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1.
J Mater Sci Mater Med ; 30(9): 103, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31493091

RESUMO

Metal-on-metal (MoM) hip arthroplasties produce abundant implant-derived wear debris composed mainly of cobalt (Co) and chromium (Cr). Cobalt-chromium (Co-Cr) wear particles are difficult to identify histologically and need to be distinguished from other wear particle types and endogenous components (e.g., haemosiderin, fibrin) which may be present in MoM periprosthetic tissues. In this study we sought to determine whether histological stains that have an affinity for metals are useful in identifying Co-Cr wear debris in MoM periprosthetic tissues. Histological sections of periprosthetic tissue from 30 failed MoM hip arthroplasties were stained with haematoxylin-eosin (HE), Solochrome Cyanine (SC), Solochrome Azurine (SA) and Perls' Prussian Blue (PB). Sections of periprosthetic tissue from 10 cases of non-MoM arthroplasties using other implant biomaterials, including titanium, ceramic, polymethylmethacrylate (PMMA) and ultra-high molecular weight polyethylene (UHMWP) were similarly analysed. Sections of 10 cases of haemosiderin-containing knee tenosynovial giant cell tumour (TSGCT) were also stained with HE, SC, SA and PB. In MoM periprosthetic tissues, SC stained metal debris in phagocytic macrophages and in the superficial necrotic zone which exhibited little or no trichrome staining for fibrin. In non-MoM periprosthetic tissues, UHMWP, PMMA, ceramic and titanium particles were not stained by SC. Prussian Blue, but not SC or SA, stained haemosiderin deposits in MoM periprosthetic tissues and TSGT. Our findings show that SC staining (most likely Cr-associated) is useful in distinguishing Co-Cr wear particles from other metal/non-metal wear particles types in histological preparations of periprosthetic tissue and that SC reliably distinguishes haemosiderin from Co-Cr wear debris.


Assuntos
Benzenossulfonatos , Corantes/farmacologia , Análise de Falha de Equipamento/métodos , Articulação do Quadril/patologia , Nanopartículas Metálicas/análise , Próteses Articulares Metal-Metal , Coloração e Rotulagem/métodos , Artroplastia de Quadril/efeitos adversos , Artroplastia de Quadril/instrumentação , Azurina/química , Azurina/farmacologia , Benzenossulfonatos/química , Benzenossulfonatos/farmacologia , Cromo/química , Corantes/síntese química , Corantes/química , Amarelo de Eosina-(YS)/química , Amarelo de Eosina-(YS)/farmacologia , Ferrocianetos/química , Ferrocianetos/farmacologia , Células Gigantes de Corpo Estranho/efeitos dos fármacos , Células Gigantes de Corpo Estranho/patologia , Hematoxilina/química , Hematoxilina/farmacologia , Articulação do Quadril/química , Articulação do Quadril/efeitos dos fármacos , Prótese de Quadril , Técnicas Histológicas/métodos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Próteses Articulares Metal-Metal/efeitos adversos , Polietilenos/análise , Polietilenos/química
2.
Int J Mol Sci ; 20(12)2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31238511

RESUMO

Raman spectroscopy, which is a suitable tool to elucidate the structural properties of intrinsically disordered proteins, was applied to investigate the changes in both the structure and the conformational heterogeneity of the DNA-binding domain (DBD) belonging to the intrinsically disordered protein p53 upon its binding to Azurin, an electron-transfer anticancer protein from Pseudomonas aeruginosa. The Raman spectra of the DBD and Azurin, isolated in solution or forming a complex, were analyzed by a combined analysis based on peak inspection, band convolution, and principal component analysis (PCA). In particular, our attention was focused on the Raman peaks of Tyrosine and Tryptophan residues, which are diagnostic markers of protein side chain environment, and on the Amide I band, of which the deconvolution allows us to extract information about α-helix, ß-sheet, and random coil contents. The results show an increase of the secondary structure content of DBD concomitantly with a decrease of its conformational heterogeneity upon its binding to Azurin. These findings suggest an Azurin-induced conformational change of DBD structure with possible implications for p53 functionality.


Assuntos
Azurina/química , DNA/química , Domínios e Motivos de Interação entre Proteínas , Análise Espectral Raman , Proteína Supressora de Tumor p53/química , Azurina/metabolismo , Sítios de Ligação , DNA/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Proteína Supressora de Tumor p53/metabolismo
3.
Mol Biol Rep ; 46(3): 3129-3140, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30937652

RESUMO

As one of the most prevalent malignancies, breast cancer still remains a significant risk for public health. Common therapeutic strategies include invasive surgery, chemotherapy and anti-herceptin antibodies. Adverse effects, drug resistance and low efficacy of current therapies necessitates the emergence of more effective platforms. Naturally released by the immune system, granzyme B activates multiple pro-apoptotic pathways by cleaving critical substrates. Bacterial cupredoxin, azurin, selectively targets cancer cells via a p53-dependent pathway. Fused by a linker, GrB-Azurin fusion protein was overexpressed in HEK293T cells, and purified by metal chromatography. SDS-PAGE, Western blotting and ELISA were performed to confirm successful expression, purification and analyze binding properties of the fusion protein. After treatment of various breast cancer cell lines with increasing concentrations of GrB-Azurin, quantitative real-time RT-PCR was used to measure relative expression of p21, Fas and DR5 pro-apoptotic genes. The results of DNA fragmentation and WST-1 cell viability assays indicated significant apoptosis induction in MDA-MB-231, MCF7 and SK-BR-3 cells, while insignificant cytotoxicity was detected on MCF 10A normal breast cells. Herein, we report the development of a novel biotherapeutic against breast cancer. Selective effectiveness of GrB-Azurin fusion protein on different breast cancer cells highlighted the potential of the designed construct as a candidate anti-cancer biodrug.


Assuntos
Azurina/genética , Granzimas/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Azurina/química , Azurina/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Granzimas/química , Granzimas/metabolismo , Células HEK293 , Humanos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
4.
Chemistry ; 25(10): 2519-2526, 2019 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-30379366

RESUMO

A computational investigation of the triplet excited states of a rhenium complex electronically coupled with a tryptophan side chain and bound to an azurin protein is presented. In particular, by using high-level molecular modeling, evidence is provided for how the electronic properties of the excited-state manifolds strongly depend on coupling with the environment. Indeed, only upon explicitly taking into account the protein environment can two stable triplet states of metal-to-ligand charge transfer or charge-separated nature be recovered. In addition, it is also demonstrated how the rhenium complex plus tryptophan system in an aqueous environment experiences too much flexibility, which prevents the two chromophores from being electronically coupled. This occurrence disables the formation of a charge-separated state. The successful strategy requires a multiscale approach of combining molecular dynamics and quantum chemistry. In this context, the strategy used to parameterize the force fields for the electronic triplet states of the metal complex is also presented.


Assuntos
Azurina/química , Complexos de Coordenação/metabolismo , Pseudomonas aeruginosa/química , Rênio/química , Água/química , Complexos de Coordenação/química , Ligantes , Modelos Moleculares
5.
Nanoscale ; 10(46): 21712-21720, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30431054

RESUMO

The incorporation of proteins as functional components in electronic junctions has received much interest recently due to their diverse bio-chemical and physical properties. However, information regarding the energies of the frontier orbitals involved in their electron transport (ETp) has remained elusive. Here we employ a new method to quantitatively determine the energy position of the molecular orbital, nearest to the Fermi level (EF) of the electrode, in the electron transfer protein Azurin. The importance of the Cu(ii) redox center of Azurin is demonstrated by measuring gate-controlled conductance switching which is absent if Azurin's copper ions are removed. Comparing different electrode materials, a higher conductance and a lower gate-induced current onset is observed for the material with smaller work function, indicating that ETp via Azurin is LUMO-mediated. We use the difference in work function to calibrate the difference in gate-induced current onset for the two electrode materials, to a specific energy level shift and find that ETp via Azurin is near resonance. Our results provide a basis for mapping and studying the role of energy level positions in (bio)molecular junctions.


Assuntos
Azurina/química , Transistores Eletrônicos , Cobre/química , Eletrodos , Transporte de Elétrons , Ouro/química , Oxirredução , Teoria Quântica
6.
Molecules ; 23(11)2018 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-30453538

RESUMO

Copper-containing nitrite reductases (CuNiRs) play a key role in the global nitrogen cycle by reducing nitrite (NO2-) to nitric oxide, a reaction that involves one electron and two protons. In typical two-domain CuNiRs, the electron is acquired from an external electron-donating partner. The recently characterised Rastonia picketti (RpNiR) system is a three-domain CuNiR, where the cupredoxin domain is tethered to a heme c domain that can function as the electron donor. The nitrite reduction starts with the binding of NO2- to the T2Cu centre, but very little is known about how NO2- binds to native RpNiR. A recent crystallographic study of an RpNiR mutant suggests that NO2- may bind via nitrogen rather than through the bidentate oxygen mode typically observed in two-domain CuNiRs. In this work we have used combined quantum mechanical/molecular mechanical (QM/MM) methods to model the binding mode of NO2- with native RpNiR in order to determine whether the N-bound or O-bound orientation is preferred. Our results indicate that binding via nitrogen or oxygen is possible for the oxidised Cu(II) state of the T2Cu centre, but in the reduced Cu(I) state the N-binding mode is energetically preferred.


Assuntos
Cobre/metabolismo , Heme/metabolismo , Simulação de Dinâmica Molecular , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Nitritos/metabolismo , Teoria Quântica , Azurina/química , Azurina/metabolismo , Cobre/química , Transporte de Elétrons , Heme/química , Modelos Moleculares , Nitritos/química , Oxirredução , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Ralstonia pickettii/enzimologia
7.
Sci Rep ; 8(1): 15669, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30353103

RESUMO

The robustness of a high-redox potential laccase has been enhanced by swapping its second cupredoxin domain with that from another fungal laccase, which introduced a pool of neutral mutations in the protein sequence without affecting enzyme functionality. The new laccase showed outstanding stability to temperature, pH (2-9) and to organic solvents, while maintaining the ability to oxidize high-redox potential substrates. By engineering the signal peptide, enzyme secretion levels in Saccharomyces cerevisiae were increased, which allowed to purify the engineered enzyme for further characterization. The purified domain-swap laccase presented higher activity in the presence of ethanol or methanol, superior half-lives at 50-70 °C, improved stability at acidic pH, and similar catalytic efficiency for DMP albeit a lower one for ABTS (due to a shift in optimum pH). A new N-glycosylation site and a putative new surface salt-bridge were evaluated as possible determinants for the improved stability by site-directed mutagenesis. Although neither seemed to be strictly responsible for the improved thermostability, the new salt bridge was found to notably contribute to the high stability of the swapped enzyme in a broad pH range. Finally, the application potential of the new laccase was demonstrated with the enzymatic treatment of kraft lignin, an industrially relevant lignin stream, at high temperature, neutral pH and short incubation times.


Assuntos
Azurina/química , Basidiomycota/química , Proteínas Fúngicas/química , Lacase/química , Engenharia de Proteínas/métodos , Saccharomyces/química , Basidiomycota/genética , Basidiomycota/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Lacase/genética , Lacase/metabolismo , Lignina/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Oxirredução , Domínios Proteicos , Saccharomyces/genética , Saccharomyces/metabolismo , Especificidade por Substrato , Temperatura Ambiente
8.
Inorg Chem ; 57(19): 12291-12302, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30226758

RESUMO

Cupredoxins are copper-dependent electron-transfer proteins that can be categorized as blue, purple, green, and red depending on the spectroscopic properties of the Cu(II) bound forms. Interestingly, despite significantly different first coordination spheres and nuclearity, all cupredoxins share a common Greek Key ß-sheet fold. We have previously reported the design of a red copper protein within a completely distinct three-helical bundle protein, α3DChC2. (1) While this design demonstrated that a ß-barrel fold was not requisite to recapitulate the properties of a native cupredoxin center, the parent peptide α3D was not sufficiently stable to allow further study through additional mutations. Here we present the design of an elongated protein GRANDα3D (GRα3D) with Δ Gu = -11.4 kcal/mol compared to the original design's -5.1 kcal/mol. Diffraction quality crystals were grown of GRα3D (a first for an α3D peptide) and solved to a resolution of 1.34 Å. Examination of this structure suggested that Glu41 might interact with the Cu in our previously reported red copper protein. The previous bis(histidine)(cysteine) site (GRα3DChC2) was designed into this new scaffold and a series of variant constructs were made to explore this hypothesis. Mutation studies around Glu41 not only prove the proposed interaction, but also enabled tuning of the constructs' hyperfine coupling constant from 160 to 127 × 10-4 cm-1. X-ray absorption spectroscopy analysis is consistent with these hyperfine coupling differences being the result of variant 4p mixing related to coordination geometry changes. These studies not only prove that an Glu41-Cu interaction leads to the α3DChC2 construct's red copper protein like spectral properties, but also exemplify the exact control one can have in a de novo construct to tune the properties of an electron-transfer Cu site.


Assuntos
Azurina/química , Bactérias/química , Cobre/química , Sequência de Aminoácidos , Azurina/síntese química , Modelos Moleculares , Nitrosomonas europaea/química , Estrutura Secundária de Proteína , Termodinâmica
9.
Cell Cycle ; 17(13): 1649-1666, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29963969

RESUMO

Membrane lipid rafts are highly ordered microdomains and essential components of plasma membranes. In this work, we demonstrate that azurin uptake by cancer cells is, in part, mediated by caveolin-1 and GM-1, lipid rafts' markers. This recognition is mediated by a surface exposed hydrophobic core displayed by azurin since the substitution of a phenylalanine residue in position 114 facing the hydrophobic cavity by alanine impacts such interactions, debilitating the uptake of azurin by cancer cells. Treating of cancer cells with azurin leads to a sequence of events: alters the lipid raft exposure at plasma membranes, causes a decrease in the plasma membrane order as examined by Laurdan two-photon imaging and leads to a decrease in the levels of caveolin-1. Caveolae, a subset of lipid rafts characterized by the presence of caveolin-1, are gaining increasing recognition as mediators in tumor progression and resistance to standard therapies. We show that azurin inhibits growth of cancer cells expressing caveolin-1, and this inhibition is only partially observed with mutant azurin. Finally, the simultaneous administration of azurin with anticancer therapeutic drugs (paclitaxel and doxorubicin) results in an enhancement in their activity, contrary to the mutated protein.


Assuntos
Antineoplásicos/farmacologia , Azurina/metabolismo , Caveolina 1/metabolismo , Gangliosídeo G(M1)/metabolismo , Fluidez de Membrana , Microdomínios da Membrana/metabolismo , Sequência de Aminoácidos , Azurina/química , Azurina/genética , Caveolina 1/química , Linhagem Celular Tumoral , Humanos , Proteínas Mutantes/metabolismo , Mutação Puntual/genética , Domínios Proteicos
10.
J Chem Theory Comput ; 14(9): 4948-4957, 2018 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-30040901

RESUMO

Understanding the regulation mechanism and molecular determinants of the reduction potential of metalloprotein is a major challenge. An ab initio quantum mechanical/molecular mechanical (QM/MM) method combining the minimum free energy path (MFEP) and fractional number of electron (FNE) approaches has been developed in our group to simulate the redox processes of large systems. The FNE scheme provides an efficient unique description for the redox process, while the MFEP method provides improved conformational sampling on complex environments such as protein in the QM/MM calculations. The reduction potentials of wild-type and seven mutants of azurin, a type 1 copper metalloprotein, were simulated with the QM/MM-MFEP+FNE approach in this paper. A range of 350 mV for the variations of the reduction potentials of these azurin proteins was reproduced faithfully with relative errors around 20 mV. The correlation between structural interactions and reduction potentials observed in simulations provides in-depth insight into the regulation of reduction potentials, which potentially can also be very useful to the engineering of metalloprotein-based electrocatalysts in artificial photosynthesis. The excellent accuracy and efficiency of the QM/MM-MFEP+FNE approach demonstrate the potential for simulations of many electron transfer processes in condensed phases and biochemical systems.


Assuntos
Azurina/química , Teoria Quântica , Azurina/classificação , Azurina/genética , Domínio Catalítico , Cristalografia por Raios X , Conformação Molecular , Mutação , Oxirredução , Pseudomonas aeruginosa/química
11.
Angew Chem Int Ed Engl ; 57(33): 10677-10682, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-29949236

RESUMO

N-heterocyclic carbene (NHC) ligands have had a major impact in homogeneous catalysis, however, their potential role in biological systems is essentially unexplored. We replaced a copper-coordinating histidine (His) in the active site of the redox enzyme azurin with exogenous dimethyl imidazolylidene. This NHC rapidly restores the type-1 Cu center, with spectroscopic properties (EPR, UV/Vis) that are identical to those from N-coordination of the His in the wild type. However, the introduction of the NHC markedly alters the redox potential of the metal, which is a key functionality of this blue copper protein. These results suggest that C-bonding for histidine is plausible and a potentially relevant bonding mode of redox-active metalloenzymes in their (transient) active states.


Assuntos
Azurina/química , Metano/análogos & derivados , Azurina/genética , Azurina/metabolismo , Domínio Catalítico , Cobre/química , Técnicas Eletroquímicas , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Heterocíclicos/química , Histidina/química , Ligantes , Metano/química , Mutagênese Sítio-Dirigida , Oxirredução , Espectrofotometria
12.
Bioelectrochemistry ; 123: 112-118, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29747129

RESUMO

The impact of different concentrations of three amino acids (cysteine, histidine and methionine) which are part of the amino acid sequence of rusticyanin on dissolution of pyrite is investigated by the application of electrochemical techniques. Cyclic voltammetric studies conducted in the anodic direction from corrosion potential have shown that in the vicinity of corrosion potential, histidine and methionine do not influence dissolution of pyrite independently on their concentrations. On the other hand, cysteine and solutions of these amino acids in the molar ratios Cys:His:Met/1:1:1 and Cys:His:Met/1:2:1 accelerate dissolution at concentrations 10-2 mol L-1 and 10-3 mol L-1. Potentiodynamic polarization measurements showed that methionine does not affect the anodic and cathodic dissolution at all concentrations, while histidine does not affect significantly on the anodic dissolution at all concentrations. Cysteine and solutions of three amino acids in the molar ratio Cys:His:Met/1:1:1 and Cys:His:Met/1:2:1 cause intensive cathodic inhibition and anodic activation at concentrations 10-2 mol L-1 and 10-3 mol L-1 respectively.


Assuntos
Azurina/química , Ferro/química , Sulfetos/química , Ácidos Sulfúricos/química , Cisteína/química , Técnicas Eletroquímicas , Eletrodos , Histidina/química , Metionina/química , Modelos Moleculares , Solubilidade
13.
Proc Natl Acad Sci U S A ; 115(24): 6129-6134, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29844178

RESUMO

We combine experimental and computational methods to address the anomalous kinetics of long-range electron transfer (ET) in mutants of Pseudomonas aeruginosa azurin. ET rates and driving forces for wild type (WT) and three N47X mutants (X = L, S, and D) of Ru(2,2'-bipyridine)2 (imidazole)(His83) azurin are reported. An enhanced ET rate for the N47L mutant suggests either an increase of the donor-acceptor (DA) electronic coupling or a decrease in the reorganization energy for the reaction. The underlying atomistic features are investigated using a recently developed nonadiabatic molecular dynamics method to simulate ET in each of the azurin mutants, revealing unexpected aspects of DA electronic coupling. In particular, WT azurin and all studied mutants exhibit more DA compression during ET (>2 Å) than previously recognized. Moreover, it is found that DA compression involves an extended network of hydrogen bonds, the fluctuations of which gate the ET reaction, such that DA compression is facilitated by transiently rupturing hydrogen bonds. It is found that the N47L mutant intrinsically disrupts this hydrogen-bond network, enabling particularly facile DA compression. This work, which reveals the surprisingly fluctional nature of ET in azurin, suggests that hydrogen-bond networks can modulate the efficiency of long-range biological ET.


Assuntos
Azurina/química , Azurina/metabolismo , Transporte de Elétrons , Ligações de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo
14.
FEBS Lett ; 592(9): 1473-1483, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29665008

RESUMO

The Neisseria gonorrhoeae bacterial cytochrome c peroxidase plays a key role in detoxifying the cells from H2 O2 by reducing it to water using the lipid-modified azurin, LAz, a small type 1 copper protein, as electron donor. Here, the interaction between these two proteins was characterized by steady-state kinetics, two-dimensional NMR and molecular docking simulations. LAz is an efficient electron donor capable of activating this enzyme. This electron transfer complex is weak with a hydrophobic character, with LAz binding close to the electron transferring heme of the enzyme. The high catalytic rate (39 ± 0.03 s-1 ) is explained by the LAz pre-orientation, due to a positive dipole moment, and by the fast-dynamic ensemble of orientations, suggested by the small chemical shifts.


Assuntos
Azurina/química , Azurina/metabolismo , Citocromo-c Peroxidase/metabolismo , Lipídeos/química , Neisseria gonorrhoeae/enzimologia , Transporte de Elétrons , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Solubilidade
15.
Sci Rep ; 8(1): 1989, 2018 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-29386517

RESUMO

Metalloproteins carry out diverse biological functions including metal transport, electron transfer, and catalysis. At present, the influence of metal cofactors on metalloprotein stability is not well understood. Here, we report the mechanical stability and unfolding pathway of azurin, a cupredoxin family protein with ß-barrel topology and type I copper-binding centre. Single-molecule force spectroscopy (SMFS) experiments reveal 2-state and 3-state unfolding pathways for apo-azurin. The intermediate in the 3-state pathway occurs at an unfolding contour length of 7.5 nm from the native state. Steered molecular dynamics (SMD) simulations show that apo-azurin unfolds via a first transition state (TS) where ß2Β-ß8 and ß7-ß8 strand pairs rupture to form the intermediate, which subsequently unfolds by the collective rupture of remaining strands. SMFS experiments on holo-azurin exhibit an additional 4-state pathway besides the 2-state and 3-state pathways. The unfolding contour length leading to the first intermediate is 6.7 nm suggesting a sequestration of ~1 nm polypeptide chain length by the copper. SMD simulations reveal atomistic details of the copper sequestration and predict a combined ß4-ß7 pair and copper coordination sphere rupture to create the third TS in the 4-state pathway. Our systematic studies provide detailed mechanistic insights on modulation of protein mechanical properties by metal-cofactors.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Azurina/química , Azurina/metabolismo , Cobre/metabolismo , Dobramento de Proteína , Fluorescência , Modelos Biológicos , Simulação de Dinâmica Molecular
16.
Angew Chem Int Ed Engl ; 57(19): 5364-5368, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29451960

RESUMO

Determining whether a protein regulates its net electrostatic charge during electron transfer (ET) will deepen our mechanistic understanding of how polypeptides tune rates and free energies of ET (e.g., by affecting reorganization energy, and/or redox potential). Charge regulation during ET has never been measured for proteins because few tools exist to measure the net charge of a folded protein in solution at different oxidation states. Herein, we used a niche analytical tool (protein charge ladders analyzed with capillary electrophoresis) to determine that the net charges of myoglobin, cytochrome c, and azurin change by 0.62±0.06, 1.19±0.02, and 0.51±0.04 units upon single ET. Computational analysis predicts that these fluctuations in charge arise from changes in the pKa  values of multiple non-coordinating residues (predominantly histidine) that involve between 0.42-0.90 eV. These results suggest that ionizable residues can tune the reactivity of redox centers by regulating the net charge of the entire protein-cofactor-solvent complex.


Assuntos
Metaloproteínas/metabolismo , Azurina/química , Azurina/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Metaloproteínas/química , Mioglobina/química , Mioglobina/metabolismo , Oxirredução , Eletricidade Estática , Termodinâmica
17.
Chemistry ; 24(3): 646-654, 2018 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-29064125

RESUMO

Fluorescent labeling of biomacromolecules enjoys increasing popularity for structural, mechanistic, and microscopic investigations. Its success hinges on the ability of the dye to alternate between bright and dark states. Förster resonance energy transfer (FRET) is an important source of fluorescence modulation. Photo-induced electron transfer (PET) may occur as well, but is often considered only when donor and acceptor are in van der Waals contact. In this study, PET is shown between a label and redox centers in oxidoreductases, which may occur over large distances. In the small blue copper protein azurin, labeled with ATTO655, PET is observed when the label is at 18.5 Å, but not when it is at 29.1 Šfrom the Cu. For CuII , PET from label to Cu occurs at a rate of (4.8±0.3)×104  s-1 and back at (0.7±0.1)×103  s-1 . With CuI the numbers are (3.3±0.7)×106  s-1 and (1.0±0.1)×104  s-1 . Reorganization energies and electronic coupling elements are in the range of 0.8-1.2 eV and 0.02-0.5 cm-1 , respectively. These data are compatible with electron transfer (ET) along a through-bond pathway although transient complex formation followed by ET cannot be ruled out. The outcome of this study is a useful guideline for experimental designs in which oxidoreductases are labelled with fluorescent dyes, with particular attention to single molecule investigations. The labelling position for FRET can be optimized to avoid reactions like PET by evaluating the structure and thermodynamics of protein and label.


Assuntos
Azurina/química , Cobre/química , Corantes Fluorescentes/química , Transporte de Elétrons , Cinética , Oxirredução , Oxirredutases/química , Espectrometria de Fluorescência , Termodinâmica
18.
ACS Nano ; 11(12): 12824-12831, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-29202236

RESUMO

The copper protein azurin, due to the peculiar coupling of its optical and vibronic properties with electron transfer (ET) and its biorecognition capabilities, is a very promising candidate for bioelectronic, bio-optoelectronic and biosensor applications. However, a complete understanding of the fundamental processes relating azurin ET and its optical and vibronic characteristics with the charge transport mechanisms occurring in proteins bound to a conductive surface, the typical scenario for a biosensor or bioelectronic component, is still lacking. We studied azurin proteins bound to a gold electrode surface by scanning tunneling microscopy combined with tip-enhanced Raman spectroscopy (STM-TERS). Robust TER spectra were obtained, and the protein's vibronic response under optical excitation in resonance with its ligand-to-metal charge transfer band was found to be affected by the tunneling parameters, indicating a direct involvement of the active site vibrations in the electron transport process.


Assuntos
Azurina/química , Metaloproteínas/química , Vibração , Transporte de Elétrons , Microscopia de Tunelamento , Modelos Moleculares , Conformação Molecular , Tamanho da Partícula , Análise Espectral Raman , Propriedades de Superfície
19.
J Am Chem Soc ; 139(43): 15337-15346, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28981262

RESUMO

Bioelectronics moves toward designing nanoscale electronic platforms that allow in vivo determinations. Such devices require interfacing complex biomolecular moieties as the sensing units to an electronic platform for signal transduction. Inevitably, a systematic design goes through a bottom-up understanding of the structurally related electrical signatures of the biomolecular circuit, which will ultimately lead us to tailor its electrical properties. Toward this aim, we show here the first example of bioengineered charge transport in a single-protein electrical contact. The results reveal that a single point-site mutation at the docking hydrophobic patch of a Cu-azurin causes minor structural distortion of the protein blue Cu site and a dramatic change in the charge transport regime of the single-protein contact, which goes from the classical Cu-mediated two-step transport in this system to a direct coherent tunneling. Our extensive spectroscopic studies and molecular-dynamics simulations show that the proteins' folding structures are preserved in the single-protein junction. The DFT-computed frontier orbital of the relevant protein segments suggests that the Cu center participation in each protein variant accounts for the different observed charge transport behavior. This work is a direct evidence of charge transport control in a protein backbone through external mutagenesis and a unique nanoscale platform to study structurally related biological electron transfer.


Assuntos
Azurina/química , Engenharia de Proteínas , Azurina/síntese química , Azurina/genética , Cobre/química , Transporte de Elétrons , Eletrônica , Simulação de Dinâmica Molecular , Mutagênese , Mutação Puntual , Dobramento de Proteína , Teoria Quântica , Análise Espectral
20.
Protein Sci ; 26(12): 2334-2341, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28960574

RESUMO

Azurin secreted by Pseudomonas aeruginosa is an anticancer bacteriocin, which preferentially enters human cancer cells and induces apoptosis or growth inhibition. It turns out that azurin is a multi-target anticancer agent interfering in the p53 signaling pathway and the non-receptor tyrosine kinases signaling pathway. This suggests that azurin exerts its anticancer activity by interacting with multiple targets and interfering in multiple steps in disease progression. Therefore, azurin could overcome resistance to therapy. Besides azurin, putative bacteriocins that possess functional properties similar to those of azurin have been identified in more bacteria species. A systematic investigation on the anticancer mechanisms of azurin and the azurin-like bacteriocins will provide more and better options in cancer therapy. In this review, we summarize how azurin and the derived peptides hijack key cellular regulators or cell surface receptors to remodel the cellular signaling networks. In particular, we highlight the necessity of determining the structure of azurin/p53 complex and investigating the influence of post-translational modifications on interactions between azurin and p53. Therapeutic applications of azurin and derived peptides are also discussed.


Assuntos
Antineoplásicos/farmacologia , Azurina/farmacologia , Proteínas de Bactérias/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/química , Azurina/química , Azurina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Humanos , Células MCF-7 , Camundongos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Pseudomonas aeruginosa/química , Proteína Supressora de Tumor p53/metabolismo
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