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1.
Spectrochim Acta A Mol Biomol Spectrosc ; 317: 124399, 2024 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-38718747

RESUMEN

Herein, a novel sandwich electrochemiluminescence (ECL) aptasensor was developed based on the resonance energy transfer (RET) with iridium complex doped silicate nanoparticles (SiO2@Ir) as energy donor and gold nanoparticles modified TiVC MXene (AuNPs@TiVC) as energy acceptor. Strong anodic ECL signal of SiO2@Ir was obtained through both co-reactant pathway and annihilation pathway. Electrochemical results showed that SiO2@Ir has good electron transfer rate and large specific surface area to immobilize more aptamers. AuNPs@TiVC apparently quenched the ECL signal of SiO2@Ir due to the ECL resonance energy transfer between them. In the presence of kanamycin (KAN), a sandwich type sensor was formed with the aptamer probes as connecters between the donor and the acceptor, resulting in the decrease of ECL intensity. Under the optimal condition, KAN could be sensitively detected in the range of 0.1 pg/mL to 10 ng/mL with a low detection limit of 24.5 fg/mL. The proposed ECL system exhibited satisfactory analytical performance, which can realize the detection of various biological molecules by adopting suitable aptamer.


Asunto(s)
Técnicas Electroquímicas , Oro , Iridio , Kanamicina , Límite de Detección , Nanopartículas del Metal , Dióxido de Silicio , Dióxido de Silicio/química , Oro/química , Nanopartículas del Metal/química , Iridio/química , Técnicas Electroquímicas/métodos , Kanamicina/análisis , Mediciones Luminiscentes/métodos , Nanosferas/química , Aptámeros de Nucleótidos/química , Titanio/química , Técnicas Biosensibles/métodos , Transferencia de Energía
2.
J Phys Chem B ; 128(21): 5201-5217, 2024 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-38756003

RESUMEN

In this study, the site energy fluctuations, energy transfer dynamics, and some spectroscopic properties of the minor light-harvesting complex CP24 in a membrane environment were determined. For this purpose, a 3 µs-long classical molecular dynamics simulation was performed for the CP24 complex. Furthermore, using the density functional tight binding/molecular mechanics molecular dynamics (DFTB/MM MD) approach, we performed excited state calculations for the chlorophyll a and chlorophyll b molecules in the complex starting from five different positions of the MD trajectory. During the extended simulations, we observed variations in the site energies of the different sets as a result of the fluctuating protein environment. In particular, a water coordination to Chl-b 608 occurred only after about 1 µs in the simulations, demonstrating dynamic changes in the environment of this pigment. From the classical and the DFTB/MM MD simulations, spectral densities and the (time-dependent) Hamiltonian of the complex were determined. Based on these results, three independent strongly coupled chlorophyll clusters were revealed within the complex. In addition, absorption and fluorescence spectra were determined together with the exciton relaxation dynamics, which reasonably well agrees with experimental time scales.


Asunto(s)
Clorofila , Complejos de Proteína Captadores de Luz , Simulación de Dinámica Molecular , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Clorofila/química , Transferencia de Energía , Clorofila A/química , Teoría Funcional de la Densidad , Espectrometría de Fluorescencia
3.
Nat Commun ; 15(1): 4437, 2024 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-38789432

RESUMEN

Photosynthetic organisms have evolved an essential energy-dependent quenching (qE) mechanism to avoid any lethal damages caused by high light. While the triggering mechanism of qE has been well addressed, candidates for quenchers are often debated. This lack of understanding is because of the tremendous difficulty in measuring intact cells using transient absorption techniques. Here, we have conducted femtosecond pump-probe measurements to characterize this photophysical reaction using micro-sized cell fractions of the green alga Chlamydomonas reinhardtii that retain physiological qE function. Combined with kinetic modeling, we have demonstrated the presence of an ultrafast excitation energy transfer (EET) pathway from Chlorophyll a (Chl a) Qy to a carotenoid (car) S1 state, therefore proposing that this carotenoid, likely lutein1, is the quencher. This work has provided an easy-to-prepare qE active thylakoid membrane system for advanced spectroscopic studies and demonstrated that the energy dissipation pathway of qE is evolutionarily conserved from green algae to land plants.


Asunto(s)
Carotenoides , Chlamydomonas reinhardtii , Transferencia de Energía , Chlamydomonas reinhardtii/metabolismo , Carotenoides/metabolismo , Carotenoides/química , Tilacoides/metabolismo , Fotosíntesis , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/genética , Clorofila A/metabolismo , Clorofila A/química , Luz , Cinética , Clorofila/metabolismo , Chlamydomonas/metabolismo
4.
An Acad Bras Cienc ; 96(1): e20230327, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38597490

RESUMEN

Aquatic macrophytes are the main autochthonous component of primary production in the Amazon Basin. Floating meadows of these plants support habitats with highly diverse animal communities. Fishes inhabiting these habitats have been assumed to use a broad range of food items and compose a particular food web. We employed carbon (δ13C) and nitrogen (δ15N) stable isotope analysis to draw the trophic structure of these habitats and to trace the energy flow by its trophic levels. Fishes and other animals from 18 independent macrophyte meadows of a floodplain lake of the Solimões River (Amazonia, Brazil) were analyzed. The food web of macrophyte meadows consists of four trophic levels above autotrophic sources. In general, primary consumers exhibited a broader range of food sources than the upper trophic levels. Some fish species depended on a large number of food sources and at the same time are consumed by several predators. The energy transfer from one trophic level to the next was then mainly accomplished by these species concentrating a high-energy flux and acting as hubs in the food web. The broad range of δ13C values observed indicates that the organisms living in the macrophyte meadows utilize a great diversity of autotrophic sources.


Asunto(s)
Pradera , Lagos , Animales , Lagos/química , Ecosistema , Cadena Alimentaria , Peces , Transferencia de Energía
5.
Anal Chim Acta ; 1303: 342508, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38609274

RESUMEN

The anti-interference ability of biosensors is critical for detection in biological samples. Fluorescence-based sensors are subject to interference from self-luminescent substances in biological matrices. Therefore, phosphorescent sensors stand out among biosensors due to their lack of self-luminescence background. In this study, a phosphorescent sensor was constructed, which can accurately detect thymidine kinase 1 (TK1) mRNA in biological samples and avoid autofluorescence interference. When there is no target, polydopamine (PDA) is used as the phosphorescence resonance energy transfer (PRET) acceptor to quench the phosphorescence of the persistently luminescent (PL) nanomaterial. When there is a target, the DNA modified by the PL nanomaterial is replaced by the hairpin H and removed away from the PDA, resulting in a rebound in phosphorescence. The phosphorescent sensor exhibits a good linear relationship in the TK1 mRNA concentration range of 0-200 nM, and the detection limit was 1.74 nM. The sensor fabricated in this study can effectively avoid interference from spontaneous fluorescence in complex biological samples, and sensitively and precisely detect TK1 mRNA in serum samples, providing a powerful tool to more accurately detect biomarkers in biological samples.


Asunto(s)
Timidina Quinasa , Transferencia de Energía , ARN Mensajero/genética , Timidina Quinasa/genética , Mediciones Luminiscentes
6.
J Hazard Mater ; 470: 134258, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38608588

RESUMEN

Photochemical active species generated from photosensitizers, e.g., dissolved organic matter (DOM), play vital roles in the transformation of micropollutants in water. Here, butanedione (BD), a redox-active moiety in DOM and widely found in nature, was employed to photo-transform naproxen (NPX) with peracetic acid (PAA) and H2O2 as contrasts. The results obtained showed that the BD exhibited more applicable on NPX degradation. It works in the lake or river water under UV and solar irradiation, and its NPX degradation efficiency was 10-30 times faster than that of PAA and H2O2. The reason for the efficient transformation of pollutants is that the BD system was proved to be a non-free radical dominated mechanism. The quantum yield of BD (Ф254 nm) was calculated to be 0.064, which indicates that photophysical process is the dominant mode of BD conversion. By adding trapping agents, direct energy transfer from 3BD* to NPX (in anoxic environment) or dissolved oxygen (in aerobic environment) was proved to play a major role (> 91 %). Additionally, the BD process reduces the toxicity of NPX and promotes microbial growth after irradiation. Overall, this study significantly deepened the understanding of the transformation between BD and micropollutants, and provided a potential BD-based process for micropollutants removal under solar irradiation.


Asunto(s)
Naproxeno , Fotólisis , Rayos Ultravioleta , Contaminantes Químicos del Agua , Naproxeno/química , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/efectos de la radiación , Transferencia de Energía , Peróxido de Hidrógeno/química , Ácido Peracético/química , Procesos Fotoquímicos
7.
Sensors (Basel) ; 24(7)2024 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-38610526

RESUMEN

Gold nanoparticles (AuNPs) are good candidates for donor material in energy transfer systems and can easily be functionalized with various ligands on the surface with Au-S bonding. Cyclodextrin (CD) forms inclusion complexes with fluorophores due to its unique structure for host-guest interaction. In this study, we fabricated ßCD-functionalized AuNPs using different lengths of thiol ligands and recognized cholesterol to confirm the energy-transfer-based turn-on fluorescence mechanism. AuNP-ßCD conjugated with various thiol ligands and quenched the fluorescein (Fl) dye, forming ßCD-Fl inclusion complexes. As the distance between AuNPs and ßCD decreased, the quenching efficiency became higher. The quenched fluorescence was recovered when the cholesterol replaced the Fl because of the stronger binding affinity of the cholesterol with ßCD. The efficiency of cholesterol recognition was also affected by the energy transfer effect because the shorter ßCD ligand had a higher fluorescence recovery. Furthermore, we fabricated a liposome with cholesterol embedded in the lipid bilayer membrane to mimic the cholesterol coexisting with lipids in human serum. These cellular cholesterols accelerated the replacement of the Fl molecules, resulting in a fluorescence recovery higher than that of pure lipid. These discoveries are expected to give guidance towards cholesterol sensors or energy-transfer-based biosensors using AuNPs.


Asunto(s)
Oro , Nanopartículas del Metal , Humanos , Colesterol , Transferencia de Energía , Fluoresceína , Compuestos de Sulfhidrilo
8.
Int J Mol Sci ; 25(7)2024 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-38612659

RESUMEN

Photosystem I (PSI) is one of the two main pigment-protein complexes where the primary steps of oxygenic photosynthesis take place. This review describes low-temperature frequency-domain experiments (absorption, emission, circular dichroism, resonant and non-resonant hole-burned spectra) and modeling efforts reported for PSI in recent years. In particular, we focus on the spectral hole-burning studies, which are not as common in photosynthesis research as the time-domain spectroscopies. Experimental and modeling data obtained for trimeric cyanobacterial Photosystem I (PSI3), PSI3 mutants, and PSI3-IsiA18 supercomplexes are analyzed to provide a more comprehensive understanding of their excitonic structure and excitation energy transfer (EET) processes. Detailed information on the excitonic structure of photosynthetic complexes is essential to determine the structure-function relationship. We will focus on the so-called "red antenna states" of cyanobacterial PSI, as these states play an important role in photochemical processes and EET pathways. The high-resolution data and modeling studies presented here provide additional information on the energetics of the lowest energy states and their chlorophyll (Chl) compositions, as well as the EET pathways and how they are altered by mutations. We present evidence that the low-energy traps observed in PSI are excitonically coupled states with significant charge-transfer (CT) character. The analysis presented for various optical spectra of PSI3 and PSI3-IsiA18 supercomplexes allowed us to make inferences about EET from the IsiA18 ring to the PSI3 core and demonstrate that the number of entry points varies between sample preparations studied by different groups. In our most recent samples, there most likely are three entry points for EET from the IsiA18 ring per the PSI core monomer, with two of these entry points likely being located next to each other. Therefore, there are nine entry points from the IsiA18 ring to the PSI3 trimer. We anticipate that the data discussed below will stimulate further research in this area, providing even more insight into the structure-based models of these important cyanobacterial photosystems.


Asunto(s)
Clorofila , Complejo de Proteína del Fotosistema I , Complejo de Proteína del Fotosistema I/genética , Dicroismo Circular , Transferencia de Energía , Frío
9.
Int J Mol Sci ; 25(7)2024 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-38612934

RESUMEN

We establish a general kinetic scheme for the energy transfer and radical-pair dynamics in photosystem I (PSI) of Chlamydomonas reinhardtii, Synechocystis PCC6803, Thermosynechococcus elongatus and Spirulina platensis grown under white-light conditions. With the help of simultaneous target analysis of transient-absorption data sets measured with two selective excitations, we resolved the spectral and kinetic properties of the different species present in PSI. WL-PSI can be described as a Bulk Chl a in equilibrium with a higher-energy Chl a, one or two Red Chl a and a reaction-center compartment (WL-RC). Three radical pairs (RPs) have been resolved with very similar properties in the four model organisms. The charge separation is virtually irreversible with a rate of ≈900 ns-1. The second rate, of RP1 → RP2, ranges from 70-90 ns-1 and the third rate, of RP2 → RP3, is ≈30 ns-1. Since RP1 and the Red Chl a are simultaneously present, resolving the RP1 properties is challenging. In Chlamydomonas reinhardtii, the excited WL-RC and Bulk Chl a compartments equilibrate with a lifetime of ≈0.28 ps, whereas the Red and the Bulk Chl a compartments equilibrate with a lifetime of ≈2.65 ps. We present a description of the thermodynamic properties of the model organisms at room temperature.


Asunto(s)
Chlamydomonas reinhardtii , Complejo de Proteína del Fotosistema I , Clorofila A , Transferencia de Energía , Cinética
10.
Biosystems ; 240: 105213, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38616011

RESUMEN

The central problem in transduction is to explain how the energy caught from sunlight by chloroplasts becomes biological work. Or to express it in different terms: how does the energy remain trapped in the biological network and not get lost through thermalization into the environment? The pathway consists of an immensely large number of steps crossing hierarchical levels - some upwards, to larger assemblies, others downwards into energy rich molecules - before fuelling an action potential or a contracting cell. Accepting the assumption that steps are executed by protein domains, we expect that transduction mechanisms are the result of conformational changes, which in turn involve rearrangements of the bonds responsible for the protein fold. But why are these essential changes so difficult to detect? In this presentation, the metabolic pathway is viewed as equivalent to an energy conduit composed of equally sized units - the protein domains - rather than a row of catalysts. The flow of energy through them occurs by the same mechanism as through the cytoplasmic medium (water). This mechanism is based on the cluster-wave model of water structure, which successfully explains the transfer of energy through the liquid medium responsible for the build up of osmotic pressure. The analogy to the line of balls called "Newton's cradle" provides a useful comparison, since there the transfer is also invisible to us because the intermediate balls are motionless. It is further proposed that the spatial arrangements of the H-bonds of the α and ß secondary structures support wave motion, with the linear and lateral forms of the groups of bonds belonging to the helices and sheets executing the longitudinal and transverse modes, respectively.


Asunto(s)
Transferencia de Energía , Transferencia de Energía/fisiología , Modelos Biológicos , Metabolismo Energético/fisiología , Humanos , Agua/metabolismo
11.
Endocrinology ; 165(6)2024 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-38679471

RESUMEN

The glycoprotein receptors, members of the large G protein-coupled receptor family, are characterized by a large extracellular domains responsible for binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays, most often using radiolabeling but also by thermal shift assays. Despite their high sensitivity, these assays require appropriate laboratory conditions and, often, purified plasma cell membranes, which do not provide information on receptor localization or activity because the assays typically focus on measuring binding only. Here, we apply bioluminescence resonance energy transfer in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc)-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (human chorionic gonadotropin or LH) fused to the enhanced green fluorescent protein. The Gluc-LHCGR, as well as other Gluc-G protein-coupled receptors such as the somatostatin and the C-X-C motif chemokine receptors, is expressed on the plasma membrane, where luminescence activity is equal to membrane receptor expression, and is fully functional. The chimeric enhanced green fluorescent protein-ligands are properly secreted from cells and able to bind and activate the wild-type LHCGR as well as the Gluc-LHCGR. Finally, bioluminescence resonance energy transfer was used to determine the interactions between clinically relevant mutations of the hormones and the LHCGR that show that this bioassay provides a fast and effective, safe, and cost-efficient tool to assist the molecular characterization of mutations in either the receptor or ligand and that it is compatible with downstream cellular assays to determine receptor activation/function.


Asunto(s)
Proteínas Fluorescentes Verdes , Unión Proteica , Humanos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Receptores de HL/metabolismo , Receptores de HL/genética , Luciferasas/metabolismo , Luciferasas/genética , Animales , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Gonadotropina Coriónica/metabolismo , Células HEK293 , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Transferencia de Energía , Glicoproteínas/metabolismo , Mediciones Luminiscentes/métodos
12.
Radiat Environ Biophys ; 63(2): 185-194, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38565701

RESUMEN

This paper describes events of anomalously high energy transfer to a micro-object by fragments of nuclei generated in nuclear interactions in the environment on board a spacecraft in flight in low-Earth orbit. An algorithm has been developed that allows for the calculation of the absorbed energy from one or more fragments - products of nuclear interaction. With this algorithm the energy distributions for a spherical micro-volume in an aqueous medium were calculated. And the resulting absorbed energy spectra from nuclear fragments and from primary cosmic rays were compared. The role of nuclear interactions in events of large energy transfers in micro-objects in the field of primary cosmic radiation has been evaluated. The calculations performed in this study showed that the energy in a micro-volume from nuclear events can be several times higher compared to the energy imparted by primary space radiation.


Asunto(s)
Radiación Cósmica , Transferencia de Energía , Algoritmos , Nave Espacial , Vuelo Espacial
13.
Spectrochim Acta A Mol Biomol Spectrosc ; 316: 124338, 2024 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-38678839

RESUMEN

In this work, the interaction between different chloro-substituted phenylurea herbicides (diuron (DIU) and chlortoluron (CHL)) and BSA were investigated and compared at three different temperatures (283 K, 298 K and 310 K) adopting UV-vis, fluorescence, and circular dichroism spectra. The quenching mechanism of the interaction was also proposed. The energy transfer between BSA and DIU/CHL was investigated. The binding sites of DIU/CHL and BSA and the variations in the microenvironment of amino acid residues were studied. The changes of the secondary structure of BSA were analyzed. The results indicate that both DIU and CHL can significantly interact with BSA, and the degree of the interaction between DIU/CHL and BSA increases with the increase of the DIU/CHL concentration. The fluorescence quenching of BSA by DIU/CHL results from the combination of static and dynamic quenching. The DIU/CHL has a weak to moderate binding affinity for BSA, and the binding stoichiometry is 1:1. Their binding processes are spontaneous, and hydrophobic interaction, hydrogen bonds and van der Waals forces are the main interaction forces. DIU/CHL has higher affinity for subdomain IIA (Site I) of BSA than subdomain IIIA (Site II), and also interacts with tryptophan more than tyrosine residues. The energy transfer can occur from BSA to DIU/CHL. By comparison, the strength of the interaction of DIU-BSA is always greater than that of CHL-BSA, and DIU can destroy the secondary structure of BSA molecules greater than CHL and thus the potential toxicity of DIU is higher due to DIU with more chlorine substituents than CHL. It is expected that this study on the interaction can offer in-depth insights into the toxicity of phenylurea herbicides, as well as their impact on human and animal health at the molecular level.


Asunto(s)
Herbicidas , Albúmina Sérica Bovina , Espectrometría de Fluorescencia , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Herbicidas/química , Herbicidas/metabolismo , Animales , Bovinos , Diurona/química , Diurona/metabolismo , Espectrofotometría Ultravioleta , Sitios de Unión , Unión Proteica , Dicroismo Circular , Transferencia de Energía , Termodinámica , Enlace de Hidrógeno
14.
Bioinspir Biomim ; 19(4)2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38663427

RESUMEN

Inspired by the vibrations of aquatic plants such as seaweed in the unsteady flow fields generated by free-surface waves, we investigate a novel device based on piezoelectric plates to harvest energy from oscillatory cross flows. Towards this end, numerical studies are conducted using a flow-structure-electric interaction model to understand the underlying physical mechanisms involved in the dynamics and energy harvesting performance of one or a pair of piezoelectric plates in an oscillatory cross flow. In a single-plate configuration, both periodic and irregular responses have been observed depending on parameters such as normalized plate stiffness and Keulegan-Carpenter number. Large power harvesting is achieved with the excitation of natural modes. Besides, when the time scale of the motion and the intrinsic time scale of the circuit are close to each other the power extraction is enhanced. In a two-plate configuration with tandem formation, the hydrodynamic interaction between the two plates can induce irregularity in the response. In terms of energy harvesting, two counteracting mechanisms have been identified, shielding and energy recovery. The shielding effect reduces plate motion and energy harvesting, whereas with the energy recovery effect one plate is able to recovery energy from the wake of another for performance enhancement. The competition between these mechanisms leads to constructive or destructive interactions between the two plates. These results suggest that for better performance the system should be excited at its natural period, which should be close to the intrinsic time scale of the circuit. Moreover, using a pair of plates in a tandem formation can further improve the energy harvesting capacity when conditions for constructive interaction are satisfied.


Asunto(s)
Algas Marinas , Algas Marinas/fisiología , Diseño de Equipo , Vibración , Hidrodinámica , Biomimética/instrumentación , Simulación por Computador , Reología , Transferencia de Energía
15.
Luminescence ; 39(4): e4736, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38590043

RESUMEN

In recent trends, radiation falls under the narrowband ultraviolet-B region (305-315 nm) widely used in phototherapy lamp applications in the treatment of skin diseases. In this paper, we report a Gd3+-doped NaYF4 luminescent material synthesized for the first time using the low-temperature co-precipitation method. It crystallized into a face-centred cubic structure, as confirmed by X-ray diffraction characterization techniques and Rietveld refinement. The photoluminescence property of the as-prepared sample shows a highly intense, sharp emission band obtained at 311 nm, which belongs to the narrowband ultraviolet-B region and corresponds to the transition of the 6P7/2→8S7/2 level of the Gd3+ ions under 272 nm excitation (8S7/2 to 6IJ). The transitions of the Gd3+ ions are detected entirely with different concentrations of Gd3+ ions. Scanning electron microscopy analysis indicated that the average particle was 288 nm. The critical distance for energy transfer was calculated to be equal to 11.5017 Å. Dipole-dipole interaction is responsible for energy transfer, as analyzed by Dexter theory. These excellent optical characteristics, together with their highly efficient and low-cost synthesis approach, indicate that synthesized NaYF4:Gd3+ phosphors have excessive potential for phototherapeutic lamp applications.


Asunto(s)
Luminiscencia , Fototerapia , Transferencia de Energía , Difracción de Rayos X , Iones
16.
Methods Mol Biol ; 2797: 253-260, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38570465

RESUMEN

Bioluminescence resonance energy transfer (BRET) is a valuable technique for studying protein-protein interactions (PPIs) within live cells (Pfleger and Eidne, Nat Methods 3:165-174, 2006). Among the various BRET methodologies, a recent addition called NanoBRET has emerged, leveraging advancements in donor and acceptor technologies (Machleidt and Woodroofe, ACS Chem Biol 10:1797-1804, 2015). In this study, we present a developed methodology designed to measure PPIs involving the RAS protein family and their effectors and interactors at the plasma membrane. By utilizing the NanoLuc and HaloTag BRET pair, we provide evidence of a saturable interaction between KRAS4b-G12D and full-length RAF1. Conversely, the RAF1 R89L mutant, known to impede RAF1 binding to active RAS, exhibits nonspecific interactions. The assay exhibits remarkable signal-to-background ratios and is highly suitable for investigating the interactions of RAS with effectors, as well as for high-throughput screening assays.


Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia , Ensayos Analíticos de Alto Rendimiento , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Transferencia de Energía , Mediciones Luminiscentes/métodos
17.
J Phys Chem B ; 128(18): 4315-4324, 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38687467

RESUMEN

Excess energy absorbed by photosystems (PSs) can result in photoinduced oxidative damage. Transfer of such energy within the core pigments of the reaction center in the form of triplet excitation is important in regulating and preserving the functionality of PSs. In the bacterial reaction center (BRC), the special pair (P) is understood to act as the electron donor in a photoinduced charge transfer process, triggering the charge separation process through the photoactive branch A pigments that experience a higher polarizing environment. At this work, triplet excitation energy transfer (TEET) in BRC is studied using a computational perspective to gain insights into the roles of the dielectric environment and interpigment orientations. We find in agreement with experimental observations that TEET proceeds through branch B. The TEET process toward branch B pigment is found to be significantly faster than the hypothetical process proceeding through branch A pigments with ps and ms time scales, respectively. Our calculations find that conformational differences play a major role in this branch asymmetry in TEET, where the dielectric environment asymmetry plays only a secondary role in directing the TEET to proceed through branch B. We also address TEET processes asserting the role of carotenoid as the final triplet energy acceptor and in a mutant form, where the branch pigments adjacent to P are replaced by bacteriopheophytins. The necessary electronic excitation energies and electronic state couplings are calculated by the recently developed polarization-consistent framework combining a screened range-separated hybrid functional and a polarizable continuum mode. The polarization-consistent potential energy surfaces are used to parametrize the quantum mechanical approach, implementing Fermi's golden rule expression of the TEET rate calculations.


Asunto(s)
Transferencia de Energía , Proteínas del Complejo del Centro de Reacción Fotosintética , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Teoría Funcional de la Densidad
18.
J Vis Exp ; (205)2024 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-38497636

RESUMEN

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.


Asunto(s)
Proteínas 14-3-3 , Transducción de Señal , Proteínas 14-3-3/genética , Transferencia de Energía , Membrana Celular , División Celular
19.
J Phys Chem Lett ; 15(11): 3149-3158, 2024 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-38478725

RESUMEN

We combine site-directed mutagenesis with picosecond time-resolved fluorescence and femtosecond transient absorption (TA) spectroscopies to identify excitation energy transfer (EET) processes between chlorophylls (Chls) and xanthophylls (Xant) in the minor antenna complex CP29 assembled inside nanodiscs, which result in quenching. When compared to WT CP29, a longer lifetime was observed in the A2 mutant, missing Chl a612, which closely interacts with Xant Lutein in site L1. Conversely, a shorter lifetime was obtained in the A5 mutant, in which the interaction between Chl a603 and Chl a609 is strengthened, shifting absorption to lower energy and enhancing Chl-Xant EET. Global analysis of TA data indicated that EET from Chl a Qy to a Car dark state S* is active in both the A2 and A5 mutants and that their rate constants are modulated by mutations. Our study provides experimental evidence that multiple Chl-Xant interactions are involved in the quenching activity of CP29.


Asunto(s)
Clorofila , Luteína , Clorofila/química , Complejos de Proteína Captadores de Luz/química , Complejo de Proteína del Fotosistema II/metabolismo , Transferencia de Energía , Xantófilas , Sitios de Unión , Mutagénesis Sitio-Dirigida
20.
Luminescence ; 39(3): e4701, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38441275

RESUMEN

In the present work, a novel n-UV convertible colour-tunable emitting phosphor was obtained based on the efficient Ce3+ -Tb3+ energy transfer in the Y10 Al2 Si3 O18 N4 host. By properly controlling the ratio of Ce3+ /Tb3+ , the colour hue of the obtained powder covered the blue and green regions, under excitation of 365 nm. The steady-state and dynamic-state luminescence measurement was performed to shed light on the related mechanism, which was justified by the electronic dipole-quadrupole dominating the related energy transfer process. Preliminary studies showed that Y10 Al2 Si3 O18 N4 :Ce3+ ,Tb3+ can be promising as an inorganic phosphor for white LED applications.


Asunto(s)
Electrónica , Color , Transferencia de Energía
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