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Daryna Smyrnova, María Del Carmen Marín, Massimo Olivucci, Arnout Ceulemans
A set formed by five reversibly-switchable fluorescent proteins (RSFPs) display spread over 40~nm in absorption maxima and only 18~nm in emission. The five proteins -- Dronpa, rsFastLime, rsKame, Padron(anionic form) and bsDronpa -- carry exactly the same chromophore and differ just in a few mutations. Thus they form an ideal set for mechanistic investigation. Starting with the results of molecular dynamics simulations we use QM/MM calculations to investigate the effects controlling the spectral tuning . In this contribution we show that the models, which are based on CASPT2//CASSCF level of QM theory, reproduce the observed absorption trend with only a limited blue-shift of 4...
May 17, 2018: Journal of Chemical Theory and Computation
Eduardo Carrascosa, James N Bull, Michael S Scholz, Neville J A Coughlan, Seth Olsen, Uta Wille, Evan John Bieske
Fluorescent proteins have revolutionised the visualisation of biological processes, prompting efforts to understand and control their intrinsic photophysics. Here we investigate the photoisomerization of deprotonated p-hydroxybenzylidene-2,3-dimethylimidazolinone anion (HBDI- ), the chromophore in green fluorescent protein and in Dronpa protein, where it plays a role in switching between fluorescent and non-fluorescent states. In the present work, isolated HBDI- molecules are switched between the Z and E forms in the gas phase in a tandem ion mobility-mass spectrometer outfitted for selecting the initial and final isomers...
May 3, 2018: Journal of Physical Chemistry Letters
Shanshan Lyu, Jing Fang, Tianyu Duan, Linglan Fu, Junqiu Liu, Hongbin Li
Exploiting the optically controlled association and dissociation behavior of a photoswitchable fluorescent protein, Dronpa145N, here we demonstrate the engineering of an optically switchable reversible protein hydrogel using Dronpa145N-based protein building blocks. Our results open the possibility to optically tune the mechanical, chemical and structural properties of protein hydrogels.
December 14, 2017: Chemical Communications: Chem Comm
Julia Sajman, Michael Trus, Daphne Atlas, Eilon Sherman
The secretory signal elicited by membrane depolarization traverses from the Ca(2+)-bound α11.2 pore-forming subunit of the L-type Ca(2+)-channel (Cav1.2) to syntaxin 1 A (Sx1A) via an intra-membrane signaling mechanism. Here, we report the use of two-color Photo-Activated-Localization-Microscopy (PALM) to determine the relation between Cav1.2 and Sx1A in single-molecule detail. We observed nanoscale co-clusters of PAmCherry-tagged Sx1A and Dronpa-tagged α11.2 at a ~1:1 ratio. PAmCherry-tagged Sx1A(C145A), or PAmCherry-tagged Sx2, an inactive Cav1...
September 12, 2017: Scientific Reports
Kathrin Brenker, Kerstin Osthof, Jianying Yang, Michael Reth
Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics...
December 30, 2016: Journal of Visualized Experiments: JoVE
Daryna Smyrnova, Kirill Zinovjev, Iñaki Tuñón, Arnout Ceulemans
The photoswitching speed of the reversibly switchable fluorescent proteins (RSFPs) from the family of green fluorescent proteins (GFPs) changes upon mutation which is of direct importance for various high-resolution techniques. Dronpa is one of the most used RSFPs. Its point mutants rsFastLime (Dronpa V157G) and rsKame (Dronpa V157L) exhibit a striking difference in their photoswitching speed. Here the QM/MM on-the-fly string method is used in order to explore the details of the thermal isomerization mechanism...
December 22, 2016: Journal of Physical Chemistry. B
Xi Zhang, Mingshu Zhang, Dong Li, Wenting He, Jianxin Peng, Eric Betzig, Pingyong Xu
Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy...
September 13, 2016: Proceedings of the National Academy of Sciences of the United States of America
Asuka Higashino, Misao Mizuno, Yasuhisa Mizutani
Dronpa is a novel photochromic fluorescent protein that exhibits fast response to light. The present article is the first report of the resonance and preresonance Raman spectra of Dronpa. We used the intensity and frequency of Raman bands to determine the structure of the Dronpa chromophore in two thermally stable photochromic states. The acid-base equilibrium in one photochromic state was observed by spectroscopic pH titration. The Raman spectra revealed that the chromophore in this state shows a protonation/deprotonation transition with a pKa of 5...
April 7, 2016: Journal of Physical Chemistry. B
Siewert Hugelier, Johan J de Rooi, Romain Bernex, Sam Duwé, Olivier Devos, Michel Sliwa, Peter Dedecker, Paul H C Eilers, Cyril Ruckebusch
In wide-field super-resolution microscopy, investigating the nanoscale structure of cellular processes, and resolving fast dynamics and morphological changes in cells requires algorithms capable of working with a high-density of emissive fluorophores. Current deconvolution algorithms estimate fluorophore density by using representations of the signal that promote sparsity of the super-resolution images via an L1-norm penalty. This penalty imposes a restriction on the sum of absolute values of the estimates of emitter brightness...
February 25, 2016: Scientific Reports
Hanbing Lin, Jian-Min Yuan
Due to high fluctuations and quantum uncertainty, the processes of single-molecules should be treated by stochastic methods. To study fluorescence time series and their statistical properties, we have applied two stochastic methods, one of which is an analytic method to study the off-time distributions of certain fluorescence transitions and the other is Gillespie's method of stochastic simulations. These methods have been applied to study the optical transition properties of two single-molecule systems, GFPmut2 and a Dronpa-like molecule, to yield results in approximate agreement with experimental observations on these systems...
March 2016: Journal of Biological Physics
Dmitry Morozov, Gerrit Groenhof
Reversibly switchable fluorescent proteins (RSFPs) are essential for high-resolution microscopy of biological samples, but the reason why these proteins are photochromic is still poorly understood. To address this problem, we performed molecular dynamics simulations of the fast switching Met159Thr mutant of the RSFP Dronpa. Our simulations revealed a ground state structural heterogeneity in the chromophore pocket that consists of three populations with one, two, or three hydrogen bonds to the phenolate moiety of the chromophore...
January 11, 2016: Angewandte Chemie
Bei Liu, Yanhong Xue, Wei Zhao, Yan Chen, Chunyan Fan, Lusheng Gu, Yongdeng Zhang, Xiang Zhang, Lei Sun, Xiaojun Huang, Wei Ding, Fei Sun, Wei Ji, Tao Xu
We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation...
2015: Scientific Reports
Daryna Smyrnova, Benjamien Moeyaert, Servaas Michielssens, Johan Hofkens, Peter Dedecker, Arnout Ceulemans
Reversibly photoswitchable fluorescent proteins (RSFPs) are highly useful probes for a range of applications including diffraction-unlimited fluorescence microscopy. It was previously shown that reversible photoswitching not only involves cis-trans isomerization and protonation-deprotonation of the chromophore but also results in a marked difference in β-barrel flexibility. In this work, we performed flexibility profiling and functional mode analysis (FMA) using molecular dynamics calculations to study how the flexibility of the RSFP β-barrel influences the photoswitching properties of several fluorescent proteins...
September 10, 2015: Journal of Physical Chemistry. B
Cyril Ruckebusch, Romain Bernex, Franco Allegrini, Michel Sliwa, Johan Hofkens, Peter Dedecker
Recent advances in fluorescence bioimaging with single-molecule sensitivity have relied on the analysis and visualization of single-molecule data obtained on smart fluorophores. We describe an alternative method to enhance the information content of densely labeled fluorescence images. Visualization is improved by representing pixels as the dissimilarities of the fluctuations of the fluorescence signals, with the dissimilarity being taken to the mean of the signals over all the pixels. Mapping pixel dissimilarity (Mappix) results in signal and information enhancement of the output images...
May 5, 2015: Analytical Chemistry
Yasuko Antoku, Peter Dedecker, Paulo S Pinheiro, Tom Vosch, Jakob Balslev Sørensen
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...
May 2015: Photochemical & Photobiological Sciences
Xi Zhang, Xuanze Chen, Zhiping Zeng, Mingshu Zhang, Yujie Sun, Peng Xi, Jianxin Peng, Pingyong Xu
Reversibly switchable fluorescent proteins (RSFPs) can be effectively used for super-resolution optical fluctuation imaging (SOFI) based on the switching and fluctuation of single molecules. Several properties of RSFPs strongly influence the quality of SOFI images. These properties include (i) the averaged fluorescence intensity in the fluctuation state, (ii) the on/off contrast ratio, (iii) the photostability, and (iv) the oligomerization tendency. The first three properties determine the fluctuation range of the imaged pixels and the SOFI signal, which are of essential importance to the spatial resolution, and the last may lead to artificial aggregation of target proteins...
March 24, 2015: ACS Nano
Andre C Stiel, Xosé Luís Deán-Ben, Yuanyuan Jiang, Vasilis Ntziachristos, Daniel Razansky, Gil G Westmeyer
Photocontrol of reversibly switchable fluorescent proteins (RSFPs) was used to program optoacoustic signal time courses that were temporally unmixed to increase the proteins' contrast-to-noise-ratios (CNRs) in optoacoustic imaging. In this way, two variants of the RSFP Dronpa with very similar optoacoustic spectra could be readily discriminated in the presence of highly absorbing blood. Addition of temporal unmixing to multispectral optoacoustic tomography (tuMSOT) in conjunction with synthetic or genetically encoded photochromic contrast agents and customized photoswitching schedules can increase the performance of multiplexed and high-contrast molecular optoacoustic imaging...
February 1, 2015: Optics Letters
Marius Kaucikas, Ann Fitzpatrick, Elana Bryan, Abelone Struve, Robert Henning, Irina Kosheleva, Vukica Srajer, Gerrit Groenhof, Jasper J Van Thor
The fluorescent protein Dronpa undergoes reversible photoswitching reactions between the bright "on" and dark "off" states via photoisomerization and proton transfer reactions. We report the room temperature crystal structure of the fast switching Met159Thr mutant of Dronpa at 2.0-Å resolution in the bright on state. Structural differences with the wild type include shifted backbone positions of strand β8 containing Thr159 as well as an altered A-C dimer interface involving strands β7, β8, β10, and β11...
March 2015: Proteins
Ilaria Testa, Elisa D'Este, Nicolai T Urban, Francisco Balzarotti, Stefan W Hell
We show that RESOLFT fluorescence nanoscopy, a low light level scanning superresolution technique employing reversibly switchable fluorescent proteins (rsFPs), is capable of dual-channel live-cell imaging that is virtually free of chromatic errors and temporal offsets. This is accomplished using rsEGFP and Dronpa, two rsFPs having similar spectra but different kinetics of switching and fluorescence emission. Our approach is demonstrated by imaging protein distributions and dynamics in living neurons and neuronal tissues...
January 14, 2015: Nano Letters
Marius Kaucikas, Martijn Tros, Jasper J van Thor
The fast-switching M159T mutant of the reversibly photoswitchable fluorescent protein Dronpa has an enhanced yield for the on-to-off reaction. The forward and reverse photoreactions proceed via cis-trans and trans-cis photoisomerization, yet protonation and deprotonation of the hydroxyphenyl oxygen of the chromophore is responsible for the majority of the resulting spectroscopic contrast. Ultrafast visible-pump, infrared-probe spectroscopy was used to detect the picosecond, nanosecond, as well as metastable millisecond intermediates...
February 12, 2015: Journal of Physical Chemistry. B
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