A5003119209- Enzyme Structure and Function
- SARS-CoV-2 and COVID-19 Research
- Monoclonal and Polyclonal Antibodies Research
- Biochemical and Molecular Research
- Protein Structure and Dynamics
- Bacteriophages and microbial interactions
- Computational Drug Discovery Methods
- Porphyrin Metabolism and Disorders
- Enzyme Production and Characterization
- Cancer, Hypoxia, and Metabolism
- Hemoglobin structure and function
- Mass Spectrometry Techniques and Applications
- Viral gastroenteritis research and epidemiology
- Metabolomics and Mass Spectrometry Studies
- Burkholderia infections and melioidosis
- Immune Cell Function and Interaction
- Metabolism and Genetic Disorders
- Spectroscopy and Quantum Chemical Studies
- Nanoparticle-Based Drug Delivery
- Ion-surface interactions and analysis
- Molecular spectroscopy and chirality
- Diamond and Carbon-based Materials Research
- Advanced Biosensing Techniques and Applications
- X-ray Diffraction in Crystallography
- Cellular Mechanics and Interactions
Diamond Light Source
2018-2025
Research Complex at Harwell
2022-2024
University of Southampton
2006-2018
University College London
2009-2011
Roland Hill (United Kingdom)
2011
The Royal Free Hospital
2009-2011
Monash University
2008
SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four (C5, H3, C1, F2) engineered homotrimers pmolar affinity the receptor binding (RBD) of spike protein. Crystal structures show C5 H3 overlap...
Abstract γ-Amino acids can play important roles in the biological activities of natural products; however, ribosomal incorporation γ-amino into peptides is challenging. Here we report how a selection campaign employing non-canonical peptide library containing cyclic γ 2,4 -amino resulted discovery very potent inhibitors SARS-CoV-2 main protease (M pro ). Two kinds acids, cis -3-aminocyclobutane carboxylic acid (γ 1 ) and (1 R ,3 S )-3-aminocyclopentane 2 ), were ribosomally introduced...
Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation controlled phosphorylation eukaryotic elongation factor 2 (eEF2), inhibits its activity and catalyzed eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation eEF2K induces independently previously known inputs into eEF2K. Here, we show that subject to hydroxylation on proline-98. Proline proline hydroxylases,...
The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of β-lactams as inhibitors bacterial nucleophilic enzymes, they are interest viral serine and cysteine proteases. We describe synthesis penicillin derivatives which potent Mpro investigate their mechanism inhibition using mass spectrometric crystallographic analyses. results suggest that have considerable potential via involving reaction with to form stable acyl-enzyme...
Diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively synthesise hydrolyse the secondary messenger cyclic dimeric GMP (c-di-GMP), both activities are often found in a single protein. Intracellular c-di-GMP levels turn regulate bacterial motility, virulence biofilm formation. We report first structure of tandem DGC-PDE fragment, which catalytic domains shown to be active. Two phosphodiesterase states distinguished by active site The structures, presence or absence c-di-GMP,...
Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their immediately useful. Laboratory-based genetic engineering methods enhance affinity, termed maturation, deliver useful reagents different areas of biology and potentially medicine. Using the receptor binding domain (RBD) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein a library, we generated closely related...
VMXi is a new high-flux microfocus macromolecular crystallography beamline at Diamond Light Source. The beamline, dedicated to fully automated and remote data collection of crystals in situ , allows rapid screening hundreds crystallization plates from multiple user groups. Its main purpose give fast feedback the complex stages crystal optimization, but it also enables small delicate samples that are particularly difficult harvest using conventional cryo-methods, grown lipidic cubic phase,...
Acidification of the extracellular and/or intracellular environment is involved in many aspects cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) a Ca2+/calmodulin-dependent that regulates translation by phosphorylating inhibiting eEF2. Here we show acidosis elicits activation eEF2K vivo, leading to enhanced phosphorylation We identify five histidine residues are crucial for during acidosis. Three them (H80, H87, H94) its calmodulin-binding site, their protonation...
A common challenge in drug design pertains to finding chemical modifications a ligand that increases its affinity the target protein. An underutilized advance is increase structural biology throughput, which has progressed from an artisanal endeavor monthly throughput of hundreds different ligands against protein modern synchrotrons. However, missing piece framework turns high-throughput crystallography data into predictive models for design. Here, we designed simple machine learning...
eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation machinery. belongs to small group of so-called ‘α-kinases’ are distinct from main eukaryotic superfamily. In addition α-kinase catalytic domain, other domains have been identified in eEF2K: CaM-binding region, N-terminal domain; C-terminal region containing several predicted α-helices (resembling SEL1 domains); and probably rather unstructured ‘linker’ connecting...
The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited challenging targets that may form fragile crystals difficult cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast the high-throughput highly automated pipelines for determination at cryogenic temperatures widely available...
Abstract Despite being fundamental to multiple biological processes, phosphorus (P) availability in marine environments is often growth-limiting, with generally low surface concentrations. Picocyanobacteria strains encode a putative ABC-type phosphite/phosphate/phosphonate transporter, phnDCE, thought provide access an alternative pool. This, however, paradoxical given most picocyanobacterial lack known phosphite degradation or carbon-phosphate lyase pathway utilise alternate pools. To...
The Omicron strains of SARS-CoV-2 pose a significant challenge to the development effective antibody-based treatments as immune evasion has compromised most available therapeutics. Therefore, in ‘arms race’ with virus, there is continuing need identify new biologics for prevention or treatment infections. Here, we report isolation nanobodies that bind BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized either Wuhan Beta proteins....
The third complementary-determining regions of the heavy-chain (CDR3H) variable (VH) some cattle antibodies are highly extended, consisting 48 or more residues. These `ultralong' CDR3Hs form β-ribbon stalks that protrude from surface antibody with a disulfide cross-linked knob region at their apex dominates antigen interactions over other CDR loops. structure Fab fragment naturally paired bovine ultralong (D08), identified by single B-cell sequencing, has been determined to 1.6 Å resolution....
Abstract The normal physiological roles of the phylogenetically conserved human plasma proteins C‐reactive protein (CRP) and serum amyloid P component (SAP) are not known. Novel drugs targeting their ligand specificities in clinical development as both have significant pathophysiological effects, SAP promoting amyloidosis CRP exacerbating ischemic injury. Both bind to phosphoethanolamine we show here that, under conditions, is bound with higher affinity by than CRP. An explanation provided...
Von Willebrand factor A (VWA) domains are versatile protein interaction with N and C termini in close proximity placing spatial constraints on overall structure. The 1.2 Å crystal structures of a collagen VI VWA domain disease-causing point mutant show C-terminal extensions that place the at opposite ends. This allows "beads-on-a-string" arrangement multiple as observed for ten N-terminal α3 chain. extension is linked to core by salt bridge two hydrophobic patches. Comparison wild-type...
Eukaryotic elongation factor 2 kinase (eEF2K) is the best-characterized member of α-kinase family. Within this group, only eEF2K and myosin heavy chain kinases (MHCKs) have known substrates. Here we studied roles specific residues, selected on basis structural data for MHCK A TRPM7, in function eEF2K. Our provide first information regarding substrate specificity α-kinases, particular residues so-called N/D loop, which appears to occupy a position structure α-kinases similar that activation...
Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from plates Diamond Light Source beamline VMXi are described. This approach enables high-quality room-temperature crystal structures to be determined multiple crystals a straightforward manner provides very rapid feedback on results of trials as well enabling serial crystallography. The value understanding structure, ligand binding, dynamics is becoming...
A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities homogenous, well diffracting microcrystals. Efficient high-throughput screening batch-grown microcrystals determination ground-state structures from different conditions thus value early stages a project. Here, highly sample-efficient methodology to measure data by raster scanning within standard situ 96-well crystallization plates described. Structures were...