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The Journal of Physical Chemistry. B Apr 2022Small values of the reorganization energy, 0.2-0.3 eV, were reported by electrochemical kinetic measurements for the half redox reaction of the redox-active protein...
Small values of the reorganization energy, 0.2-0.3 eV, were reported by electrochemical kinetic measurements for the half redox reaction of the redox-active protein azurin. This theoretical study explores possible mechanisms for the low activation barrier for electrochemical protein electron transfer: (1) electronic polarizability of the active site, (2) altering protonation states of far-away histidine residues not directly connected to the active site, and (3) a partial desolvation of the protein when attached to the electrode. The last mechanism provides the most robust explanation of the observations. Constraints imposed by the protein fold on its ability to sample the configuration space lead to the breakdown of the fluctuation-dissipation relation (FDR) and a strong separation of the Stokes-shift and variance reorganization energies. The resulting nonergodic kinetic reorganization energy observed experimentally is significantly lowered compared to predictions of standard models based on Gibbsian statistics and the FDR. The fast rate of protein electron transfer is directly related to the ability of the protein scaffold to maintain nonequilibrium statistics of electrostatic fluctuations projected on the electron-transfer reaction coordinate.
Topics: Azurin; Electron Transport; Oxidation-Reduction; Proteins; Static Electricity; Thermodynamics
PubMed: 35426669
DOI: 10.1021/acs.jpcb.2c00338 -
Cancer Diagnosis & Prognosis 2022This study aimed to present a rare case of fibrous dysplasia (FD) in a healthy young adult man with a concomitant osteoporotic vertebral compression fracture. FD is a...
BACKGROUND/AIM
This study aimed to present a rare case of fibrous dysplasia (FD) in a healthy young adult man with a concomitant osteoporotic vertebral compression fracture. FD is a benign lesion of the bone characterized by replacement of the medullary component with fibro-osseous tissue that contains abnormally arranged trabeculae of immature woven bone. Recently it has been reported that several bone tumors including FD express the receptor activator of nuclear factor-kappa B (RANK) and its ligand (RANKL). Therefore, we hypothesized that FD contributed to osteoporosis, linked by the RANK-RANKL pathway of osteoclastogenesis.
CASE REPORT
We report the case of a healthy man with monostotic femoral fibrous dysplasia (FD) with concomitant 7 thoracic vertebra compression fracture due to osteoporosis [young adult mean (YAM) was 79% in bone mineral density (BMD)]. After curettage of the FD, artificial bone grafting in the cavity, and administration of alendronate sodium, BMD improved considerably within 9 months. FD is a benign bone condition in which abnormal fibrous tissue replaces normal bone. The axis of the receptor activator of nuclear factor-kappa B (RANK) and its ligand (RANKL) has been implicated in osteoporosis pathogenesis. RANKL immunohistochemical staining was performed, and strong staining of stromal cells was observed compared to other FD cases that showed weak to moderate staining.
CONCLUSION
The presence of FD might have contributed to the low BMD due to the RANK-RANKL axis acting as osteoclastogenesis stimulator.
PubMed: 35400000
DOI: 10.21873/cdp.10082 -
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 -
The Journal of Physical Chemistry. B Apr 2022We investigate the events characterizing the steps of the unfolding pathway of blue copper metalloprotein azurin using replica exchange molecular dynamics (REMD). Our...
We investigate the events characterizing the steps of the unfolding pathway of blue copper metalloprotein azurin using replica exchange molecular dynamics (REMD). Our studies show that the unfolding of azurin begins with the melting of α-helix and β-sheets II and V. This is followed by the melting of other β-sheets and the exposure of hydrophobic protein core to the solvent, resulting in disruptions of its tertiary structure. Free energy surfaces constructed at different temperatures portray different basins that signify the stability of different melted structures in the unfolding process. The contact maps at different temperatures reveal that the strong hydrophobic interaction within the core of the protein is the vital force that renders high stability to this protein. Analysis of the individual β-sheets by looking into their amino acid sequence shows that β-sheets with charged side chains on the surface melt fast compared to others. The β-barrel of azurin is able to dynamically rearrange, and it helps the protein to preserve its hydrophobic core, holding back the native topology from melting fast. B-factor analysis shows that residues of β-sheets III, IV, and VII deviate less from their initial structure at the transition temperature.
Topics: Azurin; Copper; Hydrophobic and Hydrophilic Interactions; Metalloproteins; Molecular Dynamics Simulation; Protein Folding
PubMed: 35324174
DOI: 10.1021/acs.jpcb.2c00622 -
Biochemistry and Biophysics Reports Jul 2022The thermal unfolding of the copper redox protein azurin was studied in the presence of four different dipeptide-based ionic liquids (ILs) utilizing...
The thermal unfolding of the copper redox protein azurin was studied in the presence of four different dipeptide-based ionic liquids (ILs) utilizing tetramethylguanidinium as the cation. The four dipeptides have different sequences including the amino acids Ser and Asp: TMG-AspAsp, TMG-SerSer, TMG-SerAsp, and TMG-AspSer. Thermal unfolding curves generated from temperature-dependent fluorescence spectroscopy experiments showed that TMG-AspAsp and TMG-SerSer have minor destabilizing effects on the protein while TMG-AspSer and TMG-SerAsp strongly destabilize azurin. Red-shifted fluorescence signatures in the 25 °C correlate with the observed protein destabilization in the solutions with TMG-AspSer and TMG-SerAsp. These signals could correspond to interactions between the Asp residue in the dipeptide and the azurin Trp residue in the unfolded state. These results, supported by appropriate control experiments, suggest that dipeptide sequence-specific interactions lead to selective protein destabilization and motivate further studies of TMG-dipeptide ILs.
PubMed: 35280523
DOI: 10.1016/j.bbrep.2022.101242 -
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 the American Chemical Society Mar 2022Long-range electron tunneling through metalloproteins is facilitated by evolutionary tuning of donor-acceptor electronic couplings, formal electrochemical potentials,...
Long-range electron tunneling through metalloproteins is facilitated by evolutionary tuning of donor-acceptor electronic couplings, formal electrochemical potentials, and active-site reorganization energies. Although the minimal frustration of the folding landscape enables this tuning, residual frustration in the vicinity of the metallocofactor can allow conformational fluctuations required for protein function. We show here that the constrained copper site in wild-type azurin is governed by an intricate pattern of minimally frustrated local and distant interactions that together enable rapid electron flow to and from the protein. In contrast, sluggish electron transfer reactions (unfavorable reorganization energies) of active-site azurin variants are attributable to increased frustration near to as well as distant from the copper site, along with an exaggerated oxidation-state dependence of both minimally and highly frustrated interaction patterns.
Topics: Azurin; Copper; Electron Transport; Electrons; Pseudomonas aeruginosa
PubMed: 35171591
DOI: 10.1021/jacs.1c13454 -
Biochemical and Biophysical Research... Feb 2022EfeUOB is a siderophore-independent iron uptake mechanism in bacteria. EfeU, EfeO, and EfeB are a permease, an iron-binding or electron-transfer protein, and a...
EfeUOB is a siderophore-independent iron uptake mechanism in bacteria. EfeU, EfeO, and EfeB are a permease, an iron-binding or electron-transfer protein, and a peroxidase, respectively. A Gram-negative bacterium, Sphingomonas sp. strain A1, encodes EfeU, EfeO, EfeB together with alginate-binding protein Algp7, a truncated EfeO-like protein (EfeO), in the genome. The typical EfeO (EfeO) consists of N-terminal cupredoxin and C-terminal M75 peptidase domains. Here, we detail the structure and function of bacterial EfeB and EfeO. Crystal structures of strain A1 EfeB and Escherichia coli EfeO were determined at 2.30 Å and 1.85 Å resolutions, respectively. A molecule of heme involved in oxidase activity was bound to the C-terminal Dyp peroxidase domain of EfeB. Two domains of EfeO were connected by a short loop, and a zinc ion was bound to four residues, Glu156, Glu159, Asp173, and Glu255, in the C-terminal M75 peptidase domain. These residues formed tetrahedron geometry suitable for metal binding and are well conserved among various EfeO proteins including Algp7 (EfeO), although the metal-binding site (HxxE) is proposed in the C-terminal M75 peptidase domain. This is the first report on structure of a typical EfeO with two domains, postulating a novel metal-binding motif "ExxE-//-D-//-E" in the EfeO C-terminal M75 peptidase domain.
Topics: Amino Acid Motifs; Azurin; Bacterial Proteins; Binding Sites; Biological Transport; Cation Transport Proteins; Crystallography, X-Ray; Escherichia coli Proteins; Heme; Iron; Metals; Molecular Conformation; Oxidoreductases; Protein Binding; Protein Conformation; Protein Domains; Protein Structure, Secondary; Sphingomonas
PubMed: 35081501
DOI: 10.1016/j.bbrc.2022.01.055 -
Microorganisms Dec 2021Azurin is a bacterial-derived cupredoxin, which is mainly involved in electron transport reactions. Interest in azurin protein has risen in recent years due to its...
Azurin is a bacterial-derived cupredoxin, which is mainly involved in electron transport reactions. Interest in azurin protein has risen in recent years due to its anticancer activity and its possible applications in anticancer therapies. Nevertheless, the attention of the scientific community only focused on the azurin protein found in (, ). In this work, we performed the first comprehensive screening of all the bacterial genomes available in online repositories to assess azurin distribution in the three domains of life. The Azurin coding gene was not detected in the domains Archaea and Eucarya, whereas it was detected in phyla other than , such as , and and a phylogenetic analysis of the retrieved sequences was performed. Observed patchy distribution and phylogenetic data suggest that once it appeared in the bacterial domain, the azurin coding gene was lost in several bacterial phyla and/or anciently horizontally transferred between different phyla, even though a vertical inheritance appeared to be the major force driving the transmission of this gene. Interestingly, a shared conserved domain has been found among azurin members of all the investigated phyla. This domain is already known in as p28 domain and its importance for azurin anticancer activity has been widely explored. These findings may open a new and intriguing perspective in deciphering the azurin anticancer mechanisms and to develop new tools for treating cancer diseases.
PubMed: 35056457
DOI: 10.3390/microorganisms10010009 -
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