Practical and reliable FRET/FLIM pair of fluorescent proteins

Background: In spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings. Results: Here we screened some of the recently developed monomeric protein pairs to find the optimal combination, which would provide high dynamic range FRET changes, along with high pH- and photo-stability, fast maturation and bright fluorescence, and reliable detection in any fluorescent imaging system. Among generated FRET pairs, we have selected TagGFP-TagRFP, combining all the mentioned desirable characteristics. On the basis of this highly efficient FRET pair, we have generated a bright, high contrast, pH- and photo-stable apoptosis reporter, named CaspeR3 (Caspase 3 Reporter). Conclusion: The combined advantages suggest that the TagGFP-TagRFP is one of the most efficient green/red couples available to date for FRET/FLIM analyses to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors for various applications. CaspeR3 provides reliable detection of apoptosis, and should become a popular tool both for cell biology studies and high throughput screening assays

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 Forster resonance energy transfer applications

Single fluorescent protein-based Ca2+ sensors with increased dynamic range

<p>Abstract</p> <p>Background</p> <p>Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca²⁺sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).</p> <p>Results</p> <p>Here we report significant progress on the development of the latter type of Ca²⁺sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca²⁺concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca²⁺-free and Ca²⁺-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca²⁺response to a prolonged glutamate treatment in cortical neurons.</p> <p>Conclusion</p> <p>We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.</p

The Structure of Ca2+ Sensor Case16 Reveals the Mechanism of Reaction to Low Ca2+ Concentrations

Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca2+ sensor, Case16, in the presence of a low Ca2+ concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca2+-dependent response when only two out of four Ca2+-binding pockets of calmodulin (CaM) are occupied with Ca2+ ions. In such a “half Ca2+-bound state”, Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca2+ sensor Case12 at saturating Ca2+ concentration. Based on this structure, we postulate that cpGFP-based Ca2+ sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca2+ sensors in living cells

Practical and reliable FRET/FLIM pair of fluorescent proteins

Abstract Background In spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings. Results Here we screened some of the recently developed monomeric protein pairs to find the optimal combination, which would provide high dynamic range FRET changes, along with high pH- and photo-stability, fast maturation and bright fluorescence, and reliable detection in any fluorescent imaging system. Among generated FRET pairs, we have selected TagGFP-TagRFP, combining all the mentioned desirable characteristics. On the basis of this highly efficient FRET pair, we have generated a bright, high contrast, pH- and photo-stable apoptosis reporter, named CaspeR3 (Caspase 3 Reporter). Conclusion The combined advantages suggest that the TagGFP-TagRFP is one of the most efficient green/red couples available to date for FRET/FLIM analyses to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors for various applications. CaspeR3 provides reliable detection of apoptosis, and should become a popular tool both for cell biology studies and high throughput screening assays.</p

Single fluorescent protein-based Casensors with increased dynamic range-0

<p><b>Copyright information:</b></p><p>Taken from "Single fluorescent protein-based Casensors with increased dynamic range"</p><p>http://www.biomedcentral.com/1472-6750/7/37</p><p>BMC Biotechnology 2007;7():37-37.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1931437.</p><p></p>amino acid residues (145–148) and sensitive domains (calmodulin and M13) within Casensors

Single fluorescent protein-based Casensors with increased dynamic range-2

<p><b>Copyright information:</b></p><p>Taken from "Single fluorescent protein-based Casensors with increased dynamic range"</p><p>http://www.biomedcentral.com/1472-6750/7/37</p><p>BMC Biotechnology 2007;7():37-37.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1931437.</p><p></p>) to Caionophore A23187. c,d. HeLa cells expressing Case12 are shown before (b) and after (c) ionophore addition. e-h. Fluorescence changes of M21 (human Melanoma-derived) cells expressing Case12 in response to 100 μM ATP. Images were captured every 0.294 sec on the confocal microscope. e,f. Individual responses of two selected cells within 400 s after ATP addition. g,h. The same cells, first 60 s of response. i. PC12 cells response to 500 uM carbachol (CCH). j. PC12 cells response to 30 mM KCl. For i and j first and second arrows indicate the moments of a compound addition and of washing start, respectively