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Small Methods Feb 2022In vitro spatiotemporal control of cell differentiation is a critical issue in several biomedical fields such as stem cell therapy and regenerative medicine, as it...
In vitro spatiotemporal control of cell differentiation is a critical issue in several biomedical fields such as stem cell therapy and regenerative medicine, as it enables the generation of heterogeneous tissue structures similar to those of their native counterparts. However, the simultaneous control of both spatial and temporal cell differentiation poses important challenges, and therefore no previous studies have achieved this goal. Here, the authors develop a cell differentiation biomolecular electron controller ("Biomoletron") composed of recombinant proteins, DNA, Au nanoparticles, peptides, and an electrically released complex with retinoic acid (RA) to spatiotemporally control SH-SY5Y cell differentiation. RA is only released from the Biomoletron when the complex is electrically stimulated, thus demonstrating the temporal control of SH-SY5Y cell differentiation. Furthermore, by introducing a patterned Au substrate that allows controlling the area where the Biomoletron is immobilized, spatiotemporal differentiation of the SH-SY5Y cell is successfully achieved. Therefore, the proposed Biomoletron-mediated differentiation method provides a promising strategy for spatiotemporal cell differentiation control with applications in regenerative medicine and cell therapy.
Topics: Azurin; Cell Differentiation; Cell Line; Cell- and Tissue-Based Therapy; Cell-Penetrating Peptides; DNA; Electromagnetic Phenomena; Gold; Humans; Metal Nanoparticles; Neurons; Oligopeptides; Peptides; Regenerative Medicine; Spatio-Temporal Analysis; Tretinoin
PubMed: 35174997
DOI: 10.1002/smtd.202100912 -
Journal of Peptide Science : An... Nov 2021The p28 peptide derived from Pseudomonas aeruginosa azurin shows an anticancer activity after binding to p53 protein and is currently in Phase I of clinical trials. We...
The p28 peptide derived from Pseudomonas aeruginosa azurin shows an anticancer activity after binding to p53 protein and is currently in Phase I of clinical trials. We have studied its structure in water and in a biomimetic media and show that the peptide is unstructured in water but when studied in a biomimetic medium assumes a structure very similar to the one observed in azurin, suggesting a high propensity of this peptide to maintain this secondary structure. Analysis of p28 sequences from different bacterial species indicates conservation of the secondary structure despite amino acid replacement in different positions, suggesting that others, similar peptides could be tested for binding to p53.
Topics: Antineoplastic Agents; Azurin; Biomimetics; Peptide Fragments; Peptides; Pseudomonas aeruginosa
PubMed: 34151482
DOI: 10.1002/psc.3357 -
Proteins Mar 2024
PubMed: 37881118
DOI: 10.1002/prot.26624 -
Journal of Inorganic Biochemistry Sep 2022Type 1 copper proteins have a conserved ligand set of one cysteine and two histidines, with many proteins, such as azurin, also containing an axial methionine. While the...
Type 1 copper proteins have a conserved ligand set of one cysteine and two histidines, with many proteins, such as azurin, also containing an axial methionine. While the cysteine and methionine in azurin have been replaced with their respective isostructural analogues of unnatural amino acids to reveal their roles in tuning electronic structures and functional properties, such as reduction potentials (E°'), the histidine ligands have not been probed in this way. We herein report the substitution of His117 in azurin with three unnatural isostructural analogues, 5-nitrohistidine(Ntr), thiazolylalanine(SHis) and 1-methylhistidine(MeH) by expressed protein ligation. While UV-vis absorption and electron paramagnetic resonance spectroscopies confirm that isostructural replacement results in minimal structural change in the Cu(II) state, the E°' of these variants increases with increasing pKa of the δ nitrogens of the imidazole. This counter-intuitive relationship between E°' of the protein and pKa of the sidechain group suggests additional factors may play a role in tuning E°'.
Topics: Azurin; Copper; Cysteine; Electron Spin Resonance Spectroscopy; Histidine; Ligands; Methionine; Pseudomonas aeruginosa
PubMed: 35691263
DOI: 10.1016/j.jinorgbio.2022.111863 -
Journal of Inorganic Biochemistry May 2024Anthropogenic activities in agriculture and health use the antimicrobial properties of copper. This has led to copper accumulation in the environment and contributed to...
Anthropogenic activities in agriculture and health use the antimicrobial properties of copper. This has led to copper accumulation in the environment and contributed to the emergence of copper resistant microorganisms. Understanding bacterial copper homeostasis diversity is therefore highly relevant since it could provide valuable targets for novel antimicrobial treatments. The periplasmic CopI protein is a monodomain cupredoxin comprising several copper binding sites and is directly involved in copper resistance in bacteria. However, its structure and mechanism of action are yet to be determined. To study the different binding sites for cupric and cuprous ions and to understand their possible interactions, we have used mutants of the putative copper binding modules of CopI and spectroscopic methods to characterize their properties. We show that CopI is able to bind a cuprous ion in its central histidine/methionine-rich region and oxidize it thanks to its cupredoxin center. The resulting cupric ion can bind to a third site at the N-terminus of the protein. Nuclear magnetic resonance spectroscopy revealed that the central histidine/methionine-rich region exhibits a dynamic behavior and interacts with the cupredoxin binding region. CopI is therefore likely to participate in copper resistance by detoxifying the cuprous ions from the periplasm.
Topics: Copper; Histidine; Binding Sites; Methionine; Anti-Infective Agents; Ions; Azurin
PubMed: 38364337
DOI: 10.1016/j.jinorgbio.2024.112503 -
Metallomics : Integrated Biometal... Dec 2021The importance of copper resistance pathways in pathogenic bacteria is now well recognized, since macrophages use copper to fight bacterial infections. Additionally,...
The importance of copper resistance pathways in pathogenic bacteria is now well recognized, since macrophages use copper to fight bacterial infections. Additionally, considering the increase of antibiotic resistance, growing attention is given to the antimicrobial properties of copper. It is of primary importance to understand how bacteria deal with copper. The Cu-resistant cuproprotein CopI is present in many human bacterial pathogens and environmental bacteria and crucial under microaerobiosis (conditions for most pathogens to thrive within their host). Hence, understanding its mechanism of function is essential. CopI proteins share conserved histidine, cysteine, and methionine residues that could be ligands for different copper binding sites, among which the cupredoxin center could be involved in the protein function. Here, we demonstrated that Vibrio cholerae and Pseudomonas aeruginosa CopI restore the Cu-resistant phenotype in the Rubrivivax gelatinosus ΔcopI mutant. We identified that Cys125 (ligand in the cupredoxin center) and conserved histidines and methionines are essential for R. gelatinosus CopI (RgCopI) function. We also performed spectroscopic analyses of the purified RgCopI protein and showed that it is a green cupredoxin able to bind a maximum of three Cu(II) ions: (i) a green Cu site (CuT1.5), (ii) a type 2 Cu binding site (T2) located in the N-terminal region, and (iii) a third site with a yet unidentified location. CopI is therefore one member of the poorly described CuT1.5 center cupredoxin family. It is unique, since it is a single-domain cupredoxin with more than one Cu site involved in Cu resistance.
Topics: Azurin; Copper; Periplasm; Pseudomonas aeruginosa; Vibrio cholerae
PubMed: 34791351
DOI: 10.1093/mtomcs/mfab067 -
Small (Weinheim An Der Bergstrasse,... May 2021A central issue in protein electronics is how far the structural stability of the protein is preserved under the very high electrical field that it will experience once...
A central issue in protein electronics is how far the structural stability of the protein is preserved under the very high electrical field that it will experience once a bias voltage is applied. This question is studied on the redox protein Azurin in the solid-state Au/protein/Au junction by monitoring protein vibrations during current transport under applied bias, up to ≈1 GV m , by electrical detection of inelastic electron transport effects. Characteristic vibrational modes, such as CH stretching, amide (NH) bending, and AuS (of the bonds that connect the protein to an Au electrode), are not found to change noticeably up to 1.0 V. At >1.0 V, the NH bending and CH stretching inelastic features have disappeared, while the AuS features persist till ≈2 V, i.e., the proteins remain Au bound. Three possible causes for the disappearance of the NH and CH inelastic features at high bias, namely, i) resonance transport, ii) metallic filament formation, and iii) bond rupture leading to structural changes in the protein are proposed and tested. The results support the last option and indicate that spectrally resolved inelastic features can serve to monitor in operando structural stability of biological macromolecules while they serve as electronic current conduit.
Topics: Azurin; Electrodes; Electron Transport; Electrons; Spectrum Analysis
PubMed: 33783130
DOI: 10.1002/smll.202008218 -
The Journal of Physical Chemistry. B Sep 2022The protein, azurin, has enabled the study of the tryptophan radical. Upon UV excitation of tyrosine-deficient apoazurin and in the presence of a Co(III) electron...
The protein, azurin, has enabled the study of the tryptophan radical. Upon UV excitation of tyrosine-deficient apoazurin and in the presence of a Co(III) electron acceptor, the neutral radical (W48•) is formed. The lifetime of W48• in apoazurin is 41 s, which is shorter than the lifetime of several hours in Zn-substituted azurin. Molecular dynamics simulations revealed enhanced fluctuations of apoazurin which likely destabilize W48•. The photophysics of W48 was investigated to probe the precursor state for ET. The phosphorescence intensity was eliminated in the presence of an electron acceptor while the fluorescence was unchanged; this quenching of the phosphorescence is attributed to ET. The kinetics associated with W48• were examined with a model that incorporates intersystem crossing, ET, deprotonation, and decay of the cation radical. The estimated rate constants for ET (6 × 10 s) and deprotonation (3 × 10 s) are in agreement with a photoinduced mechanism where W48• is derived from the triplet state. The triplet as the precursor state for ET was supported by photolysis of apoazurin with 280 nm in the absence and presence of triplet-absorbing 405 nm light. Absorption bands from the neutral radical were observed only in the presence of blue light.
Topics: Apoproteins; Azurin; Kinetics; Tryptophan
PubMed: 35977067
DOI: 10.1021/acs.jpcb.2c02441 -
Phytopathology Jul 2022Soybean cyst nematode (SCN, ), one of the most devastating soybean pathogens, causes a significant yield loss in soybean production. One of the most effective ways to...
Soybean cyst nematode (SCN, ), one of the most devastating soybean pathogens, causes a significant yield loss in soybean production. One of the most effective ways to manage SCN is to grow resistant cultivars. Therefore, comparative study using resistant and susceptible soybean cultivars provides a powerful tool to identify new genes involved in soybean SCN resistance. In the present study, a transcriptome analysis was carried out using both the resistant (PI88788) and susceptible (Williams 82) soybean cultivars to characterize the responses to nematode infection. Various defense-related genes and different pathways involved in nematode resistance were recognized as being highly expressed in resistant cultivar. Promoter-GUS analysis was conducted to monitor the spatial expression pattern of the genes highly induced by nematode infection. Two nematode-inducible promoters for (encoding caffeoyl-CoA O-methyltransferase) and (encoding cupredoxin superfamily protein) were characterized, and the promoters could efficiently drive the expression of known nematode resistance genes ( or ) to affect soybean SCN resistance. Interestingly, expression of the cupredoxin family genes was upregulated not only by SCN, but also by jasmonic acid treatment. DNA sequence analysis identified that a conserved motif (GGTGCATG) with high similarity to SCNbox1 and GC-rich element is enriched in their promoter regions, suggesting its potential to serve as a nematode-responsive regulatory element. Overexpression of significantly enhanced soybean resistance to cyst nematode. Overall, our findings not only highlight the essential role of cupredoxin family genes in SCN resistance, but also offer potential functional tools to develop nematode resistance in crops.
Topics: Animals; Azurin; Cysts; Nematode Infections; Plant Diseases; Glycine max; Tylenchoidea
PubMed: 35050680
DOI: 10.1094/PHYTO-09-21-0391-R -
The Journal of Physical Chemistry. B Aug 2019Alkyl imidazolium chloride ionic liquids (ILs) have been used for numerous biochemical applications. Their hydrophobicity can be tuned by changing the alkyl chain...
Alkyl imidazolium chloride ionic liquids (ILs) have been used for numerous biochemical applications. Their hydrophobicity can be tuned by changing the alkyl chain length, and longer-chain ILs can form micelles in aqueous solution. We have investigated the effects of imidazolium chloride ILs on the structure and stability of azurin, which is a very stable Cu redox protein with both α-helix and β-sheet domains. Temperature-dependent infrared (IR) and vibrational circular dichroism spectroscopy can provide secondary-structure-specific information about how the protein is affected, and temperature-jump transient IR measurements can quantify the IL-influenced unfolding dynamics. Using these techniques, we can quantify how azurin is destabilized by 1.0 M ILs in aqueous solution. The shorter, less hydrophobic ILs, 1-butyl-3-methylimidazolium chloride and 1-hexyl-3-methylimidazolium chloride likely interact with the α-helix domain and decrease protein melting temperature from 82 °C without IL to 55 °C and disturb the overall tertiary structure, resulting in a looser, more open shape. Thermodynamic analysis indicates that protein destabilization is due to increased unfolding entropy. 1-Octyl-3-methylimidazolium chloride [OMIM]Cl, which forms micelles in solution that may partially solvate the protein, has a more significant destabilizing effect, resulting in a melting temperature of 35 °C, larger unfolding entropy, and relaxation kinetics several orders of magnitude faster than with unperturbed azurin. The temperature-independence of the relaxation time constant suggests that in the presence of [OMIM]Cl, the protein folding potential energy surface has become very smooth.
Topics: Azurin; Bacterial Proteins; Enzyme Inhibitors; Gene Expression Regulation; Imidazoles; Ionic Liquids; Kinetics; Micelles; Pseudomonas aeruginosa; Thermodynamics; Water
PubMed: 31335143
DOI: 10.1021/acs.jpcb.9b04113