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Angewandte Chemie (International Ed. in... Jun 2020The degree by which metalloproteins partially regulate net charge (Z) upon electron transfer (ET) was recently measured for the first time using "protein charge ladders"...
The degree by which metalloproteins partially regulate net charge (Z) upon electron transfer (ET) was recently measured for the first time using "protein charge ladders" of azurin, cytochrome c, and myoglobin [Angew. Chem. Int. Ed. 2018, 57(19), 5364-5368; Angew. Chem. 2018, 130, 5462-5466]. Here, we show that Cu, Zn superoxide dismutase (SOD1) is unique among proteins in its ability to resist changes in net charge upon single ET (e.g., ΔZ =0.05±0.08 per electron, compared to ΔZ =1.19±0.02). This total regulation of net charge by SOD1 is attributed to the protonation of the bridging histidine upon copper reduction, yielding redox centers that are isoelectric at both copper oxidation states. Charge regulation by SOD1 would prevent long range coulombic perturbations to residue pK 's upon ET at copper, allowing SOD1's "electrostatic loop" to attract superoxide with equal affinity (at both redox states of copper) during diffusion-limited reduction and oxidation of superoxide.
Topics: Cytochromes c; Electron Transport; Kinetics; Metalloproteins; Myoglobin; Oxidation-Reduction; Superoxide Dismutase
PubMed: 32212239
DOI: 10.1002/anie.202001452 -
The Journal of Physical Chemistry. B Feb 2024Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such...
Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a β-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the β-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the S-S bonded and S-S nonbonded () forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for azurin compared to that of the form (420 K). As observed in the case of azurin, the unfolding process of the form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual β-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.
Topics: Azurin; Copper; Metalloproteins; Disulfides; Temperature; Ions; Protein Folding
PubMed: 38236012
DOI: 10.1021/acs.jpcb.3c07089 -
Molecular Pharmaceutics Dec 2023Erythropoietin-producing hepatocellular (Eph) receptors and their ligands, ephrins, are the largest subfamily of receptor tyrosine kinases (RTKs) that have emerged as a...
Erythropoietin-producing hepatocellular (Eph) receptors and their ligands, ephrins, are the largest subfamily of receptor tyrosine kinases (RTKs) that have emerged as a new class of cancer biomarkers due to their aberrant expression in cancer progression. The activation of Eph receptors either due to their hyperexpression or via high affinity binding with their respective ephrin ligands initiates a cascade of signals that impacts cancer development and progression. In prostate cancer, the overexpression of the EphA6 receptor has been correlated with increased metastatic potential. Azurin, a small redox protein, is known to prevent tumor progression by binding to cell surface Eph receptors, inhibiting its autophosphorylation in the kinase domain and thereby disrupting Eph-ephrin signaling. Hence, a self-assembled, theranostic nanosystem of recombinant fusion protein hisEGFP-azu (80-128) was designed by conjugating enhanced green fluorescent protein (EGFP) with the C-terminal region of azurin. This design was inspired by the binding study, where the analogue of ephrinA, hisEGFP-azu (80-128) showed higher binding affinity for the EphA6 receptor than the ephrinA ligands. The hisEGFP-azu (80-128) nanosystem which assembled as nanoparticles was tested for its ability to simultaneously detect and kill the prostate cancer cells, LNCaP. This was achieved by specifically targeting EphA6 receptors overexpressed on the cancer cell surface via C-terminal peptide, azu (80-128). Herein, we report antiproliferative, apoptotic, antimigratory, and anti-invasive effects of this nanosystem on LNCaP cells, while having no similar effects on EphA6 negative human normal lung cells, WI-38.
Topics: Male; Humans; Receptors, Eph Family; Azurin; Receptor, EphA6; Precision Medicine; Prostatic Neoplasms; Ephrins
PubMed: 37906960
DOI: 10.1021/acs.molpharmaceut.3c00387 -
The Journal of Physical Chemistry. B Dec 2022One reaction step in the conductivity relay of azurin, electron transfer between the Cu-based active site and the tryptophan residue, is studied theoretically and by...
One reaction step in the conductivity relay of azurin, electron transfer between the Cu-based active site and the tryptophan residue, is studied theoretically and by classical molecular dynamics simulations. Oxidation of tryptophan results in electrowetting of this residue. This structural change makes the free energy surfaces of electron transfer nonparabolic as described by the Q-model of electron transfer. We analyze the medium dynamical effect on protein electron transfer produced by coupled Stokes-shift dynamics and the dynamics of the donor-acceptor distance modulating electron tunneling. The equilibrium donor-acceptor distance falls in the plateau region of the rate constant, where it is determined by the protein-water dynamics, and the probability of electron tunneling does not affect the rate. The crossover distance found here puts most intraprotein electron-transfer reactions under the umbrella of dynamical control. The crossover between the medium-controlled and tunneling-controlled kinetics is combined with the effect of the protein-water medium on the activation barrier to formulate principles of tunability of protein-based charge-transfer chains. The main principle in optimizing the activation barrier is the departure from the Gaussian-Gibbsian statistics of fluctuations promoting activated transitions. This is achieved either by incomplete (nonergodic) sampling, breaking the link between the Stokes-shift and variance reorganization energies, or through wetting-induced structural changes of the enzyme's active site.
Topics: Azurin; Tryptophan; Electrons; Thermodynamics; Catalytic Domain; Electron Transport; Proteins; Water
PubMed: 36459590
DOI: 10.1021/acs.jpcb.2c05258 -
Proceedings of the National Academy of... Jul 2022The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms,...
The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood-Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetyl-CoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the Ni species and requirement for ordered substrate binding in the bioorganometallic carbon-carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation.
Topics: Aldehyde Oxidoreductases; Azurin; Catalysis; Esters; Models, Chemical; Multienzyme Complexes; Nickel; Origin of Life; Sulfur Compounds
PubMed: 35858422
DOI: 10.1073/pnas.2123022119 -
Colloids and Surfaces. B, Biointerfaces Dec 2019The development of environmental friendly new procedures for the synthesis of metallic nanoparticles is one of the main objectives of nanotechnology. Plants, algae,...
The development of environmental friendly new procedures for the synthesis of metallic nanoparticles is one of the main objectives of nanotechnology. Plants, algae, fungi and bacteria for the production of nanomaterials are viable alternatives due to their low cost, the absence of toxic waste production and their highly energy efficiency. It is also known that biosynthesized silver nanoparticles (AgNPs) show higher biocompatibility compared to the chemically-synthesized ones. In previous results, biosynthesized AgNPs were obtained from the supernatant of Pseudomonas aeruginosa, and they showed a bigger antimicrobial activity against different bacterial species compared to the chemically-synthesized ones. The aim of this work was to analyze the capping of biosynthesized AgNPs using techniques such as transmission electron microscopy (TEM), infrared spectroscopy (IR), and protein identification through mass spectrometry (MS) in order to identify the compounds responsible for their formation, stability and biocompatibility. The TEM images showed that AgNPs were surrounded by an irregular coverage. The IR spectrum showed that this coverage was composed of carbohydrates and/or proteins. Different proteins were identified in the capping associated to biosynthesized AgNPs. Some proteins seem to be important for their formation (Alkyl hydroperoxide reductase and Azurin) and stabilization (Outer membrane protein OprG and Glycine zipper 2 T M domain-containing protein). The proteins identified with the capability to interact with some biomolecules can be responsible for the biocompatibility and may be responsible for the bigger antimicrobial activity than AgNPs have previously shown. These results are pioneers in the identification of proteins in the capping of biosynthesized AgNPs.
Topics: Bacterial Proteins; Biocompatible Materials; Metal Nanoparticles; Particle Size; Pseudomonas aeruginosa; Silver; Surface Properties
PubMed: 31605948
DOI: 10.1016/j.colsurfb.2019.110517 -
Probiotics and Antimicrobial Proteins Jun 2020This study proposed to investigate the effect of azurin on the major stages of pathogenesis (adhesion and invasion) of intestinal bacterial pathogens (Salmonella spp....
This study proposed to investigate the effect of azurin on the major stages of pathogenesis (adhesion and invasion) of intestinal bacterial pathogens (Salmonella spp. and Escherichia coli) and epithelial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) on the human colorectal adenocarcinoma (Caco-2) cell line. Azurin protein was produced by cloning the azurin gene into pET21a and heterologous expression in E. coli BL21. The protein was purified using affinity chromatography and confirmed by Western blotting. The purified protein was evaluated by three experiments of adhesion and invasion assays, including exclusion, competition, and replacement. Azurin was observed to significantly inhibit the attachment and invasion of S. aureus, Salmonella spp., and E. coli, while no such inhibitory effects were observed on P. aeruginosa. In fact, the protein increased the adhesion of P. aeruginosa to the cell. In conclusion, our study proposes that azurin is a potential prophylactic or preventive helper candidate to inhibit the attachment and invasion of pathogenic bacteria to host cells and reduce the progression of the infection process. Our study also reveals the involvement of azurin in bacteria-host cell interactions, providing novel and important insights toward the elucidation of its biological function in this field. Thus, this study provides new opportunities to use azurin as an adjunct therapy against critical stages of infection by a wide range of pathogenic bacteria.
Topics: Azurin; Bacteria; Bacterial Adhesion; Bacterial Infections; Caco-2 Cells; Humans; Pseudomonas aeruginosa
PubMed: 31364004
DOI: 10.1007/s12602-019-09573-2 -
PLoS Pathogens Dec 2019The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria, whose function is known to translocate substrates to eukaryotic and prokaryotic...
The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria, whose function is known to translocate substrates to eukaryotic and prokaryotic target cells to cause host damage or as a weapon for interbacterial competition. Pseudomonas aeruginosa encodes three distinct T6SS clusters (H1-, H2-, and H3-T6SS). The H1-T6SS-dependent substrates have been identified and well characterized; however, only limited information is available for the H2- and H3-T6SSs since relatively fewer substrates for them have yet been established. Here, we obtained P. aeruginosa H2-T6SS-dependent secretomes and further characterized the H2-T6SS-dependent copper (Cu2+)-binding effector azurin (Azu). Our data showed that both azu and H2-T6SS were repressed by CueR and were induced by low concentrations of Cu2+. We also identified the Azu-interacting partner OprC, a Cu2+-specific TonB-dependent outer membrane transporter. Similar to H2-T6SS genes and azu, expression of oprC was directly regulated by CueR and was induced by low Cu2+. In addition, the Azu-OprC-mediated Cu2+ transport system is critical for P. aeruginosa cells in bacterial competition and virulence. Our findings provide insights for understanding the diverse functions of T6SSs and the role of metal ions for P. aeruginosa in bacteria-bacteria competition.
Topics: Animals; Bacterial Proteins; Copper; DNA-Binding Proteins; Mice; Pseudomonas Infections; Pseudomonas aeruginosa; Type VI Secretion Systems; Virulence
PubMed: 31790504
DOI: 10.1371/journal.ppat.1008198 -
The Journal of Physical Chemistry. B Feb 2020Time-resolved phosphorescence spectra of Re(CO)(dmp) and Re(CO)(phen) chromophores (dmp = 4,7-dimethyl-1,10-phenanthroline, phen = 1,10-phenanthroline) bound to surface...
Time-resolved phosphorescence spectra of Re(CO)(dmp) and Re(CO)(phen) chromophores (dmp = 4,7-dimethyl-1,10-phenanthroline, phen = 1,10-phenanthroline) bound to surface histidines (H83, H124, and H126) of azurin mutants exhibit dynamic band maxima shifts to lower wavenumbers following 3-exponential kinetics with 1-5 and 20-100 ns major phases and a 1.1-2.5 μs minor (5-16%) phase. Observation of slow relaxation components was made possible by using an organometallic Re chromophore as a probe whose long phosphorescence lifetime extends the observation window up to ∼3 μs. Integrated emission-band areas also decay with 2- or 3-exponential kinetics; the faster decay phase(s) is relaxation-related, whereas the slowest one [360-680 ns (dmp); 90-140 ns (phen)] arises mainly from population decay. As a result of shifting bands, the emission intensity decay kinetics depend on the detection wavelength. Detailed kinetics analyses and comparisons with band-shift dynamics are needed to disentangle relaxation and population decay kinetics if they occur on comparable timescales. The dynamic phosphorescence Stokes shift in Re-azurins is caused by relaxation motions of the solvent, the protein, and solvated amino acid side chains at the Re binding site in response to chromophore electronic excitation. Comparing relaxation and decay kinetics of and suggests that electron transfer (ET) and relaxation motions in the W122 mutant are coupled. It follows that nanosecond and faster photo-induced ET steps in azurins (and likely other redox proteins) occur from unrelaxed systems; importantly, these reactions can be driven (or hindered) by structural and solvational dynamics.
Topics: Azurin; Coordination Complexes; Ligands; Luminescence; Luminescent Measurements; Mutation; Phenanthrolines; Pseudomonas aeruginosa; Rhenium
PubMed: 31935093
DOI: 10.1021/acs.jpcb.9b10802 -
Dalton Transactions (Cambridge, England... Oct 2020Synthesis and characterisation of a dithiadiaza chelator NSNS2A, as well as copper complexes thereof are reported in this paper. Solution structures of copper(i/ii)...
Synthesis and characterisation of a dithiadiaza chelator NSNS2A, as well as copper complexes thereof are reported in this paper. Solution structures of copper(i/ii) complexes were calculated using density functional theory (DFT) and validated by both NMR and EPR spectroscopy. DFT calculations revealed a switch in the orientation of tetragonal distortion upon protonation, which might be responsible for poor stability of the Cu(II)NSNS2A complex in aqueous media, whilst the same switch in tetragonal distortion was experimentally observed by changing the solvent. The chelator was radiolabeled with 64Cu and evaluated using PET/MRI in rats. Despite a favorable redox potential to stabilize the cuprous state in vivo, the 64Cu(II)NSNS2A complex showed suboptimal stability compared to its tetraazamacrocyclic analogue, 64Cu(TE2A), with a significant 64Cu uptake in the liver.
Topics: Animals; Aza Compounds; Azurin; Chelating Agents; Coordination Complexes; Copper Radioisotopes; Density Functional Theory; Electrochemical Techniques; Kidney; Liver; Macrocyclic Compounds; Magnetic Resonance Imaging; Male; Molecular Conformation; Oxidation-Reduction; Positron-Emission Tomography; Protein Binding; Radiopharmaceuticals; Rats, Wistar; Solvents; Staining and Labeling; Structure-Activity Relationship
PubMed: 32970072
DOI: 10.1039/d0dt02787a