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Frontiers in Bioscience (Landmark... Nov 2022Azurin, a bacterial cupredoxin firstly isolated from the bacterium , is considered a potential alternative therapeutic tool against different types of cancer.
BACKGROUND
Azurin, a bacterial cupredoxin firstly isolated from the bacterium , is considered a potential alternative therapeutic tool against different types of cancer.
AIMS
In this work we have explored the relationship possibly existing between azurin and colorectal cancer (CRC), in light of the evidence that microbial imbalance can lead to CRC progression.
METHODOLOGY/RESULTS
To this aim, the presence of azurin coding gene in the DNA extracted from saliva, stool, and biopsy samples of 10 CRC patients and 10 healthy controls was evaluated by real-time PCR using primers specifically designed to target the azurin coding gene from different bacterial groups. The correlation of the previously obtained microbiota data with real-time PCR results evidenced a "preferential" enrichment of seven bacterial groups in some samples than in others, even though no statistical significance was detected between controls and CRC. The subset of azurin gene-harbouring bacterial groups was representative of the entire community.
CONCLUSIONS
Despite the lack of statistical significance between healthy and diseased patients, HTS data analysis highlighted a kind of "preferential" enrichment of seven bacterial groups harbouring the azurin gene in some samples than in others.
Topics: Humans; Azurin; Genes, Bacterial; Pseudomonas aeruginosa; Microbiota
PubMed: 36472111
DOI: 10.31083/j.fbl2711305 -
Journal of Magnetic Resonance (San... Jan 2023A new deep neural network based on the WaveNet architecture (WNN) is presented, which is designed to grasp specific patterns in the NMR spectra. When trained at a fixed...
A new deep neural network based on the WaveNet architecture (WNN) is presented, which is designed to grasp specific patterns in the NMR spectra. When trained at a fixed non-uniform sampling (NUS) schedule, the WNN benefits from pattern recognition of the corresponding point spread function (PSF) pattern produced by each spectral peak resulting in the highest quality and robust reconstruction of the NUS spectra as demonstrated in simulations and exemplified in this work on 2D H-N correlation spectra of three representative globular proteins with different sizes: Ubiquitin (8.6 kDa), Azurin (14 kDa), and Malt1 (44 kDa). The pattern recognition by WNN is also demonstrated for successful virtual homo-decoupling in a 2D methyl H-C - HMQC spectrum of MALT1. We demonstrate using WNN that prior knowledge about the NUS schedule, which so far was not been fully exploited, can be used for designing new powerful NMR processing techniques that surpass the existing algorithmic methods.
Topics: Magnetic Resonance Spectroscopy; Magnetic Resonance Imaging; Neural Networks, Computer; Ubiquitin; Nuclear Magnetic Resonance, Biomolecular
PubMed: 36459916
DOI: 10.1016/j.jmr.2022.107342 -
The Journal of Physical Chemistry. B Dec 2022One reaction step in the conductivity relay of azurin, electron transfer between the Cu-based active site and the tryptophan residue, is studied theoretically and by...
One reaction step in the conductivity relay of azurin, electron transfer between the Cu-based active site and the tryptophan residue, is studied theoretically and by classical molecular dynamics simulations. Oxidation of tryptophan results in electrowetting of this residue. This structural change makes the free energy surfaces of electron transfer nonparabolic as described by the Q-model of electron transfer. We analyze the medium dynamical effect on protein electron transfer produced by coupled Stokes-shift dynamics and the dynamics of the donor-acceptor distance modulating electron tunneling. The equilibrium donor-acceptor distance falls in the plateau region of the rate constant, where it is determined by the protein-water dynamics, and the probability of electron tunneling does not affect the rate. The crossover distance found here puts most intraprotein electron-transfer reactions under the umbrella of dynamical control. The crossover between the medium-controlled and tunneling-controlled kinetics is combined with the effect of the protein-water medium on the activation barrier to formulate principles of tunability of protein-based charge-transfer chains. The main principle in optimizing the activation barrier is the departure from the Gaussian-Gibbsian statistics of fluctuations promoting activated transitions. This is achieved either by incomplete (nonergodic) sampling, breaking the link between the Stokes-shift and variance reorganization energies, or through wetting-induced structural changes of the enzyme's active site.
Topics: Azurin; Tryptophan; Electrons; Thermodynamics; Catalytic Domain; Electron Transport; Proteins; Water
PubMed: 36459590
DOI: 10.1021/acs.jpcb.2c05258 -
Journal of Biological Inorganic... Sep 2022A large number of copper binding proteins coordinate metal ions using a shared three-dimensional fold called the cupredoxin domain. This domain was originally identified... (Review)
Review
A large number of copper binding proteins coordinate metal ions using a shared three-dimensional fold called the cupredoxin domain. This domain was originally identified in Type 1 "blue copper" centers but has since proven to be a common domain architecture within an increasingly large and diverse group of copper binding domains. The cupredoxin fold has a number of qualities that make it ideal for coordinating Cu ions for purposes including electron transfer, enzyme catalysis, assembly of other copper sites, and copper sequestration. The structural core does not undergo major conformational changes upon metal binding, but variations within the coordination environment of the metal site confer a range of Cu-binding affinities, reduction potentials, and spectroscopic properties. Here, we discuss these proteins from a structural perspective, examining how variations within the overall cupredoxin fold and metal binding sites are linked to distinct spectroscopic properties and biological functions. Expanding far beyond the blue copper proteins, cupredoxin domains are used by a growing number of proteins and enzymes as a means of binding copper ions, with many more likely remaining to be identified.
Topics: Azurin; Binding Sites; Copper; Ions; Metals
PubMed: 35994119
DOI: 10.1007/s00775-022-01955-2 -
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 -
Proceedings of the National Academy of... Jul 2022The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms,...
The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood-Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetyl-CoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the Ni species and requirement for ordered substrate binding in the bioorganometallic carbon-carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation.
Topics: Aldehyde Oxidoreductases; Azurin; Catalysis; Esters; Models, Chemical; Multienzyme Complexes; Nickel; Origin of Life; Sulfur Compounds
PubMed: 35858422
DOI: 10.1073/pnas.2123022119 -
The Journal of Physical Chemistry. B Jul 2022The blue color in metalloprotein azurin has traditionally been attributed to the intense cysteine-to-Cu ligand-to-metal charge transfer transition centered at 628 nm....
The blue color in metalloprotein azurin has traditionally been attributed to the intense cysteine-to-Cu ligand-to-metal charge transfer transition centered at 628 nm. Although resonance Raman measurements of the Cu active site have implied that the LMCT transition electronically couples to the protein scaffold well beyond its primary metal-ligand coordination shell, the structural extent of this electronic coupling and visualization of the protein-mediated charge transfer dynamics have remained elusive. Here, using femtosecond broadband transient absorption and impulsive Raman spectroscopy, we provide direct evidence for a rapid relaxation between two distinct charge transfer states, having different spatial delocalization, within ∼300 fs followed by recombination of charges in subpicosecond time scales. We invoke the formation of a protein-centered radical cation, possibly Trp48 or a Phe residue, within 100 fs substantiating the long-range electronic coupling for the first time beyond the traditional copper active site. The Raman spectra of the excited CT state show the presence of protein-centric vibrations along with the vibrational modes assigned to the copper active site. Our results demonstrate a large delocalization length scale of the initially populated CT state, thereby highlighting the possibility of exploiting azurin photochemistry for energy conversion techniques.
Topics: Azurin; Catalytic Domain; Copper; Ligands; Metalloproteins
PubMed: 35797135
DOI: 10.1021/acs.jpcb.2c01427 -
Journal of Inorganic Biochemistry Sep 2022Amicyanin is a type 1 copper protein with a single tryptophan residue. Using genetic code expansion, the tryptophan was selectively replaced with the unnatural amino...
Amicyanin is a type 1 copper protein with a single tryptophan residue. Using genetic code expansion, the tryptophan was selectively replaced with the unnatural amino acid, 5-hydroxytryptophan (5-HTP). The 5-HTP substituted amicyanin exhibited absorbance at 300-320 nm, characteristic of 5-HTP and not seen in native amicyanin. The fluorescence emission maximum in 5-HTP substituted amicyanin is redshifted from 318 nm in native amicyanin to 331 nm and to 348 nm in the unfolded protein. The fluorescence quantum yield of 5-HTP substituted amicyanin mutant was much less than that of native amicyanin. Differences in intrinsic fluorescence are explained by differences in the excited states of tryptophan versus 5-HTP and the intraprotein environment. The substitution of tryptophan with 5-HTP did not affect the visible absorbance and redox potential of the copper, which is 10 Å away. In amicyanin and other cupredoxins, an unexplained quenching of the intrinsic fluorescence by the bound copper is observed. However, the fluorescence of 5-HTP substituted amicyanin is not quenched by the copper. It is shown that the mechanism of quenching in native amicyanin is Förster, or fluorescence, resonance energy transfer (FRET). This does not occur in 5-HTP substituted amicyanin because the fluorescence quantum yield is significantly lower and the red-shift of fluorescence emission maximum decreases overlap with the near UV absorbance of copper. Characterization of the distinct fluorescence properties of 5-HTP relative to tryptophan in amicyanin provides a basis for spectroscopic interrogation of the protein microenvironment using 5-HTP, and long-distance interactions with transition metals.
Topics: 5-Hydroxytryptophan; Azurin; Bacterial Proteins; Copper; Energy Transfer; Metalloproteins; Paracoccus denitrificans; Tryptophan
PubMed: 35696758
DOI: 10.1016/j.jinorgbio.2022.111895 -
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 -
The Journal of Chemical Physics May 2022Metalloproteins, known to efficiently transfer electronic charge in biological systems, recently found their utilization in nanobiotechnological devices where the...
Metalloproteins, known to efficiently transfer electronic charge in biological systems, recently found their utilization in nanobiotechnological devices where the protein is placed into direct contact with metal surfaces. The feasibility of oxidation/reduction of the protein redox sites is affected by the reorganization free energies, one of the key parameters determining the transfer rates. While their values have been measured and computed for proteins in their native environments, i.e., in aqueous solution, the reorganization free energies of dry proteins or proteins adsorbed to metal surfaces remain unknown. Here, we investigate the redox properties of blue copper protein azurin, a prototypical redox-active metalloprotein previously probed by various experimental techniques both in solution and on metal/vacuum interfaces. We used a hybrid quantum mechanical/molecular mechanical computational technique based on density functional theory to explore protein dynamics, flexibility, and corresponding reorganization free energies in aqueous solution, vacuum, and on vacuum gold interfaces. Surprisingly, the reorganization free energy only slightly decreases when azurin is dried because the loss of the hydration shell leads to larger flexibility of the protein near its redox site. At the vacuum gold surfaces, the energetics of the structure relaxation depends on the adsorption geometry; however, significant reduction of the reorganization free energy was not observed. These findings have important consequences for the charge transport mechanism in vacuum devices, showing that the free energy barriers for protein oxidation remain significant even under ultra-high vacuum conditions.
Topics: Azurin; Copper; Electron Transport; Gold; Metalloproteins; Oxidation-Reduction; Vacuum; Water
PubMed: 35525644
DOI: 10.1063/5.0085141