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1.
Nat Commun ; 12(1): 933, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568660

RESUMO

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins. Crystal structure of the peptidyl tryptophan dihydroxylase reveals a large pocket that may dock the protein substrate with the bound flavin adenine dinucleotide situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, we propose a chemical reaction mechanism of peptidyl tryptophan dihydroxylation catalyzed by the flavoprotein monooxygenase. The diversity of the tryptophylquinone-generating systems suggests convergent evolution of the peptidyl tryptophan-derived cofactors in different proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Coenzimas/metabolismo , Dipeptídeos/metabolismo , Flavoproteínas/metabolismo , Indolquinonas/metabolismo , Oxigenases de Função Mista/metabolismo , Paracoccus denitrificans/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Coenzimas/química , Dipeptídeos/química , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/química , Indolquinonas/química , Oxigenases de Função Mista/química , Paracoccus denitrificans/química , Paracoccus denitrificans/genética , Paracoccus denitrificans/metabolismo , Triptofano/química , Triptofano/metabolismo
2.
Food Chem ; 348: 129152, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-33515953

RESUMO

A red pigment was prepared by reaction of chlorogenic acid (CGA) with tryptophan (TRP) in air at pH 9 (37% w/w yield) and evaluated as food dye. The main component of pigment was formulated as an unusual benzochromeno[2,3-b]indole linked to a TRP unit, featuring a cyanine type chromophore (λmax 542, 546 nm, 1% extinction coefficient of the sodium salt = 244 ± 2). The chromophore showed a minimal pH dependence and proved stable for at least 3 h at 90 °C, both at pH 3.6 or 7.0, whereas red wine anthocyanins showed a substantial (30%) and betanin a complete abatement after 1 h at the acidic pHs. An intense coloring of different food matrices was obtained with the pigment at 0.01 % w/w. No toxicity was observed up to 0.2 mg/mL on hepatic and colonic cell lines. These data make this dye a promising alternative for red coloring of food.


Assuntos
Antocianinas/química , Ácido Clorogênico/química , Corantes de Alimentos/química , Triptofano/química , Antocianinas/farmacologia , Betacianinas/química , Betacianinas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes de Alimentos/farmacologia , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Acoplamento Oxidativo
3.
J Med Chem ; 64(1): 586-601, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33356253

RESUMO

An array of l- and d-halotryptophans with different substituents at the indole moiety was synthesized employing either enzymatic halogenation by halogenases or incorporation of haloindoles using tryptophan synthase. Introduction of these Trp derivatives into RGD peptides as a benchmark system was performed to investigate their influence on bioactivity. Halotryptophan-containing RGD peptides display increased affinity toward integrin αvß3 and enhanced selectivity over integrin α5ß1. In addition, bromotryptophan was exploited as a platform for late-stage diversification by Suzuki-Miyaura cross-coupling (SMC), resulting in new-to-nature biaryl motifs. These peptides show enhanced affinity toward αvß3, good affinity to αvß8, and remarkable selectivity over α5ß1 and αIIbß3 while featuring fluorogenic properties. Their feasibility as a probe was demonstrated in vitro. Extensive molecular dynamics simulations were undertaken to elucidate NMR and high-performance liquid chromatography (HPLC) data for these late-stage diversified cyclic RGD peptides and to further characterize their conformational preferences.


Assuntos
Halogênios/química , Oligopeptídeos/farmacologia , Triptofano/química , Cromatografia Líquida de Alta Pressão/métodos , Estudos de Viabilidade , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Relação Estrutura-Atividade
4.
J Enzyme Inhib Med Chem ; 35(1): 1539-1544, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32746637

RESUMO

Coronavirus disease 2019 (COVID-19) has been a pandemic disease of which the termination is not yet predictable. Currently, researches to develop vaccines and treatments is going on globally to cope with this disastrous disease. Main protease (3CLpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the good targets to find antiviral agents before vaccines are available. Some flavonoids are known to inhibit 3CLpro from SARS-CoV which causes SARS. Since their sequence identity is 96%, a similar approach was performed with a flavonoid library. Baicalin, herbacetin, and pectolinarin have been discovered to block the proteolytic activity of SARS-CoV-2 3CLpro. An in silico docking study showed that the binding modes of herbacetin and pectolinarin are similar to those obtained from the catalytic domain of SARS-CoV 3CLpro. However, their binding affinities are different due to the usage of whole SARS-CoV-2 3CLpro in this study. Baicalin showed an effective inhibitory activity against SARS-CoV-2 3CLpro and its docking mode is different from those of herbacetin and pectolinarin. This study suggests important scaffolds to design 3CLpro inhibitors to develop antiviral agents or health-foods and dietary supplements to cope with SARS-CoV-2.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Flavonoides/química , Pneumonia Viral/tratamento farmacológico , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Antivirais/química , Betacoronavirus , Desenho de Fármacos , Transferência Ressonante de Energia de Fluorescência , Humanos , Simulação de Acoplamento Molecular , Pandemias , Poliproteínas , Inibidores de Proteases/química , Ligação Proteica , Conformação Proteica , Espectrofotometria , Triptofano/química
5.
Comput Biol Med ; 122: 103849, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658736

RESUMO

SARS-CoV and SARS-CoV-2 do not appear to have functions of a hemagglutinin and neuraminidase. This is a mystery, because sugar binding activities appear essential to many other viruses including influenza and even most other coronaviruses in order to bind to and escape from the glycans (sugars, oligosaccharides or polysaccharides) characteristic of cell surfaces and saliva and mucin. The S1 N terminal Domains (S1-NTD) of the spike protein, largely responsible for the bulk of the characteristic knobs at the end of the spikes of SARS-CoV and SARS-CoV-2, are here predicted to be "hiding" sites for recognizing and binding glycans containing sialic acid. This may be important for infection and the ability of the virus to locate ACE2 as its known main host cell surface receptor, and if so it becomes a pharmaceutical target. It might even open up the possibility of an alternative receptor to ACE2. The prediction method developed, which uses amino acid residue sequence alone to predict domains or proteins that bind to sialic acids, is naïve, and will be advanced in future work. Nonetheless, it was surprising that such a very simple approach was so useful, and it can easily be reproduced in a very few lines of computer program to help make quick comparisons between SARS-CoV-2 sequences and to consider the effects of viral mutations.


Assuntos
Betacoronavirus/química , Biologia Computacional , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , Algoritmos , Motivos de Aminoácidos , Sítios de Ligação , Humanos , Conformação Molecular , Ácido N-Acetilneuramínico/química , Pandemias , Polissacarídeos/química , Vírus da SARS , Triptofano/química
6.
PLoS One ; 15(7): e0235407, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32649670

RESUMO

Fast scan cyclic voltammetry (FSCV) allows for real -time analysis of phasic neurotransmitter levels. Tryptophan (TRP) is an aromatic amino acid responsible for facilitating electron transfer kinetics in oxidoreductase enzymes. Previous work with TRP-modified electrodes showed increased sensitivity for cyclic voltammetry detection of dopamine (DA) when used with slower scan rates (0.05 V/s). Here, we outline an in vitro proof of concept for TRP-modified electrodes in FSCV detection of DA, and decreased sensitivity for ascorbic acid (AA). TRP-modified electrodes had a limit of detection (LOD) for DA of 2.480 ± 0.343 nM compared to 8.348 ± 0.405 nM for an uncoated electrode. Selectivity for DA/ascorbic acid (AA) was 1.107 ± 0.3643 for uncoated and 15.57 ± 4.184 for TRP-modified electrodes. Additionally, these TRP-modified electrodes demonstrated reproducibility when exposed to extended cycling. TRP-modified electrodes will provide an effective modification to increase sensitivity for DA.


Assuntos
Técnicas Biossensoriais , Dopamina/isolamento & purificação , Técnicas Eletroquímicas , Ácido Ascórbico/química , Carbono/química , Dopamina/química , Eletrodos , Cinética , Triptofano/química , Ácido Úrico/química
7.
Phys Chem Chem Phys ; 22(23): 13084-13091, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32490449

RESUMO

We report herein the first detailed study of the mechanism of redox reactions occurring during the gas-phase dissociative electron transfer of prototypical ternary [CuII(dien)M]˙2+ complexes (M, peptide). The two final products are (i) the oxidized non-zwitterionic π-centered [M]˙+ species with both the charge and spin densities delocalized over the indole ring of the tryptophan residue and with a C-terminal COOH group intact, and (ii) the complementary ion [CuI(dien)]+. Infrared multiple photon dissociation (IRMPD) action spectroscopy and low-energy collision-induced dissociation (CID) experiments, in conjunction with density functional theory (DFT) calculations, revealed the structural details of the mass-isolated precursor and product cations. Our experimental and theoretical results indicate that the doubly positively charged precursor [CuII(dien)M]˙2+ features electrostatic coordination through the anionic carboxylate end of the zwitterionic M moiety. An additional interaction exists between the indole ring of the tryptophan residue and one of the primary amino groups of the dien ligand; the DFT calculations provided the structures of the precursor ion, intermediates, and products, and enabled us to keep track of the locations of the charge and unpaired electron. The dissociative one-electron transfer reaction is initiated by a gradual transition of the M tripeptide from the zwitterionic form in [CuII(dien)M]˙2+ to the non-zwitterionic M intermediate, through a cascade of conformational changes and proton transfers. In the next step, the highest energy intermediate is formed; here, the copper center is 5-coordinate with coordination from both the carboxylic acid group and the indole ring. A subsequent switch back to 4-coordination to an intermediate IM1, where attachment to GGW occurs through the indole ring only, creates the structure that ultimately undergoes dissociation.


Assuntos
Complexos de Coordenação/química , Cobre/química , Peptídeos/química , Triptofano/química , Teoria da Densidade Funcional , Transporte de Elétrons , Estrutura Molecular , Fótons , Espectrofotometria Infravermelho , Triptofano/análogos & derivados
8.
Food Chem ; 327: 127031, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32464458

RESUMO

The 1,2-dicarbonyl compounds have received extensive attention due to their high reactivity and toxicity in vitro and in vivo. Availability of scavenging compounds may facilitate development of efficient strategies for their control. The concept of in situ generation of carbonyl trapping agents is an intriguing proposition and has been demonstrated with amino acid tryptophan. Ability of indole to undergo electrophilic aromatic substitution reaction was studied in the past with methylglyoxal. To confirm the generality of this reaction, model systems containing indole and several 1,2-dicarbonyl compounds were prepared and reacted at room temperature (RT) and at 150 °C and analyzed by ESI-qTOF-MS/MS and isotopic labeling technique. Indole showed ability to capture all the tested 1,2-dicarbonyls. Longer chain 1,2-dicarbonyls showed higher temperature dependency than shorter chain in their reactivity towards indole. Furthermore, the ability of indole to scavenge Strecker aldehydes was also demonstrated in alanine/glucose and in a bread model systems using [13C-2]indole.


Assuntos
Aldeídos/química , Indóis/química , Pão/análise , Temperatura , Triptofano/química
9.
Food Chem ; 326: 126976, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32413756

RESUMO

This study developed a novel electrochemical sensor containing nitrogen-doped ordered mesoporous carbon (NOMC) for the sensitive and selective quantification of l-tryptophan (Trp). The electro-oxidation mechanism of Trp on the NOMC/Nafion/glass carbon electrode (GCE) was first investigated, and was found to follow a two-electron/two-proton transfer mechanism. Subsequently, the analytical operation conditions were optimized. Under the optimum testing conditions, the oxidation current was found to increase linearly with Trp concentration in the ranges 0.5-70.0 µM and 70.0-200.0 µM (different slopes in each range), with the limit of detection determined to be 35.0 nM (S/N = 3). In addition, the sensor was highly selective for Trp and showed good repeatability and long-term stability. Studies of Trp in real world systems, such as an 18 amino acid mixture and an enzymatic protein hydrolysate, showed excellent recoveries (99.30-103.60%). Results suggest that NOMC/Nafion/GCE sensor has excellent performance characteristics for routine Trp analysis.


Assuntos
Carbono/química , Técnicas Eletroquímicas/métodos , Nitrogênio/química , Triptofano/análise , Polímeros de Fluorcarboneto/química , Limite de Detecção , Oxirredução , Porosidade , Proteínas/química , Triptofano/química
10.
Proc Natl Acad Sci U S A ; 117(22): 12095-12100, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32409607

RESUMO

To advance mechanistic understanding of membrane-associated peptide folding and insertion, we have studied the kinetics of three single tryptophan pHLIP (pH-Low Insertion Peptide) variants, where tryptophan residues are located near the N terminus, near the middle, and near the inserting C-terminal end of the pHLIP transmembrane helix. Single-tryptophan pHLIP variants allowed us to probe different parts of the peptide in the pathways of peptide insertion into the lipid bilayer (triggered by a pH drop) and peptide exit from the bilayer (triggered by a rise in pH). By using pH jumps of different magnitudes, we slowed down the processes and established the intermediates that helped us to understand the principles of insertion and exit. The obtained results should also aid the applications in medicine that are now entering the clinic.


Assuntos
Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Membrana Celular/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Bicamadas Lipídicas/química , Lipossomos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Dobramento de Proteína , Termodinâmica , Triptofano/química , Triptofano/genética
11.
PLoS One ; 15(5): e0232846, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32380514

RESUMO

The structure of lactose permease, stabilized in a periplasmic open conformation by two Gly to Trp replacements (LacYww) and complexed with a nanobody directed against this conformation, provides the highest resolution structure of the symporter. The nanobody binds in a different manner than two other nanobodies made against the same mutant, which also bind to the same general region on the periplasmic side. This region of the protein may represent an immune hotspot. The CDR3 loop of the nanobody is held by hydrogen bonds in a conformation that partially blocks access to the substrate-binding site. As a result, kon and koff for galactoside binding to either LacY or the double mutant complexed with the nanobody are lower than for the other two LacY/nanobody complexes though the Kd values are similar, reflecting the fact that the nanobodies rigidify structures along the pathway. While the wild-type LacY/nanobody complex clearly stabilizes a similar 'extracellular open' conformation in solution, judged by binding kinetics, the complex with wild-type LacY did not yet crystallize, suggesting the nanobody does not bind strongly enough to shift the equilibrium to stabilize a periplasmic side-open conformation suitable for crystallization. However, the similarity of the galactoside binding kinetics for the nanobody-bound complexes with wild type LacY and with LacYWW indicates that they have similar structures, showing that the reported co-structures reliably show nanobody interactions with LacY.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Transporte de Monossacarídeos/química , Anticorpos de Domínio Único/química , Simportadores/química , Substituição de Aminoácidos , Reações Antígeno-Anticorpo , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Galactose/metabolismo , Glicina/química , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/imunologia , Mutação de Sentido Incorreto , Mutação Puntual , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Anticorpos de Domínio Único/imunologia , Relação Estrutura-Atividade , Simportadores/genética , Simportadores/imunologia , Tiogalactosídeos/química , Triptofano/química
12.
Biochim Biophys Acta Biomembr ; 1862(9): 183363, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32450141

RESUMO

Melatonin is a neurohormone that has been shown to be protective in Alzheimer's diseases against amyloid-ß (Aß) toxicity, which involves interaction of Aß with neuronal membrane. Non-specific interactions of melatonin with cell membrane may play a physiological role in this process by preserving membrane fluidity. In the brain, melatonin is derived from the amino acid tryptophan through a pathway that includes serotonin and N-acetylserotonin (NAS). How these molecules affect the membrane properties is not understood. In this work, we studied interactions of melatonin and its metabolic precursors tryptophan, serotonin and NAS with dipalmitoylphosphatidylcholine (DPPC) monolayers at the air-water interface using Langmuir monolayer technique. Analysis of compression isotherms, phase transitions and compressibility moduli indicate that all four molecules alter the DPPC monolayer properties in a structure and concentration dependent manner. This effect was most pronounced for melatonin followed by NAS. Melatonin and NAS both decreased the compressibility modulus and shifted the LE/LC phase transition suggesting an increase in the membrane fluidity. Tryptophan and serotonin caused less pronounced effects on the DPPC isotherm. These differences suggest different interaction mechanisms and may be attributed to the interplay between electrostatic and hydrophobic interactions of these molecules with the zwitterionic DPPC headgroups which correlate with water solubility and oil partition coefficients (LogS and LogP) of each the four molecules. The results here demonstrate how the physiochemical properties of indoles can affect lipid membranes which may shed light on the functional significance of these important neurochemicals and the neuroprotective mechanisms of melatonin.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Melatonina/química , Fluidez de Membrana , Membranas Artificiais , Serotonina/química , Triptofano/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Doença de Alzheimer/metabolismo , Humanos , Melatonina/metabolismo , Serotonina/metabolismo , Triptofano/metabolismo
13.
J Chromatogr A ; 1622: 461147, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32450989

RESUMO

Molecularly imprinted polymers coated magnetic particles (Fe3O4@MIPs) were prepared and used as adsorbents in solid phase extraction for efficient enantioseparation of racemic tryptophan (Trp) in aqueous medium. The amino-modified magnetic particles (Fe3O4-NH2) were first synthesized by one-pot hydrothermal method. Then the template molecules (L-Trp) were assembled on the surface of Fe3O4-NH2. Finally, Fe3O4@MIPs were prepared via a sol-gel method using L-Trp@Fe3O4-NH2 complex as matrix, 3-aminopropyltriethoxylsilane and n-octyltriethoxysilane as functional monomers. The as-prepared Fe3O4@MIPs were spherical with an average diameter about 149 ± 6.0 nm. The thickness of MIPs layer was approximately 3.5 ± 2.3 nm. The adsorption isotherms data of Fe3O4@MIPs toward L-Trp and D-Trp were well described by the Langmuir model. The maximum adsorption capacities of Fe3O4@MIPs for L-Trp and D-Trp were calculated to be 17.2 ± 0.34 mg/g and 7.2 ± 0.19 mg/g, respectively. The material exhibited good selectivity toward L-Trp with imprinting factor of 5.6. Excitingly, the enantiomeric excess (ee) of Trp in supernatant after adsorption of racemic Trp by Fe3O4@MIPs was as high as 100%. The result suggests that the imprinted caves in Fe3O4@MIPs are highly matched with L-Trp molecule in space structure and spatial arrangement of active functional groups. The work also demonstrates that sol-gel technology has great potential in preparation of MIPs for chiral separation.


Assuntos
Fenômenos Magnéticos , Impressão Molecular/métodos , Polímeros/síntese química , Triptofano/química , Triptofano/isolamento & purificação , Água/química , Adsorção , Compostos Férricos/química , Concentração de Íons de Hidrogênio , Polímeros/química , Reprodutibilidade dos Testes , Extração em Fase Sólida/métodos , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Estereoisomerismo , Temperatura , Fatores de Tempo
14.
Arch Biochem Biophys ; 687: 108388, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32343975

RESUMO

The active sites of metalloproteins may be mimicked by designing peptides that bind to their respective metal ions. Studying the binding of protein ligands to metal ions along with the associated structural changes is important in understanding metal uptake, transport and electron transfer functions of proteins. Copper-binding metalloprotein azurin is a 128-residue electron transfer protein with a redox-active copper cofactor. Here, we report the copper-binding associated spectroscopic and structural properties of peptide loops (11 and 13 residues) from the copper-binding site of azurin. These peptides develop a ß-turn upon copper-binding with a 1:1 Cu2+:peptide stoichiometry as seen in circular dichroism and exhibit electronic transitions centered at 340 nm and 540 nm. Further addition of copper develops a helical feature along with a shift in the absorption maxima to ~360 nm and ~580 nm at 2:1 Cu2+:peptide stoichiometry, indicating stoichiometric dependence of copper-binding geometry. Mass spectrometry indicates the copper-binding to cysteine, histidine and methionine in the peptide with 1:1 stoichiometry, and interestingly, dimerization through a disulfide linkage at 2:1 stoichiometry, as observed previously for denatured azurin. Fluorescence quenching studies on peptides with tryptophan further confirm the copper-binding induced changes in the two peptides are bi-phasic.


Assuntos
Azurina/metabolismo , Cobre/metabolismo , Fragmentos de Peptídeos/metabolismo , Conformação Proteica/efeitos dos fármacos , Azurina/química , Domínio Catalítico , Cobre/química , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Fragmentos de Peptídeos/química , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Triptofano/química
15.
Inorg Chem ; 59(8): 5243-5246, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32255347

RESUMO

The Anderson-type hexamolybdoaluminate functionalized with lauric acid (LA), (TBA)3[Al(OH)3Mo6O18{(OCH2)3CNHCOC11H23}]·9H2O (TBA-AlMo6-LA, where TBA = tetrabutylammonium), was prepared via two synthetic routes and characterized by thermogravimetric and elemental analyses, mass spectrometry, IR and 1H NMR spectroscopy, and powder and single-crystal X-ray diffraction. The interaction of TBA-AlMo6-LA with human serum albumin (HSA) was investigated via fluorescence and circular dichroism spectroscopy. The results revealed that TBA-AlMo6-LA binds strongly to HSA (63% quenching at an HSA/TBA-AlMo6-LA ratio of 1:1), exhibiting static quenching. In contrast to TBA-AlMo6-LA, the nonfunctionalized polyoxometalate, Na3(H2O)6[Al(OH)6Mo6O18]·2H2O (AlMo6), showed weak binding toward HSA (22% quenching at a HSA/AlMo6 ratio of 1:25). HSA binding was confirmed by X-ray structure analysis of the HSA-Myr-AlMo6-LA complex (Myr = myristate). These results provide a promising lead for the design of novel polyoxometalate-based hybrids that are able to exploit HSA as a delivery vehicle to improve their pharmacokinetics and bioactivity.


Assuntos
Compostos de Alumínio/metabolismo , Ácidos Láuricos/metabolismo , Albumina Sérica Humana/metabolismo , Compostos de Alumínio/síntese química , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Ácidos Láuricos/síntese química , Molibdênio/química , Ligação Proteica , Albumina Sérica Humana/química , Espectrometria de Fluorescência , Triptofano/química
16.
Biochim Biophys Acta Biomembr ; 1862(7): 183280, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32220553

RESUMO

Short linear antimicrobial peptides are attractive templates for developing new antibiotics. Here, it is described a study of the interaction between two short Trp-rich peptides, horine and verine-L, and model membranes. Isothermal titration calorimetry studies showed that the affinity of these peptides towards large unilamellar vesicles (LUV) having a lipid composition mimicking the lipid composition of S. aureus membranes is ca. 30-fold higher than that towards E. coli mimetics. The former interaction is driven by enthalpy and entropy, while the latter case is driven by entropy, suggesting differences in the forces that play a role in the binding to the two types of model membranes. Upon membrane binding the peptides acquired different conformations according to circular dichroism (CD) studies; however, in both cases CD studies indicated stacked W-residues. Peptide-induced membrane permeabilization, lipid flip-flop, molecular packing at the membrane-water interface, and lateral lipid segregation were observed in all cases. However, the extent of these peptide-induced changes on membrane properties was always higher in S. aureus than E. coli mimetics. Both peptides seem to act via a similar mechanism of membrane permeabilization of S. aureus membrane mimetics, while their mechanisms seem to differ in the case of E. coli. This may be the result of differences in both the peptides´ structure and the membrane lipid composition between both types of bacteria.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Conformação Molecular , Sequência de Aminoácidos/genética , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Biomimética , Calorimetria , Dicroísmo Circular , Escherichia coli/química , Escherichia coli/patogenicidade , Humanos , Staphylococcus aureus/química , Staphylococcus aureus/patogenicidade , Termodinâmica , Triptofano/química , Triptofano/genética , Lipossomas Unilamelares/química
17.
Biochim Biophys Acta Biomembr ; 1862(6): 183235, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126232

RESUMO

The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.


Assuntos
Lipídeos/química , Receptor A2A de Adenosina/química , Estireno/química , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Maleatos/química , Pichia/química , Conformação Proteica , Espectrometria de Fluorescência/métodos , Triazinas/farmacologia , Triazóis/farmacologia , Triptofano/química
18.
Biophys Chem ; 259: 106337, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32126442

RESUMO

LOV2 (Light-Oxygen-Voltage) domain from Avena sativa phototropin 1 (AsLOV2) belongs to the superfamily of PAS (Per-Arnt-Sim) domains, members of which function as signaling sensors. AsLOV2 undergoes a conformational change upon blue-light absorption by its FMN cofactor. AsLOV2 wild type (wt) is intensively studied as a photo-switchable element in conjugation with various proteins. On the other hand, its variant AsLOV2 with replaced cysteinyl residue C450, which is critical for the forming a covalent adduct with FMN upon irradiation, forms a precursor for some recently developed genetically encoded photosensitizers. In the presented work, we investigated conformational properties of AsLOV2 wt and its variant C450A by circular dichroism, tryptophan and FMN fluorescence, and differential scanning calorimetry in dependence on pH and temperature. We show that both variants are similarly sensitive towards pH of solvent. On the other hand, the mutation C450A leads to a more stable AsLOV2 variant in comparison with the wild type. Thermal transitions of the AsLOV2 proteins monitored by circular dichroism indicate the presence of significant residual structure in thermally-denatured states of both proteins in the pH range from 4 to 9. Both pH- and thermal- transitions of AsLOV2 are accompanied by FMN leaching to solvent. Higher stability, reversibility of thermal transitions, and efficiency of FMN rebinding in the case of C450A variant suggest that the cofactor release may be modulated by suitable mutations in combination with a suitable physicochemical perturbation. These findings can have implications for a design of genetically encoded photosensitizers.


Assuntos
Fototropinas/química , Proteínas de Plantas/química , Substituição de Aminoácidos , Avena/química , Avena/metabolismo , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Mononucleotídeo de Flavina/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Triptofano/química
19.
PLoS Biol ; 18(3): e3000618, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32182233

RESUMO

Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.


Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/farmacologia , Membrana Celular/metabolismo , Animais , Sítios de Ligação , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/genética , Células Cultivadas , Cristalografia por Raios X , Feminino , Gangliosídeos/metabolismo , Lipídeos de Membrana/metabolismo , Camundongos , Músculo Esquelético/efeitos dos fármacos , Mutação , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Paralisia/induzido quimicamente , Engenharia de Proteínas , Ratos Transgênicos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Sinaptotagminas/metabolismo , Triptofano/química , Triptofano/metabolismo
20.
J Phys Chem Lett ; 11(7): 2408-2413, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32134666

RESUMO

Tyrosine and tryptophan play critical roles in facilitating proton-coupled electron transfer (PCET) processes essential to life. The local protein environment is anticipated to modulate the thermodynamics of amino acid radicals to achieve controlled, unidirectional PCET. Herein, square-wave voltammetry was employed to investigate the electrostatic effects on the redox properties of tryptophan in two variants of the protein azurin. Each variant contains a single redox-active tryptophan, W48 or W108, in a unique and buried protein environment. These tryptophan residues exhibit reversible square-wave voltammograms. A Pourbaix plot, representing the reduction potentials versus pH, is presented for the non-H-bonded W48, which has potentials comparable to those of tryptophan in solution. The reduction potentials of W108 are seen to be increased by more than 100 mV across the same pH range. Molecular dynamics shows that, despite its buried indole ring, the N-H of W108 hydrogen bonds with a water cluster, while W48 is completely excluded from interactions with water or polar groups. These redox properties provide insight into the role of the protein in tuning the reactivity of tryptophan radicals, a requirement for controlled biological PCET.


Assuntos
Azurina/química , Elétrons , Radicais Livres/química , Triptofano/química , Simulação de Dinâmica Molecular , Oxirredução , Eletricidade Estática
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