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Inorganic Chemistry Apr 2022The interplay between the primary and secondary coordination spheres in biological metal sites plays an essential role in controlling their properties. Some of the...
The interplay between the primary and secondary coordination spheres in biological metal sites plays an essential role in controlling their properties. Some of the clearest examples of this are from copper sites in blue and purple copper proteins. Many such proteins contain methionine (Met) in the primary coordination sphere as a weakly bound ligand to Cu. While the effects of replacing the coordinated Met are understood, less so is the importance of its second-sphere interactions. In this combined informatics and experimental study, we first present a bioinformatics investigation of the second-sphere environments in biological Met-Cu motifs. The most common interaction is between the Met-CH and the π-face of a phenylalanine (Phe) (81% of surveyed structures), tyrosine (Tyr) (11%), and tryptophan (Trp) (8%). In most cases, the Met-CH also forms a contact with a π-face of one of a Cu-ligating histidine-imidazole. Such interactions are widely distributed in different Cu proteins. Second, to explore the impact of the second-sphere interactions of Met, a series of artificial azurin proteins were produced where the native Phe15 was replaced with Tyr or Trp. The proteins were characterized using optical and magnetic resonance spectroscopies, X-ray diffraction, electrochemistry, and an investigation of the time-resolved electron-transfer kinetics of photosensitizer-modified proteins. The influence of the Cu-Met-Aro interaction on azurin's physical properties is subtle, and the hallmarks of the azurin blue copper site are maintained. In the Phe15Trp variant, the mutation to Phe15 induces changes in Cu properties that are comparable to replacement of the weak Met ligand. The broader impacts of these widely distributed interactions are discussed.
Topics: Azurin; Copper; Ligands; Methionine; Models, Molecular; Proteins; Tryptophan; Tyrosine
PubMed: 35347989
DOI: 10.1021/acs.inorgchem.2c00030 -
Preparative Biochemistry & Biotechnology 2021Azurin, which is a bacterial secondary metabolite has been attracted as a potential anticancer agent in recent years because induced death of cancer cells and inhibited...
Azurin, which is a bacterial secondary metabolite has been attracted as a potential anticancer agent in recent years because induced death of cancer cells and inhibited their growth. In this study, the production of azurin under the control of the alcohol oxidase promoter which is frequently used in the expression system was performed. The azurin gene amplified from genomic DNA and inserted into the pPICZαA was cloned in cells. Then, a linearized recombinant vector was transferred to the X-33 cells. Antibiotic resistance test and colony PCR were performed for the selection of multicopy transformants. Protein expression capacities of selected transformants were compared at the end of 48 h incubation. Both extracellular and intracellular protein expressions were observed in all of them by Western blot analysis. The relative expression levels of both intracellular and extracellular protein that belongs to the first clone were higher than the others. On the other hand, it was seen that the 4th clone had the highest protein secretion ability. The molecular mass of the extracellular azurin protein which is produced by recombinant clones was found to be about 20 kDa. This is the first report on azurin expression in .
Topics: Azurin; Gene Expression; Pseudomonas aeruginosa; Recombinant Proteins; Saccharomycetales
PubMed: 33346686
DOI: 10.1080/10826068.2020.1855444 -
Biochemical and Biophysical Research... Jul 2021Proteins with hetero-bimetallic metal centers can catalyze important reactions and are challenging to design. Azurin is a mononuclear copper center that has been...
Proteins with hetero-bimetallic metal centers can catalyze important reactions and are challenging to design. Azurin is a mononuclear copper center that has been extensively studied for electron transfer. Here we inserted the lanthanide binding tag (LBT), which binds lanthanide with sub μM affinity, into the copper binding loop of azurin, while keeping the type 1 copper center unperturbed. The resulting protein, Az-LBT, which has two metal bonding centers, shows strong luminescence upon coordination with Tb and luminescence quenching upon Cu binding. The in vitro luminescence quenching has high metal specificity and a limit-of-detection of 0.65 μM for Cu. With the low background from lanthanide's long luminescence lifetime, bacterial cells expressing Az-LBT in the periplasm also shows sensitivity for metal sensing.
Topics: Azurin; Bacteria; Binding Sites; Biosensing Techniques; Catalysis; Copper; Lanthanoid Series Elements; Luminescence; Models, Molecular; Protein Domains
PubMed: 34004515
DOI: 10.1016/j.bbrc.2021.05.013 -
Advances in Microbial Physiology 2020Absorbance measurements on intact chemolithotrophic microorganisms that respire aerobically on soluble iron are described that used a novel integrating cavity absorption... (Review)
Review
Absorbance measurements on intact chemolithotrophic microorganisms that respire aerobically on soluble iron are described that used a novel integrating cavity absorption meter to eliminate the effects of light scattering on the experimental results. Steady state kinetic measurements on ferric iron production by intact cells revealed that the Michaelis Menten equation described the initial rates of product formation for at least 8 different chemolithotrophic microorganisms in 6 phyla distributed equally among the archaea and the Gram negative and Gram positive eubacteria. Cell-monitored turnover measurements during aerobic respiration on soluble iron by the same 12 intact microorganisms revealed six different patterns of iron-dependent absorbance changes, suggesting that there may be at least six different sets of prosthetic groups and biomolecules that can accomplish aerobic respiration on soluble iron. Detailed kinetic studies revealed that the 3-component iron respiratory chain of Acidithiobacillus ferrooxidans functioned as an ensemble with a single macroscopic rate constant when the iron-reduced proteins were oxidized in the presence of excess molecular oxygen. The principal member of this 3-component system was a cupredoxin called rusticyanin that was present in the periplasm of At. ferrooxidans at an approximate concentration of 350 mg/mL, an observation that provides new insights into the crowded environments in the periplasms of Gram negative eubacteria that conduct electrons across their periplasm. The ability to conduct direct spectrophotometric measurements under noninvasive physiological conditions represents a new and powerful approach to examine the rates and extents of biological events in situ without disrupting the complexity of the live cellular environment.
Topics: Acidithiobacillus; Archaea; Azurin; Bacteria; Cell Respiration; Electron Transport; Iron; Kinetics; Oxidation-Reduction; Spectrum Analysis
PubMed: 32408948
DOI: 10.1016/bs.ampbs.2020.01.003 -
Nanotechnology Dec 2019Hybrid semiconductor nanostructures have attracted tremendous response due to their unique properties and applications in nano-optoelectronics and sensors. Here, we...
Hybrid semiconductor nanostructures have attracted tremendous response due to their unique properties and applications in nano-optoelectronics and sensors. Here, we fabricated a back-gated transistor based on 300 nm channel of the Azurin-TiO hybrid nanostructure, whose enhanced performance is attributed to the synergetic effect of the metal oxide and azurin. Surface potential mapping under the dark and light condition using Kelvin probe force microscopy, gives the perfect correlation of band gap estimation for Azurin, TiO and Azurin-TiO nanostructures. The extracted parameters of the transistor exhibit the majority carrier mobility of 2.26 cm V s, Schottky barrier height of 133.56 meV and low off current (6 × 10 A). The photodetector showed the high spectral response of 8.7 × 10 A W and detectivity of 6.4 × 10 Jones for 260 nm wavelength, at an applied gate bias of 5 V. The short carrier transit time (3 μs) and large recombination time (0.4 s) with multiple recirculations of photo generated carries facilitate the high gain of 2.6 × 10. A significant rejection ratio (R /R ) of 56.2 at V = 5 V and the linear dynamic range of 45.75 dB for 260 nm wavelength is achieved. The obtained rise and fall time of the photodetector is 0.52 s, and 0.65 s, respectively. This study suggests the applicability of Azurin-TiO hybrid nanostructures with high performance for the biocompatible optoelectronic devices.
PubMed: 31476745
DOI: 10.1088/1361-6528/ab4082 -
Bioengineering (Basel, Switzerland) Oct 2023Compared to chemical drugs, therapeutic proteins exhibit higher specificity and activity and are generally well-tolerated by the human body. However, the limitations,...
Compared to chemical drugs, therapeutic proteins exhibit higher specificity and activity and are generally well-tolerated by the human body. However, the limitations, such as poor stability both in vivo and in vitro as well as difficulties in penetrating cell membranes, hinder their widespread application. To overcome the challenges, a highly efficient protocol was developed and implemented for the recombinant expression of the therapeutic protein azurin and secretion into minicells derived from probiotic Nissle 1917. The novel coupled production with a delivery system of therapeutic proteins based on minicells was obtained through purification to enhance protein activity, circulation characteristics, and targeting specificity. This protein drug carrier integrates the production of carrier materials and the loading of expression proteins. The drug carrier also protects the encapsulated polypeptide drugs from enzymatic or gastric acid degradation until they are released. Nissle 1917-derived minicells have natural targeting to colon cancer cells, low toxicity, and can accumulate for a long time after penetrating tumor tissue. This self-produced protein drug delivery system simplified the process of protein preparation, and its inhibitory effect on different types of colon cancer cells was verified by CCK-8 cytotoxicity assay, cancer cell invasion, and migration assay. This work provided a simple method to prepare minicell drug delivery systems for protein drug production and a novel approach for the transport of recombinant protein drugs.
PubMed: 37892918
DOI: 10.3390/bioengineering10101188 -
World Neurosurgery Jun 2023The microscope is important in neurosurgery, but it is not exempt from limitations. The exoscope has emerged as an alternative because it offers better 3-dimensional... (Review)
Review
BACKGROUND
The microscope is important in neurosurgery, but it is not exempt from limitations. The exoscope has emerged as an alternative because it offers better 3-dimensional (3D) visualization and better ergonomics. We present our initial experience in vascular pathology using 3D exoscopy at the Dos de Mayo National Hospital to show the viability of the 3D exoscope in vascular microsurgery. We also provide a review of the literature.
METHODS
In this work, the Kinevo 900 exoscope was used in 3 patients with cerebral (2) and spinal (1) vascular pathology. We evaluated the image quality, equipment management, ergonomics, educational utility, and 3D glasses and recorded the characteristics of the cases. We reviewed the experience of other authors as well.
RESULTS
Three patients underwent surgery: 1 occipital cavernoma, 1 cerebral dural fistula, and 1 spinal dural fistula. Excellent 3D visualization with Zeiss Kinevo 900 exoscope (Carl Zeiss, Germany), surgical comfort, and educational utility occurred, and there were no complications.
CONCLUSIONS
Our experience and that of other authors suggests that the 3D exoscope shows excellent visualization, better ergonomics, and an innovative educational experience. Vascular microsurgery can be performed safely and effectively.
Topics: Humans; Neurosurgical Procedures; Microsurgery; Microscopy; Germany
PubMed: 36871654
DOI: 10.1016/j.wneu.2023.02.120 -
Preventing Chronic Disease Jun 2023A transformative change grounded in a commitment to antiracism and racial and health equity is underway at the University of California, Berkeley, School of Public...
A transformative change grounded in a commitment to antiracism and racial and health equity is underway at the University of California, Berkeley, School of Public Health. Responding to a confluence of national, state, and local circumstances, bold leadership, and a moral and disciplinary imperative to name and address racism as a root cause of health inequities, our community united around a common vision of becoming an antiracist institution. Berkeley Public Health has a long history of efforts supporting diversity, equity, inclusion, belonging, and justice. Building upon those efforts, we pursued an institution-wide initiative, one that creates a more equitable and inclusive school of public health that models and supports the development of future public health leaders, practitioners, scholars, and educators. Grounded in the principles of cultural humility, we recognized that our vision was a journey, not a destination. This article describes our efforts from June 2020 through June 2022 in developing and implementing ARC4JSTC (Anti-racist Community for Justice and Social Transformative Change), a comprehensive, multiyear antiracist change initiative encompassing faculty and workforce development, student experience, curriculum and pedagogy, community engagement outreach, and business processes. Our work is data informed, grounded in principles of change management, and focused on building internal capacity to promote long-term change. Our discussion of lessons learned and next steps helps to inform our ongoing work and antiracist institutional change efforts at other schools and programs of public health.
Topics: Humans; Public Health; Racism; Curriculum; Health Equity; Social Justice
PubMed: 37290006
DOI: 10.5888/pcd20.220370 -
ACS Physical Chemistry Au Jan 2023Tryptophan is one of few residues that participates in biological electron transfer reactions. Upon substitution of the native Cu center with Zn in the blue-copper...
Tryptophan is one of few residues that participates in biological electron transfer reactions. Upon substitution of the native Cu center with Zn in the blue-copper protein azurin, a long-lived tryptophan neutral radical can be photogenerated. We report the following quantum yield values for Zn-substituted azurin in the presence of the electron acceptor Cu(II)-azurin: formation of the tryptophan neutral radical (Φ), electron transfer (Φ), fluorescence (Φ), and phosphorescence (Φ), as well as the efficiency of proton transfer of the cation radical (Φ). Increasing the concentration of the electron acceptor increased Φ and Φ values and decreased Φ without affecting Φ. At all concentrations of the acceptor, the value of Φ was nearly unity. These observations indicate that the phosphorescent triplet state is the parent state of electron transfer and that nearly all electron transfer events lead to proton loss. Similar results regarding the parent state were obtained with a different electron acceptor, [Co(NH)Cl]; however, Stern-Volmer graphs revealed that [Co(NH)Cl] was a more effective phosphorescence quencher ( = 230 000 M) compared to Cu(II)-azurin ( = 88 000 M). Competition experiments in the presence of both [Co(NH)Cl] and Cu(II)-azurin suggested that [Co(NH)Cl] is the preferred electron acceptor. Implications of these results in terms of quenching mechanisms are discussed.
PubMed: 36718260
DOI: 10.1021/acsphyschemau.2c00042 -
Analytical Methods : Advancing Methods... May 2024This work describes an analytical procedure, single particle-inductively coupled plasma-time-of-flight-mass spectrometry (SP-ICP-TOF-MS), that was developed to determine...
This work describes an analytical procedure, single particle-inductively coupled plasma-time-of-flight-mass spectrometry (SP-ICP-TOF-MS), that was developed to determine the platinum binding efficiency of protein-coated magnetic microparticles. SP-ICP-TOF-MS is advantageous due to its ability to quasi-simultaneously detect all nuclides (Li-Pu), allowing for both platinum and iron (composition of magnetic microparticles) to be measured concurrently. This method subsequently allows for the differentiation between bound and unbound platinum. The 1 μm magnetic microparticles were fully characterized for their iron concentration, particle concentration, and trace element composition by bulk digestion-ICP-MS and SP-ICP-TOF-MS. The results of both approaches agreed with the certificate values. Using the single particle methodology the platinum loading was quantified to be to 0.18 ± 0.02 fg per particle and 0.32 ± 0.02 fg per particle, for the streptavidin-coated and azurin-coated microparticles, respectively. Both streptavidin-coated and the azurin-coated microparticles had a particle-platinum association of >65%. Platinum bound samples were also analyzed bulk digestion-based ICP-MS. The bulk ICP-MS results overestimated platinum loading due to free platinum in the samples. This highlights the importance of single particle analysis for a closer inspection of platinum binding performance. The SP-ICP-TOF-MS approach offers advantages over typical bulk digestion methods by eliminating laborious sample preparation, enabling differentiation between bound/unbound platinum in a solution, and quantification of platinum on a particle-by-particle basis. The procedure presented here enables quantification of metal content per particle, which could be broadly implemented for other single particle applications.
Topics: Platinum; Mass Spectrometry; Microspheres; Iron; Streptavidin; Particle Size; Magnetite Nanoparticles
PubMed: 38639200
DOI: 10.1039/d4ay00268g