The Experts below are selected from a list of 234 Experts worldwide ranked by ideXlab platform
Johan Hofkens - One of the best experts on this subject based on the ideXlab platform.
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green to red photoconvertible Dronpa mutant for multimodal super resolution fluorescence microscopy
ACS Nano, 2014Co-Authors: Benjamien Moeyaert, Ngan Nguyen Bich, Hideaki Mizuno, Johan Hofkens, Luc Van Meervelt, Elke De Zitter, Susana Rocha, Koen Clays, Peter DedeckerAbstract:Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent protein Dronpa that matures up to three times faster while retaining its interesting photochromic features. Using a combined evolutionary and structure-driven rational design strategy, we developed a green-to-red photoconvertible ffDronpa mutant, called pcDronpa, and explored different optimization strategies that resulted in its improved version, pcDronpa2. This fluorescent probe combines a high brightness with low photobleaching and photoblinking. We herein show that, despite its tetrameric nature, pcDronpa2 allows for multimodal subdiffraction imaging by sequentially imaging a given sample using both super-resolution fluctuation imaging and localization microscopy.
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engineering characterization and usage of a green to red photoconvertible Dronpa mutant
Biophysical Journal, 2013Co-Authors: Benjamien Moeyaert, Ngan Nguyen Bich, Peter Dedecker, Hideaki Mizuno, Luc Van Meervelt, Johan HofkensAbstract:The development of fluorescent proteins (FPs) and recent advances in diffraction-unlimited far-field optical microscopy have truly revolutionized our understanding of life and disease. Especially, genetic fusions of photoactivatable FPs have allowed the visualization of biological events at the nanometer scale. However, for these complicated microscopy schemes, one does not merely need a fluorescing species but rather an intelligent probe. Of remarkable interest are the photoswitchable FPs, that can be reversibly switched off and on, and the photoconvertible FPs that can be irreversibly converted from a green- to a red-emitting state.We previously reported on the rational design of a four-way optical highlighter based on Dendra2, a green-to-red photoconvertible probe (Adam et al, Chemistry & Biology, 2011). This protein, which we called NijiFP, is not only green-to-red photoconvertible, but also shows reversible photoswitching in both states. In the present work, we inverted this question and engineered a similar optical highlighter probe, this time by engineering green-to-red photoconversion properties into the photoswitchable protein Dronpa.We made ffDronpa, a Dronpa mutant that is formed up to three times as fast as Dronpa, while retaining the interesting photochromic features of Dronpa. Using rational and random mutagenesis, we transformed our fast folding Dronpa to pcDronpa. This Dronpa mutant combines Dronpa's photochromism with the feature of being photoconvertible to a red-emitting state. pcDronpa was studied in detail structurally and spectroscopically and was applied in several microscopic settings.The addition of pcDronpa to the repertoire of intelligent probes creates new opportunities for protein design and fluorescence imaging. Our example demonstrates that it is possible to elegantly introduce photodynamic behaviors in a single protein scaffold, in this case Dronpa. This work will open up new possibilities for rational and random design of FPs and their complicated photodynamic properties.
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structural basis for the influence of a single mutation k145n on the oligomerization and photoswitching rate of Dronpa
Acta Crystallographica Section D-biological Crystallography, 2012Co-Authors: Ngan Nguyen Bich, Benjamien Moeyaert, Kristof Van Hecke, Peter Dedecker, Hideaki Mizuno, Johan Hofkens, Luc Van MeerveltAbstract:The crystal structure of the on-state of PDM1-4, a single-mutation variant of the photochromic fluorescent protein Dronpa, is reported at 1.95 A resolution. PDM1-4 is a Dronpa variant that possesses a slower off-switching rate than Dronpa and thus can effectively increase the image resolution in subdiffraction optical microscopy, although the precise molecular basis for this change has not been elucidated. This work shows that the Lys145Asn mutation in PDM1-4 stabilizes the interface available for dimerization, facilitating oligomerization of the protein. No significant changes were observed in the chromophore environment of PDM1-4 compared with Dronpa, and the ensemble absorption and emission properties of PDM1-4 were highly similar to those of Dronpa. It is proposed that the slower off-switching rate in PDM1-4 is caused by a decrease in the potential flexibility of certain β-strands caused by oligomerization along the AC interface.
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how is cis trans isomerization controlled in Dronpa mutants a replica exchange molecular dynamics study
Journal of Chemical Theory and Computation, 2008Co-Authors: Samuel L C Moors, Peter Dedecker, Johan Hofkens, Cristina Flors, Servaas Michielssens, Arnout CeulemansAbstract:The reversibly photoactivatable green fluorescent protein analog Dronpa holds great promise as a marker for various new cellular imaging applications. Using a replica exchange method which combines both Hamiltonian and temperature exchanges, the ground-state dynamics of Dronpa and two mutants with increased switching kinetics, Val157Gly and Met159Thr, were compared. The dominant chromophore state was found to be the cis isomer in all three proteins. The simulation data suggest that both mutations strongly increase the chromophore flexibility and cis-trans isomerization rate. We identify three key amino acids, Val157, Met159, and Phe173, which are able to impede the bottom hula-twist transition path, depending on their position and rotameric state. We believe our insights will help to understand the switching process and provide useful information for the design of new variants with improved fluorescence properties.
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subdiffraction imaging through the selective donut mode depletion of thermally stable photoswitchable fluorophores numerical analysis and application to the fluorescent protein Dronpa
Journal of the American Chemical Society, 2007Co-Authors: Peter Dedecker, Hideaki Mizuno, Junichi Hotta, Ryoko Ando, Cristina Flors, Hiroshi Ujii, And Atsushi Miyawaki, Michel Sliwa, Maarten B J Roeffaers, Johan HofkensAbstract:The fast and reversible on/off switching of the fluorescence emission of the GFP-like fluorescent protein Dronpa has attracted considerable interest for applications in subdiffraction imaging. In this paper we study the use of a donut-mode beam in combination with two more overlapping laser beams to increase the imaging resolution through selective switching to the nonfluorescent photoswitched state. We devise and run a series of numerical simulations to determine suitable photophysical parameters of prospective, thermally stable photoswitchable molecules, in terms of photoswitching quantum yields, fatigue resistance, and possible presence of transient nonfluorescent states. Many of our findings are applicable to other measurements that make use of donut beams, and these guidelines can be used in the synthesis and screening of novel photoswitchable compounds. We experimentally demonstrate the possibility of obtaining increased resolution by making use of the efficient and thermally stable Dronpa photoswit...
Hideaki Mizuno - One of the best experts on this subject based on the ideXlab platform.
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green to red photoconvertible Dronpa mutant for multimodal super resolution fluorescence microscopy
ACS Nano, 2014Co-Authors: Benjamien Moeyaert, Ngan Nguyen Bich, Hideaki Mizuno, Johan Hofkens, Luc Van Meervelt, Elke De Zitter, Susana Rocha, Koen Clays, Peter DedeckerAbstract:Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent protein Dronpa that matures up to three times faster while retaining its interesting photochromic features. Using a combined evolutionary and structure-driven rational design strategy, we developed a green-to-red photoconvertible ffDronpa mutant, called pcDronpa, and explored different optimization strategies that resulted in its improved version, pcDronpa2. This fluorescent probe combines a high brightness with low photobleaching and photoblinking. We herein show that, despite its tetrameric nature, pcDronpa2 allows for multimodal subdiffraction imaging by sequentially imaging a given sample using both super-resolution fluctuation imaging and localization microscopy.
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engineering characterization and usage of a green to red photoconvertible Dronpa mutant
Biophysical Journal, 2013Co-Authors: Benjamien Moeyaert, Ngan Nguyen Bich, Peter Dedecker, Hideaki Mizuno, Luc Van Meervelt, Johan HofkensAbstract:The development of fluorescent proteins (FPs) and recent advances in diffraction-unlimited far-field optical microscopy have truly revolutionized our understanding of life and disease. Especially, genetic fusions of photoactivatable FPs have allowed the visualization of biological events at the nanometer scale. However, for these complicated microscopy schemes, one does not merely need a fluorescing species but rather an intelligent probe. Of remarkable interest are the photoswitchable FPs, that can be reversibly switched off and on, and the photoconvertible FPs that can be irreversibly converted from a green- to a red-emitting state.We previously reported on the rational design of a four-way optical highlighter based on Dendra2, a green-to-red photoconvertible probe (Adam et al, Chemistry & Biology, 2011). This protein, which we called NijiFP, is not only green-to-red photoconvertible, but also shows reversible photoswitching in both states. In the present work, we inverted this question and engineered a similar optical highlighter probe, this time by engineering green-to-red photoconversion properties into the photoswitchable protein Dronpa.We made ffDronpa, a Dronpa mutant that is formed up to three times as fast as Dronpa, while retaining the interesting photochromic features of Dronpa. Using rational and random mutagenesis, we transformed our fast folding Dronpa to pcDronpa. This Dronpa mutant combines Dronpa's photochromism with the feature of being photoconvertible to a red-emitting state. pcDronpa was studied in detail structurally and spectroscopically and was applied in several microscopic settings.The addition of pcDronpa to the repertoire of intelligent probes creates new opportunities for protein design and fluorescence imaging. Our example demonstrates that it is possible to elegantly introduce photodynamic behaviors in a single protein scaffold, in this case Dronpa. This work will open up new possibilities for rational and random design of FPs and their complicated photodynamic properties.
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structural basis for the influence of a single mutation k145n on the oligomerization and photoswitching rate of Dronpa
Acta Crystallographica Section D-biological Crystallography, 2012Co-Authors: Ngan Nguyen Bich, Benjamien Moeyaert, Kristof Van Hecke, Peter Dedecker, Hideaki Mizuno, Johan Hofkens, Luc Van MeerveltAbstract:The crystal structure of the on-state of PDM1-4, a single-mutation variant of the photochromic fluorescent protein Dronpa, is reported at 1.95 A resolution. PDM1-4 is a Dronpa variant that possesses a slower off-switching rate than Dronpa and thus can effectively increase the image resolution in subdiffraction optical microscopy, although the precise molecular basis for this change has not been elucidated. This work shows that the Lys145Asn mutation in PDM1-4 stabilizes the interface available for dimerization, facilitating oligomerization of the protein. No significant changes were observed in the chromophore environment of PDM1-4 compared with Dronpa, and the ensemble absorption and emission properties of PDM1-4 were highly similar to those of Dronpa. It is proposed that the slower off-switching rate in PDM1-4 is caused by a decrease in the potential flexibility of certain β-strands caused by oligomerization along the AC interface.
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primary events of photodynamics in reversible photoswitching fluorescent protein Dronpa
Journal of Physical Chemistry Letters, 2010Co-Authors: Lung Wa Chung, Hideaki Mizuno, Atsushi Miyawaki, Keiji MorokumaAbstract:Reversible photoswitching fluorescent protein Dronpa can reversibly switch between fluorescent on-state and nonfluorescent off-state by two radiations. The primary events of photodynamics in Dronpa were elucidated by nonadiabatic ONIOM (CASSCF:AMBER) molecular dynamics simulations. All radiationless decay processes are found mainly to result from one bond flip of a bridge C−C bond of the chromophore in the protein, regardless of its protonation state or conformation, rather than hula twisting. In the off-state protein, trans−cis photoisomerization of the neutral trans chromophore takes place via a rotation around the imidazolinone ring. In the wild-type on-state protein, the anionic cis chromophore mostly remains planar for at least 20 ps. In contrast, in the H193T mutant on-state, faster decay via a rotation of the phenoxy ring or imidazolinone ring of the anionic cis chromophore was found, suggesting that flexibility of the chromophore and its immediate protein environment is the key to radiationless de...
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a theoretical study on the nature of on and off states of reversibly photoswitching fluorescent protein Dronpa absorption emission protonation and raman
Journal of Physical Chemistry B, 2010Co-Authors: Lung Wa Chung, Hideaki Mizuno, Atsushi Miyawaki, Keiji MorokumaAbstract:A GFP-like fluorescent protein, Dronpa, which was engineered from a coral Pectiniidae, was found to display perfect photochromic properties; the fluorescent “on”-state and nonfluorescent “off”-state of Dronpa can be reversibly switched by irradiation of two different wavelengths of light. To understand the detailed mechanism of the reversible photoswitching process at the atomic level, we performed QM and ONIOM(QM:MM) calculations to study the nature of the proposed on-state and off-state. Several high-level QM methods (TD-B3LYP, CASSCF, CASPT2, and SAC-CI) were employed to compute the vertical absorption and emission energies in the gas phase for four different protonation states as well as two conformations. The vertical absorption and emission energies of the on- and off-states in the proteins were further studied by the ONIOM(QM:MM) calculations. The ONIOM calculations on the absorption and emission suggest the neutral trans form is the off-state and the anionic cis form is the on-state. The dominant ...
Peter Dedecker - One of the best experts on this subject based on the ideXlab platform.
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spatial distribution and temporal evolution of Dronpa fused snap25 clusters in adrenal chromaffin cells
Photochemical and Photobiological Sciences, 2015Co-Authors: Yasuko Antoku, Peter Dedecker, Paulo S Pinheiro, Tom Vosch, Jakob B SorensenAbstract:Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (Soluble NSF Attachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information. Here, we have imaged localized fluorescence bursts of Dronpa-fused SNAP-25 molecules in live chromaffin cells by Total Internal Reflection Fluorescence (TIRF) imaging. We find that this method allows tracking protein cluster dynamics over relatively long times (∼20 min.), partly due to the diffusion into the TIRF field of fresh molecules, making possible the simultaneous identification of cluster size, location and temporal evolution. The results indicate that the Dronpa-fused SNAP-25 clusters display rich dynamics, going from staying constant to disappearing and reappearing in specific cluster domains within minutes.
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green to red photoconvertible Dronpa mutant for multimodal super resolution fluorescence microscopy
ACS Nano, 2014Co-Authors: Benjamien Moeyaert, Ngan Nguyen Bich, Hideaki Mizuno, Johan Hofkens, Luc Van Meervelt, Elke De Zitter, Susana Rocha, Koen Clays, Peter DedeckerAbstract:Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent protein Dronpa that matures up to three times faster while retaining its interesting photochromic features. Using a combined evolutionary and structure-driven rational design strategy, we developed a green-to-red photoconvertible ffDronpa mutant, called pcDronpa, and explored different optimization strategies that resulted in its improved version, pcDronpa2. This fluorescent probe combines a high brightness with low photobleaching and photoblinking. We herein show that, despite its tetrameric nature, pcDronpa2 allows for multimodal subdiffraction imaging by sequentially imaging a given sample using both super-resolution fluctuation imaging and localization microscopy.
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engineering characterization and usage of a green to red photoconvertible Dronpa mutant
Biophysical Journal, 2013Co-Authors: Benjamien Moeyaert, Ngan Nguyen Bich, Peter Dedecker, Hideaki Mizuno, Luc Van Meervelt, Johan HofkensAbstract:The development of fluorescent proteins (FPs) and recent advances in diffraction-unlimited far-field optical microscopy have truly revolutionized our understanding of life and disease. Especially, genetic fusions of photoactivatable FPs have allowed the visualization of biological events at the nanometer scale. However, for these complicated microscopy schemes, one does not merely need a fluorescing species but rather an intelligent probe. Of remarkable interest are the photoswitchable FPs, that can be reversibly switched off and on, and the photoconvertible FPs that can be irreversibly converted from a green- to a red-emitting state.We previously reported on the rational design of a four-way optical highlighter based on Dendra2, a green-to-red photoconvertible probe (Adam et al, Chemistry & Biology, 2011). This protein, which we called NijiFP, is not only green-to-red photoconvertible, but also shows reversible photoswitching in both states. In the present work, we inverted this question and engineered a similar optical highlighter probe, this time by engineering green-to-red photoconversion properties into the photoswitchable protein Dronpa.We made ffDronpa, a Dronpa mutant that is formed up to three times as fast as Dronpa, while retaining the interesting photochromic features of Dronpa. Using rational and random mutagenesis, we transformed our fast folding Dronpa to pcDronpa. This Dronpa mutant combines Dronpa's photochromism with the feature of being photoconvertible to a red-emitting state. pcDronpa was studied in detail structurally and spectroscopically and was applied in several microscopic settings.The addition of pcDronpa to the repertoire of intelligent probes creates new opportunities for protein design and fluorescence imaging. Our example demonstrates that it is possible to elegantly introduce photodynamic behaviors in a single protein scaffold, in this case Dronpa. This work will open up new possibilities for rational and random design of FPs and their complicated photodynamic properties.
-
structural basis for the influence of a single mutation k145n on the oligomerization and photoswitching rate of Dronpa
Acta Crystallographica Section D-biological Crystallography, 2012Co-Authors: Ngan Nguyen Bich, Benjamien Moeyaert, Kristof Van Hecke, Peter Dedecker, Hideaki Mizuno, Johan Hofkens, Luc Van MeerveltAbstract:The crystal structure of the on-state of PDM1-4, a single-mutation variant of the photochromic fluorescent protein Dronpa, is reported at 1.95 A resolution. PDM1-4 is a Dronpa variant that possesses a slower off-switching rate than Dronpa and thus can effectively increase the image resolution in subdiffraction optical microscopy, although the precise molecular basis for this change has not been elucidated. This work shows that the Lys145Asn mutation in PDM1-4 stabilizes the interface available for dimerization, facilitating oligomerization of the protein. No significant changes were observed in the chromophore environment of PDM1-4 compared with Dronpa, and the ensemble absorption and emission properties of PDM1-4 were highly similar to those of Dronpa. It is proposed that the slower off-switching rate in PDM1-4 is caused by a decrease in the potential flexibility of certain β-strands caused by oligomerization along the AC interface.
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how is cis trans isomerization controlled in Dronpa mutants a replica exchange molecular dynamics study
Journal of Chemical Theory and Computation, 2008Co-Authors: Samuel L C Moors, Peter Dedecker, Johan Hofkens, Cristina Flors, Servaas Michielssens, Arnout CeulemansAbstract:The reversibly photoactivatable green fluorescent protein analog Dronpa holds great promise as a marker for various new cellular imaging applications. Using a replica exchange method which combines both Hamiltonian and temperature exchanges, the ground-state dynamics of Dronpa and two mutants with increased switching kinetics, Val157Gly and Met159Thr, were compared. The dominant chromophore state was found to be the cis isomer in all three proteins. The simulation data suggest that both mutations strongly increase the chromophore flexibility and cis-trans isomerization rate. We identify three key amino acids, Val157, Met159, and Phe173, which are able to impede the bottom hula-twist transition path, depending on their position and rotameric state. We believe our insights will help to understand the switching process and provide useful information for the design of new variants with improved fluorescence properties.
Ryoko Ando - One of the best experts on this subject based on the ideXlab platform.
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subdiffraction imaging through the selective donut mode depletion of thermally stable photoswitchable fluorophores numerical analysis and application to the fluorescent protein Dronpa
Journal of the American Chemical Society, 2007Co-Authors: Peter Dedecker, Hideaki Mizuno, Junichi Hotta, Ryoko Ando, Cristina Flors, Hiroshi Ujii, And Atsushi Miyawaki, Michel Sliwa, Maarten B J Roeffaers, Johan HofkensAbstract:The fast and reversible on/off switching of the fluorescence emission of the GFP-like fluorescent protein Dronpa has attracted considerable interest for applications in subdiffraction imaging. In this paper we study the use of a donut-mode beam in combination with two more overlapping laser beams to increase the imaging resolution through selective switching to the nonfluorescent photoswitched state. We devise and run a series of numerical simulations to determine suitable photophysical parameters of prospective, thermally stable photoswitchable molecules, in terms of photoswitching quantum yields, fatigue resistance, and possible presence of transient nonfluorescent states. Many of our findings are applicable to other measurements that make use of donut beams, and these guidelines can be used in the synthesis and screening of novel photoswitchable compounds. We experimentally demonstrate the possibility of obtaining increased resolution by making use of the efficient and thermally stable Dronpa photoswit...
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a stroboscopic approach for fast photoactivation localization microscopy with Dronpa mutants
Journal of the American Chemical Society, 2007Co-Authors: Cristina Flors, Peter Dedecker, Hideaki Mizuno, Junichi Hotta, Ryoko Ando, Hiroshi Ujii, And Atsushi Miyawaki, Johan HofkensAbstract:The photophysical properties and photoswitching scheme of the reversible photoswitchable green fluorescent protein-like fluorescent proteins Dronpa-2 and Dronpa-3 were investigated by means of ensemble and single-molecule fluorescence spectroscopy and compared to those of the precursor protein Dronpa. The faster response to light and the faster dark recovery of the new mutants observed in bulk also hold at the single-molecule level. Analysis of the single-molecule traces allows us to extract the efficiencies and rate constants of the pathways involved in the forward and backward switching, and we find important differences when comparing the mutants to Dronpa. We rationalize our results in terms of a higher conformational freedom of the chromophore in the protein environment provided by the β-can. This thorough understanding of the photophysical parameters has allowed us to optimize the acquisition parameters for camera-based sub-diffraction-limit imaging with these photochromic proteins. We show that Dro...
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highlighted generation of fluorescence signals using simultaneous two color irradiation on Dronpa mutants
Biophysical Journal, 2007Co-Authors: Ryoko Ando, Hideaki Mizuno, Johan Hofkens, Cristina Flors, Atsushi MiyawakiAbstract:Dronpa absorbs blue light and emits bright green fluorescence. It can also be converted by strong irradiation at 490 nm to a nonfluorescent state, which can then be switched back to the original emissive state with irradiation at 400 nm. Through semirandom mutagenesis studies, we have developed two mutants of Dronpa that show efficient photoswitching kinetics. Compared to Dronpa, the mutants can be turned off by blue light more efficiently. Thus, excitation with an argon laser line (488 nm) makes the mutants quickly become dark such that no substantial fluorescence signals can be observed. Excitation with a violet laser diode (405 nm) also produces no fluorescence signals. Simultaneous 488- and 405-nm irradiation, however, results in a rapid oscillation between the two states, thereby keeping the emissive state population large enough to produce sufficiently bright fluorescence signals.
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ultrafast excited state dynamics of the photoswitchable protein Dronpa
Journal of the American Chemical Society, 2007Co-Authors: Eduard Fron, Hideaki Mizuno, Ryoko Ando, Satoshi Habuchi, Cristina Flors, And Atsushi Miyawaki, Gerd Schweitzer, Frans C De Schryver, Johan HofkensAbstract:Dronpa is a photoswitchable protein from the family of green fluorescent proteins (GFPs). Photoswitching involves forward and backward proton transfer between a bright deprotonated form (B) and a dark protonated form (A2). We have used femtosecond transient absorption spectroscopy to determine the kinetics of the first step of the photoconversion from the dark to the bright form, which we have measured as 4 ps. The 2-fold isotope effect on the kinetics of this process shows that excited-state proton transfer (ESPT) is involved in this step. It was also demonstrated that the acid-induced protonated form A1 and the photoconverted protonated form A2 are two distinct spectroscopic species.
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fast and reversible photoswitching of the fluorescent protein Dronpa as evidenced by fluorescence correlation spectroscopy
Biophysical Journal, 2006Co-Authors: Peter Dedecker, Junichi Hotta, Ryoko Ando, Atsushi Miyawaki, Yves Engelborghs, Johan HofkensAbstract:Controlling molecular properties through photoirradiation holds great promise for its potential for noninvasive and selective manipulation of matter. Photochromism has been observed for several different molecules, including green fluorescent proteins, and recently the discovery of a novel photoswitchable green fluorescent protein called Dronpa was reported. Dronpa displays reversible and highly efficient on/off photoswitching of its fluorescence emission, and reversible switching of immobilized single molecules of Dronpa with response times faster than 20 ms was demonstrated. In this Letter, we expand these observations to freely diffusing molecules by using fluorescence correlation spectroscopy with simultaneous excitation at 488 and 405 nm. By varying the intensity of irradiation at 405 nm, we demonstrate the reversible photoswitching of Dronpa under these conditions, and from the obtained autocorrelation functions we conclude that this photoswitching can occur within tens of microseconds.
Atsushi Miyawaki - One of the best experts on this subject based on the ideXlab platform.
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primary events of photodynamics in reversible photoswitching fluorescent protein Dronpa
Journal of Physical Chemistry Letters, 2010Co-Authors: Lung Wa Chung, Hideaki Mizuno, Atsushi Miyawaki, Keiji MorokumaAbstract:Reversible photoswitching fluorescent protein Dronpa can reversibly switch between fluorescent on-state and nonfluorescent off-state by two radiations. The primary events of photodynamics in Dronpa were elucidated by nonadiabatic ONIOM (CASSCF:AMBER) molecular dynamics simulations. All radiationless decay processes are found mainly to result from one bond flip of a bridge C−C bond of the chromophore in the protein, regardless of its protonation state or conformation, rather than hula twisting. In the off-state protein, trans−cis photoisomerization of the neutral trans chromophore takes place via a rotation around the imidazolinone ring. In the wild-type on-state protein, the anionic cis chromophore mostly remains planar for at least 20 ps. In contrast, in the H193T mutant on-state, faster decay via a rotation of the phenoxy ring or imidazolinone ring of the anionic cis chromophore was found, suggesting that flexibility of the chromophore and its immediate protein environment is the key to radiationless de...
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a theoretical study on the nature of on and off states of reversibly photoswitching fluorescent protein Dronpa absorption emission protonation and raman
Journal of Physical Chemistry B, 2010Co-Authors: Lung Wa Chung, Hideaki Mizuno, Atsushi Miyawaki, Keiji MorokumaAbstract:A GFP-like fluorescent protein, Dronpa, which was engineered from a coral Pectiniidae, was found to display perfect photochromic properties; the fluorescent “on”-state and nonfluorescent “off”-state of Dronpa can be reversibly switched by irradiation of two different wavelengths of light. To understand the detailed mechanism of the reversible photoswitching process at the atomic level, we performed QM and ONIOM(QM:MM) calculations to study the nature of the proposed on-state and off-state. Several high-level QM methods (TD-B3LYP, CASSCF, CASPT2, and SAC-CI) were employed to compute the vertical absorption and emission energies in the gas phase for four different protonation states as well as two conformations. The vertical absorption and emission energies of the on- and off-states in the proteins were further studied by the ONIOM(QM:MM) calculations. The ONIOM calculations on the absorption and emission suggest the neutral trans form is the off-state and the anionic cis form is the on-state. The dominant ...
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highlighted generation of fluorescence signals using simultaneous two color irradiation on Dronpa mutants
Biophysical Journal, 2007Co-Authors: Ryoko Ando, Hideaki Mizuno, Johan Hofkens, Cristina Flors, Atsushi MiyawakiAbstract:Dronpa absorbs blue light and emits bright green fluorescence. It can also be converted by strong irradiation at 490 nm to a nonfluorescent state, which can then be switched back to the original emissive state with irradiation at 400 nm. Through semirandom mutagenesis studies, we have developed two mutants of Dronpa that show efficient photoswitching kinetics. Compared to Dronpa, the mutants can be turned off by blue light more efficiently. Thus, excitation with an argon laser line (488 nm) makes the mutants quickly become dark such that no substantial fluorescence signals can be observed. Excitation with a violet laser diode (405 nm) also produces no fluorescence signals. Simultaneous 488- and 405-nm irradiation, however, results in a rapid oscillation between the two states, thereby keeping the emissive state population large enough to produce sufficiently bright fluorescence signals.
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fast and reversible photoswitching of the fluorescent protein Dronpa as evidenced by fluorescence correlation spectroscopy
Biophysical Journal, 2006Co-Authors: Peter Dedecker, Junichi Hotta, Ryoko Ando, Atsushi Miyawaki, Yves Engelborghs, Johan HofkensAbstract:Controlling molecular properties through photoirradiation holds great promise for its potential for noninvasive and selective manipulation of matter. Photochromism has been observed for several different molecules, including green fluorescent proteins, and recently the discovery of a novel photoswitchable green fluorescent protein called Dronpa was reported. Dronpa displays reversible and highly efficient on/off photoswitching of its fluorescence emission, and reversible switching of immobilized single molecules of Dronpa with response times faster than 20 ms was demonstrated. In this Letter, we expand these observations to freely diffusing molecules by using fluorescence correlation spectroscopy with simultaneous excitation at 488 and 405 nm. By varying the intensity of irradiation at 405 nm, we demonstrate the reversible photoswitching of Dronpa under these conditions, and from the obtained autocorrelation functions we conclude that this photoswitching can occur within tens of microseconds.
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photo induced protonation deprotonation in the gfp like fluorescent protein Dronpa mechanism responsible for the reversible photoswitching
Photochemical and Photobiological Sciences, 2006Co-Authors: Satoshi Habuchi, Peter Dedecker, Hideaki Mizuno, Johan Hofkens, Junichi Hotta, Ryoko Ando, Atsushi Miyawaki, Cristina FlorsAbstract:Recently, reversible photoswitching in bulk samples or in individual molecules of Dronpa, a mutant of a green fluorescent protein (GFP)-like fluorescent protein, has been demonstrated. Intense irradiation at 488 nm changed Dronpa in a dim protonated form, and weak irradiation at 405 nm restored it to the bright deprotonated form. Here, we report on the mechanism of photoswitching of Dronpa by means of ensemble and single-molecule spectroscopy. Ensemble spectroscopy shows that the photoswitching can be described, in first approximation, by a three-state model including a deprotonated (B), a protonated (A1), and a photoswitched protonated (A2) forms of the chromophore. While the B and the A1 forms are in a ground state acid–base equilibrium, the B and the A2 forms are reversibly photoswitched upon irradiation with 488 and 405 nm light. At the single-molecule level, the on-times in fluorescence intensity trajectories excited at 488 nm decrease with increasing the excitation power, consistent with the photoswitching from the B to A2 form. The on-times agree well with expected values, which are calculated based on the ensemble spectroscopic properties of Dronpa. The fluorescence trajectory obtained with simultaneous dual-color excitation at 488 and 405 nm demonstrates reversible photoswitching between the B and the A2 forms at the single-molecule level. The efficiency of the photoswitching from the A2 to B form increased with increasing the excitation power of the 405 nm light. Our results demonstrate that Dronpa holds its outstanding photoswitching properties, based on a photo-induced protonation/deprotonation process, even at the single-molecule level.
