Mechanistic insights into reversible photoactivation in proteins of the GFP family.
ABSTRACT: Light-controlled modification of the fluorescence emission properties of proteins of the GFP family is of crucial importance for many imaging applications including superresolution microscopy. Here, we have studied the reversibly photoswitchable fluorescent protein mIrisGFP using optical spectroscopy. By analyzing the pH dependence of isomerization and protonation equilibria and the isomerization kinetics, we have obtained insight into the coupling of the chromophore to the surrounding protein moiety and a better understanding of the photoswitching mechanism. A different acid-base environment of the chromophore's protonating group in its two isomeric forms, which can be inferred from the x-ray structures of IrisFP, is key to the photoswitching function and ensures that isomerization and protonation are correlated. Amino acids near the chromophore, especially Glu212, rearrange upon isomerization, and Glu212 protonation modulates the chromophore pK(a). In mIrisGFP, the cis chromophore protonates in two steps, with pK(cis) of 5.3 and 6, which is much lower than pK(trans) (>10). Based on these results, we have put forward a mechanistic scheme that explains how the combination of isomeric and acid-base properties of the chromophore in its protein environment can produce negative and positive photoswitching modes.
Project description:Recent advances in fluorescent proteins (FPs) have generated a remarkable family of optical highlighters with special light responses. Among them, Dronpa exhibits a unique capability of reversible light-regulated on-off switching. However, the environmental dependence of this photochromism is largely unexplored. Herein we report that the photoswitching kinetics of the chromophore inside Dronpa is actually slowed down by increasing medium viscosity outside Dronpa. This finding is a special example of an FP where the environment can exert a hydrodynamic effect on the internal chromophore. We attribute this effect to protein-flexibility mediated coupling where the chromophore's cis-trans isomerization during photoswitching is accompanied by conformational motion of a part of the protein ?-barrel whose dynamics should be hindered by medium friction. Consistent with this mechanism, the photoswitching kinetics of Dronpa-3, a structurally more flexible mutant, is found to exhibit a more pronounced viscosity dependence. Furthermore, we mapped out spatial distributions of microviscosity in live cells experienced by a histone protein using the photoswitching kinetics of Dronpa-3 fusion as a contrast mechanism. This unique reporter should provide protein-specific information about the crowded intracellular environments by offering a genetically encoded microviscosity probe, which did not exist with normal FPs before.
Project description:rsTagRFP is the first monomeric red fluorescent protein (FP) with reversibly photoswitchable absorbance spectra. The switching is realized by irradiation of rsTagRFP with blue (440 nm) and yellow (567 nm) light, turning the protein fluorescence ON and OFF, respectively. It is perhaps the most useful probe in this color class that has yet been reported. Because of the photoswitchable absorbance, rsTagRFP can be used as an acceptor in photochromic Förster resonance energy transfer. Yellow FPs, YPet and mVenus, are demonstrated to be excellent photochromic Förster resonance energy transfer donors for the rsTagRFP acceptor in its fusion constructs. Analysis of X-ray structures has shown that photoswitching of rsTagRFP is accompanied by cis-trans isomerization and protonation/deprotonation of the chromophore, with the deprotonated cis- and protonated trans-isomers corresponding to its ON and OFF states, respectively. Unlike in other photoswitchable FPs, both conformers of rsTagRFP chromophore are essentially coplanar. Two other peculiarities of the rsTagRFP chromophore are an essentially hydrophobic environment of its p-hydroxyphenyl site and the absence of direct hydrogen bonding between this moiety and the protein scaffold. The influence of the immediate environment on rsTagRFP chromophore was probed by site-directed mutagenesis. Residues Glu145 and His197 were found to participate in protonation/deprotonation of the chromophore accompanying the photoswitching of rsTagRFP fluorescence, whereas residues Met160 and Leu174 were shown to spatially restrict chromophore isomerization, favoring its radiative decay.
Project description:Reversibly photoswitchable fluorescent proteins (RSFPs) are fluorescent proteins whose fluorescence, upon excitation at a certain wavelength, can be switched on or off by light in a reversible manner. In the last 10 years, many new RSFPs have been developed and novel applications in cell imaging discovered that rely on their photoswitching properties. This review will describe research on the mechanisms of reversible photoswitching and recent applications using RSFPs. While cis-trans isomerization of the chromophore is believed to be the general mechanism for most RSFPs, structural studies reveal diversity in the details of photoswitching mechanisms, including different effects of protonation, chromophore planarity, and pocket flexibility. Applications of RSFPs include new types of live-cell superresolution imaging, tracking of protein movements and interactions, information storage, and optical control of protein activity.
Project description:Fluorescent proteins have been widely used as genetically encodable fusion tags for biological imaging. Recently, a new class of fluorescent proteins was discovered that can be reversibly light-switched between a fluorescent and a non-fluorescent state. Such proteins can not only provide nanoscale resolution in far-field fluorescence optical microscopy much below the diffraction limit, but also hold promise for other nanotechnological applications, such as optical data storage. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements to their properties requires a detailed understanding of the molecular switching mechanism, which is currently unknown. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 at the atomic level by multiconfigurational ab initio (CASSCF) calculations and QM/MM excited state molecular dynamics simulations with explicit surface hopping. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Accordingly, changes in the protonation state of the chromophore and some proximal amino acids lead to different photochemical states, which all turn out to be essential for the photoswitching mechanism. These photochemical states are (i) a neutral chromophore, which can trans-cis photoisomerize, (ii) an anionic chromophore, which rapidly undergoes radiationless decay after excitation, and (iii) a putative fluorescent zwitterionic chromophore. The overall stability of the different protonation states is controlled by the isomeric state of the chromophore. We finally propose that radiation-induced decarboxylation of the glutamic acid Glu215 blocks the proton transfer pathways that enable the deactivation of the zwitterionic chromophore and thus leads to irreversible fluorescence. We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data. The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins.
Project description:Reversibly switchable fluorescent proteins can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with the light of two different wavelengths. The molecular basis of the switching process remains a controversial topic. Padron0.9 is a reversibly switchable fluorescent protein with "positive" switching characteristics, exhibiting excellent spectroscopic properties. Its chromophore is formed by the amino acids Cys-Tyr-Gly. We obtained high resolution x-ray structures of Padron0.9 in both the fluorescent and the nonfluorescent states and used the structural information for molecular dynamics simulations. We found that in Padron0.9 the chromophore undergoes a cis-trans isomerization upon photoswitching. The molecular dynamics simulations clarified the protonation states of the amino acid residues within the chromophore pocket that influence the protonation state of the chromophore. We conclude that a light driven cis-trans isomerization of the chromophore appears to be the fundamental switching mechanism in all photochromic fluorescent proteins known to date. Distinct absorption cross-sections for the switching wavelengths in the fluorescent and the nonfluorescent state are not essential for efficient photochromism in fluorescent proteins, although they may facilitate the switching process.
Project description:Dronpa is a novel GFP-like fluorescent protein with exceptional light-controlled switching properties. It may be reversibly switched between a fluorescent on-state and a nonfluorescent off-state by irradiation with light. To elucidate the molecular basis of the switching mechanism, we generated reversibly switchable Dronpa protein crystals. Using these crystals we determined the elusive dark-state structure of Dronpa at 1.95-A resolution. We found that the photoswitching results in a cis-trans isomerization of the chromophore accompanied by complex structural rearrangements of four nearby amino acid residues. Because of this cascade of intramolecular events, the chromophore is exposed to distinct electrostatic surface potentials, which are likely to influence the protonation equilibria at the chromophore. We suggest a comprehensive model for the light-induced switching mechanism, connecting a cascade of structural rearrangements with different protonation states of the chromophore.
Project description:Photoactivatable fluorescent proteins (FPs) are powerful fluorescent highlighters in live cell imaging and offer perspectives for optical nanoscopy and the development of biophotonic devices. Two types of photoactivation are currently being distinguished, reversible photoswitching between fluorescent and nonfluorescent forms and irreversible photoconversion. Here, we have combined crystallography and (in crystallo) spectroscopy to characterize the Phe-173-Ser mutant of the tetrameric variant of EosFP, named IrisFP, which incorporates both types of phototransformations. In its green fluorescent state, IrisFP displays reversible photoswitching, which involves cis-trans isomerization of the chromophore. Like its parent protein EosFP, IrisFP also photoconverts irreversibly to a red-emitting state under violet light because of an extension of the conjugated pi-electron system of the chromophore, accompanied by a cleavage of the polypeptide backbone. The red form of IrisFP exhibits a second reversible photoswitching process, which may also involve cis-trans isomerization of the chromophore. Therefore, IrisFP displays altogether 3 distinct photoactivation processes. The possibility to engineer and precisely control multiple phototransformations in photoactivatable FPs offers exciting perspectives for the extension of the fluorescent protein toolkit.
Project description:Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the ?s timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.
Project description:One of the distinctive features of eubacterial retinal-based proton pumps, proteorhodopsins, xanthorhodopsin, and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in Escherichia coli, especially features that depend on Asp-His interaction, the protonation state of the key aspartate, Asp85, and its ability to accept a proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH-dependent properties of ESR. In the H57M mutant, a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum with an increase in the pH from 5 to 8 indicate deprotonation of the counterion with a pK(a) of 6.3, which is also the pK(a) at which the M intermediate is observed in the photocycle of the protein solubilized in detergent [dodecyl maltoside (DDM)]. This is in contrast with the case for the wild-type protein, for which the same experiments show that the major fraction of Asp85 is deprotonated at pH >3 and that it protonates only at low pH, with a pK(a) of 2.3. The M intermediate in the wild-type photocycle accumulates only at high pH, with an apparent pK(a) of 9, via deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR, the pK(a) values for M formation and spectral shifts are 2-3 pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild-type protein versus the H57M mutant indicate that there is strong Asp-His interaction, which substantially lowers the pK(a) of Asp85 by stabilizing its deprotonated state.
Project description:Attachment of retinal to opsin forms the chromophore N-retinylidene, which isomerizes during photoactivation of rhodopsins. To test whether isomerization is crucial, custom-tailored chromophores lacking the ?-ionone ring and any isomerizable bonds were incorporated in vivo into the opsin of a blind mutant of the eukaryote Chlamydomonas reinhardtii. The analogs restored phototaxis with the anticipated action spectra, ruling out the need for isomerization in photoactivation. To further elucidate photoactivation, responses to chromophores formed from naphthalene aldehydes were studied. The resulting action spectral shifts suggest that charge separation within the excited chromophore leads to electric field-induced polarization of nearby amino acid residues and altered hydrogen bonding. This redistribution of charge facilitates the reported multiple bond rotations and protein rearrangements of rhodopsin activation. These results provide insight into the activation of rhodopsins and related GPCRs.