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Sensors (Basel, Switzerland) Dec 2020The tumor suppressor p53 protein plays a crucial role in many biological processes. The presence of abnormal concentrations of wild-type p53, or some of its mutants, can... (Review)
Review
The tumor suppressor p53 protein plays a crucial role in many biological processes. The presence of abnormal concentrations of wild-type p53, or some of its mutants, can be indicative of a pathological cancer state. p53 represents therefore a valuable biomarker for tumor screening approaches and development of suitable biosensors for its detection deserves a high interest in early diagnostics. Here, we revisit our experimental approaches, combining Surface Enhanced Raman Spectroscopy (SERS) and nanotechnological materials, for ultrasensitive detection of wild-type and mutated p53, in the perspective to develop biosensors to be used in clinical diagnostics. The Raman marker is provided by a small molecule (4-ATP) acting as a bridge between gold nanoparticles (NPs) and a protein biomolecule. The Azurin copper protein and specific antibodies of p53 were used as a capture element for p53 (wild-type and its mutants). The developed approaches allowed us to reach a detection level of p53 down to 10 M in both buffer and serum. The implementation of the method in a biosensor device, together with some possible developments are discussed.
Topics: Gold; Humans; Metal Nanoparticles; Neoplasms; Spectrum Analysis, Raman; Tumor Suppressor Protein p53
PubMed: 33327383
DOI: 10.3390/s20247153 -
Annual Review of Biochemistry 1996Electron-transfer (ET) reactions are key steps in a diverse array of biological transformations ranging from photosynthesis to aerobic respiration. A powerful... (Review)
Review
Electron-transfer (ET) reactions are key steps in a diverse array of biological transformations ranging from photosynthesis to aerobic respiration. A powerful theoretical formalism has been developed that describes ET rates in terms of two parameters: the nuclear reorganization energy (lambda) and the electronic-coupling strength (HAB). Studies of ET reactions in ruthenium-modified proteins have probed lambda and HAB in several metalloproteins (cytochrome c, myoglobin, azurin). This work has shown that protein reorganization energies are sensitive to the medium surrounding the redox sites and that an aqueous environment, in particular, leads to large reorganization energies. Analyses of electronic-coupling strengths suggest that the efficiency of long-range ET depends on the protein secondary structure: beta sheets appear to mediate coupling more efficiently than alpha-helical structures, and hydrogen bonds play a critical role in both.
Topics: Electrons; Proteins
PubMed: 8811189
DOI: 10.1146/annurev.bi.65.070196.002541 -
Iranian Journal of Microbiology Apr 2019The aim of this study was to evaluate the antibacterial and antibiofilm activity of recombinant Azurin from against different bacterial species.
BACKGROUND AND OBJECTIVES
The aim of this study was to evaluate the antibacterial and antibiofilm activity of recombinant Azurin from against different bacterial species.
MATERIALS AND METHODS
The gene was cloned in the pET21a vector. The pET21a- construct was transformed into BL21. The recombinant Azurin was expressed and purified using affinity chromatography and confirmed by Western blotting. The cytotoxicity of rAzurin was assessed on peripheral blood mononuclear cells. Antibacterial and antibiofilm activity of rAzurin with different concentrations were determined by micro-broth dilution and crystal violet methods, respectively. The effect of rAzurin on bacterial species was statistically analyzed by t-test and spearman correlation.
RESULTS
The identity of purified protein was confirmed by blotting and distinguished as a 14 kDa band on 15% SDS-PAGE. The IC50 of rAzurin on Peripheral Blood Mononuclear Cell (PBMC) was determined as 377.91±0.5 μg/mL in 24 h. and displayed the most sensitivity to rAzurin (27.5 and 55 μg/mL, respectively) and the highest resistance (220 μg/mL) was displayed by and The MIC for other species was 110 μg/mL. The Minimum Biofilm Inhibition Concentration (MBIC) was determined as 220 μg/mL for and , 300 μg/mL for and and 440 μg/mL for the other species. The antimicrobial effect of rAzurin on bacterial species were significant (p value<0.05) and correlation coefficient was negative.
CONCLUSION
The rAzurin appears to be an appropriate choice and a new strategy for prevention of bacterial infection. It inhibits bacterial growth and biofilm formation and candidates as antimicrobial peptides.
PubMed: 31341572
DOI: No ID Found -
Journal of Biomedicine & Biotechnology 2012Since in most cases biological macromolecular systems including solvent water molecules are remarkably large, the computational costs of performing ab initio... (Review)
Review
Since in most cases biological macromolecular systems including solvent water molecules are remarkably large, the computational costs of performing ab initio calculations for the entire structures are prohibitive. Accordingly, QM calculations that are jointed with MM calculations are crucial to evaluate the long-range electrostatic interactions, which significantly affect the electronic structures of biological macromolecules. A UNIX-shell-based interface program connecting the quantum mechanics (QMs) and molecular mechanics (MMs) calculation engines, GAMESS and AMBER, was developed in our lab. The system was applied to a metalloenzyme, azurin, and PU.1-DNA complex; thereby, the significance of the environmental effects on the electronic structures of the site of interest was elucidated. Subsequently, hybrid QM/MM molecular dynamics (MD) simulation using the calculation system was employed for investigation of mechanisms of hydrolysis (editing reaction) in leucyl-tRNA synthetase complexed with the misaminoacylated tRNA(Leu), and a novel mechanism of the enzymatic reaction was revealed. Thus, our interface program can play a critical role as a powerful tool for state-of-the-art sophisticated hybrid ab initio QM/MM MD simulations of large systems, such as biological macromolecules.
Topics: Azurin; Catalytic Domain; Computational Biology; Hydrolysis; Leucine-tRNA Ligase; Models, Biological; Molecular Dynamics Simulation; Quantum Theory; Software
PubMed: 22536015
DOI: 10.1155/2012/236157 -
Bioinformation 2022Oral cancer is becoming more common, and it threatens to be a serious worldwide medical issue. Hence, it is of interest to elucidate the networks between proteins and...
Oral cancer is becoming more common, and it threatens to be a serious worldwide medical issue. Hence, it is of interest to elucidate the networks between proteins and biologically active compounds, as well as their functional annotations, and cell signaling pathways. The online STRING software was used to create a molecular genetics interaction network named AZURIN on oral bacterial proteins. We also used the cystoscope software to identify 11 nodes and 16 edges with an average node order of 2.91. Thus, we document data on the interaction of protein networks with other proteins for identifying potential therapeutic drug candidates linked to oral disease.
PubMed: 37323560
DOI: 10.6026/97320630018724 -
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 -
Bioengineered 2014Cancer is one of the most deadly diseases worldwide. In the last three decades many efforts have been made focused on understanding how cancer grows and responds to... (Review)
Review
Cancer is one of the most deadly diseases worldwide. In the last three decades many efforts have been made focused on understanding how cancer grows and responds to drugs. The dominant drug-development paradigm has been the "one drug, one target." Based on that, the two main targeted therapies developed to combat cancer include the use of tyrosine kinase inhibitors and monoclonal antibodies. Development of drug resistance and side effects represent the major limiting factors for their use in cancer treatment. Nowadays, a new paradigm for cancer drug discovery is emerging wherein multi-targeted approaches gain ground in cancer therapy. Therefore, to overcome resistance to therapy, it is clear that a new generation of drugs is urgently needed. Here, regarding the concept of multi-targeted therapy, we discuss the challenges of using bacterial proteins and peptides as a new generation of effective anti-cancer drugs.
Topics: Antibodies, Monoclonal; Antineoplastic Agents; Azurin; Bacterial Proteins; Drug Discovery; Drug Resistance, Neoplasm; Protein Conformation; Signal Transduction; Transcriptome
PubMed: 24875003
DOI: 10.4161/bioe.29266 -
Proceedings of the National Academy of... Mar 2021Hole hopping through tryptophan/tyrosine chains enables rapid unidirectional charge transport over long distances. We have elucidated structural and dynamical factors...
Hole hopping through tryptophan/tyrosine chains enables rapid unidirectional charge transport over long distances. We have elucidated structural and dynamical factors controlling hopping speed and efficiency in two modified azurin constructs that include a rhenium(I) sensitizer, Re(His)(CO)(dmp), and one or two tryptophans (W, W). Experimental kinetics investigations showed that the two closely spaced (3 to 4 Å) intervening tryptophans dramatically accelerated long-range electron transfer (ET) from Cu to the photoexcited sensitizer. In our theoretical work, we found that time-dependent density-functional theory (TDDFT) quantum mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) trajectories of low-lying triplet excited states of Re(His)(CO)(dmp)-W(-W) exhibited crossings between sensitizer-localized (*Re) and charge-separated [Re(His)(CO)(dmp)/(W or W)] (CS1 or CS2) states. Our analysis revealed that the distances, angles, and mutual orientations of ET-active cofactors fluctuate in a relatively narrow range in which the cofactors are strongly coupled, enabling adiabatic ET. Water-dominated electrostatic field fluctuations bring *Re and CS1 states to a crossing where *Re(CO)(dmp)←W ET occurs, and CS1 becomes the lowest triplet state. ET is promoted by solvation dynamics around *Re(CO)(dmp)(W); and CS1 is stabilized by Re(dmp)/W electron/hole interaction and enhanced W solvation. The second hop, W←W, is facilitated by water fluctuations near the W/W unit, taking place when the electrostatic potential at W drops well below that at W Insufficient solvation and reorganization around W make W←W ET endergonic, shifting the equilibrium toward W and decreasing the charge-separation yield. We suggest that multiscale TDDFT/MM/MD is a suitable technique to model the simultaneous evolution of photogenerated excited-state manifolds.
Topics: Azurin; Electron Transport; Electrons; Molecular Dynamics Simulation; Oxidation-Reduction; Photochemistry; Pseudomonas aeruginosa; Quantum Theory; Rhenium; Static Electricity; Tryptophan; Water
PubMed: 33836608
DOI: 10.1073/pnas.2024627118 -
ISRN Neurology 2012The current therapy for glioblastoma multiforme involves total surgical resection followed by combination of radiation therapy and temozolomide. Unfortunately, the...
The current therapy for glioblastoma multiforme involves total surgical resection followed by combination of radiation therapy and temozolomide. Unfortunately, the efficacy for such current therapy is limited, and newer approaches are sorely needed to treat this deadly disease. We have recently described the isolation of bacterial proteins and peptides with anticancer activity. In phase I human clinical trials, one such peptide, p28, derived from a bacterial protein azurin, showed partial and complete regression of tumors in several patients among 15 advanced-stage cancer patients with refractory metastatic tumors where the tumors were no longer responsive to current conventional drugs. An azurin-like protein called Laz derived from Neisseria meningitides demonstrates efficient entry and high cytotoxicity towards glioblastoma cells. Laz differs from azurin in having an additional 39-amino-acid peptide called an H.8 epitope, which allows entry and high cytotoxicity towards glioblastoma cells. Since p28 has been shown to have very little toxicity and high anti-tumor activity in advanced-stage cancer patients, it will be worthwhile to explore the use of H.8-p28, H.8-azurin, and Laz in toxicity studies and glioblastoma therapy in preclinical and human clinical trials.
PubMed: 22462021
DOI: 10.5402/2012/642345 -
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