through association of charged headgroups amino acid side chains, rendering the protonation status both partners important. Here we use native mass spectrometry to explore lipid binding as a function charge outer membrane porin F (OmpF). We find that anionic phosphatidylglycerol (POPG) or zwitterionic phosphatidylcholine (POPC) OmpF is sensitive electrospray polarity while effects are less pronounced for other in mitochondrial...
Mapping the interaction sites between membrane-spanning proteins is a key challenge in structural biology. In this study carbene-footprinting approach was developed and applied to identify interfacial of trimeric, integral membrane protein, OmpF, solubilised micelles. The diazirine-based footprinting probe effectively sequestered by, incorporated into, micelles, thus leading efficient labelling regions protein upon irradiation at 349 nm. Areas associated with protein-protein interactions...
Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound and non-specific, annular interactions is challenging problem. Applying native mass spectrometry to three membrane protein complexes different lipid-binding properties, we explore the ability detergents compete lipids environments. We show that positions on presenilin homologue protease are subject constant exchange detergent. By contrast, detergent-resistant at...
Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary understanding functional roles these biomolecules. Recent research expanding application native MS towards membrane proteins directly from isolated preparations or purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; latter enables therapeutic targets screened against multiplexed compound libraries. In...
Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used remove detergent, however, often causes unfolding and dissociation. As case for soluble proteins, electrospray positive ion mode is most study of membrane proteins. Here we show several distinct advantages employing negative mode. Negative polarity can yield lower average charge states proteins saccharide detergents, with enhanced peak resolution...
Abstract Redox-regulated effector systems that counteract oxidative stress are essential for all forms of life. Here we uncover a new paradigm sensing centred on the hydrophobic core sensor protein. RsrA is an archetypal zinc-binding anti-sigma factor responds to disulfide in cytoplasm Actinobacteria. We show utilizes its bind sigma σ R preventing association with RNA polymerase, and zinc plays central role maintaining this high-affinity complex. Oxidation limited by rate release, which...