Phototransformable fluorescent proteins: which one for which application? (original) (raw)
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Phototransformable fluorescent proteins: Future challenges
Current Opinion in Chemical Biology, 2014
In fluorescence microscopy, the photophysical properties of the fluorescent markers play a fundamental role. The beauty of phototransformable fluorescent proteins (PTFPs) is that some of these properties can be precisely controlled by light. A wide range of PTFPs have been developed in recent years, including photoactivatable, photoconvertible and photoswitchable fluorescent proteins. These smart labels triggered a plethora of advanced fluorescence methods to scrutinize biological cells or organisms dynamically, quantitatively and with unprecedented resolution. Despite continuous improvements, PTFPs still suffer from limitations, and mechanistic questions remain as to how these proteins precisely work.
ABSTRACTFluorescent proteins, while essential for bioimaging, are limited to visualizing cellular localization without offering additional functionality. We report for the first time a strategy to expand the chemical, structural, and functional diversity of fluorescent proteins by harnessing light to induce red fluorescence in a previously non-fluorescent protein. We accomplish this by inducing the transfer of the genetically encoded chromophore from a photocleavable protein (PhoCl1) to a non-fluorescent kinase (MjRibK) inducing red fluorescence in the latter. We have employed analytical and spectroscopic techniques to validate the presence of red fluorescence inMjRibK. Furthermore, molecular dynamics simulations were carried out to investigate the amino acid residues ofMjRibK involved in the generation of red fluorescence. Finally, we demonstrate the ability of the red fluorescentMjRibK to operate as a cyclable high-temperature sensor. We anticipate that this light-induced chromoph...
Photoactivatable and Photoconvertible Fluorescent Probes for Protein Labeling
ACS Chemical Biology, 2010
Photosensitive probes are powerful tools to study cellular processes with high temporal and spatial resolution. However, most synthetic fluorophores suited for biomolecular imaging have not been converted yet to appropriate photosensitive analogues. Here we describe a generally applicable strategy for the generation of photoactivatable and photoconvertible fluorescent probes that can be selectively coupled to SNAP-tag fusion proteins in living cells. Photoactivatable versions of fluorescein and Cy3 as well as a photoconvertible Cy5-Cy3 probe were prepared and coupled to selected proteins on the cell surface, in the cytosol, and in the nucleus of cells. In proof-of-principle experiments, the photoactivatable Cy3 probe was used to characterize the mobility of a lipid-anchored cell surface protein and of a G protein coupled receptor (GPCR). This work establishes a generally applicable strategy for the generation of a large variety of different photosensitive fluorophores with tailor-made properties for biomolecular imaging. ARTICLE www.acschemicalbiology.org VOL.5 NO.5 • ACS CHEMICAL BIOLOGY
Green to red photoconversion of GFP for protein tracking in vivo
Scientific Reports, 2015
A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.
A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization
Journal of Molecular Biology, 2010
The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequencealignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells.
Photoconvertible fluorescent protein EosFP: Biophysical properties and cell biology applications
Photochemistry and Photobiology, 2006
EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30"C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37°C. A variety of applications in cellular imaging, developmental biology and automated highcontent screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.
Advances in engineering of fluorescent proteins and photoactivatable proteins with red emission
Current Opinion in Chemical Biology, 2010
Monomeric fluorescent proteins of different colors are widely used to study behavior and targeting of proteins in living cells. Fluorescent proteins that irreversibly change their spectral properties in response to light irradiation of a specific wavelength, or photoactivate, have become increasingly popular to image intracellular dynamics and super-resolution protein localization. Until recently, however, no optimized monomeric red fluorescent proteins and red photoactivatable proteins have been available. Furthermore, monomeric fluorescent proteins, which change emission from blue to red simply with time, so-called fluorescent timers, were developed to study protein age and turnover. Understanding of chemical mechanisms of the chromophore maturation or photoactivation into a red form will further advance engineering of fluorescent timers and photoactivatable proteins with enhanced and novel properties.
Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light
Nature Biotechnology, 2006
Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes 1,2. In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed 3. Except for tetrameric KFP1 (ref. 4), all known PAFPs, including PA-GFP 5 , Kaede 6 , EosFP 7 , PS-CFP 8 , Dronpa 9 , PA-mRFP1 10 and KikGR 11 require light in the UV-violet spectral region for activation through one-photon excitation-such light can be phototoxic to some biological systems 12. Here, we report a monomeric PAFP, Dendra, derived from octocoral Dendronephthya sp. and capable of 1,000-to 4,500-fold photoconversion from green to red fluorescent states in response to either visible blue or UV-violet light. Dendra represents the first PAFP, which is simultaneously monomeric, efficiently matures at 37 1C, demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. We demonstrate the suitability of Dendra for protein labeling and tracking to quantitatively study dynamics of fibrillarin and vimentin in mammalian cells.
Generation of photoactivatable fluorescent protein from photoconvertible ancestor
Russian Journal of Bioorganic Chemistry, 2017
DendFP protein from Dendronephthya sp. converts from the green to red fluorescent state under UV light. We have obtained the mutant variant of the protein, which, in contrast to original DendFP, tends to be phototransformed from the nonfluorescent form to the green fluorescent state.
The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins
Trends in biochemical sciences, 2016
Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.