RESUMEN
Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.
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Rodopsinas Microbianas , Bases de Schiff , Animales , Hidrógeno , Enlace de Hidrógeno , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genética , Bases de Schiff/química , Análisis EspectralRESUMEN
Fluorescence Cross-Correlation Spectroscopy (FCCS) is a well-established and useful tool in physics and chemistry. Furthermore, due to its hybrid character of being a bulk assay at a single molecular level, it found many applications in biophysics and molecular biochemistry. Examples may be investigating kinetics and dynamics of chemical and biochemical reactions such as protein-ligand-, protein-protein-binding, fast conformational changes, and intracellular transportation. Also, it was utilized to characterize larger structures such as lipid vesicles and multi-protein complexes. A two-photon excitation source makes FCCS relatively easy-to-use and easy-to-maintain. Combining this technique with fluorescence lifetime analysis results in a versatile biophysical tool that can be used to solve many biological problems, as even small changes in the local environment, like pH or salt concentration, can be monitored if appropriate fluorophores are used. An example of its use for membrane docking and fusion assays is described in Chap. 13 . In this chapter, we want to give the reader a simple, detailed step-by-step guide of how to set up such a tool.
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Colorantes Fluorescentes , Fotones , Fluorescencia , Colorantes Fluorescentes/química , Cinética , Unión Proteica , Espectrometría de Fluorescencia/métodosRESUMEN
Watching events of membrane fusion in real time and distinguishing between intermediate steps of these events is useful for mechanistic insights but at the same time a challenging task. In this chapter, we describe how to use fluorescence cross-correlation spectroscopy and Förster-resonance energy transfer to resolve the tethering and fusion of membranes by SNARE proteins (syntaxin-1, SNAP-25, and synaptobrevin-2) as an example. The given protocols can easily be adapted to other membrane proteins to investigate their ability to tether or even fuse vesicular membrane.
Asunto(s)
Liposomas , Fusión de Membrana , Transferencia Resonante de Energía de Fluorescencia , Liposomas/química , Proteínas SNARE/metabolismo , Espectrometría de Fluorescencia , Sintaxina 1RESUMEN
In addition to (bacterio)chlorophylls, (B)Chls, photosynthetic pigment-protein complexes bind carotenoids (Cars) that fulfil various important functions which are not fully understood, yet. However, certain excited states of Cars are optically one-photon forbidden ("dark") and can potentially undergo excitation energy transfer (EET) to (B)Chls following two-photon absorption (TPA). The amount of EET is reflected by the differences in TPA and two-photon excitation (TPE) spectra of a complex (multi-pigment) system. Since it is technically and analytically demanding to resolve optically forbidden states, different studies reported varying contributions of Cars and Chls to TPE/TPA spectra. In a study using well-defined 1 : 1 Car-tetrapyrrole dyads TPE contributions of tetrapyrrole molecules, including Chls, and Cars were measured. In these experiments, TPE of Cars dominated over Chl a TPE in a broad wavelength range. Another study suggested only minor contributions of Cars to TPE spectra of pigment-protein complexes such as the plant main light-harvesting complex (LHCII), in particular for wavelengths longer than â¼600/1200 nm. By joining forces and a combined analysis of all available data by both teams, we try to resolve this apparent contradiction. Here, we demonstrate that reconstruction of a wide spectral range of TPE for LHCII and photosystem I (PSI) requires both, significant Car and Chl contributions. Direct comparison of TPE spectra obtained in both studies demonstrates a good agreement of the primary data. We conclude that in TPE spectra of LHCII and PSI, the contribution of Chls is dominating above 600/1200 nm, whereas the contributions of forbidden Car states increase particularly at wavelengths shorter than 600/1200 nm. Estimates of Car contributions to TPA as well as TPE spectra are given for various wavelengths.
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Carotenoides/química , Clorofila/química , Complejos de Proteína Captadores de Luz/química , Fotones , Análisis Espectral/métodosRESUMEN
We developed three bathochromic, green-light activatable, photolabile protecting groups based on a nitrodibenzofuran (NDBF) core with D-π-A push-pull structures. Variation of donor substituents (D) at the favored ring position enabled us to observe their impact on the photolysis quantum yields. Comparing our new azetidinyl-NDBF (Az-NDBF) photolabile protecting group with our earlier published DMA-NDBF, we obtained insight into its excitation-specific photochemistry. While the "two-photon-only" cage DMA-NDBF was inert against one-photon excitation (1PE) in the visible spectral range, we were able to efficiently release glutamic acid from azetidinyl-NDBF with irradiation at 420 and 530â nm. Thus, a minimal change (a cyclization adding only one carbon atom) resulted in a drastically changed photochemical behavior, which enables photolysis in the green part of the spectrum.
RESUMEN
Based on nitrodibenzofuran (NDBF) a new photocage with higher two-photon action cross section and red-shifted absorption was developed. Due to calculations, a dimethylamino functionality (DMA) was added at ring position 7. The uncaging of nucleobases after two-photon excitation (2PE) could be visualized via double-strand displacement in a hydrogel. With this assay we achieved three-dimensional photorelease of DMA-NDBF-protected DNA orthogonal to NDBF-protected strands. While being an excellent 2P-cage, DMA-NDBF is surprisingly stable under visible-light one-photon excitation (1PE). This case of excitation-specific photochemistry enhances the scope of orthogonal photoregulation.
RESUMEN
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.
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Exocitosis/fisiología , Membranas Intracelulares/fisiología , Fusión de Membrana , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Animales , Bovinos , Cricetulus , Unión Proteica , Proteolípidos , RatasRESUMEN
The HIV-1 envelope gp120/gp41 trimer mediates viral membrane fusion. After cluster of differentiation-4 recognition, gp120 detaches from the virus, exposing gp41 which triggers fusion. During the fusion process, gp41 may not remain trimeric, which could have functional importance. Here, we probe the reversible association of full length gp41 (minus the cytoplasmic domain) in detergent micelles (with probes attached to transmembrane domain) by fluorescence resonance energy transfer (FRET) with a µm dissociation constant. This is compared with other methods. A gp41-targeted fusion inhibitor must interfere with this transition, and monomeric, partially monomeric or trimeric states all present potential binding epitopes. The gp41 self-association is a valid drug target model and FRET, a potential high-throughput assay system, could be used to screen drug libraries.
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Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , Multimerización de Proteína , Transferencia Resonante de Energía de Fluorescencia , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , MicelasRESUMEN
When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm)(2) image nanoareas can provide subdiffraction resolution (super resolution by polarization demodulation, SPoD). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, ExPAN). This shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. Our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.
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Polarización de Fluorescencia/métodos , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Algoritmos , Animales , Células Cultivadas , Simulación por Computador , Células Epiteliales/metabolismo , Diseño de Equipo , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/metabolismo , Microtúbulos/ultraestructura , Modelos Teóricos , Nanosferas/química , Distribución Normal , Fotones , Potoroidae , Programas InformáticosRESUMEN
Many aspects in the regulation of photosynthetic light-harvesting of plants are still quite poorly understood. For example, it is still a matter of debate which physical mechanism(s) results in the regulation and dissipation of excess energy in high light. Many researchers agree that electronic interactions between chlorophylls (Chl) and certain states of carotenoids are involved in these mechanisms. However, in particular, the role of the first excited state of carotenoids (Car S1) is not easily revealed, because of its optical forbidden character. The use of two-photon excitation is an elegant approach to address directly this state and to investigate the energy transfer in the direction Car S1 â Chl. Meanwhile, it has been applied to a large variety of systems starting from simple carotenoid-tetrapyrrole model compounds up to entire plants. Here, we present a systematic summary of the observations obtained by two-photon excitation about Car S1 â Chl energy transfer in systems with increasing complexity and the correlation to fluorescence quenching. We compare these observations directly with the energy transfer in the opposite direction, Chl â Car S1, for the same systems as obtained in pump-probe studies. We discuss what surprising aspects of this comparison led us to the suggestion that quenching excitonic Car-Chl interactions could contribute to the regulation of light harvesting, and how this suggestion can be connected to other models proposed.
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Carotenoides/metabolismo , Clorofila/metabolismo , Fenómenos Fisiológicos de las Plantas , Metabolismo Energético , Indoles/metabolismo , Isoindoles , Luz , Complejos de Proteína Captadores de Luz/metabolismo , Modelos BiológicosRESUMEN
Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction, we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.
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Membrana Dobles de Lípidos/metabolismo , Liposomas , Fusión de Membrana , Proteínas SNARE/metabolismo , Animales , Membrana Dobles de Lípidos/química , Liposomas/química , Liposomas/metabolismo , Unión Proteica , Conformación Proteica , Ratas , Proteínas SNARE/química , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
In two recent studies, energy transfer was reported in certain phthalocyanine-carotenoid dyads between the optically forbidden first excited state of carotenoids (Car S(1)) and phthalocyanines (Pcs) in the direction Pc â Car S(1) (Kloz et al., J Am Chem Soc 133:7007-7015, 2011) as well as in the direction Car S(1) â Pc (Liao et al., J Phys Chem A 115:4082-4091, 2011). In this article, we show that the extent of this energy transfer in both directions is closely correlated in these dyads. This correlation and the additional observation that Car S(1) is instantaneously populated after Pc excitation provides evidence that in these compounds excitonic interactions can occur. Besides pure energy transfer and electron transfer, this is the third type of tetrapyrrole-carotenoid interaction that has been shown to occur in these model compounds and that has previously been proposed as a photosynthetic regulation mechanism. We discuss the implications of these models for photosynthetic regulation. The findings are also discussed in the context of a model in which both electronic states are disordered and in which the strength of the electronic coupling determines whether energy transfer, excitonic coupling, or electron transfer occurs.
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Carotenoides/química , Transferencia de Energía , Indoles/química , Fotosíntesis/fisiología , Carotenoides/síntesis química , Transporte de Electrón , Indoles/síntesis química , Isoindoles , Cinética , Luz , Análisis EspectralRESUMEN
Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S(1)) performed on LHCII gels shows that the extent of electronic interactions between Car S(1) and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).
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Arabidopsis/fisiología , Luteína/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Arabidopsis/metabolismo , Sitios de Unión , Cromatografía Líquida de Alta Presión , Espectrometría RamanRESUMEN
Electronic interactions between the first excited states (S(1)) of carotenoids (Car) of different conjugation lengths (8-11 double bonds) and phthalocyanines (Pc) in different Car-Pc dyad molecules were investigated by two-photon spectroscopy and compared with Car S(1)-chlorophyll (Chl) interactions in photosynthetic light harvesting complexes (LHCs). The observation of Chl/Pc fluorescence after selective two-photon excitation of the Car S(1) state allowed sensitive monitoring of the flow of energy between Car S(1) and Pc or Chl. It is found that two-photon excitation excites to about 80% to 100% exclusively the carotenoid state Car S(1) and that only a small fraction of direct tetrapyrrole two-photon excitation occurs. Amide-linked Car-Pc dyads in tetrahydrofuran demonstrate a molecular gear shift mechanism in that effective Car S(1) â Pc energy transfer is observed in a dyad with 9 double bonds in the carotenoid, whereas in similar dyads with 11 double bonds in the carotenoid, the Pc fluorescence is strongly quenched by Pc â Car S(1) energy transfer. In phenylamino-linked Car-Pc dyads in toluene extremely large electronic interactions between the Car S(1) state and Pc were observed, particularly in the case of a dyad in which the carotenoid contained 10 double bonds. This observation together with previous findings in the same system provides strong evidence for excitonic Car S(1)-Pc Q(y) interactions. Very similar results were observed with photosynthetic LHC II complexes in the past, supporting an important role of such interactions in photosynthetic down-regulation.
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Carotenoides/química , Electrones , Fotones , Teoría Cuántica , Tetrapirroles/química , Clorofila/química , Luz , Complejos de Proteína Captadores de Luz/química , Estructura Molecular , EstereoisomerismoRESUMEN
Recently, excitonic carotenoid-chlorophyll interactions have been proposed as a simple but effective model for the down-regulation of photosynthesis in plants. The model was proposed on the basis of quenching-correlated electronic carotenoid-chlorophyll interactions (Car S(1) â Chl) determined by Car S(1) two-photon excitation and red-shifted absorption bands. However, if excitonic interactions are indeed responsible for this effect, a simultaneous correlation of quenching with increased energy transfer in the opposite direction, Chl Q(y) â Car S(1), should be observed. Here we present a systematic study on the correlation of Car S(1) â Chl and Chl â Car S(1) energy transfer with the occurrence of red-shifted bands and quenching in isolated LHCII. We found a direct correlation between all four phenomena, supporting our conclusion that excitonic Car S(1)-Chl interactions provide low-lying states serving as energy traps and dissipative valves for excess excitation energy.
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Complejos de Proteína Captadores de Luz/química , Clorofila/química , Transferencia de Energía , Modelos Moleculares , Espectrometría de FluorescenciaRESUMEN
Recently, it has been shown that 2-photon fluorescence correlation spectroscopy of single glycosylated 20-nm fluorescent spheres allows measurement of the relative carbohydrate binding affinities of unlabeled proteins and that these modified spheres can mimic the glycocalix of cell or virus surfaces. An especially useful extension would be the analysis of mixtures of nanospheres that each contain different fluorescent labels and are thus differentially "encoded." If the surfaces of these encoded nanospheres are modified with various receptors, many different biomolecule-surface interactions and concurrent reactions can be measured quickly and simultaneously in a single-reaction vessel. An essential prerequisite for this general assay principle is the ability to identify with an accuracy of nearly 100% any encoded nanosphere present in a mixture on a single-particle level. Here the authors present a method that indeed allows certain identification of differently encoded nanospheres during single transits through the focal volume of a microscope objective (ø approximately 200-500 nm) in aqueous solution. This opens the way for using the encoded nanospheres in 1-well measurements of a large variety of biomolecular receptor-ligand interactions, inhibition and concurrent reactions, and thus either for testing the behavior of ligands in a mimicked complex biomolecular environment or for a fast simultaneous measurement of a multitude of receptor-ligand interactions.
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Nanosferas , Nanotecnología/métodos , Colorantes Fluorescentes/química , Ligandos , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Tamaño de la Partícula , Unión Proteica , Espectrometría de Fluorescencia/métodos , EspectrofotometríaRESUMEN
Neuronal exocytosis is mediated by the SNARE proteins synaptobrevin 2/VAMP, syntaxin 1A, and SNAP-25A. While it is well-established that these proteins mediate membrane fusion after reconstitution in artificial membranes, it has so far been difficult to monitor intermediate stages of the reaction. Using a confocal two-photon setup, we applied fluorescence cross-correlation spectroscopy (FCCS) and fluorescence lifetime analysis to discriminate between docking and fusion of liposomes. We show that liposome populations that are either non-interacting, or are undergoing docking and fusion, as well as multiple interactions can be quantitatively discriminated without the need for immobilizing the lipid bilayers. When liposomes containing a stabilized syntaxin 1A/SNAP-25A complex were mixed with liposomes containing synaptobrevin 2, we observed that rapid docking precedes fusion. Accordingly, docked intermediates accumulated in the initial phase of the reaction. Furthermore, rapid formation of multiple docked states was observed with on average four liposomes interacting with each other. When liposomes of different sizes were compared, only the rate of lipid mixing depended on the liposome size but not the rate of docking. Our results show that under appropriate conditions a docked state, mediated by trans-SNARE interactions, can be isolated that constitutes an intermediate in the fusion pathway.