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1.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806656

RESUMO

Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can strongly influence ligand binding and unbinding processes. Here, we explored the use of a classical optical spectroscopic technique, red-edge excitation shift spectroscopy (REES) to determine the number, population, and energetics associated with ligand-bound states in protein-ligand complexes. We describe a statistical mechanical model of a two-level fluorescent ligand located amongst a finite number of discrete protein microstates. We relate the progressive emission red shift with red-edge excitation to thermodynamic parameters underlying the protein-ligand free energy landscape and to photo-physical parameters relating to the fluorescent ligand. We applied the theoretical model to published red-edge excitation shift data from small molecule inhibitor-kinase complexes. The derived thermodynamic parameters allowed dissection of the energetic contribution of intermediate bound states to inhibitor-kinase interactions.


Assuntos
Proteínas/química , Espectrometria de Fluorescência/métodos , Polarização de Fluorescência/métodos , Corantes Fluorescentes/química , Ligantes , Bibliotecas de Moléculas Pequenas/química , Termodinâmica
2.
Biosens Bioelectron ; 178: 113049, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33540323

RESUMO

Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/µL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.


Assuntos
Técnicas Biossensoriais/métodos , /diagnóstico , Polarização de Fluorescência/métodos , /isolamento & purificação , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/estatística & dados numéricos , /instrumentação , Sistemas CRISPR-Cas , Desenho de Equipamento , Polarização de Fluorescência/instrumentação , Polarização de Fluorescência/estatística & dados numéricos , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito/estatística & dados numéricos , Processamento de Sinais Assistido por Computador , Razão Sinal-Ruído
3.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443163

RESUMO

The tumor-suppressor p53 is a critical regulator of the cellular response to DNA damage and is tightly regulated by posttranslational modifications. Thr55 in the AD2 interaction motif of the N-terminal transactivation domain functions as a phosphorylation-dependent regulatory switch that modulates p53 activity. Thr55 is constitutively phosphorylated, becomes dephosphorylated upon DNA damage, and is subsequently rephosphorylated to facilitate dissociation of p53 from promoters and inactivate p53-mediated transcription. Using NMR and fluorescence spectroscopy, we show that Thr55 phosphorylation inhibits DNA-binding by enhancing competitive interactions between the disordered AD2 motif and the structured DNA-binding domain (DBD). Nonphosphorylated p53 exhibits positive cooperativity in binding DNA as a tetramer. Upon phosphorylation of Thr55, cooperativity is abolished and p53 binds initially to cognate DNA sites as a dimer. As the concentration of phosphorylated p53 is further increased, a second dimer binds and causes p53 to dissociate from the DNA, resulting in a bell-shaped binding curve. This autoinhibition is driven by favorable interactions between the DNA-binding surface of the DBD and the multiple phosphorylated AD2 motifs within the tetramer. These interactions are augmented by additional phosphorylation of Ser46 and are fine-tuned by the proline-rich domain (PRD). Removal of the PRD strengthens the AD2-DBD interaction and leads to autoinhibition of DNA binding even in the absence of Thr55 phosphorylation. This study reveals the molecular mechanism by which the phosphorylation status of Thr55 modulates DNA binding and controls both activation and termination of p53-mediated transcriptional programs at different stages of the cellular DNA damage response.


Assuntos
Proteínas de Ligação a DNA/química , Domínios Proteicos Ricos em Prolina , Proteína Supressora de Tumor p53/química , Sítios de Ligação , DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Polarização de Fluorescência , Expressão Gênica , Espectroscopia de Ressonância Magnética , Mutação , Fosforilação , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes , Deleção de Sequência , Espectrometria de Fluorescência , Proteína Supressora de Tumor p53/genética
4.
Methods Mol Biol ; 2251: 215-223, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481243

RESUMO

Phosphoinositides play important roles in the regulation of protein recruitment at specialized membrane domains, protein activity, and membrane dynamics. Phosphoinositide-protein interplay occurs via multiple mechanisms and proteins associate with membranes through different binding patterns. Determinations of membrane-binding mode and membrane penetration depth of proteins in lipid bilayer are thus important steps in characterizing the molecular mechanisms of membrane-protein interactions. Here, we show two standard in vitro assays using liposomes, diphenylhexatriene (DPH) anisotropy, and fluorescence quenching by brominated lipids to determine membrane penetration of proteins into lipid bilayer. These methods will provide useful tools to study membrane-protein association and uncover molecular details of protein-lipid interplay, which are important for understanding biological functions of membrane-associated proteins and membrane dynamics.


Assuntos
Polarização de Fluorescência/métodos , Fluidez de Membrana/fisiologia , Espectrometria de Fluorescência/métodos , Animais , Difenilexatrieno/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Bicamadas Lipídicas/química , Lipossomos/química , Proteínas de Membrana/análise , Proteínas de Membrana/química , Membranas/química , Fosfatidilcolinas/química , Fosfatidilinositóis/análise , Fosfatidilinositóis/química
5.
Nat Commun ; 12(1): 548, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483497

RESUMO

Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Nucleotídeos/metabolismo , Actinas/química , Algoritmos , Animais , Polarização de Fluorescência , Corantes Fluorescentes/química , Hidrólise , Cinética , Modelos Teóricos , Proteínas Musculares/química , Nucleotídeos/química , Ligação Proteica , Coelhos , Termodinâmica
6.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33361153

RESUMO

Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR-PXR and potentially several other nuclear receptor heterodimers.


Assuntos
Receptor de Pregnano X/química , Receptores X Retinoide/química , Xenobióticos , Animais , Linhagem Celular , Cristalografia por Raios X , Dimerização , Polarização de Fluorescência , Regulação da Expressão Gênica , Humanos , Ligantes , Luciferases/genética , Luciferases/metabolismo , Modelos Químicos , Receptor de Pregnano X/metabolismo , Receptores X Retinoide/metabolismo , Xenobióticos/química , Xenobióticos/metabolismo , Xenobióticos/farmacologia , Xenopus
7.
Methods Mol Biol ; 2130: 3-18, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33284432

RESUMO

Stochastic diffusion of a solution of fluorophores after photoselection reduces the polarization of emission, or fluorescence anisotropy. Because this randomization process is slower for larger molecules, fluorescence anisotropy is effective for measuring the kinetics of protein-binding events. Here, we describe how to use the technique to carry out real-time observations in vitro of the cyanobacterial circadian clock.


Assuntos
Relógios Circadianos , Cianobactérias/metabolismo , Cianobactérias/genética , Polarização de Fluorescência/métodos
8.
PLoS One ; 15(12): e0244613, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382810

RESUMO

Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. PTEN, a lipid phosphatase involved in phosphatidylinositol 3-kinase (PI3K) pathway, contains such a short motif located at the extreme C-terminus capable to recognize PDZ domains. It has been shown that the acetylation of this motif could modulate the interaction with several PDZ domains. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and competitive fluorescence polarization technique to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN. We substantially extended the previous knowledge towards the 266 known human PDZ domains, generating the full PDZome-binding profile of the PTEN PBM. We confirmed that inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile. A numerical specificity index is also introduced as an attempt to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity.


Assuntos
Mutação , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/metabolismo , Acetilação , Sítios de Ligação , Polarização de Fluorescência , Humanos , Domínios PDZ , PTEN Fosfo-Hidrolase/genética , Ligação Proteica
9.
Sensors (Basel) ; 20(24)2020 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-33322750

RESUMO

Fluorescence polarization holds considerable promise for bioanalytical systems because it allows the detection of selective interactions in real time and a choice of fluorophores, the detection of which the biosample matrix does not influence; thus, their choice simplifies and accelerates the preparation of samples. For decades, these possibilities were successfully applied in fluorescence polarization immunoassays based on differences in the polarization of fluorophore emissions excited by plane-polarized light, whether in a free state or as part of an immune complex. However, the results of recent studies demonstrate the efficacy of fluorescence polarization as a detected signal in many bioanalytical methods. This review summarizes and comparatively characterizes these developments. It considers the integration of fluorescence polarization with the use of alternative receptor molecules and various fluorophores; different schemes for the formation of detectable complexes and the amplification of the signals generated by them. New techniques for the detection of metal ions, nucleic acids, and enzymatic reactions based on fluorescence polarization are also considered.


Assuntos
Bioensaio , Corantes Fluorescentes , Ácidos Nucleicos , Polarização de Fluorescência , Metais
10.
J Environ Sci (China) ; 97: 19-24, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32933735

RESUMO

Bisphenol A (BPA) is one of the environmental endocrine disruptors (EDCs), and BPA contamination in environment can cause high risks to human health. Rapid determination of BPA on sites is in high demand in environmental analysis. Taking advantage of aptamers as affinity ligands and fluorescence anisotropy (FA) analysis, we developed a simple and rapid FA assay for BPA by employing a single tetramethylrhodamine (TMR) labeled short 35-mer DNA aptamer against BPA. The assay is based on the BPA-binding induced conformation change of TMR-labeled aptamer and alteration of interaction between TMR and guanine bases, resulting in change of FA signals. We screened the FA change of aptamer probes having TMR label on a specific site of the aptamer upon BPA addition. The aptamer with a TMR label on the 22nd T base showed large FA-decreasing response to BPA and maintained good binding affinity to BPA. By using this TMR-labeled aptamer, we achieved FA detection of BPA with a detection limit of 0.5 µmol/L under the optimized conditions. This assay was selective towards BPA and enabled the detection of BPA spiked in tap water sample, showing the potential applications on water samples.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Compostos Benzidrílicos , Polarização de Fluorescência , Humanos , Limite de Detecção , Fenóis
11.
Nat Commun ; 11(1): 4115, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807795

RESUMO

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms.


Assuntos
Anticorpos/metabolismo , Fator de Transcrição STAT3/metabolismo , Células A549 , Anticorpos/genética , Western Blotting , Calorimetria , Cristalografia por Raios X , Citometria de Fluxo , Polarização de Fluorescência , Imunofluorescência , Humanos , Espectrometria de Massas , Ligação Proteica , Domínios Proteicos/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Biologia Sintética
12.
Proc Natl Acad Sci U S A ; 117(31): 18431-18438, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690700

RESUMO

Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against ∼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Polarização de Fluorescência/métodos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Influenza Humana/virologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antivirais/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Bibliotecas de Moléculas Pequenas/química
13.
Nat Chem Biol ; 16(9): 997-1005, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32514184

RESUMO

Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse-small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1-a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1's catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.


Assuntos
Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Lisofosfolipase/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Descoberta de Drogas , Ativadores de Enzimas/farmacocinética , Polarização de Fluorescência , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Resistência à Insulina , Lisofosfolipase/química , Lisofosfolipase/genética , Masculino , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Obesos , Simulação de Dinâmica Molecular , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacocinética , Relação Estrutura-Atividade
14.
Nat Chem Biol ; 16(5): 529-537, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32152540

RESUMO

Combination antiretroviral therapy has transformed HIV-1 infection, once a fatal illness, into a manageable chronic condition. Drug resistance, severe side effects and treatment noncompliance bring challenges to combination antiretroviral therapy implementation in clinical settings and indicate the need for additional molecular targets. Here, we have identified several small-molecule fusion inhibitors, guided by a neutralizing antibody, against an extensively studied vaccine target-the membrane proximal external region (MPER) of the HIV-1 envelope spike. These compounds specifically inhibit the HIV-1 envelope-mediated membrane fusion by blocking CD4-induced conformational changes. An NMR structure of one compound complexed with a trimeric MPER construct reveals that the compound partially inserts into a hydrophobic pocket formed exclusively by the MPER residues, thereby stabilizing its prefusion conformation. These results suggest that the MPER is a potential therapeutic target for developing fusion inhibitors and that strategies employing an antibody-guided search for novel therapeutics may be applied to other human diseases.


Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Internalização do Vírus/efeitos dos fármacos , Sítios de Ligação , Antígenos CD4/metabolismo , Membrana Celular/metabolismo , Dequalínio/química , Dequalínio/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Polarização de Fluorescência , Células HEK293 , Proteína gp41 do Envelope de HIV/genética , HIV-1/patogenicidade , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Mutação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
15.
J Agric Food Chem ; 68(14): 4277-4283, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32182058

RESUMO

Ochratoxin A (OTA), a common mycotoxin, has attracted great concern as many foodstuffs can suffer from OTA contamination; OTA causes harmful effects on human and animals. Rapid and sensitive detection of OTA is demanded in many fields for agricultural product quality, food safety, and health. Aptamer fluorescence polarization/anisotropy (FP/FA) assays integrate advantages of nucleic acid aptamers (e.g., easy preparation, high stability, and low cost) and FP/FA analysis (e.g., high sensitivity, rapidity, simplicity, and robustness). Here, we report the preparation of lissamine rhodamine B labeled OTA and developed competitive aptamer fluorescence anisotropy (FA) assays for OTA with signal-off or signal-on responses by using this fluorescently labeled probe. In the signal-off FA assay, the binding between the fluorescent probe and aptamer gave a large FA signal due to molecular volume increase, and the fluorescent probe was displaced from the aptamer in the presence of OTA target, causing FA to decrease. To further enhance the FA change in the signal-off assay, large-sized streptavidin was conjugated on the aptamer, and this assay allowed for a detection limit of 2.5 nM and a more remarkable FA decrease. Furthermore, we found that the fluorescent probe could interact with Tween 20, which caused the fluorescent probe to show a higher FA value than that of the aptamer-fluorescent probe complex. A signal-on FA assay was achieved in the binding buffer containing 0.1% Tween 20, with a detection limit of 10 nM. Signal-off and signal-on FA methods both were selective and enabled detection of OTA spiked in red wine samples, showing capability for target analysis in complex sample matrix.


Assuntos
Aptâmeros de Nucleotídeos/química , Corantes Fluorescentes/química , Ocratoxinas/análise , Rodaminas/química , Ligação Competitiva , Técnicas Biossensoriais , Polarização de Fluorescência/métodos , Contaminação de Alimentos/prevenção & controle , Humanos , Limite de Detecção , Polissorbatos/química , Sensibilidade e Especificidade , Estreptavidina/química , Vinho/análise
16.
Sci Rep ; 10(1): 5595, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221374

RESUMO

Introduction of microfluidic mixing technique opens a new door for preparation of the liposomes and lipid-based nanoparticles by on-chip technologies that are applicable in a laboratory and industrial scale. This study demonstrates the role of phospholipid bilayer fragment as the key intermediate in the mechanism of liposome formation by microfluidic mixing in the channel with "herring-bone" geometry used with the instrument NanoAssemblr. The fluidity of the lipid bilayer expressed as fluorescence anisotropy of the probe N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) was found to be the basic parameter affecting the final size of formed liposomes prepared by microfluidic mixing of an ethanol solution of lipids and water phase. Both saturated and unsaturated lipids together with various content of cholesterol were used for liposome preparation and it was demonstrated, that an increase in fluidity results in a decrease of liposome size as analyzed by DLS. Gadolinium chelating lipids were used to visualize the fine structure of liposomes and bilayer fragments by CryoTEM. Experimental data and theoretical calculations are in good accordance with the theory of lipid disc micelle vesiculation.


Assuntos
Lipossomos/síntese química , Fluidez de Membrana , Microfluídica/métodos , Nanoestruturas , Materiais Biocompatíveis/metabolismo , Resina de Colestiramina/metabolismo , Polarização de Fluorescência , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação
17.
Talanta ; 211: 120730, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32070579

RESUMO

Recently, various inorganic nanomaterials have been used as fluorescence anisotropy (FA) enhancers for biosensing successfully. However, most of them are size-uncontrollable and possess an intensive fluorescence quenching ability, which will seriously reduce the accuracy and sensitivity of FA method. Herein, we report a two-dimensional DNA nanosheet (DNS) without fluorescence quenching effect as a novel FA amplification platform. In our strategy, fluorophore-labeled probe DNA (pDNA) is linked onto the DNS surface through the hybridization with the handle DNA (hDNA) that extended from the DNS, resulting in the significantly enhanced FA value. After the addition of target, the pDNA was released from the DNS surface due to the high affinity between the hDNA and target, and the FA was decreased. Thus, target could be detected by the significantly decreased FA value. The linear range was 10-50 nM and the limit of detection was 8 nM for the single-stranded DNA detection. This new method is general and has been also successfully applied for the detection of ATP and thrombin sensitively. Our method improved the accuracy of FA assay and has great potential to detect series of biological analytes in complex biosensing systems.


Assuntos
Trifosfato de Adenosina/análise , Técnicas Biossensoriais/métodos , DNA/química , Polarização de Fluorescência/métodos , Corantes Fluorescentes/química , Nanoestruturas/química , Trombina/análise , DNA de Cadeia Simples/análise , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência
18.
Eur J Med Chem ; 191: 112150, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32105981

RESUMO

Since the discovery and early characterization of the histamine H3 receptor (H3R) in the 1980's, predominantly imidazole-based agonists were presented to the scientific community such as Nα-methylhistamine (Nα-MeHA) or (R)-α-methylhistamine ((R)α-MeHA). Whereas therapeutic applications have been prompted for H3R agonists such as treatment of pain, asthma and obesity, several drawbacks associated with imidazole-containing ligands makes the search for new agonists for this receptor demanding. Accordingly, high interest arose after publication of several pyrrolidindione-based, highly affine H3R agonists within this journal that avoid the imidazole moiety and thus, presenting a novel type of potential pharmacophores (Ghoshal, Anirban et al., 2018). In our present study performed in two independent laboratories, we further evaluated the exposed lead-compound (EC50 = 0.1 nM) of the previous research project with regards to pharmacological behavior at H3R. Thereby, no binding affinity was observed in neither [3H]Nα-MeHA nor bodilisant displacement assays that contradicts the previously published activity. Additional functional exploration employing GTPγ[35S], cAMP-accumulation assay and cAMP response element (CRE)-driven reporter gene assays exhibited slight partial agonist properties of such pyrrolidindiones but acting apart from the reported concentration range. We conclude, that the previously reported actions of such pyrrolidindiones result from an overestimation based on the method of measurement and thus, we cast doubt on the new pharmacophores with H3R agonist activity.


Assuntos
Pirrolidinonas/farmacologia , Receptores Histamínicos H3/metabolismo , Relação Dose-Resposta a Droga , Polarização de Fluorescência , Células HEK293 , Humanos , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Pirrolidinonas/síntese química , Pirrolidinonas/química , Relação Estrutura-Atividade
19.
Nat Commun ; 11(1): 21, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911590

RESUMO

Monitoring of protein oligomerization has benefited greatly from Förster Resonance Energy Transfer (FRET) measurements. Although donors and acceptors are typically fluorescent molecules with different spectra, homo-FRET can occur between fluorescent molecules of the same type if the emission spectrum overlaps with the absorption spectrum. Here, we describe homo-FRET measurements by monitoring anisotropy changes in photoswitchable fluorescent proteins while photoswitching to the off state. These offer the capability to estimate anisotropy in the same specimen during homo-FRET as well as non-FRET conditions. We demonstrate photoswitching anisotropy FRET (psAFRET) with a number of test chimeras and example oligomeric complexes inside living cells. We also present an equation derived from FRET and anisotropy equations which converts anisotropy changes into a factor we call delta r FRET (drFRET). This is analogous to an energy transfer efficiency and allows experiments performed on a given homo-FRET pair to be more easily compared across different optical configurations.


Assuntos
Polarização de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/química , Humanos , Ligação Proteica
20.
J Biol Chem ; 295(10): 2959-2973, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31969393

RESUMO

Human leukocyte antigen-DM (HLA-DM) is an integral component of the major histocompatibility complex class II (MHCII) antigen-processing and -presentation pathway. HLA-DM shapes the immune system by differentially catalyzing peptide exchange on MHCII molecules, thereby editing the peptide-MHCII (pMHCII) repertoire by imposing a bias on the foreign and self-derived peptide cargos that are presented on the cell surface for immune surveillance and tolerance induction by CD4+ T cells. To better understand DM selectivity, here we developed a real-time fluorescence anisotropy assay to delineate the pMHCII intrinsic stability, DM-binding affinity, and catalytic turnover, independent kinetic parameters of HLA-DM enzymatic activity. We analyzed prominent pMHCII contacts by differentiating the kinetic parameters in pMHCII homologs, observing that peptide interactions throughout the MHCII-binding cleft influence both the rate of peptide dissociation from the DM-pMHCII catalytic complex and the binding affinity of HLA-DM for a pMHCII. We show that the intrinsic stability of a pMHCII linearly correlates with DM catalytic turnover, but is nonlinearly correlated with its binding affinity. Surprisingly, interactions at the peptides N terminus up to and including MHCII position one (P1) anchor affected the catalytic turnover, suggesting that the active DM-pMHCII catalytic complex operates on pMHCII complexes with full peptide occupancy. Furthermore, interactions at the peptide C terminus modulated DM-binding affinity, suggesting distal communication between peptide interactions with the MHCII and the DM-pMHCII binding interface. Our results imply an intimate linkage between the DM-pMHCII interface and peptide-MHCII interactions throughout the peptide-binding cleft.


Assuntos
Antígenos HLA-D/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Apresentação do Antígeno , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cristalografia por Raios X , Drosophila/metabolismo , Polarização de Fluorescência , Corantes Fluorescentes/química , Células HEK293 , Antígenos HLA-D/genética , Antígenos HLA-D/imunologia , Humanos , Cinética , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
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