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Biomolecules Oct 2019In the growing field of biomolecular electronics, blue-copper Azurin stands out as one of the most widely studied protein in single-molecule contacts. Interestingly,...
In the growing field of biomolecular electronics, blue-copper Azurin stands out as one of the most widely studied protein in single-molecule contacts. Interestingly, despite the paramount importance of the structure/dynamics of molecular contacts in their transport properties, these factors remain largely unexplored from the theoretical point of view in the context of single Azurin junctions. Here we address this issue using all-atom Molecular Dynamics (MD) of Pseudomonas Aeruginosa Azurin adsorbed to a Au(111) substrate. In particular, we focus on the structure and dynamics of the free/adsorbed protein and how these properties are altered upon single-point mutations. The results revealed that wild-type Azurin adsorbs on Au(111) along two well defined configurations: one tethered via cysteine groups and the other via the hydrophobic pocket surrounding the Cu 2 + . Surprisingly, our simulations revealed that single amino-acid mutations gave rise to a quenching of protein vibrations ultimately resulting in its overall stiffening. Given the role of amino-acid vibrations and reorientation in the dehydration process at the protein-water-substrate interface, we suggest that this might have an effect on the adsorption process of the mutant, giving rise to new adsorption configurations.
Topics: Adsorption; Amino Acids; Azurin; Molecular Dynamics Simulation; Mutation; Protein Conformation; Water
PubMed: 31618974
DOI: 10.3390/biom9100611 -
The Journal of Physical Chemistry. B Jan 2024Electron transfer (ET) between neutral and cationic tryptophan residues in the azurin construct [Re(H126)(CO)(dmp)](W124)(W122)Cu (dmp = 4,7-Me-1,10-phenanthroline) was...
Electron transfer (ET) between neutral and cationic tryptophan residues in the azurin construct [Re(H126)(CO)(dmp)](W124)(W122)Cu (dmp = 4,7-Me-1,10-phenanthroline) was investigated by Born-Oppenheimer quantum-mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) simulations. We focused on W124 ← W122 ET, which is the middle step of the photochemical hole-hopping process *Re(CO)(dmp) ← W124 ← W122 ← Cu, where sequential hopping amounts to nearly 10,000-fold acceleration over single-step tunneling (. , , 192-200). In accordance with experiments, UKS-DFT QM/MM/MD simulations identified forward and reverse steps of W124 ↔ W122 ET equilibrium, as well as back ET Re(CO)(dmp) → W124 that restores *Re(CO)(dmp). Strong electronic coupling between the two indoles (≥40 meV in the crossing region) makes the productive W124 ← W122 ET adiabatic. Energies of the two redox states are driven to degeneracy by fluctuations of the electrostatic potential at the two indoles, mainly caused by water solvation, with contributions from the protein dynamics in the W122 vicinity. ET probability depends on the orientation of Re(CO)(dmp) relative to W124 and its rotation diminishes the hopping yield. Comparison with hole hopping in natural systems reveals structural and dynamics factors that are important for designing efficient hole-hopping processes.
Topics: Azurin; Tryptophan; Oxidation-Reduction; Electron Transport; Indoles
PubMed: 38145895
DOI: 10.1021/acs.jpcb.3c06568 -
The New Phytologist Feb 2021The SKU5 similar (SKS) genes encode a family of multi-copper-oxidase-like proteins with cupredoxin domains similar to those in laccase and ascorbate oxidase. Although...
The SKU5 similar (SKS) genes encode a family of multi-copper-oxidase-like proteins with cupredoxin domains similar to those in laccase and ascorbate oxidase. Although SKS proteins are known to function in root growth and cotyledon vascular patterning in Arabidopsis, their role in plant reproductive processes is poorly understood. Here, we identified a seed mutant of maize (Zea mays), generated by ethyl methane sulfonate (EMS) mutagenesis, that we designated defective kernel-zk1 (dek-zk1). The mutant produced small, shriveled kernels with an aberrant basal endosperm transfer layer (BETL) and placento-chalazal (PC) layer and irregular starch granules. Map-based cloning revealed that Dek-zk1 encodes an SKU5 similar 13 (GenBank: ONM36900.1), so it was named ZmSKS13. ZmSKS13 comprises a paralogous pair with Zm00001d012524, but the transcript abundance of ZmSKS13 in developing kernels is 15 times higher than that of Zm00001d012524, resulting in dek-zk1 mutation conveying a distinct kernel phenotype. ZmSKS13 loss of function led to overaccumulation of reactive oxygen species (ROS) and severe DNA damage in the nucellus and BETL and PC layer cells, and exogenous antioxidants significantly alleviated the defects of the mutant kernels. Our results thus demonstrate that ZmSKS13 is a novel regulator that plays a crucial role in kernel development in maize through the modulation of ROS homeostasis.
Topics: Azurin; Gene Expression Regulation, Plant; Homeostasis; Oxidation-Reduction; Plant Proteins; Zea mays
PubMed: 33034042
DOI: 10.1111/nph.16988 -
Nanoscale Advances Jul 2019The NIR excitation and large anti-Stokes shift of upconversion nanoparticles (UCNPs) have made them an ideal choice as biological nanoprobes. A key challenge in the...
The NIR excitation and large anti-Stokes shift of upconversion nanoparticles (UCNPs) have made them an ideal choice as biological nanoprobes. A key challenge in the field is to confer biorecognition units to UCNPs so that they can be used to probe specific targets in biological systems. While various agents have been combined with UCNPs to meet such a challenge, most studies are limited to small molecules, while biomolecules such as metalloproteins that possess much higher affinity and selectivity for metal ions have not been explored. Herein we demonstrate that fusion of zwitterion-coated UCNPs with azurin, a member of a family of redox-active copper proteins called cupredoxins that play important roles in diverse biological functions, can serve as an ideal platform for the label-free upconversion luminescence sensing of Cu with a ratiometric response. The selectively binding of apo-azurin with Cu induce a significant absorbance at about 625 nm, and hence decreases the red emission of the UCNPs. In contrast, the green emission of the UCNPs remains constant and acts as an internal standard reference for the ratiometric sensing of Cu. This approach opens a new window for the development of assays for biosensing based on a combination of specific metalloproteins with UCNPs.
PubMed: 32195453
DOI: 10.1039/c9na00168a -
ACS Omega Jun 2023Redox-active amino acid residues are at the heart of biological electron-transfer reactions. They play important roles in natural protein functions and are implicated in...
Redox-active amino acid residues are at the heart of biological electron-transfer reactions. They play important roles in natural protein functions and are implicated in disease states (e.g., oxidative-stress-associated disorders). Tryptophan (Trp) is one such redox-active amino acid residue, and it has long been known to serve a functional role in proteins. Broadly speaking, there is still much to learn about the local features that make some Trp redox active and others inactive. Herein, we describe a new protein model system where we investigate how a methionine (Met) residue proximal to a redox-active Trp affects its reactivity and spectroscopy. We use an artificial variant of azurin from to produce these models. We employ a series of UV-visible spectroscopy, electrochemistry, electron paramagnetic resonance, and density functional theory experiments to demonstrate the effect that placing Met near Trp radicals has in the context of redox proteins. The introduction of Met proximal to Trp lowers its reduction potential by ca. 30 mV and causes clear shifts in the optical spectra of the corresponding radicals. While the effect may be small, it is significant enough to be a way for natural systems to tune Trp reactivity.
PubMed: 37305310
DOI: 10.1021/acsomega.3c01589 -
Frontiers in Cell and Developmental... 2020Recently, cell-based therapies have been explored as a strategy to enhance the specificity of anticancer therapeutic agents. In this perspective, human mesenchymal...
Recently, cell-based therapies have been explored as a strategy to enhance the specificity of anticancer therapeutic agents. In this perspective, human mesenchymal stromal cells (MSC) hold a promising future as cell delivery systems for anticancer proteins due to their unique biological features. In this study, we engineered human MSC to secrete a human codon-optimized version of azurin (hazu), a bacterial protein that has demonstrated anticancer activity toward different cancer models both and . To this end, microporation was used to deliver plasmid DNA encoding azurin into MSC derived from bone marrow (BM) and umbilical cord matrix (UCM), leading to expression and secretion of hazu to the conditioned medium (CM). Engineered hazu-MSC were shown to preserve tumor tropism toward breast (MCF-7) and lung (A549) cancer cell lines, comparable to non-modified MSC. Azurin was detected in the CM of transfected MSC and, upon treatment with hazu-MSC-CM, we observed a decrease in cancer cell proliferation, migration, and invasion, and an increase in cell death for both cancer cell lines. Moreover, expression of azurin caused no changes in MSC expression profile of cytokines relevant in the context of cancer progression, thus suggesting that the antitumoral effects induced by hazu-MSC secretome might be due to the presence of azurin independently. In conclusion, data shown herein indicate that MSC-produced azurin in a CM configuration elicits an anticancer effect.
PubMed: 32733876
DOI: 10.3389/fcell.2020.00471 -
Marine Drugs Jan 2024Biofilm is accountable for nosocomial infections and chronic illness, making it a serious economic and public health problem. , thanks to its ability to form biofilm and...
Biofilm is accountable for nosocomial infections and chronic illness, making it a serious economic and public health problem. , thanks to its ability to form biofilm and colonize biomaterials, represents the most frequent causative agent involved in biofilm-associated infections of medical devices. Therefore, the research of new molecules able to interfere with biofilm formation has a remarkable interest. In the present work, the attention was focused on sp. TAE6080, an Antarctic marine bacterium able to produce and secrete an effective antibiofilm compound. The molecule responsible for this activity was purified by an activity-guided approach and identified by LC-MS/MS. Results indicated the active protein was a periplasmic protein similar to the PAO1 azurin, named cold-azurin. The cold-azurin was recombinantly produced in and purified. The recombinant protein was able to impair attachment to the polystyrene surface and effectively prevent biofilm formation.
Topics: Pseudomonas; Azurin; Anti-Bacterial Agents; Antarctic Regions; Escherichia coli; Chromatography, Liquid; Tandem Mass Spectrometry; Biofilms; Pseudomonas aeruginosa; Staphylococcus epidermidis
PubMed: 38393032
DOI: 10.3390/md22020061 -
Journal of Inorganic Biochemistry Jul 2024Motivated by the ambition to establish an enzyme-driven bioleaching pathway for copper extraction, properties of the Type-1 copper protein rusticyanin from...
Kinetic, electrochemical and spectral characterization of bacterial and archaeal rusticyanins; unexpected stability issues and consequences for applications in biotechnology.
Motivated by the ambition to establish an enzyme-driven bioleaching pathway for copper extraction, properties of the Type-1 copper protein rusticyanin from Acidithiobacillus ferrooxidans (AfR) were compared with those from an ancestral form of this enzyme (N0) and an archaeal enzyme identified in Ferroplasma acidiphilum (FaR). While both N0 and FaR show redox potentials similar to that of AfR their electron transport rates were significantly slower. The lack of a correlation between the redox potentials and electron transfer rates indicates that AfR and its associated electron transfer chain evolved to specifically facilitate the efficient conversion of the energy of iron oxidation to ATP formation. In F. acidiphilum this pathway is not as efficient unless it is up-regulated by an as of yet unknown mechanism. In addition, while the electrochemical properties of AfR were consistent with previous data, previously unreported behavior was found leading to a form that is associated with a partially unfolded form of the protein. The cyclic voltammetry (CV) response of AfR immobilized onto an electrode showed limited stability, which may be connected to the presence of the partially unfolded state of this protein. Insights gained in this study may thus inform the engineering of optimized rusticyanin variants for bioleaching processes as well as enzyme-catalyzed solubilization of copper-containing ores such as chalcopyrite.
Topics: Kinetics; Electrochemistry; Azurin; Actinobacteria; Thermoplasmales; Electron Spin Resonance Spectroscopy; Models, Molecular; Protein Structure, Tertiary; Iron; Oxidation-Reduction; Biotechnology; Protein Stability; Conserved Sequence
PubMed: 38593609
DOI: 10.1016/j.jinorgbio.2024.112539 -
AMB Express Mar 2020Salmonella typhimurium VNP-20009 (VNP) is a non-pathogenic attenuated strain, which, as a facultative anaerobe, preferentially accumulates in hypoxic regions of solid...
Salmonella typhimurium VNP-20009 (VNP) is a non-pathogenic attenuated strain, which, as a facultative anaerobe, preferentially accumulates in hypoxic regions of solid tumors. Here, VNP was utilized as a delivery vehicle of the anti-tumor protein Lipidated azurin, Laz, which is produced by the meningitis-causing bacterium Neisseria meningitides. In brain cancer cells, Laz has been demonstrated to induce apoptosis through an interaction with the tumor suppressor protein p53. In this study, the laz gene, including its signal sequence, was cloned downstream of a hypoxia inducible promoter (HIP-1), before being electroporated into VNP. Successful ectopic expression and export of the Laz protein by VNP under hypoxic conditions were confirmed by Western blot analysis of the cell-free culture medium. Effective expression of Laz by VNP was investigated in two glioblastoma cell lines: LN-229 and U-373, with the latter line carrying a mutated version of p53; as well as in the breast cancer line MCF-7. Cytotoxicity of the VNP-Laz was assessed by determining the fluorescence of the apoptotic marker caspases 3/7. Compared to the purified Laz, VNP-Laz, significantly induced apoptosis in MCF-7, LN-229 and, to a much lower extent in U-373 cells, suggesting a p53-linked mechanism. Our results might represent a new approach of targeted gene delivery and suggest a potential application in brain tumor therapy.
PubMed: 32221741
DOI: 10.1186/s13568-020-00995-8 -
Journal of the American Chemical Society Sep 2020Blue copper proteins have a constrained Cu(II) geometry that has proven difficult to recapitulate outside native cupredoxin folds. Previous work has successfully...
Blue copper proteins have a constrained Cu(II) geometry that has proven difficult to recapitulate outside native cupredoxin folds. Previous work has successfully designed green copper proteins which could be tuned blue using exogenous ligands, but the question of how one can create a self-contained blue copper site within a de novo scaffold, especially one removed from a cupredoxin fold, remained. We have recently reported a red copper protein site within a three helical bundle scaffold which we later revisited and determined to be a nitrosocyanin mimic, with a CuHisCysGlu binding site. We now report efforts to rationally design this construct toward either green or blue copper chromophores using mutation strategies that have proven successful in native cupredoxins. By rotating the metal binding site, we created a de novo green copper protein. This in turn was converted to a blue copper protein by an axial methionine. Following this rational sequence, we have successfully created red, green, and blue copper proteins within an alpha helical fold, enabling comparisons for the first time of their structure and function disconnected from the overall cupredoxin fold.
Topics: Azurin; Binding Sites; Copper; Electrochemical Techniques; Models, Molecular; X-Ray Absorption Spectroscopy
PubMed: 32786767
DOI: 10.1021/jacs.0c04757