Nadya G. Gurskaya

Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light

Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes. In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed. Except for tetrameric KFP1 (ref. 4), all known PAFPs, including PA-GFP, Kaede, EosFP, PS-CFP, Dronpa, PA-mRFP1 and KikGR require light in the UV-violet spectral region for activation through one-photon excitation—such light can be phototoxic to some biological systems. 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 °C, 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.

Diversity and evolution of the green fluorescent protein family

The family of proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria exhibits striking diversity of features, including several different types of autocatalytically synthesized chromophores. Here we report 11 new members of the family, among which there are 3 red-emitters possessing unusual features, and discuss the similarity relationships within the family in structural, spectroscopic, and evolutionary terms. Phylogenetic analysis has shown that GFP-like proteins from representatives of subclass Zoantharia fall into at least four distinct clades, each clade containing proteins of more than one emission color. This topology suggests multiple recent events of color conversion. Combining this result with previous mutagenesis and structural data, we propose that (i) different chromophore structures are alternative products synthesized within a similar autocatalytic environment, and (ii) the phylogenetic pattern and color diversity in reef Anthozoa is a result of a balance between selection for GFP-like proteins of particular colors and mutation pressure driving the color conversions.

GFP-like chromoproteins as a source of far-red fluorescent proteins.

We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins. An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far‐red FPs (emission λ max=615–640 nm). Moreover, coupled site‐directed and random mutagenesis of the chromoprotein from H. crispa resulted in a unique far‐red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi‐color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi‐color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.

Intra-axonal translation and retrograde trafficking of CREB promotes neuronal survival

During development of the nervous system, axons and growth cones contain mRNAs such as β-actin, cofilin and RhoA, which are locally translated in response to guidance cues. Intra-axonal translation of these mRNAs results in local morphological responses; however, other functions of intra-axonal mRNA translation remain unknown. Here, we show that axons of developing mammalian neurons contain mRNA encoding the cAMP-responsive element (CRE)-binding protein (CREB). CREB is translated within axons in response to nerve growth factor (NGF) and is retrogradely trafficked to the cell body. In neurons that are selectively deficient in axonal CREB transcripts, increases in nuclear pCREB, CRE-mediated transcription and neuronal survival elicited by axonal application of NGF are abolished, indicating a signalling function for axonally synthesized CREB. These studies identify a signalling role for axonally derived CREB, and indicate that signal-dependent synthesis and retrograde trafficking of transcription factors enables specific transcriptional responses to signalling events at distal axons.

A strategy for the generation of non-aggregating mutants of Anthozoa fluorescent proteins

Recently, we cloned several fluorescent proteins of different colors homologous to Aequorea victoria green fluorescent protein, which have great biotechnological potential as in vivo markers of gene expression. However, later investigations revealed severe drawbacks in the use of novel fluorescent proteins (FPs), in particular, the formation of tetramers (tetramerization) and high molecular weight aggregates (aggregation). In this report, we employ a mutagenic approach to resolve the problem of aggregation. The elimination of basic residues located near the N‐termini of FPs results in the generation of non‐aggregating versions of several FPs, specifically, drFP583 (DsRed), DsRed‐Timer, ds/drFP616, zFP506, zFP538, amFP486, and asFP595.

Red Fluorescent Protein with Reversibly Photoswitchable Absorbance for Photochromic FRET

We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF states exhibit absorbance maxima at 567 and 440 nm, respectively. Due to the photoswitchable absorbance, rsTagRFP can be used as an acceptor for a photochromic Förster resonance energy transfer (pcFRET). The photochromic acceptor facilitates determination of a protein-protein interaction by providing an internal control for FRET. Using pcFRET with EYFP as a donor, we observed an interaction between epidermal growth factor receptor and growth factor receptor-binding protein 2 in live cells by detecting the modulation of both the fluorescence intensity and lifetime of the EYFP donor upon the ON-OFF photoswitching of the rsTagRFP acceptor.

Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the β-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu68 and Ser119, located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.

Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina.

Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.

Method for real-time monitoring of protein degradation at the single cell level

Protein degradation is important in practically all aspects of cellular physi-ology (1). Together with transcription and translation, proteolysis ensures maintenance and rapid regulation of individual protein concentration in living cells. The half-life for different proteins varies between a few minutes and days. Moreover, decay rate for many proteins change drastically throughout the cell cycle or in response to external stimuli.Two methods are commonly used to determine a protein’s half-life, namely radioactive pulse-chase analysis and cycloheximide chase (2). Pulse-chase analysis provides minimal distortion of normal cell physiology. The main disadvantages of this method are its laboriousness and necessity for radio-labeling. In contrast to pulse-chase analysis, cycloheximide chase strongly affects cellular metabolism that should be considered a serious disadvantage for this approach. Importantly, both methods do not allow real-time measurements at the single cell level.The use of green fluorescent protein (GFP) provided an opportunity to apply fluorescence microscopy and flow cytometry to protein degradation analysis (3–6). However, to extract information regarding protein degra-dation using GFP, one should block the synthesis of new GFP molecules (e.g., by cycloheximide) (3,4). Even in presence of cycloheximide, residual GFP chromophore maturation affects estimation of degradation rate, especially when maturation and degra-dation half-times are comparable.Since 2002, a number of so-called photoactivatable fluorescent proteins (PAFPs) have been developed (7). Most commonly, local PAFP activation is used to visualize protein movement within cells. Here we propose to use PAFP activation within a whole cell to monitor protein degradation (Figure 1). Indeed, while steady-state fluorescence intensity of an FP-tagged protein depends on both synthesis and degradation rates, photo-activation creates a fluorescent signal that depends only on protein degra-dation (fluorescence bleaching during observation should be taken into account and made allowance for). Thus, time-lapse imaging of activated PAFP allows quantification of the tagged protein degradation process. Also, this protocol can be potentially adapted for PAFP photoactivation in large cell culture samples and their further analysis by flow cytometry or microplate readers.To demonstrate the usability of the method proposed, we used the green-to-red photoconvertible fluorescent protein Dendra2 (Evrogen, Moscow, Russia), which is a commercially available improved version of Dendra (8). In the dark, Dendra2 matures up to the green fluorescent state with excitation-emission maxima at 490 and 507 nm, respectively. Upon maturation, it can be converted into a red-emitting protein (excitation-emission at 553/573 nm) by irradiation with violet (e.g., 405 nm) or blue (e.g., 488 nm) light. Due to its monomeric state, Dendra2 can be safely used for protein labeling. In contrast to other PAFPs, Dendra2 can be activated with blue light, which is less damaging compared with ultraviolet (UV) or violet light. Using a well-established assay based on dithionite reduction of chromo-phore of urea-denatured fluorescent protein followed by dilution of the sample and maturation of the fluorescent protein starting from the native polypeptide (9), we measured Dendra2 maturation half-time as 90 min at 37°C.Similar to GFP, Dendra2 was found to be a long-lived protein. In HeLa and HEK 293 cells, we observed no decrease in Dendra2 green fluorescence for several hours after the addition of cycloheximide (not shown). To photoconvert Dendra2 throughout the experiments, we applied 10–20 s of irradiation with blue light from a 100 W Hg-lamp using the GFP filter set. As a result, the green signal decreased while clearly detectable red fluores-cence appeared. Then, we monitored red fluorescence at 37°C in the confocal mode (Leica DMIRE2 TCS SP2 micro-scope; Leica Microsystems GmbH, Wetzlar, Germany) using the 543-nm laser line. Practically no decay of red fluorescence after Dendra2 photocon-version was observed (Figure 2A), demonstrating very high stability of this protein in living cells. Next we tested the influence of peptides or proteins known to determine fast degra-

Color transitions in coral's fluorescent proteins by site-directed mutagenesis

BackgroundGreen Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (~ 485 nm), green (~ 505 nm), yellow (~ 540 nm), and red (>580 nm) emitters.ResultsHere we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence.ConclusionsSeveral amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals.