A5028431484- Bacterial Genetics and Biotechnology
- RNA and protein synthesis mechanisms
- Protein Structure and Dynamics
- Lipid Membrane Structure and Behavior
- Enzyme Structure and Function
- Escherichia coli research studies
- Advanced biosensing and bioanalysis techniques
- Chemical Synthesis and Analysis
- Bacteriophages and microbial interactions
- Electron Spin Resonance Studies
- Antibiotic Resistance in Bacteria
- Advanced NMR Techniques and Applications
- Spectroscopy and Quantum Chemical Studies
- Click Chemistry and Applications
- Advanced Electron Microscopy Techniques and Applications
- NF-κB Signaling Pathways
- Force Microscopy Techniques and Applications
- Machine Learning in Bioinformatics
- Viral Infectious Diseases and Gene Expression in Insects
- Biotin and Related Studies
- Genomics and Phylogenetic Studies
- Advanced X-ray Imaging Techniques
- ATP Synthase and ATPases Research
- Biochemical and Structural Characterization
- Human Resource Development and Performance Evaluation
Institute of Structural and Molecular Biology
2018-2024
University of Leeds
2016-2024
University of Oxford
2020-2021
University of Bath
2015
Transmembrane β-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes form stable pores, but the range pore properties that be achieved by repurposing natural TMBs is limited. We leverage power de novo computational design coupled with a "hypothesis, design, test" approach to determine TMB principles, notably, importance negative slow β-sheet assembly. new eight-stranded TMBs, no homology known...
Abstract The β‐barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram‐negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all obtained under conditions which lack the unique features complexity (OM). Here, we use Pulsed Electron‐Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate ensemble complex E. coli cells. We show that...
Abstract The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli comprises core domain and two peptidylprolyl isomerase domains (P1 P2), but its mechanisms of client binding function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET molecular dynamics simulations to map the site(s) on interrogate conformational OMP recognition. We demonstrate that samples an array...
Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane insertion. In bacteria, this mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis cytoplasmic SecA, trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order dissect complex reaction pathway. The results show that both signal sequence mature components pre-protein,...
Abstract The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of proteins (OMPs) by membrane-embedded β-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM periplasmic chaperone SurA, but how SurA work together ensure successful OMP delivery remains unclear. Here, guided AlphaFold2 models, we use disulphide bond engineering in an attempt trap act BAM, solve...
Analysing protein complexes by chemical crosslinking-mass spectrometry (XL-MS) is limited the side-chain reactivities and sizes of available crosslinkers, their slow reaction rates, difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label reactive cysteine introduced into bait protein, residue-unbiased diazirine-based photoactivatable trap its interacting...
Abstract The β-barrel assembly machinery (BAM) catalyses the folding and insertion of outer membrane proteins (OMPs) into membranes Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble cryoEM structures E. coli BamABCDE complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported single-molecule FRET measurements, describe a range motions BAM complex, mostly localised within periplasmic region major subunit BamA....
Abstract The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the assembly machinery (BAM). How lateral opening major subunit BamA assists OMP folding, and contribution disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) two disulphide-crosslinked variants (lid-locked (LL), POTRA-5-locked (P5L)) dissect these roles. Despite being lethal vivo, show that all complexes catalyse vitro, albeit...
The outer-membrane protein TolC from Escherichia coli belongs to an extensive superfamily whose members are found throughout the didermal, Gram-negative bacterial lineages. serves as activated exit duct in multi-drug efflux pumps and secretion machinery. Many homologs bear a lipid modification on N-terminus that embeds into inner leaflet of outer membrane appears have been conserved feature for millions years; however, moiety is absent entirely E. TolC. We discovered lipoprotein YbjP...
Abstract Analysing protein complexes by chemical crosslinking‐mass spectrometry (XL‐MS) is limited the side‐chain reactivities and sizes of available crosslinkers, their slow reaction rates, difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label reactive cysteine introduced into bait protein, residue‐unbiased diazirine‐based photoactivatable trap its...
The β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all obtained under conditions which lack the unique features complexity (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate ensemble complex
Abstract Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane insertion. This mediated, the majority of proteins, by highly conserved Sec machinery. The bacterial translocon – SecY MK EG resides in plasma membrane, where driven through rounds ATP hydrolysis cytoplasmic SecA ATPase, proton motive force (PMF). We have used single molecule Förster resonance energy transfer (FRET) alongside combination confocal total internal reflection...
Abstract The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli comprises core domain and two peptidylprolyl isomerase domains (P1 P2), but how it binds its OMP clients the mechanism(s) of action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET molecular dynamics simulations to map client binding site(s) on interrogate conformational chaperone’s recognition. We demonstrate that samples...
The β-barrel assembly machine (BAM) is essential for folding outer membrane proteins (OMPs) into the (OM) of Gram-negative bacteria. Structures BAM have been solved using X-ray crystallography and cryoEM, but conformation in highly crowded native OM remained unsolved. Using EPR spectroscopy intact E. coli cells, structure situ has now revealed Research Article (e202218783) by Sheena Radford, Christos Pliotas et al., both alone presence antibacterial, darobactin B. Image: Jeroen Claus...