The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Arsenio M Fialho - One of the best experts on this subject based on the ideXlab platform.
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exploring the anticancer potential of the bacterial protein Azurin
microbiology 2016 Vol. 2 Pages 292-303, 2016Co-Authors: Arsenio M Fialho, Nuno Bernardes, A M ChakrabartyAbstract:Bacterial proteins and their derivative peptides have emerged as promising anticancer agents. Nowadays they represent a valuable set of candidate drugs with different origins and modes of action. Among these, monomeric cupredoxins, which are metalloproteins involved in the electron transport chain of prokaryotes, have been shown to possess potent anticancer activities. In particular, much attention has been focused on Azurin produced by the pathogenic bacterium Pseudomonas aeruginosa. More recently, several in vitro and in vivo studies have reported the multi-targeting anticancer properties of Azurin. Moreover, p28, a peptide derived from Azurin, has completed two phase I clinical trials in cancer patients with promising results. In this updated review, we examine the current knowledge regarding Azurin’s modes of action as an anticancer therapeutic protein. We also review the clinical trial results of p28 emphasizing findings that make it suited (alone or in combination) as a therapeutic agent for cancer treatment. Finally we discuss and address the challenges of using the human microbiome to discover novel and unique therapeutic cupredoxin-like proteins.
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modulation of membrane properties of lung cancer cells by Azurin enhances the sensitivity to egfr targeted therapy and decreased β1 integrin mediated adhesion
Cell Cycle, 2016Co-Authors: Nuno Bernardes, Sofia Abreu, Filomena A Carvalho, Fabio Fernandes, Nuno C Santos, Arsenio M FialhoAbstract:In lung cancer, the Epidermal Growth Factor Receptor (EGFR) is one of the main targets for clinical management of this disease. The effectiveness of therapies toward this receptor has already been linked to the expression of integrin receptor subunit β1 in NSCLC A549 cells. In this work we demonstrate that Azurin, an anticancer therapeutic protein originated from bacterial cells, controls the levels of integrin β1 and its appropriate membrane localization, impairing the intracellular signaling cascades downstream these receptors and the invasiveness of cells. We show evidences that Azurin when combined with gefitinib and erlotinib, tyrosine kinase inhibitors which targets specifically the EGFR, enhances the sensitivity of these lung cancer cells to these molecules. The broad effect of Azurin at the cell surface level was examined by Atomic Force Microscopy. The Young 's module (E) shows that the stiffness of A549 lung cancer cells decreased with exposure to Azurin and also gefitinib, suggesting that the alterations in the membrane properties may be the basis of the broad anticancer activity of this protein. Overall, these results show that Azurin may be relevant as an adjuvant to improve the effects of other anticancer agents already in clinical use, to which patients often develop resistance hampering its full therapeutic response.
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high throughput molecular profiling of a p cadherin overexpressing breast cancer model reveals new targets for the anti cancer bacterial protein Azurin
The International Journal of Biochemistry & Cell Biology, 2014Co-Authors: Nuno Bernardes, Ana Sofia Ribeiro, Sofia Abreu, Andre Filipe Vieira, Laura Carreto, Manuel A S Santos, Raquel Seruca, Joana Paredes, Arsenio M FialhoAbstract:Abstract Azurin is a bacterial protein from Pseudomonas aeruginosa which exerts an inhibitory activity in cancer cells. In P-cadherin-overexpressing models, a bad prognosis marker in breast cancer increasing invasion and other malignant features, Azurin decreases the invasion of cancer cells. We performed a microarray analysis to compare the expression profile of Azurin treated cells with different P-cadherin expression levels. Azurin up-regulated apoptosis mediated by p53 protein, endocytosis and vesicle-mediated transport. In the contrary, in invasive MCF-7/AZ.Pcad cells, Azurin decreased the expression of genes associated with cell surface receptors and signal transduction, as well as biological adhesion. Further, Azurin decreased adhesion of cells to proteins from the extracellular matrix (ECM) and altered protein expression of integrins α6, β4 and β1 and interfered with the ability of these cells to form mammospheres. Altogether, our results further enlighten the anti-cancer effects mediated by Azurin in P-cadherin overexpression breast cancer models.
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the bacterial protein Azurin impairs invasion and fak src signaling in p cadherin overexpressing breast cancer cell models
PLOS ONE, 2013Co-Authors: Nuno Bernardes, Ana Sofia Ribeiro, Sofia Abreu, Raquel Seruca, Joana Paredes, Bruna Mota, Rute G Matos, Cecilia M Arraiano, Arsenio M FialhoAbstract:P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of Azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of Azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by Azurin. Azurin (50–100 µM) also caused a specific decrease on P-cadherin protein levels from 30–50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of Azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that Azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, Azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.
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Azurin like protein blocks invasion of toxoplasma gondii through potential interactions with parasite surface antigen sag1
Antimicrobial Agents and Chemotherapy, 2008Co-Authors: Arunasalam Naguleswaran, Arsenio M Fialho, Anita Chaudhari, Chang Soo Hong, A M Chakrabarty, William J SullivanAbstract:Some pathogenic bacteria produce factors that have evolved a capacity to neutralize competing microbes. The cupredoxin family protein Azurin, produced by Pseudomonas aeruginosa, exhibits a remarkable ability to impede invasion of a number of diverse intracellular pathogens, including the human AIDS virus human immunodeficiency virus type 1 and the protozoan parasite Plasmodium falciparum (which causes malaria). Here we report that Azurin and an Azurin-like protein (Laz) from gonococci/meningococci have activity against Toxoplasma, an apicomplexan parasite that causes opportunistic infection in immunocompromised individuals. We demonstrate that the mechanism of action for Laz involves interfering with the ability of Toxoplasma to adhere to host cells. Computer structural analysis reveals that Azurin shares structural features with the predominant surface antigen SAG1, which is known to play an important role in parasite attachment. Interestingly, Azurin also has structural similarities to a monoclonal antibody to SAG1. Surface plasmon resonance binding studies validate that SAG1 interacts strongly with Laz and, to lesser extent, Azurin. Moreover, Toxoplasma mutants lacking SAG1 are not as susceptible to the growth-inhibitory effects of Laz. Collectively, our data show that Toxoplasma adhesion can be significantly impaired by Laz, and to some extent by Azurin, via interactions with SAG1. These observations indicate that Laz can serve as an important tool in the study of host-pathogen interactions and is worthy of further study for development into potential therapeutic agents.
Nuno Bernardes - One of the best experts on this subject based on the ideXlab platform.
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exploring the anticancer potential of the bacterial protein Azurin
microbiology 2016 Vol. 2 Pages 292-303, 2016Co-Authors: Arsenio M Fialho, Nuno Bernardes, A M ChakrabartyAbstract:Bacterial proteins and their derivative peptides have emerged as promising anticancer agents. Nowadays they represent a valuable set of candidate drugs with different origins and modes of action. Among these, monomeric cupredoxins, which are metalloproteins involved in the electron transport chain of prokaryotes, have been shown to possess potent anticancer activities. In particular, much attention has been focused on Azurin produced by the pathogenic bacterium Pseudomonas aeruginosa. More recently, several in vitro and in vivo studies have reported the multi-targeting anticancer properties of Azurin. Moreover, p28, a peptide derived from Azurin, has completed two phase I clinical trials in cancer patients with promising results. In this updated review, we examine the current knowledge regarding Azurin’s modes of action as an anticancer therapeutic protein. We also review the clinical trial results of p28 emphasizing findings that make it suited (alone or in combination) as a therapeutic agent for cancer treatment. Finally we discuss and address the challenges of using the human microbiome to discover novel and unique therapeutic cupredoxin-like proteins.
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modulation of membrane properties of lung cancer cells by Azurin enhances the sensitivity to egfr targeted therapy and decreased β1 integrin mediated adhesion
Cell Cycle, 2016Co-Authors: Nuno Bernardes, Sofia Abreu, Filomena A Carvalho, Fabio Fernandes, Nuno C Santos, Arsenio M FialhoAbstract:In lung cancer, the Epidermal Growth Factor Receptor (EGFR) is one of the main targets for clinical management of this disease. The effectiveness of therapies toward this receptor has already been linked to the expression of integrin receptor subunit β1 in NSCLC A549 cells. In this work we demonstrate that Azurin, an anticancer therapeutic protein originated from bacterial cells, controls the levels of integrin β1 and its appropriate membrane localization, impairing the intracellular signaling cascades downstream these receptors and the invasiveness of cells. We show evidences that Azurin when combined with gefitinib and erlotinib, tyrosine kinase inhibitors which targets specifically the EGFR, enhances the sensitivity of these lung cancer cells to these molecules. The broad effect of Azurin at the cell surface level was examined by Atomic Force Microscopy. The Young 's module (E) shows that the stiffness of A549 lung cancer cells decreased with exposure to Azurin and also gefitinib, suggesting that the alterations in the membrane properties may be the basis of the broad anticancer activity of this protein. Overall, these results show that Azurin may be relevant as an adjuvant to improve the effects of other anticancer agents already in clinical use, to which patients often develop resistance hampering its full therapeutic response.
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high throughput molecular profiling of a p cadherin overexpressing breast cancer model reveals new targets for the anti cancer bacterial protein Azurin
The International Journal of Biochemistry & Cell Biology, 2014Co-Authors: Nuno Bernardes, Ana Sofia Ribeiro, Sofia Abreu, Andre Filipe Vieira, Laura Carreto, Manuel A S Santos, Raquel Seruca, Joana Paredes, Arsenio M FialhoAbstract:Abstract Azurin is a bacterial protein from Pseudomonas aeruginosa which exerts an inhibitory activity in cancer cells. In P-cadherin-overexpressing models, a bad prognosis marker in breast cancer increasing invasion and other malignant features, Azurin decreases the invasion of cancer cells. We performed a microarray analysis to compare the expression profile of Azurin treated cells with different P-cadherin expression levels. Azurin up-regulated apoptosis mediated by p53 protein, endocytosis and vesicle-mediated transport. In the contrary, in invasive MCF-7/AZ.Pcad cells, Azurin decreased the expression of genes associated with cell surface receptors and signal transduction, as well as biological adhesion. Further, Azurin decreased adhesion of cells to proteins from the extracellular matrix (ECM) and altered protein expression of integrins α6, β4 and β1 and interfered with the ability of these cells to form mammospheres. Altogether, our results further enlighten the anti-cancer effects mediated by Azurin in P-cadherin overexpression breast cancer models.
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the bacterial protein Azurin impairs invasion and fak src signaling in p cadherin overexpressing breast cancer cell models
PLOS ONE, 2013Co-Authors: Nuno Bernardes, Ana Sofia Ribeiro, Sofia Abreu, Raquel Seruca, Joana Paredes, Bruna Mota, Rute G Matos, Cecilia M Arraiano, Arsenio M FialhoAbstract:P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of Azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of Azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by Azurin. Azurin (50–100 µM) also caused a specific decrease on P-cadherin protein levels from 30–50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of Azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that Azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, Azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.
Jens Ulstrup - One of the best experts on this subject based on the ideXlab platform.
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long range interfacial electrochemical electron transfer of pseudomonas aeruginosa Azurin gold nanoparticle hybrid systems
Journal of Physical Chemistry C, 2009Co-Authors: Palle Skovhus Jensen, Qijin Chi, Jingdong Zhang, Jens UlstrupAbstract:We have prepared a “hybrid” of the blue copper protein Azurin (Pseudomonas aeruginosa) and a 3 nm gold nanoparticle (AuNP). The AuNP/Azurin hybrid was assembled on a Au(111)-electrode surface in a two-step process. The AuNP was first attached to the Au(111) electrode via Au−S chemisorption of a 4,4′-biphenyldithiol (4,4′-BPDT) monolayer. This was followed by 1-decanethiol modification of the bound AuNP and hydrophobic binding of Azurin to the AuNP. The Au(111)/AuNP/Azurin system was characterized by atomic force microscopy (AFM), cyclic voltammetry (CV), and in situ electrochemical scanning tunneling microscopy (in situ STM). AFM and STM point to the feasibility of preparing both dense and sparsely populated AuNP monolayers. CV shows two pairs of voltammetric peaks at high scan rates, both around the Azurin equilibrium potential. One pair of redox peaks follows closely that of Azurin hydrophobically immobilized directly on a Au(111)/1-tetradecanethiol reference surface. The other pair, tentatively assigne...
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long range interfacial electron transfer of metalloproteins based on molecular wiring assemblies
Faraday Discussions, 2006Co-Authors: Qijin Chi, Jingdong Zhang, Palle Skovhus Jensen, Hans Erik Molager Christensen, Jens UlstrupAbstract:We address some physical features associated with long-range interfacial electron transfer (ET) of metalloproteins in both electrochemical and electrochemical scanning tunneling microscopy (ECSTM) configurations, which offer a brief foundation for understanding of the ET mechanisms. These features are illustrated experimentally by new developments of two systems with the blue copper protein Azurin and enzyme nitrite reductase as model metalloproteins. Azurin and nitrite reductase were assembled on Au(111) surfaces by molecular wiring to establish effective electronic coupling between the redox centers in the proteins and the electrode surface for ET and biological electrocatalysis. With such assemblies, interfacial ET proceeds through chemically defined and well oriented sites and parallels biological ET. In the case of Azurin, the ET properties can be characterized comprehensively and even down to the single-molecule level with direct observation of redox-gated electron tunnelling resonance. Molecular wiring using a π-conjugated thiol is suitable for assembling monolayers of the enzyme with catalytic activity well-retained. The catalytic mechanism involves multiple-ET steps including both intramolecular and interfacial processes. Interestingly, ET appears to exhibit a substrate-gated pattern observed preliminarily in both voltammetry and ECSTM.
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long range protein electron transfer observed at the single molecule level in situ mapping of redox gated tunneling resonance
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Qijin Chi, Ole Farver, Jens UlstrupAbstract:A biomimetic long-range electron transfer (ET) system consisting of the blue copper protein Azurin, a tunneling barrier bridge, and a gold single-crystal electrode was designed on the basis of molecular wiring self-assembly principles. This system is sufficiently stable and sensitive in a quasi-biological environment, suitable for detailed observations of long-range protein interfacial ET at the nanoscale and single-molecule levels. Because Azurin is located at clearly identifiable fixed sites in well controlled orientation, the ET configuration parallels biological ET. The ET is nonadiabatic, and the rate constants display tunneling features with distance-decay factors of 0.83 and 0.91 A–1 in H2O and D2O, respectively. Redox-gated tunneling resonance is observed in situ at the single-molecule level by using electrochemical scanning tunneling microscopy, exhibiting an asymmetric dependence on the redox potential. Maximum resonance appears around the equilibrium redox potential of Azurin with an on/off current ratio of ≈9. Simulation analyses, based on a two-step interfacial ET model for the scanning tunneling microscopy redox process, were performed and provide quantitative information for rational understanding of the ET mechanism.
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ordered assembly and controlled electron transfer of the blue copper protein Azurin at gold 111 single crystal substrates
Journal of Physical Chemistry B, 2001Co-Authors: Qijin Chi, Jingdong Zhang, Jens Enevold Thaulov Andersen, Jens UlstrupAbstract:We have shown that Pseudomonas aeruginosa Azurin can be immobilized on alkanethiol monolayers self-assembled on Au(111). Immobilization is achieved through hydrophobic interactions between the hydrophobic area around the copper atom in Azurin and methyl heads of alkanethiol to form submonolayers or monolayers. In this orientation mode Azurin molecules on Au(111) are oriented with the redox center (copper atom) facing the electrode surface. This is opposite to the orientation of Azurin on bare gold which is via a surface disulfide group such as recently reported. Scanning tunneling microscopy (STM) with molecular resolution reveals that both well-ordered alkanethiol and protein adlayers are present. Adsorbed Azurin molecules exhibit high stability and retain electron transfer (ET) function. Long-range interfacial ET between Azurin and Au(111) across variable-length alkanethiol bridges was systematically investigated by different electrochemical techniques. Distance-dependent ET can be controlled by adjusti...
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molecular monolayers and interfacial electron transfer of pseudomonas aeruginosa Azurin on au 111
Journal of the American Chemical Society, 2000Co-Authors: Qijin Chi, Jingdong Zhang, Jens Enevold Thaulov Andersen, Gerard W Canters, Jens Ulrik Nielsen, Esben Peter Friis, Ib Chorkendorff, Jens UlstrupAbstract:We provide a comprehensive approach to the formation and characterization of molecular monolayers of the blue copper protein Pseudomonas aeruginosa Azurin on Au(111) in aqueous ammonium acetate solution. Main issues are adsorption patterns, reductive desorption, properties of the double layer, and long-range electrochemical electron transfer between the electrode and the copper center. Voltammetry, electrochemical impedance spectroscopy (EIS), in situ scanning tunneling microscopy (STM), and X-ray photoelectron spectroscopy (XPS) have been employed to disclose features of these issues. Zn-substituted Azurin, cystine, and 1-butanethiol are investigated for comparison. Cyclic voltammetric and capacitance measurements show qualitatively that Azurin is adsorbed at submicromolar concentrations over a broad potential range. The characteristics of reductive desorption suggest that Azurin is adsorbed via its disulfide group to form a monolayer. The adsorption of this protein on Au(111) via a gold−sulfur binding m...
Jeongwoo Choi - One of the best experts on this subject based on the ideXlab platform.
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recombinant Azurin cdse zns hybrid structures for nanoscale resistive random access memory device
Biosensors and Bioelectronics, 2017Co-Authors: Ajay Kumar Yagati, Sanguk Kim, Taek Lee, Junhong Min, Jeongwoo ChoiAbstract:In the present study, we developed a biohybrid material composed of recombinant Azurin and CdSe-ZnS quantum dot to perform as a resistive random access memory (ReRAM) device. Site specific amino acid sequences were introduced in Azurin to bind with the surface of CdSe-ZnS nanoparticle allowing the formation of a hybrid and voltage-driven switching enabled to develop a resistive random access memory (ReRAM) device. The analytical measurements confirmed that the Azurin and CdSe-ZnS nanoparticles were well conjugated and formed into a single hybrid. Further, reversible, bistable switching along with repeatable writing-reading-erasing processes on individual Azurin/CdSe-ZnS hybrid at nanoscale was achieved on the hybrid device. The device was programmed tested for 50 cycles with an ON/OFF ratio and measured to be of three orders of magnitude. The developed device shown good stability and repeatability and operates at low voltages thus makes it promising candidate for future memory device applications.
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fusion protein bilayer fabrication composed of recombinant Azurin cytochrome p450 by the sortase mediated ligation method
Colloids and Surfaces B: Biointerfaces, 2014Co-Authors: Taek Lee, Junhong Min, Hidehiko Hirakawa, Teruyuki Nagamune, Jeongwoo ChoiAbstract:Recently, the fabrication of protein bilayer has been required for the development of protein or enzyme complex formation. In the present study, we fabricated a fusion protein bilayer composed of recombinant Azurin-cytochrome P450, which was synthesized by a site-specific sortase-mediated ligation method. The Pseudomonas aeruginosa Azurin was modified by DNA recombinant technique, for enzymatic ligation and immobilization. The Pseudomonas putida cytochrome P450 was also modified for enzymatic ligation. The recombinant metalloproteins were conjugated via the sortase A. The conjugation was confirmed by SDS-PAGE and UV-vis. Then, the prepared fusion protein was immobilized on Au substrate, by the self-assembly method. The Azu-P450 (recombinant Azurin-cytochrome P450) fusion protein layer was confirmed by AFM (Atomic Force Microscopy) and SERS (Surface-enhanced Raman Spectroscopy), to confirm the fusion protein bilayer orientation. Moreover, the electrochemical property of Azu-P450 was observed by cyclic voltammetry (CV). As a result, the Azu-P450 fusion protein bilayer shows good orientation on the Au substrate. Also, the original redox property of this fusion protein bilayer has been well maintained. The proposed fusion protein bilayer can.
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signal enhancement of electrochemical biomemory device composed of recombinant Azurin gold nanoparticle
Electroanalysis, 2011Co-Authors: Taek Lee, Junhong Min, Siyoul Yoo, Yongho Chung, Jeongwoo ChoiAbstract:A signal-enhanced biomemory device was developed by introducing cysteine-modified Azurin/gold nanoparticle (GNP) heterolayers. The proposed recombinant Azurin/GNP heterolayers provided an enhanced electron transfer between recombinant Azurin/GNP and the Au surface, which stored the charges in the fabricated heterolayer. The fabricated recombinant Azurin/GNP heterolayers was investigated by atomic force microscopy (AFM) and surface plasmon resonance (SPR) spectroscopy. Cyclic voltammetry (CV) was performed to examine the current signal-amplified electrochemical property. In this analysis, the recombinant Azurin/GNP heterolayers produced current that was 5 times greater than the recombinant Azurin monolayer. These redox potentials were then used to obtain the ‘write step’ and ‘erase step’. Using these parameters, the biomemory function of the device was verified by chronoamperometry (CA).
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multifunctional 4 bit biomemory chip consisting of recombinant Azurin variants
Biomaterials, 2011Co-Authors: Taek Lee, Sanguk Kim, Junhong Min, Jeongwoo ChoiAbstract:We developed a multi-functional 4-bit biomemory chip that consisted of recombinant Azurin variants. The Azurin was modified to introduce cysteine-residues. In addition, the Cu ion in this recombinant Azurin protein was substituted with various other metal ions such as Co, Mn, Fe and Ni ion to allow the protein to perform various memory functions. Each metal-substituted recombinant protein was directly self-assembled attached onto Au surface via the thiol group of the cysteine. UV–VIS spectroscopy was performed to confirm the metal substitution. Atomic force microscopy was used to measure the film organization. Also, the 4 different Azurin variants were investigated to assess the electrochemical behavior. Cyclic voltammetry and an open circuit potential indicated that the Azurin variants had different redox peaks and specific open circuit potential values. Using these parameters, memory function was verified by chronoamperometry and open circuit potential amperometry. Therefore, a multi-bit biomemory chip was successfully developed. The results presented here provide a new approach, concept and material combination for the development of biomemory systems using recombinant protein. If a low electrochemical signal from a few single proteins could be achieved, it may be possible to substitute silicon-based memory devices with biological-based memory devices.
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nanoscale film formation of recombinant Azurin variants with various cysteine residues on gold substrate for bioelectronic device
Journal of Nanoscience and Nanotechnology, 2010Co-Authors: Sanguk Kim, Jinho Lee, Taek Lee, Junhong Min, Jeongwoo ChoiAbstract:Nanoscale film fabrication of recombinant Azurin variants with various cysteine residues on gold substrate was developed without any surface modification for bioelectronic device. We have modified Azurin with different number of cysteine residues at its amino acid chain based on site-directed mutagenesis. The resulting recombinant protein, Azurin, retained its original redox property in the same manner as native Azurin. Recombinant Azurin was immobilized on Au substrate by strong affinity between thiol of cysteine and gold. The orientations of recombinant Azurin with various cysteine residues immobilized on the Au substrate were analyzed by fluorescence microscope, scanning tunneling microscope, and surface plasmon resonance. Our data revealed that binding activity of recombinant Azurin with three cysteine residues on the Au substrate significantly increased in comparison to single residue Azurin. Immobilization method of highly oriented recombinant Azurin based on cysteine-modification could be useful for the nanoscale film fabrication of nanobiochip.
Gerard W Canters - One of the best experts on this subject based on the ideXlab platform.
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electron tunnelling through Azurin is mediated by the active site cu ion
Chemical Physics Letters, 2003Co-Authors: Andrea Alessandrini, Gerard W Canters, Mimmo Gerunda, Ph M Verbeet, Paolo FacciAbstract:Abstract Cu– and Zn–Azurin chemisorbed on Au(1 1 1) have been comparatively investigated by electrochemical scanning tunnelling microscopy in buffer solution. Cu–Azurin shows a marked tunnelling current resonance upon substrate potential at −0.21 V (vs SCE), whereas Zn counterparts do not. These data, discussed in terms of current theories on electron tunnelling through redox adsorbates, demonstrate the role of the electroactive metal ion present in the active site in assisting electron transfer via this metalloprotein.
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molecular monolayers and interfacial electron transfer of pseudomonas aeruginosa Azurin on au 111
Journal of the American Chemical Society, 2000Co-Authors: Qijin Chi, Jingdong Zhang, Jens Enevold Thaulov Andersen, Gerard W Canters, Jens Ulrik Nielsen, Esben Peter Friis, Ib Chorkendorff, Jens UlstrupAbstract:We provide a comprehensive approach to the formation and characterization of molecular monolayers of the blue copper protein Pseudomonas aeruginosa Azurin on Au(111) in aqueous ammonium acetate solution. Main issues are adsorption patterns, reductive desorption, properties of the double layer, and long-range electrochemical electron transfer between the electrode and the copper center. Voltammetry, electrochemical impedance spectroscopy (EIS), in situ scanning tunneling microscopy (STM), and X-ray photoelectron spectroscopy (XPS) have been employed to disclose features of these issues. Zn-substituted Azurin, cystine, and 1-butanethiol are investigated for comparison. Cyclic voltammetric and capacitance measurements show qualitatively that Azurin is adsorbed at submicromolar concentrations over a broad potential range. The characteristics of reductive desorption suggest that Azurin is adsorbed via its disulfide group to form a monolayer. The adsorption of this protein on Au(111) via a gold−sulfur binding m...
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in vivo studies disprove an obligatory role of Azurin in denitrification in pseudomonas aeruginosa and show that azu expression is under control of rpos and anr
Microbiology, 1997Co-Authors: Erik Vijgenboom, Julie E Busch, Gerard W CantersAbstract:Summary: The role of the blue copper protein Azurin and cytochrome C551 as the possible electron donors to nitrite reductase in the dissimilatory nitrate reduction pathway in Pseudomonas aeruginosa have been investigated. It was shown by an in vivo approach with mutant strains of P. aeruginosa deficient in one or both of these electron-transfer proteins that cytochrome C551, but not Azurin, is functional in this pathway. Expression studies demonstrated the presence of Azurin in both aerobic and anaerobic cultures. A sharp increase in Azurin expression was observed when cultures were shifted from exponential to stationary phase. The stationary-phase sigma factor, σs, was shown to be responsible for this induction. In addition, one of the two promoters transcribing the azu gene was regulated by the anaerobic transcriptional regulator ANR. An Azurin-deficient mutant was more sensitive to hydrogen peroxide and paraquat than the wild-type P. aeruginosa. These results suggest a physiological role of Azurin in stress situations like those encountered in the transition to the stationary phase.
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effect of lysine ionization on the structure and electrochemical behaviour of the met44 lys mutant of the blue copper protein Azurin from pseudomonas aeruginosa
FEBS Journal, 1993Co-Authors: Mart Van De Kamp, Gerard W Canters, Colin R Andrew, Joann Sandersloehr, Christopher J Bender, Jack PeisachAbstract:The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface patch of Azurin from Pseudomonas aeruginosa by a lysine residue were studied as a function of the ionization state of the lysine. In the pH range 5 -8, the optical absorption, resonance Raman, EPR and electron spin-echo envelope modulation spectroscopic properties of wild-type and Met44+Lys (M44K) Azurin are very similar, indicating that the Cu-site geometry has been maintained. At higher pH, the deprotonation of Lys44 in M44K Azurin (pK, 9-10) is accompanied by changes in the optical-absorption maxima (614 nm and 450 nm instead of 625 nm and 470 nm) and in the EPR gll value (2.298 instead of 2.241), indicative of a change in the bonding interactions of Cu at high pH. The strong pH dependence of the electron self-exchange rate of M44K Azurin supports the assignment of Lys44 as the ionizable group and demonstrates the importance of the hydrophobic patch for electron transfer. The pH dependence of the midpoint potentials of wild-type and M44K Azurin can be accounted for by the ionizations of His35 and His83 and by the additional electrostatic effect of the mutation.
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x ray analysis and spectroscopic characterization of m121q Azurin a copper site model for stellacyanin
Journal of Molecular Biology, 1993Co-Authors: Antonio Romero, Herbert Nar, Robert Huber, Albrecht Messerschmidt, Carla W G Hoitink, Gerard W CantersAbstract:The dependence of the properties of the Azurin blue copper site on the nature of the axial ligand at position 121 was tested by site-directed mutagenesis. This residue was substituted for a glutamine, the purported fourth copper ligand in the related protein stellacyanin. M121Q Azurin was isolated and purified from Escherichia coli and characterized by spectroscopic methods. The mutant copper site has the ultra-violet-vis and electron paramagnetic resonance (EPR) characteristics of a type I site, but the spectroscopic details differ significantly from wild-type (wt) Azurin. The X and S-band EPR spectra of M121Q Azurin can be well stimulated with the parameters for stellacyanin, indicating that the copper sites of both proteins in the oxidized state are similar. The midpoint potential of M121Q is 263 mV, 25 mV lower than for wt Azurin. The reactivity of the mutant was probed by measuring the electron self exchange rate by nuclear magnetic resonance spectroscopy. The rate was 8 x 10(3) mol-1 s-1, almost two orders of magnitude lower than the value for wt Azurin (5 x 10(5) mol-1 s-1). Detailed structural information on the M121Q Cu(II)-site was obtained by X-ray analysis of M121Q Azurin crystals at 1.9 A resolution. The histidine and cysteine copper ligand distances and angles in the equatorial plane around the copper are very similar to the wt protein. Gln121 is co-ordinated in a monodentate fashion via its side-chain oxygen atom at a distance of 2.26 A. The distance between copper and the carbonyl group of Gly45 is increased from 3.13 A (wt) to 3.37 A resulting in a distorted tetrahedral N2SO copper co-ordination. The possible significance of these results for the structure of the copper site of stellacyanin, the only small blue copper protein lacking a methionine ligand, is discussed. Conformational changes with respect to the wt Azurin are seen in some of the connecting loops between secondary structure elements, in the mutation site and in the beta-strand 2a. The side-chains involved in the hydrophobic patch surrounding His117 are subject to large changes in their conformations. In contrast to wt Azurin, the copper site in M121Q Azurin undergoes significant structural changes on reduction.(ABSTRACT TRUNCATED AT 400 WORDS)
