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Journal of the American Chemical Society Sep 2023Much progress has been made in understanding the roles of the secondary coordination sphere (SCS) in tuning redox potentials of metalloproteins. In contrast, the impact...
Much progress has been made in understanding the roles of the secondary coordination sphere (SCS) in tuning redox potentials of metalloproteins. In contrast, the impact of SCS on reactivity is much less understood. A primary example is how copper proteins can promote -nitrosylation (SNO), which is one of the most important dynamic post-translational modifications, and is crucial in regulating nitric oxide storage and transportation. Specifically, the factors that instill Cu with -nitrosylating capabilities and modulate activity are not well understood. To address this issue, we investigated the influence of the primary and secondary coordination sphere on Cu-catalyzed -nitrosylation by developing a series of azurin variants with varying catalytic capabilities. We have employed a multidimensional approach involving electronic absorption, S and Cu K-edge XAS, EPR, and resonance Raman spectroscopies together with QM/MM computational analysis to examine the relationships between structure and molecular mechanism in this reaction. Our findings have revealed that kinetic competency is correlated with three balancing factors, namely Cu-S bond strength, Cu spin localization, and relative S(p) vs S(p) contributions to the ground state. Together, these results support a reaction pathway that proceeds through the attack of the Cu-S bond rather than electrophilic addition to Cu or radical attack of S. The insights gained from this work provide not only a deeper understanding of SNO in biology but also a basis for designing artificial and tunable SNO enzymes to regulate NO and prevent diseases due to SNO dysregulation.
Topics: Azurin; Copper; Metalloproteins; Catalysis; Electronics
PubMed: 37696009
DOI: 10.1021/jacs.3c07399 -
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 -
3 Biotech Jul 2019In the present study, the simultaneous application of gene of and - antigen on the induction of immune responses against breast cancer tumors was investigated in...
In the present study, the simultaneous application of gene of and - antigen on the induction of immune responses against breast cancer tumors was investigated in BALB/c mice. The pBudCE4.1-azurin-MAM-A recombinant vector was generated and prepared at a large scale. This recombinant vector alone or combined with chitosan nanoparticles was infused into the hip muscle of animals. Animals were divided into the "prevention" and "therapy" categories. The animals of prevention category were first, immunized by a recombinant vector and then exposed to chemical cancer inducers; while the animals in the therapy category were first treated with chemical compounds and then infused by a recombinant plasmid. The tumor tissues, infusion sites, and blood specimens were collected and examined by serological, molecular, and histological tests. The breast tumor incidence in the infused animals by recombinant plasmid alone or combined with nanoparticles (in both prevention and therapy categories) compared with infused mice by empty pBudCE4.1 vector was significantly decreased (< 0.05). These results were supported by histological studies using H&E staining. The ELISA and q-real-time PCR techniques showed the range of IFN-γ, IL-12, IL-4, and IL-17A cytokines in the infused mice by recombinant vector alone or combined with nanoparticles compared to the healthy mice and infused animals by intact pBudCE4.1 were significantly increased (< 0.05). Accordingly, the expression of the tumor markers , , and were significantly decreased in treated mice either by the sole recombinant vector or combined with nanoparticles (< 0.05). These findings indicated that pBudCE4.1-azurin-MAM-A recombinant vector plays an essential role against the formation and expansion of breast tumors in the animal model. In addition, this recombinant vector is safe and has the proper ability to stimulate the immune system. In addition, the chitosan nanoparticle represents a promising adjuvant for DNA vaccine delivery, which improves the immune system stimulation and boosts the vaccine performance.
PubMed: 31245235
DOI: 10.1007/s13205-019-1804-7 -
The Journal of Physical Chemistry... Dec 2023We conducted a theoretical study of electron transport through junctions of the blue-copper azurin from . We found that single-site hopping can lead to either higher or...
We conducted a theoretical study of electron transport through junctions of the blue-copper azurin from . We found that single-site hopping can lead to either higher or lower current values compared to fully coherent transport. This depends on the structural details of the junctions as well as the alignment of the protein orbitals. Moreover, we show how the asymmetry of the curves can be affected by the position of the tip in the junction and that, under specific conditions, such a hopping mechanism is consistent with a fairly low temperature dependence of the current. Finally, we show that increasing the number of hopping sites leads to higher hopping currents. Our findings, from fully quantum calculations, provide deep insight to help guide the interpretation of experimental curves on highly complex systems.
PubMed: 38059566
DOI: 10.1021/acs.jpclett.3c02702 -
Biomolecules Apr 2023Due to the similarity in the basic coordination behavior of their mono-charged cations, silver biochemistry is known to be linked to that of copper in biological...
Due to the similarity in the basic coordination behavior of their mono-charged cations, silver biochemistry is known to be linked to that of copper in biological systems. Still, Cu/ is an essential micronutrient in many organisms, while no known biological process requires silver. In human cells, copper regulation and trafficking is strictly controlled by complex systems including many cytosolic copper chaperones, whereas some bacteria exploit the so-called "blue copper" proteins. Therefore, evaluating the controlling factors of the competition between these two metal cations is of enormous interest. By employing the tools of computational chemistry, we aim to delineate the extent to which Ag might be able to compete with the endogenous copper in its Type I (T1Cu) proteins, and where and if, alternatively, it is handled uniquely. The effect of the surrounding media (dielectric constant) and the type, number, and composition of amino acid residues are taken into account when modelling the reactions in the present study. The obtained results clearly indicate the susceptibility of the T1Cu proteins to a silver attack due to the favorable composition and geometry of the metal-binding centers, along with the similarity between the Ag/Cu-containing structures. Furthermore, by exploring intriguing questions of both metals' coordination chemistry, an important background for understanding the metabolism and biotransformation of silver in organisms is provided.
Topics: Humans; Copper; Silver
PubMed: 37189429
DOI: 10.3390/biom13040681 -
Infection and Drug Resistance 2020The purpose of this study was to analyze the sequence of gene in relation to its expression in strains isolated from different clinical specimens of burn patients....
AIM
The purpose of this study was to analyze the sequence of gene in relation to its expression in strains isolated from different clinical specimens of burn patients. Moreover, in silico sequence analysis of gene using globally reported sequences was intended.
MATERIALS AND METHODS
Fifty-nine multidrug-resistant isolates were selected from different clinical specimens of patients suffering from burn wound infections in two university hospitals and subjected to antibacterial susceptibility testing. The frequency and genetic diversity of the gene was determined by polymerase chain reaction (PCR) and Sanger sequencing. The gene sequences were compared with the sequence data from other countries. The expression level of gene in isolates with different sequences from different clinical specimens was evaluated by real-time PCR.
RESULTS AND CONCLUSION
About 98%-100% of the isolates were resistant to gentamicin, tobramycin, cefoxitin, ciprofloxacin, amikacin, and imipenem, while 100% and 23.9% of the isolates were susceptible to colistin and ceftazidime, respectively. Only eight point mutations were detected with amino acid substitutions in only two positions (81 and 102). In global analysis, 93% of strains showed missense mutation at positions 81 (alanine to threonine). The majority (81%) of Iranian strains were allocated to two major clusters distinct from the rest of world, which may suggest that strains from Iran have made a distinct genetic stockpile through point mutations which has established them separate from the other counties. However, 19% were distributed in different clusters together with the strains from different countries of North and South America, Europe, South and East Asia. The expression level of the gene was statistically higher in the isolates collected from the blood of burns patients with systemic infection compared to the isolates collected from other specimens (wound, catheter and tissue), which shows a positive correlation between gene expression and increased pathogenicity and capability for dissemination. This study may open new insight about genetic variation and significance in pathogenesis.
PubMed: 32765002
DOI: 10.2147/IDR.S248043 -
Journal of the American Society For... May 2020Ultraviolet photodissociation (UVPD) has emerged as a promising tool to characterize proteins with regard to not only their primary sequences and post-translational...
Ultraviolet photodissociation (UVPD) has emerged as a promising tool to characterize proteins with regard to not only their primary sequences and post-translational modifications, but also their tertiary structures. In this study, three metal-binding proteins, Staphylococcal nuclease, azurin, and calmodulin, are used to demonstrate the use of UVPD to elucidate metal-binding regions via comparisons between the fragmentation patterns of apo (metal-free) and holo (metal-bound) proteins. The binding of staphylococcal nuclease to calcium was evaluated, in addition to a series of lanthanide(III) ions which are expected to bind in a similar manner as calcium. On the basis of comparative analysis of the UVPD spectra, the binding region for calcium and the lanthanide ions was determined to extend from residues 40-50, aligning with the known crystal structure. Similar analysis was performed for both azurin (interrogating copper and silver binding) and calmodulin (four calcium binding sites). This work demonstrates the utility of UVPD methods for determining and analyzing the metal binding sites of a variety of classes of proteins.
Topics: Amino Acid Sequence; Azurin; Binding Sites; Calcium; Calmodulin; Lanthanoid Series Elements; Metals; Micrococcal Nuclease; Models, Molecular; Spectrometry, Mass, Electrospray Ionization
PubMed: 32275426
DOI: 10.1021/jasms.0c00066 -
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 -
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 -
Chemical Science May 2024Native chemical ligation (NCL) has been playing an increasingly important role in chemical protein synthesis (CPS). More efficient ligation methods that circumvent the...
Native chemical ligation (NCL) has been playing an increasingly important role in chemical protein synthesis (CPS). More efficient ligation methods that circumvent the requirement of a peptidyl thioester and thiol additive-which allow the following desulfurization or refolding in one pot-are urgently needed for the synthesis of more complex protein targets and in large quantities. Herein, we discover that the weak acyl donor peptidyl -acyl pyrazole can be activated by azole reagents like 3-methylpyrazole or imidazole to facilitate its ligation directly with an N-terminal cysteine peptide. As it requires no thioester or thiol additive, this ligation strategy can be conveniently combined with metal-free desulfurization (MFD) or oxidative protein folding to allow various one-pot protocols. The utility and generality of the strategy are showcased by the total synthesis of ubiquitin an N-to-C sequential ligation-MFD strategy, the semi-synthesis of the copper protein azurin, and the efficient assembly of a sulfated hirudin variant and the cyclotide kalata B1, all in a one-pot fashion.
PubMed: 38817582
DOI: 10.1039/d3sc06697e