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Physical Chemistry Chemical Physics :... May 2023As the field of nanoelectronics based on biomolecules such as peptides and proteins rapidly grows, there is a need for robust computational methods able to reliably...
As the field of nanoelectronics based on biomolecules such as peptides and proteins rapidly grows, there is a need for robust computational methods able to reliably predict charge transfer properties at bio/metallic interfaces. Traditionally, hybrid quantum-mechanical/molecular-mechanical techniques are employed for systems where the electron hopping transfer mechanism is applicable to determine physical parameters controlling the thermodynamics and kinetics of charge transfer processes. However, these approaches are limited by a relatively high computational cost when extensive sampling of a configurational space is required, like in the case of soft biomatter. For these applications, semi-empirical approaches such as the perturbed matrix method (PMM) have been developed and successfully used to study charge-transfer processes in biomolecules. Here, we explore the performance of PMM on prototypical redox-active protein azurin in various environments, from solution to vacuum interfaces with gold surfaces and protein junction. We systematically benchmarked the robustness and convergence of the method with respect to the quantum-centre size, size of the Hamiltonian, number of samples, and level of theory. We show that PMM can adequately capture all the trends associated with the structural and electronic changes related to azurin oxidation at bio/metallic interfaces.
Topics: Azurin; Electron Transport; Oxidation-Reduction; Proteins; Peptides
PubMed: 37097130
DOI: 10.1039/d3cp00197k -
Accounts of Chemical Research May 2023"What I cannot create, I do not understand"─Richard Feynman. This sentiment motivates the entire field of artificial metalloenzymes. Naturally occurring enzymes...
"What I cannot create, I do not understand"─Richard Feynman. This sentiment motivates the entire field of artificial metalloenzymes. Naturally occurring enzymes catalyze reactions with efficiencies, rates, and selectivity that generally cannot be achieved in synthetic systems. Many of these processes represent vital building blocks for a sustainable society, including CO conversion, nitrogen fixation, water oxidation, and liquid fuel synthesis. Our inability as chemists to fully reproduce the functionality of naturally occurring enzymes implicates yet-unknown contributors to reactivity. To identify these properties, it is necessary to consider all of the components of naturally occurring metalloenzymes, from the active site metal(s) to large-scale dynamics. In this Account, we describe the holistic development of a metalloprotein-based model that functionally reproduces the acetyl coenzyme A synthase (ACS) enzyme.ACS catalyzes the synthesis of a thioester, acetyl coenzyme A, from gaseous carbon monoxide, a methyl group donated by a cobalt corrinoid protein, and coenzyme A. The active site of ACS contains a bimetallic nickel site coupled to a [4Fe-4S] cluster. This reaction mimics Monsanto's acetic acid synthesis and represents an ancient process for incorporating inorganic carbon into cellular biomass through the primordial Wood-Ljungdahl metabolic pathway. From a sustainability standpoint, the reversible conversion of C substrates into an acetyl group and selective downstream transfer to a thiolate nucleophile offer opportunities to expand this reactivity to the anthropogenic synthesis of liquid fuels. However, substantial gaps in our understanding of the ACS catalytic mechanism coupled with the enzyme's oxygen sensitivity and general instability have limited these applications. It is our hope that development of an artificial metalloenzyme that carries out ACS-like reactions will advance our mechanistic understanding and enable synthesis of robust compounds with the capacity for similar reactivity.To construct this model, we first focused on the catalytic proximal nickel (Ni) site, which has a single metal center bound by three bridging cysteine residues in a "Y"-shaped arrangement. With an initial emphasis on reproducing the general structure of a low-coordinate metal binding site, the type I cupredoxin, azurin, was selected as the protein scaffold, and a nickel center was incorporated into the mononuclear site. Using numerous spectroscopic and computational techniques, including electron paramagnetic resonance (EPR) spectroscopy, nickel-substituted azurin was shown to have similar electronic and geometric structures to the Ni center in ACS. A substrate access channel was installed, and both carbon monoxide and a methyl group were shown to bind individually to the reduced Ni center. The elusive EPR-active S = 1/2 Ni-CH species, which has never been detected in native ACS, was observed in the azurin-based model, establishing the capacity of a biological Ni species to support two-electron organometallic reactions. Pulsed EPR studies on the S = 1/2 Ni-CH species in azurin suggested a noncanonical electronic structure with an inverted ligand field, which was proposed to prevent irreversible site degradation. This model azurin protein was ultimately shown to perform carbon-carbon and carbon-sulfur bond formation using sequential, ordered substrate addition for selective, stoichiometric thioester synthesis. X-ray spectroscopic methods were used to provide characterization of the remaining catalytic intermediates, resolving some debate over key mechanistic details.The overall approach and strategies that we employed for the successful construction of a functional protein-based model of ACS are described in this Account. We anticipate that these principles can be adapted across diverse metalloenzyme classes, providing essential mechanistic details and guiding the development of next-generation, functional artificial metalloenzymes.
Topics: Azurin; Acetyl Coenzyme A; Nickel; Carbon Monoxide; Metalloproteins; Electron Spin Resonance Spectroscopy
PubMed: 37042748
DOI: 10.1021/acs.accounts.2c00824 -
International Journal of Biological... May 2023To maintain life, charge transfer processes must be efficient to allow electrons to migrate across distances as large as 30-50 Å within a timescale from picoseconds to...
To maintain life, charge transfer processes must be efficient to allow electrons to migrate across distances as large as 30-50 Å within a timescale from picoseconds to milliseconds, and the free-energy cost should not exceed one electron volt. By employing local ionization and local affinity energies, we calculated the pathway for electron and electron-hole transport, respectively. The pathway is then used to calculate both the driving force and the activation energy. The electronic coupling is calculated using configuration interaction procedure. When the charge acceptor is not known, as in oxidative stress, the charge transport terminals are found using Monte-Carlo simulation. These parameters were used to calculate the rate described by Marcus theory. Our approach has been elaborately explained using the famous androstane example and then applied to two proteins: electron transport in azurin protein and hole-hopping migration route from the heme center of cytochrome c peroxidase to its surface. This model gives an effective method to calculate the charge transport pathway and the free-energy profile within 0.1 eV from the experimental measurements and electronic coupling within 3 meV.
Topics: Electron Transport; Proteins; Azurin; Computer Simulation
PubMed: 36948333
DOI: 10.1016/j.ijbiomac.2023.124065 -
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 -
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 -
Communications Biology Jan 2023Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in...
Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in human cancers. Thus, p53 becomes one of the most promising targets for anticancer treatment. A bacterial effector protein azurin triggers tumor suppression by stabilizing p53 and elevating its basal level. However, the structural and mechanistic basis of azurin-mediated tumor suppression remains elusive. Here we report the atomic details of azurin-mediated p53 stabilization by combining X-ray crystallography with nuclear magnetic resonance. Structural and mutagenic analysis reveals that the p28 region of azurin, which corresponds to a therapeutic peptide, significantly contributes to p53 binding. This binding stabilizes p53 by disrupting COP1-mediated p53 ubiquitination and degradation. Using the structure-based design, we obtain several affinity-enhancing mutants that enable amplifying the effect of azurin-induced apoptosis. Our findings highlight how the structure of the azurin-p53 complex can be leveraged to design azurin derivatives for cancer therapy.
Topics: Humans; Azurin; Bacterial Proteins; Peptides; Tumor Suppressor Protein p53; Ubiquitination
PubMed: 36650277
DOI: 10.1038/s42003-023-04458-1 -
Communications Biology Jan 2023Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in...
Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in cancer, and therapies that incorporate bacteria have been tried in various types of cancer. We previously demonstrated that cupredoxin azurin secreted by the opportunistic pathogen Pseudomonas aeruginosa, enters human cancer cells and induces apoptotic death. However, the physiological interactions between P. aeruginosa and humans and their role in tumor homeostasis are largely unknown. Here, we show that P. aeruginosa upregulated azurin secretion in response to increasing numbers of and proximity to cancer cells. Conversely, cancer cells upregulated aldolase A secretion in response to increasing proximity to P. aeruginosa, which also correlated with enhanced P. aeruginosa adherence to cancer cells. Additionally, we show that cancer patients had detectable P. aeruginosa and azurin in their tumors and exhibited increased overall survival when they did, and that azurin administration reduced tumor growth in transgenic mice. Our results suggest host-bacterial symbiotic mutualism acting as a diverse adjunct to the host defense system via inter-kingdom communication mediated by the evolutionarily conserved proteins azurin and human aldolase A. This improved understanding of the symbiotic relationship of bacteria with humans indicates the potential contribution to tumor homeostasis.
Topics: Mice; Animals; Humans; Azurin; Pseudomonas aeruginosa; Fructose-Bisphosphate Aldolase; Neoplasms; Cell Physiological Phenomena
PubMed: 36609683
DOI: 10.1038/s42003-022-04395-5 -
Sarcoma 2022We report a retrospective case series analysis of clinical outcomes of patients with soft tissue sarcoma around the elbow.
BACKGROUND
We report a retrospective case series analysis of clinical outcomes of patients with soft tissue sarcoma around the elbow.
METHODS
Twenty-two patients underwent surgical tumor excision between January 1999 and May 2017, with a mean follow-up of 85.2 months.
RESULTS
Six tumors were localized in the upper arm, nine in the elbow, and seven in the forearm. Sixteen tumors were deep-seated, and six were superficially located. Fifteen patients underwent wide excision, including one amputation, and 18 achieved (81.8%) negative margins histologically. Two local recurrences and four distant metastases developed. The mean Musculoskeletal Tumor Society score was 92.0% (range, 33.3-100). The 5-year local recurrence-free survival rate, metastasis-free survival rate, and overall survival rate were 90.0%, 77.0%, and 79.7%, respectively.
CONCLUSIONS
Local control and limb function can have favorable outcomes when the tumor excised has a histologically negative margin without sacrificing the major structure.
PubMed: 36573098
DOI: 10.1155/2022/1087726 -
The Journal of Physical Chemistry. B Jan 2023Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference...
Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference in electrochemical potentials for two tryptophan residues in azurin with distinct hydrogen-bonding environments. Here, we test whether reducing the side chain bulk at position Phe110 to Leu, Ser, or Ala impacts the electrochemical potentials (°) for tryptophan at position 48. X-ray diffraction confirmed the influx of crystallographically resolved water molecules for both the F110A and F110L tyrosine free azurin mutants. The local environments of W48 in all azurin mutants were further evaluated by UV resonance Raman (UVRR) spectroscopy to probe the impact of mutations on hydrogen bonding and polarity. A correlation between the frequency of the ω17 mode─considered a vibrational marker for hydrogen bonding─and ° is proposed. However, the trend is opposite to the expectation from a previous study on small molecules. Density functional theory calculations suggest that the ω17 mode reflects hydrogen bonding as well as local polarity. Further, the UVRR data reveal different intensity/frequency shifts of the ω9/ω10 vibrational modes that characterize the local H-bonding environments of tryptophan. The cumulative data support that the presence of water increases ° and reveal properties of the protein microenvironment surrounding tryptophan.
Topics: Azurin; Tryptophan; Oxidation-Reduction; Hydrogen; Water
PubMed: 36542812
DOI: 10.1021/acs.jpcb.2c06677 -
Proteins May 2023Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order...
Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key β-barrel, β-sandwich, β-α-β, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.
Topics: Azurin; Copper; Thermodynamics; Protein Folding; Zinc; Kinetics; Protein Denaturation
PubMed: 36511110
DOI: 10.1002/prot.26454