Conversion of red fluorescent protein into a bright blue probe

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in F?rster resonance energy transfer applications.     

Constructing Photoactivatable Protein with Genetically Encoded Photocaged Glutamic Acid

Genetically replacing an essential residue with the corresponding photocaged analogues via genetic code expansion (GCE) constitutes a useful and unique strategy to directly and effectively generate photoactivatable proteins. However, the application of this strategy is severely hampered by the limited number of encoded photocaged proteinogenic amino acids. Herein, we report the genetic incorporation of photocaged glutamic acid analogues in E. coli and mammalian cells and demonstrate their use in constructing photoactivatable variants of various fluorescent proteins and SpyCatcher. We believe genetically encoded photocaged Glu would significantly promote the design and application of photoactivatable proteins in many areas.     

Rapid, photoactivatable turn-on fluorescent probes based on an intramolecular photoclick reaction

Photoactivatable fluorescent probes are invaluable tools for the study of biological processes with high resolution in space and time. Numerous strategies have been developed in generating photoactivatable fluorescent probes, most of which rely on the photo-"uncaging" and photoisomerization reactions. To broaden photoactivation modalities, here we report a new strategy in which the fluorophore is generated in situ through an intramolecular tetrazole-alkene cycloaddition reaction ("photoclick chemistry"). By conjugating a specific microtubule-binding taxoid core to the tetrazole/alkene prefluorophores, robust photoactivatable fluorescent probes were obtained with fast photoactivation (～1 min) and high fluorescence turn-on ratio (up to 112-fold) in acetonitrile/PBS (1:1). Highly efficient photoactivation of the taxoid-tetrazoles inside the mammalian cells was also observed under a confocal fluorescence microscope when the treated cells were exposed to either a metal halide lamp light passing through a 300/395 filter or a 405 nm laser beam. Furthermore, a spatially controlled fluorescent labeling of microtubules in live CHO cells was demonstrated with a long-wavelength photoactivatable taxoid-tetrazole probe. Because of its modular design and tunability of the photoactivation efficiency and photophysical properties, this intramolecular photoclick reaction based approach should provide a versatile platform for designing photoactivatable fluorescent probes for various biological processes.     

Highly activatable and environment-insensitive optical highlighters for selective spatiotemporal imaging of target proteins

Optical highlighters are photoactivatable fluorescent molecules that exhibit pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength and intensity. Here, we present a novel design strategy for a new class of caged BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophores, based on the use of photoremovable protecting groups (PRPGs) with high reduction potentials that serve as both a photosensitive unit and a fluorescence quencher via photoinduced electron transfer (PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated, with activation ratios exceeding 600-fold in aqueous solutions. We then combined this photoactivatable fluorophore with a SNAP (mutant of O(6)-alkylguanine DNA alkyltransferase) ligand to obtain a small-molecule-based optical highlighter for visualization of protein dynamics, using the well-established SNAP tag technology. As proof of concept, we demonstrate spatiotemporal imaging of the fusion protein of epidermal growth factor receptor (EGFR) with SNAP tag in living cells. We also demonstrate highlighting of cells of interest in live zebrafish embryos, using the fusion protein of histone 2A with SNAP tag.     

Green-red flashers to accelerate biology

Photoactivatable fluorescent proteins are now widely used for cell and protein tracking and super-resolution optical imaging. In this issue, Adam et?al. (2011) report a general approach to introduce photochromism into green-to-red photoconvertible proteins and describe new photoactivatable protein with a complex four-state flasher-like behavior and advanced characteristics.     

Chemically induced photoswitching of fluorescent probes--a general concept for super-resolution microscopy

We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.     

Rational design of photoconvertible and biphotochromic fluorescent proteins for advanced microscopy applications

Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent protein combining these two types of phototransformations. The introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging. However, the spectroscopic properties of IrisFP are far from being optimal and its tetrameric organization complicates its use as a fusion tag. Here, we demonstrate how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins and develop and characterize a new set of such enhanced optical highlighters derived from mEosFP and Dendra2. We present in particular NijiFP, a promising new fluorescent protein with photoconvertible and biphotochromic properties that make it ideal for advanced fluorescence-based imaging applications.     

Patterned two-photon photoactivation illuminates spatial reorganization in live cells

Photoactivatable fluorescent proteins offer the possibility to optically tag and track the location of molecules in their bright state with high spatial and temporal resolution. Several reports of patterned photoactivation have emerged since the development of a photoactivatable variant of the green fluorescent protein (PaGFP) and the demonstration of two-photon activation of PaGFP. To date, however, there have been few methods developed to quantify the spatial reorganization of the photoactivated population. Here we report on the use of singular value decomposition (SVD) to track the time-dependent distribution of fluorophores after photoactivation. The method was used to describe live-cell actin cytoskeleton behavior in primary murine T-cells, in which a dynamic cytoskeleton is responsible for the reorganization of membrane proteins in response to antigen peptide recognition. The method was also used to observe immortalized simian kidney (Cos-7) cells, in which the cytoskeleton is more stable. Both cell types were transfected with PaGFP fused to the F-actin binding domain of utrophin (UtrCH). Photoactivation patterns were written in the samples with a pair of galvanometric scanning mirrors in circular patterns that were analyzed by transforming the images into a time series of radial distribution profiles. The time-evolution of the profiles was well-described by the first two SVD component states. For T-cells, we find that actin filaments are highly mobile. Inward transport from the photoactivation region was observed and occurred on a 1-2 s time scale, which is consistent with retrograde cycling. For Cos-7 cells, we find that the actin is relatively stationary and does not undergo significant centripetal flow as expected for a resting fibroblast. The combination of patterned photoactivation and SVD analysis offers a unique way to measure spatial redistribution dynamics within live cells.     

Rational Design of the β-Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing**

Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the β-bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the β-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.     

Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry

Fluorescent proteins (FPs) are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. In this paper, we use molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variant. We employ implicit ligand sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. We also show that these pathways can be blocked or altered and barrel fluctuations can be reduced by strategic amino acid substitutions.     

Modulation of Protein Dimerization by a Supramolecular Host–Guest System

Two sets of cyan and yellow fluorescent proteins, monomeric analogues, and analogues with a weak affinity for dimerization were functionalized with supramolecular host–guest molecules by expressed protein ligation. The host–guest elements induce selective assembly of the monomeric variants into a supramolecular heterodimer. For the second set of analogues, the supramolecular host–guest system acts in cooperation with the intrinsic affinity between the two proteins, resulting in the induction of a selective protein–protein heterodimerization at a more dilute concentration. Additionally, the supramolecular host–guest system allows locking of the two proteins in a covalent heterodimer through the facilitated and selective formation of a reversible disulfide linkage. For the monomeric analogues this results in a strong increase of the energy transfer between the proteins. The protein heterodimerization can be reversed in a stepwise fashion. The trajectory of the disassembly process differs for the monomeric and dimerizing set of proteins. The results highlight that supramolecular elements connected to proteins can both be used to facilitate the interaction between two proteins without intrinsic affinity and to stabilize weak protein–protein interactions at concentrations below those determined by the actual affinity of the proteins alone. The subsequent covalent linkage between the proteins generates a stable protein dimer as a single species. The action of the supramolecular elements in concert with the proteins thus allows the generation of protein architectures with specific properties and compositions.     

Synthesis and biological activity of photoactivatable N-ras peptides and proteins

A modular strategy for the assembly of farnesylated N-Ras heptapeptides carrying a photoactivatable benzophenone (BP) group within the lipid residue is described. This strategy is based on the fragment condensation of a N-terminal hexapeptide synthesized on the solid support with a cysteine methyl ester which is modified with different farnesyl analogues, incorporating the photophor. At the N-terminus of the peptides different functional groups can be attached, e.g., biotin for product enrichment and detection after photoactivation or a maleimido (MIC) linker, allowing for the coupling to proteins carrying a C-terminal free cysteine. Using this strategy, 24 peptides were synthesized, incorporating farnesyl analogues with four different chain lengths. Two of these photoactivatable conjugates were ligated to oncogenic human N-RasG12V Delta 181. A cellular transformation assay revealed that the semisynthetic proteins retain their biological activity despite the photolabel. The first photolabeling experiments with a geranyl-BP-labeled N-Ras construct and the farnesyl-sensitive guanine nucleotide exchange factor hSos1 indicate that this photoaffinity labeling system can be particularly useful for studying protein-protein interactions, e.g., the participation of the farnesyl group in Ras signaling, which is still discussed with controversy.     

A FRET-Facilitated Photoswitching Using an Orange Fluorescent Protein with the Fast Photoconversion Kinetics

Fluorescent proteins photoswitchable with noncytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via F?rster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells.     

Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores

Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.     

Bioorthogonal chemistry:a covalent strategy for the study of biological systems

The development of genetically encoded,wavelength-tunable fluorescent proteins has provided a powerful imaging tool to the study of protein dynamics and functions in cellular and organismal biology.However,many biological functions are not directly encoded in the protein primary sequence,e.g.,dynamic regulation afforded by protein posttranslational modifications such as phosphorylation.To meet this challenge,an emerging field of bioorthogonal chemistry has promised to offer a versatile strategy to selective...     

Trans-cis isomerization is responsible for the red-shifted fluorescence in variants of the red fluorescent protein eqFP611

An important class of red fluorescent proteins (RFPs) feature a 2-iminomethyl-5-(4-hydroxybenzylidene)imidazolinone chromophore. Among these proteins, eqFP611 has the chromophore in a coplanar trans orientation, whereas the cis isomer is preferred by other RFPs such as DsRed and its variants. In the photoactivatable protein asFP595, the chromophore can even be switched from the nonfluorescent trans to the fluorescent cis state by light. By using X-ray crystallography, we have determined the structure of dimeric eqFP611 at high resolution (up to 1.1 A). In the far-red emitting eqFP611 variant d2RFP630, which carries an additional Asn143Ser mutation, the chromophore resides predominantly (approximately 80%) in the cis isomeric state, and in RFP639, which has Asn143Ser and Ser158Cys mutations, the chromophore is found completely in the cis form. The pronounced red shift of excitation and emission maxima of RFP639 can thus unambiguously be assigned to trans-cis isomerization of the chromophore. Among RFPs, eqFP611 is thus unique because its chromophore is highly fluorescent in both the cis and trans isomeric forms.     

Resonance enhancement of two-photon absorption in fluorescent proteins

We measure two-photon absorption (2PA) spectra of wild-type green fluorescent protein, cyan fluorescent protein, and monomeric red fluorescent protein in absolute cross section values in a wide spectral range (lambda2PA = 550 - 1300 nm), and find, for the first time to our knowledge, a new S0 --> Sn 2PA transition in all three proteins in the short-wavelength region. This transition is strongly resonantly enhanced, showing 2PA cross section values of approximately 20-160 GM, which are at least 2-4 times higher than those measured in the lowest energy (S0 --> S1) transition of corresponding proteins. We also show that the change of permanent dipole moment upon S0 --> S1 excitation (|Deltamu10|) can be deduced from 2PA cross section, providing a new tool for fast evaluation of |Deltamu10| in physiological conditions.     

Development of a Versatile Protein Labeling Tool for Live-Cell Imaging Using Fluorescent β-Lactamase Inhibitors

To understand the function of protein in live cells, real-time monitoring of protein dynamics and sensing of their surrounding environment are important methods. Fluorescent labeling tools are thus needed that possess fast labeling kinetics, high efficiency, and long-term stability. We developed a versatile chemical protein-labeling tool based on fluorophore-conjugated diazabicyclooctane β-lactamase inhibitors (BLIs) and wild-type TEM-1 β-lactamase protein tag. The fluorescent probes efficiently formed a stable carbamoylated complex with β-lactamase, and the labeled proteins were visualized over a long period of time in live cells. Moreover, use of an α-fluorinated carboxylate ester-based BLI prodrug enabled the probe to permeate cell membranes and stably label intracellular proteins after unexpected spontaneous ester hydrolysis. Lastly, combining the labeling tool with a pH-activatable fluorescent probe allowed visual monitoring of lysosomal protein translocation during autophagy.     

Solid-phase synthesis of succinylhydroxamate peptides: functionalized matrix metalloproteinase inhibitors

[reaction: see text] A novel solid-phase synthesis strategy toward succinylhydroxamate peptides, using an appropriately protected hydroxamate building block, is described. Rapid and efficient access is gained to amine-functionalized peptides, which can be decorated with, for instance, a fluorescent label. In addition, we demonstrate an on-resin synthesis of a biotinylated photoactivatable hydroxamate peptide, which can be used as an activity-based probe for matrix metalloproteinases and ADAMs.