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The LABEXTRACT plant extract bank, featuring diverse members of the Myrtaceae family from Brazilian hot spot regions, provides a promising avenue for bioprospection. Given the pivotal roles of the Spike protein and 3CLpro and PLpro proteases in SARS-CoV-2 infection, this study delves into the correlations between the Myrtaceae species from the Atlantic Forest and these targets, as well as an antiviral activity through both in vitro and in silico analyses. The results uncovered notable inhibitory effects, with Eugenia prasina and E. mosenii standing out, while E. mosenii proved to be multitarget, presenting inhibition values above 72% in the three targets analyzed. All extracts inhibited viral replication in Calu-3 cells (EC50 was lower than 8.3 µg·mL-1). Chemometric analyses, through LC-MS/MS, encompassing prediction models and molecular networking, identified potential active compounds, such as myrtucommulones, described in the literature for their antiviral activity. Docking analyses showed that one undescribed myrtucommulone (m/z 841 [M - H]-) had a higher fitness score when interacting with the targets of this study, including ACE2, Spike, PLpro and 3CLpro of SARS-CoV-2. Also, the study concludes that Myrtaceae extracts, particularly from E. mosenii and E. prasina, exhibit promising inhibitory effects against crucial stages in SARS-CoV-2 infection. Compounds like myrtucommulones emerge as potential anti-SARS-CoV-2 agents, warranting further exploration.
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Professor Carlos Gutiérrez-Merino, a prominent scientist working in the complex realm of biological membranes, has made significant theoretical and experimental contributions to the field. Contemporaneous with the development of the fluid-mosaic model of Singer and Nicolson, the Förster resonance energy transfer (FRET) approach has become an invaluable tool for studying molecular interactions in membranes, providing structural insights on a scale of 1-10 nm and remaining important alongside evolving perspectives on membrane structures. In the last few decades, Gutiérrez-Merino's work has covered multiple facets in the field of FRET, with his contributions producing significant advances in quantitative membrane biology. His more recent experimental work expanded the ground concepts of FRET to high-resolution cell imaging. Commencing in the late 1980s, a series of collaborations between Gutiérrez-Merino and the authors involved research visits and joint investigations focused on the nicotinic acetylcholine receptor and its relation to membrane lipids, fostering a lasting friendship.
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Lipídeos de Membrana , Receptores Nicotínicos , Membrana Celular/metabolismo , Lipídeos de Membrana/química , Transferência Ressonante de Energia de Fluorescência , Membranas/metabolismo , Receptores Nicotínicos/metabolismoRESUMO
In multicellular organisms, metabolic coordination across multiple tissues and cell types is essential to satisfy regionalized energetic requirements and respond coherently to changing environmental conditions. However, most metabolic assays require the destruction of the biological sample, with a concomitant loss of spatial information. Fluorescent metabolic sensors and probes are among the most user-friendly techniques for collecting metabolic information with spatial resolution. In a previous work, we have adapted to an animal system, Drosophila melanogaster, genetically encoded metabolic FRET-based sensors that had been previously developed in single-cell systems. These sensors provide semi-quantitative data on the stationary concentrations of key metabolites of the bioenergetic metabolism: lactate, pyruvate, and 2-oxoglutarate. The use of these sensors in intact organs required the development of an image processing method that minimizes the contribution of spatially complex autofluorescence patterns, that would obscure the FRET signals. In this article, we show step by step how to design FRET-based sensor experiments and how to process the fluorescence signal to obtain reliable FRET values.
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Drosophila melanogaster , Transferência Ressonante de Energia de Fluorescência , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Processamento de Imagem Assistida por Computador/métodos , Metabolismo Energético , Ácido PirúvicoRESUMO
Fluorescence Resonance Energy Transfer (FRET)-based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super-resolved FRET imaging based on single-molecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.
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DNA , Transferência Ressonante de Energia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , DNA/química , Microscopia Confocal , Imagem Individual de MoléculaRESUMO
Actinomycetes are versatile about their metabolism, displaying high capacity to produce bioactive metabolites. Enzymes from actinomycetes represent new opportunities for industrial applications. However, proteases from actinomycetes are poorly described by literature. Thereby, to verify proteolytic potential of actinomycetes, the present study aimed the investigation of bacterial isolates from Caatinga and Atlantic Forest rhizosphere. Fluorescence resonance energy transfer (FRET) peptide libraries were adopted for the evaluations, since they are faster and more qualitative methods, if compared with others described by most reports. A total of 52 microorganisms were inoculated in different culture media (PMB, potato dextrose agar, brain heart infusion agar, Starch Casein Agar and Reasoner's 2A agar), temperatures (12, 20, 30, 37, 45 and 60°C), and saline conditions (0-4 M NaCl), during 7 days. The actinomycetes named as AC 01, 02 and 52 were selected and showed enzymatic abilities under the peptide probes Abz-KLRSSKQ-EDDnp and Abz-KLYSSKQ-EDDnp, achieving enhanced performance at 30 °C. Biochemical parameters were established, showing a predominance of alkaline proteases with activity under saline conditions. Secreted proteases hydrolysed preferentially polar uncharged residues (Y and N) and positively charged groups (R). Phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid inhibited the proteins, a characteristic of serine (AC 01 e 02) and metalloproteases (AC 52). All selected strains belonged to Streptomyces genera. In summary, actinomycete strains with halophilic proteolytic abilities were selected, which improve possibilities for their use in detergent formulations, food processing, waste management and industrial bioconversion. It is important to highlight that this is the first report using FRET libraries for proteolytic screening from Caatinga and Atlantic Forest actinobacteria.
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Actinobacteria , Peptídeo Hidrolases/metabolismo , Actinomyces , Ágar/metabolismo , Solo , Meios de Cultura/metabolismoRESUMO
Fluorescent proteins are standard tools for addressing biological questions in a cell biology laboratory. The genetic tagging of protein of interest with fluorescent proteins opens the opportunity to follow them in vivo and to understand their interactions and dynamics. In addition, the latest advances in optical microscopy image acquisition and processing allow us to study many cellular processes in vivo. Techniques such as fluorescence lifetime microscopy and hyperspectral imaging provide valuable tools for understanding fluorescent protein interactions and their photophysics. Finally, fluorescence fluctuation analysis opens the possibility to address questions of molecular diffusion, protein-protein interactions, and oligomerization, among others, yielding quantitative information on the subject of study. This chapter will cover some of the more important advances in cutting-edge technologies and methods that, combined with fluorescent proteins, open new frontiers for biological studies.
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Corantes , Proteínas , Fenômenos Fisiológicos Celulares , Microscopia de Fluorescência/métodosRESUMO
A new benzodithiophene and benzotriazole-based terpolymer bearing a fluorescein derivative as a side group was synthesized and studied for organic solar cell (OSC) applications. This side group was covalently bounded to the backbone through an n-hexyl chain to induce the intramolecular Förster Resonance Energy Transfer (FRET) process and thus improve the photovoltaic performance of the polymeric material. The polymer exhibited good solubility in common organic chlorinated solvents as well as thermal stability (TDT10% > 360 °C). Photophysical measurements demonstrated the occurrence of the FRET phenomenon between the lateral group and the terpolymer. The terpolymer exhibited an absorption band centered at 501 nm, an optical bandgap of 2.02 eV, and HOMO and LUMO energy levels of −5.30 eV and −3.28 eV, respectively. A preliminary study on terpolymer-based OSC devices showed a low power-conversion efficiency (PCE) but a higher performance than devices based on an analogous polymer without the fluorescein derivative. These results mean that the design presented here is a promising strategy to improve the performance of polymers used in OSCs.
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Energia Solar , Transferência Ressonante de Energia de Fluorescência , Tiofenos , Fluoresceína , PolímerosRESUMO
Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment.
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Nanotecnologia , Oligonucleotídeos , Microscopia de Fluorescência/métodosRESUMO
Erwin London dedicated considerable effort to understanding lipid interactions with membrane-resident proteins and how these interactions shaped the formation and maintenance of lipid phases and domains. In this endeavor, he developed ad hoc techniques that greatly contributed to advancements in the field. We have employed and/or modified/extended some of his methodological approaches and applied them to investigate lipid interaction with the nicotinic acetylcholine receptor (nAChR) protein, the paradigm member of the superfamily of rapid pentameric ligand-gated ion channels (pLGIC). Our experimental systems ranged from purified receptor protein reconstituted into synthetic lipid membranes having known effects on receptor function, to cellular systems subjected to modification of their lipid content, e.g., varying cholesterol levels. We have often employed fluorescence techniques, including fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and of nAChR intrinsic fluorescence by nitroxide spin-labeled phospholipids, DPH anisotropy, excimer formation of pyrene-phosphatidylcholine, and Förster resonance energy transfer (FRET) from the protein moiety to the extrinsic probes Laurdan, DPH, or pyrene-phospholipid to characterize various biophysical properties of lipid-receptor interactions. Some of these strategies are revisited in this review. Special attention is devoted to the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the nAChR. The receptor protein was shown to organize its PA-containing immediate microenvironment into microdomains with high lateral packing density and rigidity. PA and cholesterol appear to compete for the same binding sites on the nAChR protein.
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Canais Iônicos de Abertura Ativada por Ligante , Receptores Nicotínicos , Animais , Receptores Nicotínicos/química , Torpedo/metabolismo , Difenilexatrieno , Londres , Fosfatidilcolinas/metabolismo , Colesterol/química , Ácidos Fosfatídicos/metabolismo , PirenosRESUMO
Genetically encoded FRET sensors for revealing local concentrations of second messengers in living cells have enormously contributed to our understanding of physiological and pathological processes. However, the development of sensors remains an intricate process. Using simulation techniques, we recently introduced a new architecture to measure intracellular concentrations of cAMP named CUTie, which works as a FRET tag for arbitrary targeting domains. Although our method showed quasi-quantitative predictive power in the design of cAMP and cGMP sensors, it remains intricate and requires specific computational skills. Here, we provide a simplified computer-aided protocol to design tailor-made CUTie sensors based on arbitrary cyclic nucleotide-binding domains. As a proof of concept, we applied this method to construct a new CUTie sensor with a significantly higher cAMP sensitivity (EC50 = 460 nM).This simple protocol, which integrates our previous experience, only requires free web servers and can be straightforwardly used to create cAMP sensors adapted to the physicochemical characteristics of known cyclic nucleotide-binding domains.
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AMP Cíclico , Pedestres , AMP Cíclico/química , GMP Cíclico , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Sistemas do Segundo MensageiroRESUMO
In this work, the Förster resonance energy transfer (FRET) between carbon dots (CDs) as energy donors and riboflavin (RF) as an energy acceptor was optimized and the main parameters that characterize the FRET process were determined. The results were successfully applied in the development of an ultrasensitive ratiometric fluorescent sensor for the selective and sensitive determination of RF in different beverages. Water-soluble CDs with a high quantum yield (54%) were synthesized by a facile and direct microwave-assisted technique. The CDs were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), Zeta potential, and UV-visible and molecular fluorescence spectroscopy. The study of the FRET process at two donor concentrations showed that the energy transfer efficiency decreases as the donor concentration increases, confirming its dependence on the acceptor:donor ratio in nanoparticle-based systems. The results show the importance of optimizing the FRET process conditions to improve the corresponding output signal. The variation in the ratiometric signal with the concentration of RF showed linearity in a concentration range of 0 to 11 µM with R2 = 0.9973 and a detection limit of 0.025 µM. The developed nanosensor showed good selectivity over other possible types of interference. The sensor was then applied for the determination of RF in beverage samples using the standard addition method with recoveries between 96% and 106%. Preliminary cytocompatibility tests carried out with breast cancer cells (MDA-MB-231) revealed the nanosensor to be cytocompatible in its working concentration regime, even after long incubation times with cells. Altogether, the developed RF determination method was found to be fast, low-cost, highly sensitive, and selective and can be extended to other samples of interest in the biological and food sectors. Moreover, thanks to its long-lasting cytocompatibility, the developed platform can also be envisaged for other applications of biological interest, such as intracellular sensing and staining for live cell microscopy.
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Understanding signal propagation across biological networks requires to simultaneously monitor the dynamics of several nodes to uncover correlations masked by inherent intercellular variability. To monitor the enzymatic activity of more than two components over short time scales has proven challenging. Exploiting the narrow spectral width of homo-FRET-based biosensors, up to three activities can be imaged through fluorescence polarization anisotropy microscopy. We introduce Caspase Activity Sensor by Polarization Anisotropy Multiplexing (CASPAM) a single-plasmid triple-modality reporter of key nodes of the apoptotic network. Apoptosis provides an ideal molecular framework to study interactions between its three composing pathways (intrinsic, extrinsic, and effector). We characterized the biosensor performance and demonstrated the advantages that equimolar expression has in both simplifying experimental procedure and reducing observable variation, thus enabling robust data-driven modeling. Tools like CASPAM become essential to analyze molecular pathways where multiple nodes need to be simultaneously monitored.
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Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Anisotropia , Caspases/genética , Microscopia de FluorescênciaRESUMO
Ternary quantum dots (QDs) are novel nanomaterials that can be used in chemical analysis due their unique physicochemical and spectroscopic properties. These properties are size-dependent and can be adjusted in the synthetic protocol modifying the reaction medium, time, source of heat, and the ligand used for stabilization. In the last decade, several spectroscopic methods have been developed for the analysis of organic and inorganic analytes in biological, drug, environmental, and food samples, in which different sensing schemes have been applied using ternary quantum dots. This review addresses the different synthetic approaches of ternary quantum dots, the sensing mechanisms involved in the analyte detection, and the predominant areas in which these nanomaterials are used.
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The RNA Degradosome (RNAD) is a multi-enzyme complex, which performs important functions in post-transcriptional regulation in Escherichia coli with the assistance of regulatory sRNAs and the RNA chaperone Hfq. Although the interaction of the canonical RNAD components with RNase E has been extensively studied, the dynamic nature of the interactions in vivo remains largely unknown. In this work, we explored the rearrangements upon glucose stress using fluorescence energy transfer (hetero-FRET). Results revealed differences in the proximity of the canonical components with 1% (55.5 mM) glucose concentration, with the helicase RhlB and the glycolytic enzyme Enolase exhibiting the largest changes to the C-terminus of RNase E, followed by PNPase. We quantified ptsG mRNA decay and SgrS sRNA synthesis as they mediate bacterial adaptation to glucose stress conditions. We propose that once the mRNA degradation is completed, the RhlB, Enolase and PNPase decrease their proximity to the C-terminus of RNase E. Based on the results, we present a model where the canonical components of the RNAD coalesce when the bacteria is under glucose-6-phosphate stress and associate it with RNA decay. Our results demonstrate that FRET is a helpful tool to study conformational rearrangements in enzymatic complexes in bacteria in vivo.
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Escherichia coli/metabolismo , Glucose/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Estabilidade de RNA/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Estresse Fisiológico/genéticaRESUMO
The detection of small molecules in living cells using genetically encoded FRET sensors has revolutionized our understanding of signaling pathways at the sub-cellular level. However, engineering fluorescent proteins and specific binding domains to create new sensors remains challenging because of the difficulties associated with the large size of the polypeptides involved, and their intrinsically huge conformational variability. Indeed, FRET sensors' design still relies on vague structural notions, and trial and error combinations of linkers and protein modules. We recently designed a FRET sensor for the second messenger cAMP named CUTie (Cyclic nucleotide Universal Tag for imaging experiments), which granted sub-micrometer resolution in living cells. Here we apply a combination of sequence/structure analysis to produce a new-generation FRET sensor for the second messenger cGMP based on Protein kinase G I (PKGI), which we named CUTie2. Coarse-grained molecular dynamics simulations achieved an exhaustive sampling of the relevant spatio-temporal coordinates providing a quasi-quantitative prediction of the FRET efficiency, as confirmed by in vitro experiments. Moreover, biochemical characterization showed that the cGMP binding module maintains virtually the same affinity and selectivity for its ligand thant the full-length protein. The computational approach proposed here is easily generalizable to other allosteric protein modules, providing a cost effective-strategy for the custom design of FRET sensors.
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The acute rise in interstitial K+ that accompanies neural activity couples the energy demand of neurons to the metabolism of astrocytes. The effects of elevated K+ on astrocytes include activation of aerobic glycolysis, inhibition of mitochondrial respiration and the release of lactate. Using a genetically encoded FRET glucose sensor and a novel protocol based on 3-O-methylglucose trans-acceleration and numerical simulation of glucose dynamics, we report that extracellular K+ is also a potent and reversible modulator of the astrocytic glucose transporter GLUT1. In cultured mouse astrocytes, the stimulatory effect developed within seconds, engaged both the influx and efflux modes of the transporter, and was detected even at 1 mM incremental K+ . The modulation of GLUT1 explains how astrocytes are able to maintain their glucose pool in the face of strong glycolysis stimulation. We propose that the stimulation of GLUT1 by K+ supports the production of lactate by astrocytes and the timely delivery of glucose to active neurons.
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Astrócitos , Glicólise , Animais , Glucose , Transportador de Glucose Tipo 1/genética , Ácido Láctico , CamundongosRESUMO
Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II-III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.
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Bacillus cereus/metabolismo , Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Manduca/efeitos dos fármacos , Controle Biológico de Vetores , Animais , Bacillus cereus/genética , Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/metabolismo , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Manduca/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Mutação , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Relação Estrutura-AtividadeRESUMO
Lactate, the product of aerobic glycolysis, plays a dual role as fuel and intercellular signal in inflammation, immune evasion, and tumor progression. The production of lactate by macrophages has been associated with their polarization and function. Here we describe imaging protocols to characterize the metabolism of cultured human macrophages using a genetically encoded fluorescent sensor-specific for lactate. By superfusing cultures with increasing lactate concentrations and pharmacological inhibitors, it is possible to estimate the kinetic parameters of monocarboxylate transporter 4 (MCT4) and lactate production. Practical advice is given regarding sensor expression, imaging, and data analysis. The spatiotemporal resolution of this technique is amenable to the study of fast events at the single-cell level in different immune and other cell types.
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Ácido Láctico/metabolismo , Macrófagos/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Células THP-1/metabolismoRESUMO
The use of fixed fibroblasts from familial and sporadic Alzheimer's disease patients has previously indicated an upregulation of mitochondria-ER contacts (MERCs) as a hallmark of Alzheimer's disease. Despite its potential significance, the relevance of these results is limited because they were not extended to live neurons. Here we performed a dynamic in vivo analysis of MERCs in hippocampal neurons from McGill-R-Thy1-APP transgenic rats, a model of Alzheimer's disease-like amyloid pathology. Live FRET imaging of neurons from transgenic rats revealed perturbed 'lipid-MERCs' (gap width <10â nm), while 'Ca2+-MERCs' (10-20â nm gap width) were unchanged. In situ TEM showed no significant differences in the lipid-MERCs:total MERCs or lipid-MERCs:mitochondria ratios; however, the average length of lipid-MERCs was significantly decreased in neurons from transgenic rats as compared to controls. In accordance with FRET results, untargeted lipidomics showed significant decreases in levels of 12 lipids and bioenergetic analysis revealed respiratory dysfunction of mitochondria from transgenic rats. Thus, our results reveal changes in MERC structures coupled with impaired mitochondrial functions in Alzheimer's disease-related neurons.This article has an associated First Person interview with the first author of the paper.
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Doença de Alzheimer , Retículo Endoplasmático , Mitocôndrias , Neurônios , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos TransgênicosRESUMO
Fluorescent probes are employed to investigate natural and model membranes. It is important to know probe location and extent of perturbations they cause into the lipid bilayer. Förster Resonance Energy Transfer (FRET) is a useful tool to investigate phenomena involving plasma membranes, and reports in literature used relatively large fluorophores like 1,6-diphenylhexatriene, located at the center of the hydrophobic region, 4-aminophthalimide-based molecules located at lipid/water interfaces and BODIPY-labeled phosphatidylcholine. In this work we explored FRET process in 1,2-dimyristoyl-L-α-GPC large unilamellar vesicles, in gel and fluid phase, using as donor the very small group o-Abz bound to hexadecyl chain (2-amino-N-hexadecyl-benzamide - AHBA) and 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) labeled lipids as acceptor. From the intensity decay of donor in presence of acceptors, the FRET efficiency was calculated, and used to fit the model proposed by Fung and Stryer to that efficiency. Using lipid bilayer structural data, the procedure allowed the determination of Förster distance for each donor-acceptor pair in vesicles, without imposing any value for the orientational factor κ2. From distance distributions between o-Abz in AHBA and NBD in lipid bilayer obtained using the program CONTIN, we obtained donor-acceptor populations having different separation distances. The populations reflect the occurrence of FRET involving probes in the same or in opposite leaflet. A dynamic picture emerged showing how relative position of the probes is dependent on the structural thermal phase of the DMPC bilayer. The results emphasize the need of careful analysis in order to understand processes involving fluorescent probes in model membranes.