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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 -
The Journal of Physical Chemistry. B Aug 2019Isotopologues are valuable vibrational probes that shift features in a vibrational spectrum while preserving the electronic structure of the molecule. We report the...
Isotopologues are valuable vibrational probes that shift features in a vibrational spectrum while preserving the electronic structure of the molecule. We report the vibrational and electronic spectra of perdeuterated tryptophan in solution (l-Trp-), as Trp48- in azurin, and as the photogenerated neutral tryptophan radical, Trp48-, in azurin. The UV resonance Raman bands of the perdeuterated closed-shell tryptophan in solution and in azurin are lower in frequency relative to the protiated counterpart. The observed decrease in frequencies of l-Trp- bands relative to l-Trp- enables the analysis of vibrational markers of other amino acids, e.g., phenylalanine, that overlap with some modes of l-Trp-. The Raman intensities vary between l-Trp- and l-Trp-; these differences likely reflect modifications in normal mode composition upon perdeuteration. Analysis of the W3, W6, and W17 modes suggests that the W3 mode retains its utility as a conformational marker; however, the H-bond markers W6 and W17 appear to be less sensitive upon perdeuteration. The neutral tryptophan radical, Trp48-, was generated in azurin with a slightly lower radical quantum yield than for Trp48-. The visible resonance Raman spectrum of Trp48- is different from that of Trp48-, especially in terms of relative intensities, and all assignable peaks decreased in frequency upon perdeuteration. The absorption and emission spectra of the perdeuterated closed-shell and radical species exhibited hypsochromic shifts of less than 1 nm relative to the protiated species. The data presented here indicate that l-Trp- is a valuable probe of vibrational structure, with minimal modification of photoreactivity and photophysics compared to l-Trp-.
Topics: Azurin; Models, Molecular; Protein Conformation; Spectrum Analysis, Raman; Tryptophan
PubMed: 31313925
DOI: 10.1021/acs.jpcb.9b04655 -
Journal of Peptide Science : An... May 2021Cell-penetrating peptides (CPPs) can transport various cargoes through membranes of live cells. Since the first generations of CPPs suffered from insufficient cell and... (Review)
Review
Cell-penetrating peptides (CPPs) can transport various cargoes through membranes of live cells. Since the first generations of CPPs suffered from insufficient cell and tissue selectivity, stability against proteases, and escape from endosomes, a new generation of peptides, with optimized properties, was developed. These are either derived from natural sources or created through the combination of multivalent structures. The second method allows achieving high internalization efficiency, high cell and tissue selectivity, and release from endosomes via hybrid structures, combining sequences for endosomal release, homing sequences, and sequences for activation at the target tissue and for local delivery of cargoes. CPPs with innate tumor selectivity include azurin, crotamine, maurocalcine, lycosin-I, buffalo cathelicidin, and peptide CB5005. Some of them can penetrate the membranes of live cells and influence intracellular signaling pathways, thereby exerting cytotoxic effects against tumor cells. To obtain multilayer penetration and stabilization against proteolytic degradation, as well as for better handling, CPPs are often conjugated to nanoparticles. A special problem for tumor treatment is the efficiency of drug transport through three-dimensional cell cultures. Therefore, the capability of CPPs to deliver the drug even to the innermost tissues is of crucial importance. Notably, the ability of certain CPPs to penetrate barriers such as skin, the blood-brain barrier (BBB), and cornea or conjunctiva of eyes enabled the replacement of dangerous and painful injections with soothing sprays, creams, and drops. However, it is difficult to rank the efficacy of CPPs because transport efficiency and tissue selectivity depend not only on the CPP itself but also on the target tissue or organ, as well as on the cargo and method of CPP-cargo coupling. Therefore, the present review describes some examples of new-generation CPPs and aims to provide advice on how to find or create the right CPP for a given task.
Topics: Animals; Antineoplastic Agents; Cell-Penetrating Peptides; Humans; Neoplasms
PubMed: 33615648
DOI: 10.1002/psc.3300 -
Inorganic Chemistry Jul 2021Metalloproteins are an important class of proteins involved in metal uptake, transport, and electron-transfer reactions. Mimicking the active sites of these proteins... (Comparative Study)
Comparative Study
Metalloproteins are an important class of proteins involved in metal uptake, transport, and electron-transfer reactions. Mimicking the active sites of these proteins through miniaturization is an active area of research with applications in biotechnology and medicine. Azurin is a 128-residue copper-binding cupredoxin protein involved in electron-transfer reactions. Previous studies have reported on the copper-binding-induced spectroscopic and structural properties of peptide loops (11 and 13 residues) from azurin. These azurin peptides exhibited novel stoichiometries. However, the underlying mechanism of fluorescence quenching upon copper binding remains to be understood, whether it is due to electron transfer, energy transfer, or both. Here, we report nickel-binding-associated spectroscopic and structural properties of the azurin peptides. They develop a β-turn upon nickel binding as seen in circular dichroism and exhibit electronic transitions centered at 270 and 450 nm. Unlike copper, which exhibited 1:1 and 1:2 peptide:metal stoichiometries, nickel exhibited only a 1:1 stoichiometry. Tryptophan-containing peptides showed fluorescence quenching upon nickel binding, which is due to electron transfer. These results further suggest that the quenching in copper-bound peptides is also due to electron transfer, which could not be ascertained in previous studies. Overall, azurin peptides provide a platform for studying metal-induced structural and spectroscopic properties using transition-metal ions.
Topics: Azurin; Binding Sites; Copper; Fluorescence; Metalloproteins; Nickel; Peptides
PubMed: 34137603
DOI: 10.1021/acs.inorgchem.1c01007 -
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 -
Sensors (Basel, Switzerland) Nov 2017Surface Plasmon Resonance (SPR) is a powerful technique to study the kinetics of biomolecules undergoing biorecognition processes, particularly suited for... (Review)
Review
Surface Plasmon Resonance (SPR) is a powerful technique to study the kinetics of biomolecules undergoing biorecognition processes, particularly suited for protein-protein interactions of biomedical interest. The potentiality of SPR was exploited to sense the interactions occurring within the network of the tumor suppressor p53, which is crucial for maintaining genome integrity and whose function is inactivated, mainly by down regulation or by mutation, in the majority of human tumors. This study includes p53 down-regulators, p53 mutants and also the p53 family members, p63 and p73, which could vicariate p53 protective function. Furthermore, the application of SPR was extended to sense the interaction of p53 with anti-cancer drugs, which might restore p53 function. An extended review of previous published work and unpublished kinetic data is provided, dealing with the interaction between the p53 family members, or their mutants and two anticancer molecules, Azurin and its cell-penetrating peptide, p28. All the kinetic results are discussed in connection with those obtained by a complementary approach operating at the single molecule level, namely Atomic Force Spectroscopy and the related literature data. The overview of the SPR kinetic results may significantly contribute to a deeper understanding of the interactions within p53 network, also in the perspective of designing suitable anticancer drugs.
Topics: Azurin; Humans; Microscopy, Atomic Force; Protein Binding; Surface Plasmon Resonance; Tumor Suppressor Protein p53
PubMed: 29156626
DOI: 10.3390/s17112680 -
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 -
Molecular Therapy Oncolytics Mar 2017Treatment of aggressive glioblastoma brain tumors is challenging, largely due to diffusion barriers preventing efficient drug dosing to tumors. To overcome these...
Treatment of aggressive glioblastoma brain tumors is challenging, largely due to diffusion barriers preventing efficient drug dosing to tumors. To overcome these barriers, bacterial carriers that are actively motile and programmed to migrate and localize to tumor zones were designed. These carriers can induce apoptosis via hypoxia-controlled expression of a tumor suppressor protein p53 and a pro-apoptotic drug, Azurin. In a xenograft model of human glioblastoma in rats, bacterial carrier therapy conferred a significant survival benefit with 19% overall long-term survival of >100 days in treated animals relative to a median survival of 26 days in control untreated animals. Histological and proteomic analyses were performed to elucidate the safety and efficacy of these carriers, showing an absence of systemic toxicity and a restored neural environment in treated responders. In the treated non-responders, proteomic analysis revealed competing mechanisms of pro-apoptotic and drug-resistant activity. This bacterial carrier opens a versatile avenue to overcome diffusion barriers in glioblastoma by virtue of its active motility in extracellular space and can lead to tailored therapies via tumor-specific expression of tumoricidal proteins.
PubMed: 28345020
DOI: 10.1016/j.omto.2016.12.003 -
International Journal of Molecular... Dec 2018Cancer is a multi-process disease where different mechanisms exist in parallel to ensure cell survival and constant adaptation to the extracellular environment. To adapt... (Review)
Review
Cancer is a multi-process disease where different mechanisms exist in parallel to ensure cell survival and constant adaptation to the extracellular environment. To adapt rapidly, cancer cells re-arrange their plasma membranes to sustain proliferation, avoid apoptosis and resist anticancer drugs. In this review, we discuss novel approaches based on the modifications and manipulations that new classes of molecules can exert in the plasma membrane lateral organization and order of cancer cells, affecting growth factor signaling, invasiveness, and drug resistance. Furthermore, we present azurin, an anticancer protein from bacterial origin, as a new approach in the development of therapeutic strategies that target the cell membrane to improve the existing standard therapies.
Topics: Animals; Antineoplastic Agents; Apoptosis; Biophysical Phenomena; Cell Membrane; Humans; Molecular Targeted Therapy; Neoplasms
PubMed: 30518103
DOI: 10.3390/ijms19123871 -
Biomacromolecules Dec 2020Azulitox as a new fusion polypeptide with cancer cell specificity and phototoxicity was generated and is composed of a photosensitizer domain and the cell-penetrating...
Azulitox as a new fusion polypeptide with cancer cell specificity and phototoxicity was generated and is composed of a photosensitizer domain and the cell-penetrating peptide P28. The photosensitizer domain (EcFbFP) was derived from a bacterial blue-light receptor, which belongs to the family of light-oxygen-voltage proteins and produces reactive oxygen species (ROS) upon excitation. P28 is derived from the cupredoxin protein azurin that is known to specifically penetrate cancer cells and bind to the tumor suppressor protein p53. We show that the P28 domain specifically directs and translocates the fused photosensitizer into cancer cells. Under blue-light illumination, Azulitox significantly induced cytotoxicity. Compared to the extracellular application of EcFbFP, Azulitox caused death to about 90% of cells, as monitored by flow cytometry, which also directly correlated with the amount of ROS produced in the cells. Azulitox may open new avenues toward targeted polypeptide-photosensitizer-based photodynamic therapies with reduced systemic toxicity compared to conventional photosensitizers.
Topics: Antineoplastic Agents; Neoplasms; Peptide Fragments; Photochemotherapy; Photosensitizing Agents; Pseudomonas aeruginosa; Tumor Suppressor Protein p53
PubMed: 33140635
DOI: 10.1021/acs.biomac.0c01216