A5087887712- Protein Structure and Dynamics
- Enzyme-mediated dye degradation
- Endoplasmic Reticulum Stress and Disease
- Enzyme Catalysis and Immobilization
- Protein purification and stability
- Protein Interaction Studies and Fluorescence Analysis
- Microplastics and Plastic Pollution
- Enzyme Structure and Function
- Surfactants and Colloidal Systems
- Effects and risks of endocrine disrupting chemicals
- Autophagy in Disease and Therapy
- Microbial Metabolism and Applications
- Biotin and Related Studies
- Photosynthetic Processes and Mechanisms
- Metal-Catalyzed Oxygenation Mechanisms
- Microbial Metabolic Engineering and Bioproduction
- Polymer Surface Interaction Studies
- Advanced Fluorescence Microscopy Techniques
- Advanced Polymer Synthesis and Characterization
- Analytical Chemistry and Chromatography
- Redox biology and oxidative stress
- Biochemical and biochemical processes
- biodegradable polymer synthesis and properties
- Heme Oxygenase-1 and Carbon Monoxide
- Cell Adhesion Molecules Research
University of Algarve
2016-2025
UK Dementia Research Institute
2021-2024
University of Cambridge
2013-2024
For Mar Centro de Formação Profissional das Pescas e do Mar
2020
Hospital de Faro EPE
2020
Algarve Biomedical Center
2015-2018
NIHR Cambridge Biomedical Research Centre
2013-2017
Wellcome Trust
2016-2017
Wellcome/MRC Institute of Metabolic Science
2016
Instituto Superior Técnico
2001-2015
Interfering with disulfide bond formation impedes protein folding and promotes endoplasmic reticulum (ER) stress. Due to limitations in measurement techniques, the relationships of altered thiol redox ER stress have been difficult assess. We report that fluorescent lifetime measurements circumvented crippling dimness an ER-tuned redox-responsive probe (roGFPiE), faithfully tracking activity major ER-localized isomerase, PDI. In vivo imaging by time-correlated single-photon counting (TCSPC)...
Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during maturation or degradation of secreted proteins. To critically examine widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed modified form cytosolic glutathione-degrading enzyme, ChaC1, lumen. ChaC1CtoS purged eliciting expected kinetic defect oxidation an ER-localized...
The endoplasmic reticulum (ER)-localized peroxiredoxin 4 (PRDX4) supports disulfide bond formation in eukaryotic cells lacking oxidase 1 (ERO1). source of peroxide that fuels PRDX4-mediated has remained a mystery, because ERO1 is believed to be major producer hydrogen (H2O2) the ER lumen. We report on simple kinetic technique track H2O2 equilibration between cellular compartments, suggesting relatively isolated from cytosolic or mitochondrial pools. Furthermore, expression an ER-adapted...
Protein synthesis is supported by cellular machineries that ensure polypeptides fold to their native conformation, whilst eliminating misfolded, aggregation prone species. underlies pathologies including neurodegeneration. Aggregates' formation antagonised molecular chaperones, with cytoplasmic machinery resolving insoluble protein aggregates. However, it unknown whether an analogous disaggregation system exists in the Endoplasmic Reticulum (ER) where ~30% of proteome synthesised. Here we...
Abstract C -glycosides are natural products with important biological activities but recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) recently identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze oxidation of concomitant reduction O 2 H . This is followed by C-C acid/base-assisted bond cleavage in two-step -deglycosylation pathways. Soil and gut microorganisms have different oxidative enzymes, details their catalytic mechanisms largely...
Abstract Induced pluripotent stem cells (iPSCs) hold large potential in regenerative medicine due to their pluripotency and unlimited self‐renewal capacity without the ethical issues of embryonic cells. To provide quality‐controlled iPSCs for clinical therapies, it is essential develop safe cryopreservation protocols long‐term storage, preferably amenable scale‐up automation. We have compared impact two different freezing geometries (bottom‐up conventional radial freezing) on viability...
Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded load imposed by diverse physiological challenges including compromise the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting rapid and reversible shift towards more reducing poise. Calcium depletion induced ER alterations are relevant conditions associated with such response of pancreatic...
The fate of hydrogen peroxide (H2O2) in the endoplasmic reticulum (ER) has been inferred indirectly from activity ER-localized thiol oxidases and peroxiredoxins, vitro, consequences their genetic manipulation, vivo. Over years hints have suggested that glutathione, puzzlingly abundant ER lumen, might a role reducing heavy burden H2O2 produced by luminal enzymatic machinery for disulfide bond formation. However, limitations existing organelle-targeted probes rendered them inert...
Protein stability arises from a combination of factors which are often difficult to rationalise. Therefore its improvement is better addressed through directed evolution than by rational design approaches. In this study, five rounds mutagenesis/recombination followed high-throughput screening (≈10,000 clones) yielded the hit 1B6 showing 300-fold higher half life at 50°C that exhibited homodimeric wild type PpAzoR azoreductase Pseudomonas putida MET94. The characterization using fluorescence,...
Freezing of protein solutions is required for many applications such as storage, transport, or lyophilization; however, freezing has inherent risks integrity. It difficult to study stability below the temperature because phase separation constrains solute concentration in solution. In this work, we developed an isochoric method aggregation at -5, -10, -15, and -20 °C. Lowering point a fixed volume prevents aqueous solution from freezing, pressure rises until equilibrium (P,T) reached....
The effect of trehalose (0.5 M) on the thermal stability cutinase in alkaline pH range was studied. unfolding induced by increasing temperature analyzed absence and presence according to a two-state model (which assumes that only folded unfolded states were present). Trehalose delays reversible unfolding. midpoint transition (Tm) increases 4.0 degrees C 2. 6 at 9.2 10.5, respectively, trehalose. At occurs higher temperatures (Tm is 52.6 compared 42.0 10.5) refolding yield around 80% obtained...
The unfolding of cutinase at pH 4.5 was induced by increasing the temperature and guanidine hydrochloride concentration in presence potassium chloride, trehalose, mannosylglycerate salt. Protein thermal approached a two-state process, since transitions were coincident within experimental error when assessed near-ultraviolet (UV) difference, tryptophyl, 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence spectroscopy. Trehalose 0.5 M increased which 50% is unfolded 3°C. Unfolding clearly...
Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability enzyme cutinase was studied. Thermal unfolding reveals that delays thermal unfolding, thus increasing temperature at midpoint by 7.2 degrees . Despite this stabilizing effect, also favors pathways lead irreversible denaturation. Stopped-flow kinetics folding measured introduced experimental variable assess mechanism thermodynamics protein stabilization...
Conjugation of fluorescently-labelled DNA onto gold nanorods produces strongly emitting nano-assemblies, but only tip-selective functionalization affords an effective emission enhancement.
DyP-type peroxidases (DyPs) are microbial enzymes that catalyze the oxidation of a wide range substrates, including synthetic dyes, lignin-derived compounds, and metals, such as Mn2+ Fe2+, have enormous biotechnological potential in biorefineries. However, many questions on molecular basis enzyme function stability remain unanswered. In this work, high-resolution structures PpDyP wild-type two engineered variants (6E10 29E4) generated by directed evolution were obtained. The X-ray crystal...