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Here we present a method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. It exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method reduces photobleaching effects 1.5-9.2-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins.
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Dreiklang is a reversibly photoswitchable fluorescent protein used as a probe in advanced fluorescence imaging. It undergoes a unique and still poorly understood photoswitching mechanism based on the reversible addition of a water molecule to the chromophore. We report the first comprehensive study of the dynamics of this reaction by transient absorption spectroscopy from 100 fs to seconds in the original Dreiklang protein and two point variants. The picture that emerges from our work is that of a competition between photoswitching and nonproductive reaction pathways. We found that photoswitching had a low quantum yield of 0.4%. It involves electron transfer from a tyrosine residue (Tyr203) to the chromophore and is completed in 33 ns. Nonproductive deactivation pathways comprise recombination of a charge transfer intermediate, excited-state proton transfer from the chromophore to a histidine residue (His145), and decay to the ground state via micro-/millisecond-lived intermediates.
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Protones , Proteínas Luminiscentes/química , Análisis Espectral , Proteínas Fluorescentes Verdes/químicaRESUMEN
We introduce HIGHLIGHT as a simple and general strategy to selectively image a reversibly photoactivatable fluorescent label associated with a given kinetics. The label is submitted to sine-wave illumination of large amplitude, which generates oscillations of its concentration and fluorescence at higher harmonic frequencies. For singularizing a label, HIGHLIGHT uses specific frequencies and mean light intensities associated with resonances of the amplitudes of concentration and fluorescence oscillations at harmonic frequencies. Several non-redundant resonant observables are simultaneously retrieved from a single experiment with phase-sensitive detection. HIGHLIGHT is used for selective imaging of four spectrally similar fluorescent proteins that had not been discriminated so far. Moreover, labels out of targeted locations can be discarded in an inhomogeneous spatial profile of illumination. HIGHLIGHT opens roads for simplified optical setups at reduced cost and easier maintenance.
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Luz , Fluorescencia , Procesos FotoquímicosRESUMEN
Photosensitization of organogold intermediates is an emerging field in catalysis. In this context, an access to 2,3-disubstituted indoles from o-alkynyl aniline and iodoalkyne derivatives via a gold-catalyzed sequence under visible-light irradiation and in the absence of an exogenous photocatalyst was uncovered. A wide scope of the process is observed. Of note, 2-iodo-ynamides can be used as electrophiles in this cross-coupling reaction. The resulting N-alkynyl indoles lend themselves to post-functionalization affording valuable scaffolds, notably benzo[a]carbazoles. Mechanistic studies converge on the fact that a potassium sulfonyl amide generates emissive aggregates in the reaction medium. Static quenching of these aggregates by a vinylgold(I) intermediate yields to an excited state of the latter, which can react with an electrophile via oxidative addition and reductive elimination to forge the key C-C bond. This reactant-induced photoactivation of an organogold intermediate opens rich perspectives in the field of cross-coupling reactions.
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Due to its sensitivity and versatility, fluorescence is widely used to detect specifically labeled biomolecules. However, fluorescence is currently limited by label discrimination, which suffers from the broad full width of the absorption/emission bands and the narrow lifetime distribution of the bright fluorophores. We overcome this limitation by introducing extra kinetic dimensions through illuminations of reversibly photoswitchable fluorophores (RSFs) at different light intensities. In this expanded space, each RSF is characterized by a chromatic aberration-free kinetic fingerprint of photochemical reactivity, which can be recovered with limited hardware, excellent photon budget, and minimal data processing. This fingerprint was used to identify and discriminate up to 20 among 22 spectrally similar reversibly photoswitchable fluorescent proteins (RSFPs) in less than 1s. This strategy opens promising perspectives for expanding the multiplexing capabilities of fluorescence imaging.
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Colorantes Fluorescentes , Imagen Óptica , Cinética , Luz , Microscopía Fluorescente/métodosRESUMEN
Fluorescence imaging has become a powerful tool for observations in biology. Yet it has also encountered limitations to overcome optical interferences of ambient light, autofluorescence, and spectrally interfering fluorophores. In this account, we first examine the current approaches which address these limitations. Then we more specifically report on Out-of-Phase Imaging after Optical Modulation (OPIOM), which has proved attractive for highly selective multiplexed fluorescence imaging even under adverse optical conditions. After exposing the OPIOM principle, we detail the protocols for successful OPIOM implementation.
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Técnica del Anticuerpo Fluorescente/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Imagen Óptica/métodos , Algoritmos , Animales , Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador , Luz , Modelos Teóricos , Coloración y EtiquetadoRESUMEN
NewPHL is a recently discovered subgroup of ancestral DNA photolyases. Its domain architecture displays pronounced differences from that of canonical photolyases, in particular at the level of the characteristic electron transfer chain, which is limited to merely two tryptophans, instead of the "classical" three or four. Using transient absorption spectroscopy, we show that the dynamics of photoreduction of the oxidized FAD cofactor in the NewPHL begins similarly as that in canonical photolyases, i.e., with a sub-ps primary reduction of the excited FAD cofactor by an adjacent tryptophan, followed by migration of the electron hole towards the second tryptophan in the tens of ps regime. However, the resulting tryptophanyl radical then undergoes an unprecedentedly fast deprotonation in less than 100 ps in the NewPHL. In spite of the stabilization effect of this deprotonation, almost complete charge recombination follows in two phases of ~ 950 ps and ~ 50 ns. Such a rapid recombination of the radical pair implies that the first FAD photoreduction step, i.e., conversion of the fully oxidized to the semi-quinone state, should be rather difficult in vivo. We hence suggest that the flavin chromophore likely switches only between its semi-reduced and fully reduced form in NewPHL under physiological conditions.
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Desoxirribodipirimidina Fotoliasa/metabolismo , Termodinámica , Desoxirribodipirimidina Fotoliasa/química , Electrones , Flavinas/química , Flavinas/metabolismo , Oxidación-Reducción , Procesos Fotoquímicos , Triptófano/química , Triptófano/metabolismoRESUMEN
Interrogating living cells requires sensitive imaging of a large number of components in real time. The state-of-the-art of multiplexed imaging is usually limited to a few components. This review reports on the promise and the challenges of dynamic contrast to overcome this limitation.
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The well-established oxidative addition-reductive elimination pathway is the most followed one in transition metal-catalysed cross-coupling reactions. While readily occurring with a series of transition metals, gold(I) complexes have shown some reluctance to undergo oxidative addition unless special sets of ligands on gold(I), reagents or reaction conditions are used. Here we show that under visible-light irradiation, an iridium photocatalyst triggers-via triplet sensitization-the oxidative addition of an alkynyl iodide onto a vinylgold(I) intermediate to deliver C(sp)2-C(sp) coupling products after reductive elimination. Mechanistic and modelling studies support that an energy-transfer event takes place, rather than a redox pathway. This particular mode of activation in gold homogenous catalysis was applied in several dual catalytic processes. Alkynylbenzofuran derivatives were obtained from o-alkynylphenols and iodoalkynes in the presence of catalytic gold(I) and iridium(III) complexes under blue light-emitting diode irradiation.
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The animal-like cryptochrome of Chlamydomonas reinhardtii (CraCRY) is a recently discovered photoreceptor that controls the transcriptional profile and sexual life cycle of this alga by both blue and red light. CraCRY has the uncommon feature of efficient formation and longevity of the semireduced neutral form of its FAD cofactor upon blue light illumination. Tyrosine Y373 plays a crucial role by elongating , as fourth member, the electron transfer (ET) chain found in most other cryptochromes and DNA photolyases, which comprises a conserved tryptophan triad. Here, we report the full mechanism of light-induced FADH⢠formation in CraCRY using transient absorption spectroscopy from hundreds of femtoseconds to seconds. Electron transfer starts from ultrafast reduction of excited FAD to FADâ¢- by the proximal tryptophan (0.4 ps) and is followed by delocalized migration of the produced WHâ¢+ radical along the tryptophan triad (â¼4 and â¼50 ps). Oxidation of Y373 by coupled ET to WHâ¢+ and deprotonation then proceeds in â¼800 ps, without any significant kinetic isotope effect, nor a pH effect between pH 6.5 and 9.0. The FADâ¢-/Y373⢠pair is formed with high quantum yield (â¼60%); its intrinsic decay by recombination is slow (â¼50 ms), favoring reduction of Y373⢠by extrinsic agents and protonation of FADâ¢- to form the long-lived, red-light absorbing FADH⢠species. Possible mechanisms of tyrosine oxidation by ultrafast proton-coupled ET in CraCRY, a process about 40 times faster than the archetypal tyrosine-Z oxidation in photosystem II, are discussed in detail.
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Chlamydomonas reinhardtii/metabolismo , Criptocromos/metabolismo , Proteínas de Plantas/metabolismo , Tirosina/metabolismo , Chlamydomonas reinhardtii/química , Criptocromos/química , Transporte de Electrón , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Luz , Modelos Moleculares , Oxidación-Reducción , Proteínas de Plantas/química , Protones , Tirosina/químicaRESUMEN
Macroscale fluorescence imaging is increasingly used to observe biological samples. However, it may suffer from spectral interferences that originate from ambient light or autofluorescence of the sample or its support. In this manuscript, we built a simple and inexpensive fluorescence macroscope, which has been used to evaluate the performance of Speed OPIOM (Out of Phase Imaging after Optical Modulation), which is a reference-free dynamic contrast protocol, to selectively image reversibly photoswitchable fluorophores as labels against detrimental autofluorescence and ambient light. By tuning the intensity and radial frequency of the modulated illumination to the Speed OPIOM resonance and adopting a phase-sensitive detection scheme that ensures noise rejection, we enhanced the sensitivity and the signal-to-noise ratio for fluorescence detection in blot assays by factors of 50 and 10, respectively, over direct fluorescence observation under constant illumination. Then, we overcame the strong autofluorescence of growth media that are currently used in microbiology and realized multiplexed fluorescence observation of colonies of spectrally similar fluorescent bacteria with a unique configuration of excitation and emission wavelengths. Finally, we easily discriminated fluorescent labels from the autofluorescent and reflective background in labeled leaves, even under the interference of incident light at intensities that are comparable to sunlight. The proposed approach is expected to find multiple applications, from biological assays to outdoor observations, in fluorescence macroimaging.
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Class II photolyases utilize for the photoreduction of their flavin cofactor (FAD) a completely different tryptophan triad than most other photolyases and cryptochromes. To counter sped-up back electron transfer, they evolved an unusually fast deprotonation of the distal tryptophanyl radical cation (WHË+) that is produced after excitation of the flavin. We studied the primary aspects of oxidized FAD photoreduction by ultrafast transient absorption spectroscopy, using the class II photolyase from Methanosarcina mazei. With a time constant of 9.2 ps, the initial reduction step of the excited flavin by the proximal W381 tryptophan proceeds almost twentyfold slower than in other photolyases carrying oxidized FAD, most likely because of the larger distance between the flavin and the proximal tryptophan. The thus formed W381HË+ radical is tracked by transient anisotropy measurements to migrate in 29 ps with delocalization over several members of the tryptophan triad. This 29 ps phase also includes the decay of a small fraction of excited flavin, reacting on a slower timescale, and partial recombination of the FADË-/WHË+ radical pair. A final kinetic phase in 230 ps is assigned to the deprotonation of W388HË+ that occurs in competition with partial charge recombination. Interestingly, we show by comparison with the Y345F mutant that this last phase additionally involves oxidation of the Y345 phenolic group by W388HË+, producing a small amount of neutral tyrosyl radical (YOË). The rate of this electron transfer step is about six orders of magnitude faster than the corresponding oxidation of Y345 by the deprotonated W388Ë radical. Unlike conventional photolyases, where the electron hole accumulates on the distal tryptophan before the much slower tryptophanyl deprotonation, our data show that delocalized hole transport is concomitantly concluded by ultrafast deprotonation of W388HË+.
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Desoxirribodipirimidina Fotoliasa/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Methanosarcina/enzimología , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/genética , Flavina-Adenina Dinucleótido/química , Cinética , Methanosarcina/química , Methanosarcina/genética , Methanosarcina/metabolismo , Modelos Moleculares , Oxidación-Reducción , Procesos Fotoquímicos , Mutación Puntual , Conformación Proteica , ProtonesRESUMEN
Photolyases are flavoenzymes repairing UV-induced lesions in DNA, which may be activated by a photoreduction of their FAD cofactor. In most photolyases, this photoreduction proceeds by electron transfer along a chain of three tryptophan (Trp) residues, connecting the flavin to the protein surface. Much less studied, animal (6-4) photolyases (repairing pyrimidine-pyrimidone (6-4) photoproducts) are particularly interesting as they were recently shown to have a longer electron transfer chain, counting four Trp residues. Using femtosecond polarized transient absorption spectroscopy, we performed a detailed analysis of the photoactivation reaction in the (6-4) photolyase of Xenopus laevis with oxidized FAD. We showed that the excited flavin is very quickly reduced (â¼0.5 ps) by a nearby tryptophan residue, yielding FADË- and WHË+ radicals. Subsequent kinetic steps in the picosecond regime were assigned to the migration of the positive charge along the Trp tetrad, in competition with charge recombination. We propose that the positive charge is actually delocalized over various Trp residues during most of the dynamics and that charge recombination essentially occurs through the proximal tryptophanyl radical. Oxidation of the fourth tryptophan is thought to be reached about as fast as that of the third one (â¼40 ps), based on a comparison with a mutant protein lacking the distal Trp, implying ultrafast electron transfer between these two residues. This unusual mechanism sheds light on the rich diversity of electron transfer pathways found in various photolyases, and evolution-related cryptochromes alike.
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Because of growing applications in advanced fluorescence imaging, the mechanisms and dynamics of photoinduced reactions in reversibly photoswitchable fluorescent proteins are currently attracting much interest. We report the first time-resolved study of the photoswitching of Dreiklang, so far the only fluorescent protein to undergo reversible photoinduced chromophore hydration. Using broadband femtosecond transient absorption spectroscopy, we show that the reaction is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state in 100 fs. This primary step is accompanied by coherent oscillations that we assign to its coupling with a low-frequency mode, possibly a deformation of the chromophore hydrogen bond network. A ground-state intermediate is formed in the picosecond-nanosecond regime that we tentatively assign to the deprotonated water adduct. We suggest that proton ejection from the phenol group leads to a charge transfer from the phenol to the imidazolinone ring, which triggers imidazolinone protonation by nearby Glu222 and catalyzes the addition of the water molecule.
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The competition between excited-state proton transfer (ESPT) and torsion plays a central role in the photophysics of fluorescent proteins of the green fluorescent protein (GFP) family and their chromophores. Here, it was investigated in a single GFP chromophore analogue bearing o-hydroxy and p-diethylamino substituents, OHIM. The light-induced dynamics of OHIM was studied by femtosecond transient absorption spectroscopy, at different pH. We found that the photophysics of OHIM is determined by the electron-donating character of the diethylamino group: torsional relaxation dominates when the diethylamino group is neutral, whereas ultrafast ESPT followed by cis/trans isomerization and ground-state reprotonation are observed when the diethylamino group is protonated and therefore inactive as an electron donor.
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Proteínas Fluorescentes Verdes/química , Simulación de Dinámica Molecular , Protones , Electrones , Concentración de Iones de Hidrógeno , Estructura MolecularRESUMEN
Non-invasive separation-free protocols are attractive for analyzing complex mixtures. To increase selectivity, an analysis under kinetic control, through exploitation of the photochemical reactivity of labeling contrast agents, is described. The simple protocol is applied in optical fluorescence microscopy, where autofluorescence, light scattering, as well as spectral crowding presents limitations. Introduced herein is OPIOM (out-of-phase imaging after optical modulation), which exploits the rich kinetic signature of a photoswitching fluorescent probe to increase selectively and quantitatively its contrast. Filtering the specific contribution of the probe only requires phase-sensitive detection upon matching the photoswitching dynamics of the probe and the intensity and frequency of a modulated monochromatic light excitation. After in vitro validation, we applied OPIOM for selective imaging in mammalian cells and zebrafish, thus opening attractive perspectives for multiplexed observations in biological samples.
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Colorantes Fluorescentes/química , Imagen Óptica , Animales , Células HEK293 , Humanos , Cinética , Microscopía Fluorescente , Procesos Fotoquímicos , Pez CebraRESUMEN
Dronpa is a photochromic green fluorescent protein (GFP) homologue used as a probe in super-resolution microscopy. It is known that the photochromic reaction involves cis/trans isomerization of the chromophore and protonation/deprotonation of its phenol group, but the sequence in time of the two steps and their characteristic time scales are still the subject of much debate. We report here a comprehensive UV-visible transient absorption spectroscopy study of the photoactivation mechanism of Dronpa, covering all relevant time scales from â¼100 fs to milliseconds. The Dronpa-2 variant was also studied and showed the same behavior. By carefully controlling the excitation energy to avoid multiphoton processes, we could measure both the spectrum and the anisotropy of the first photoactivation intermediate. We show that the observed few nanometer blue-shift of this intermediate is characteristic for a neutral cis chromophore, and that its anisotropy of â¼0.2 is in good agreement with the reorientation of the transition dipole moment expected upon isomerization. These data constitute the first clear evidence that trans â cis isomerization of the chromophore precedes its deprotonation and occurs on the picosecond time scale, concomitantly to the excited-state decay. We found the deprotonation step to follow in â¼10 µs and lead directly from the neutral cis intermediate to the final state.
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Proteínas Fluorescentes Verdes/química , Protones , Rayos Ultravioleta , Modelos Moleculares , Conformación Proteica , EstereoisomerismoRESUMEN
G-protein-coupled receptor homo-oligomerization has been increasingly reported. However, little is known regarding the relationship between activation of the receptor and its association/conformational states. The mammalian olfactory receptors (ORs) belong to the G protein-coupled receptor superfamily. In this study, the homo-oligomerization status of the human OR1740 receptor and its involvement in receptor activation upon odorant ligand binding were addressed by co-immunoprecipitation and bioluminescence resonance energy transfer approaches using crude membranes or membranes from different cellular compartments. For the first time, our data clearly show that mammalian ORs constitutively self-associate into homodimers at the plasma membrane level. This study also demonstrates that ligand binding mediates a conformational change and promotes an inactive state of the OR dimers at high ligand concentrations. These findings support and validate our previously proposed model of OR activation/inactivation based on the tripartite odorant-binding protein-odorant-OR partnership.
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Transferencia de Energía , Mediciones Luminiscentes , Multimerización de Proteína , Receptores Odorantes/metabolismo , Humanos , Unión Proteica , Conformación Proteica , Receptores Odorantes/químicaRESUMEN
The tendency of GFP-like fluorescent proteins to dimerize in vitro is a permanent concern as it may lead to artifacts in FRET imaging applications. However, we have found recently that CFP and YFP (the couple of GFP variants mostly used in FRET studies) show no trace of association in the cytosol of living cells up to millimolar concentrations. In this study, we investigated the oligomerization properties of purified CFP, by fluorescence anisotropy and sedimentation velocity. Surprisingly, we found that CFP has a much weaker homoaffinity than other fluorescent proteins (K(d) ≥ 3 × 10(-3) M), and that this is due to the constitutive N146I mutation, originally introduced into CFP to improve its brightness.