A5061858006- Bacteriophages and microbial interactions
- RNA and protein synthesis mechanisms
- Bacterial Genetics and Biotechnology
- Protein Structure and Dynamics
- Viral gastroenteritis research and epidemiology
- Plant Virus Research Studies
- Photosynthetic Processes and Mechanisms
- ATP Synthase and ATPases Research
- Enzyme Structure and Function
- Lipid Membrane Structure and Behavior
- RNA modifications and cancer
- Cancer Research and Treatments
- Genomics and Phylogenetic Studies
- T-cell and B-cell Immunology
- Animal Virus Infections Studies
- Mass Spectrometry Techniques and Applications
- Biochemical and Molecular Research
- Spectroscopy and Quantum Chemical Studies
- Virus-based gene therapy research
- RNA Research and Splicing
- Peptidase Inhibition and Analysis
- HIV Research and Treatment
- Protein purification and stability
- Mosquito-borne diseases and control
- Immunotherapy and Immune Responses
University of Leeds
2014-2024
University of South Bohemia in České Budějovice
2018-2024
Universidade Estadual Paulista (Unesp)
2024
Institute of Structural and Molecular Biology
2012-2021
Hackensack Meridian Health
2020-2021
Center for Discovery
2021
Bayshore Community Hospital
2020
Easton Hospital
2018
Sewanee: The University of the South
2018
Imperial College London
2014
Abstract Raman spectroscopy has become a versatile tool in protein science and biotechnology. Recent advances spectral assignments vibrational theory, examples of use structural biology selected industrial applications are discussed. New insights into folding, assembly aggregation were obtained by classical spectroscopy. been used to characterize intrinsically unstructured proteins. The improved instrument sensitivity made it possible difference enzyme–substrate interactions. Specifically,...
Killed or inactivated vaccines targeting intracellular bacterial and protozoal pathogens are notoriously ineffective at generating protective immunity. For example, vaccination with heat-killed Listeria monocytogenes (HKLM) is not protective, although infection live L. induces long-lived, CD8 T cell–mediated We demonstrate that HKLM immunization primes memory lymphocyte populations that, substantial in size, providing protection from subsequent infection. In contrast to infection, which...
Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using single molecule fluorescence assay monitor conformation assembly real time, two from differing structural families, we discovered that two-stage process. Initially, the genomic RNAs undergo rapid dramatic (approximately 20–30%) collapse of solution conformations upon...
Significance Oligomers formed en route to amyloid fibrils are thought be the perpetrators of toxicity in many disorders. How contribute disease, however, is less clear. Here, using β 2 -micoglobulin (β m) as a model system, we show that stability highly pH-dependent, with mild acidification enhancing formation fibril-derived nonnative oligomers disrupt membranes and alter cellular function. Enhancing fibril by incubation molecular chaperone, hsp70, or cross-linking, protects against...
The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, drive for translocation comes from ATP hydrolysis cytosolic motor-protein SecA, in concert with proton motive force (PMF). However, mechanism through which SecA coupled to directional movement SecYEG unclear. Here, we combine all-atom molecular dynamics (MD) simulations single molecule FRET and biochemical assays. We show that binding causes opening SecY-channel at long range, while...
Relative to other extrinsic factors, the effects of hydrodynamic flow fields on protein stability and conformation remain poorly understood. Flow-induced remodeling and/or aggregation is observed both in Nature during large-scale industrial manufacture proteins. Despite its ubiquity, relationships between type magnitude flow, a protein's structure stability, resultant propensity are unclear. Here, we assess defined quantified field dominated by extensional BSA, β2-microglobulin (β2m),...
The formation of a protective protein container is an essential step in the life-cycle most viruses. In case single-stranded (ss)RNA viruses, this occurs parallel with genome packaging co-assembly process. Previously, it had been thought that process can be explained entirely by electrostatics. Inspired recent single-molecule fluorescence experiments recapitulate RNA specificity seen vivo for two model we present alternative theory, which recognizes important cooperative roles played...
Significance Single-stranded RNA viruses self-assemble protective protein containers around their cognate genomes rapidly and efficiently at low concentrations. encapsidation in vivo occurs preferentially with the genome, contrast to many vitro reassembly experiments. We describe molecular detail how this specificity efficiency is accomplished using multiple contacts between coat proteins dispersed packaging signals viral genome. The sequences relative positioning of are important for...
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,...
Significance Viruses composed of a shell coat proteins enclosing ssRNA genomes are among the simplest biological entities. Their lifecycles include range processes, such as specific genome encapsidation and efficient capsid self-assembly. Until recently, these were not linked, but we have shown that many viruses in this class encode multiple, degenerate RNA sequence/structure motifs bind cognate collectively. This simultaneously ensures packaging virion assembly via an RNA-encoded...
Long RNA molecules are at the core of gene regulation across all kingdoms life, while also serving as genomes in viruses. Few studies have addressed basic physical properties long single-stranded RNAs. RNAs with nonrepeating sequences usually adopt highly ramified secondary structures and better described branched polymers. To test whether a polymer model can estimate overall sizes large RNAs, we employed fluorescence correlation spectroscopy to examine hydrodynamic radii broad spectrum...
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....
Twinfilin is a ubiquitous and abundant actin monomer–binding protein that composed of two ADF-H domains. To elucidate the role twinfilin in dynamics, we examined interactions mouse its isolated domains with G-actin. Wild-type binds ADP-G-actin higher affinity (K D = 0.05 μM) than ATP-G-actin 0.47 under physiological ionic conditions forms relatively stable (k off 1.8 s −1 ) complex ADP-G-actin. Data from native PAGE size exclusion chromatography coupled light scattering suggest competes...
Using real-time re-programmable signal processing we connect acousto-optic steering and back-focal-plane interferometric position detection in optical tweezers to create a fast feedback controlled instrument. When trapping 3μm latex beads water find that proportional-gain position-clamping increases the effective lateral trap stiffness ∼13-fold. A theoretical power spectrum for bead fluctuations during position-clamped is derived agrees with experimental data. The loop delay, ∼19μs our...
The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via protein baseplate. structure from C. was investigated by using combination cryo-electron microscopy X-ray diffraction compared with that Chlorobi species. Cryo-electron tomography revealed thin for which distinct crystalline baseplate lattice visualized in high-resolution...
Long RNAs often exist as multiple conformers in equilibrium. For the genomes of single-stranded RNA viruses, one these must include a compacted state allowing to be confined within virion. We have used single molecule fluorescence correlation spectroscopy monitor conformations viral and sub-fragments absence presence coat proteins. Cognate RNA-coat protein interactions two model viruses cause rapid collapse hydrodynamic radii their respective RNAs. This is caused by binding at sites on that...
Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production during aerobic respiration. They catalyze the reduction of terminal electron acceptor, oxygen, utilize Gibbs free to transport protons across a membrane generate proton (ΔpH) electrochemical gradient termed motive force (PMF), which provides driving adenosine triphosphate (ATP) synthesis. Excessive PMF is known limit turnover HCOs, but molecular mechanism this regulatory feedback remains...
Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on interior of infectious MS2 virions overlaps with known peptides bind high affinity translational operator, TR, within phage genome. The data also reveal sequences genomic fragments in contact coat shell. These show remarkable overlap previous predictions based hypothesis virion assembly is mediated by multiple sequences-specific contacts at sites termed Packaging Signals (PSs). PSs are variations...