Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP
© Zapata-Hommer and Griesbeck; licensee BioMed Central Ltd. 2003
Received: 3 March 2003
Accepted: 22 May 2003
Published: 22 May 2003
The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET). However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.
By directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag. Its absorption spectrum is pH-stable and the pKa of its emission is 4.9, making it very resistant to pH perturbation inside cells.
"T-Sapphire" and its circular permutations can be used as labels of proteins or cellular structures and as FRET donors in combination with red-fluorescent acceptor proteins such as DsRed, making it possible to completely separate donor and acceptor excitation and emission in intensity-based FRET experiments.
Mutants of the green fluorescent protein (GFP) have been exploited for various applications in biochemistry and cell biology, serving as reporters of gene expression, protein labels and since recently also as active indicators of physiological signals . Especially fluorescence resonance energy transfer (FRET) between suitable GFPs has received widespread attention as it allows, in principle, to monitor protein conformations and protein-protein interactions inside living cells . Currently the most preferred donor-acceptor combination is CFP-YFP (Cyan Fluorescent Protein-Yellow Fluorescent Protein). One drawback of this combination, however, is the long emission tail of the donor CFP that overlaps with the YFP emission. While this is tolerable for genetic probes in which donor and acceptor are concatenated to each other within a single gene construct, this poses a serious problem when studying interactions between two different proteins labelled with the corresponding donor and acceptor GFP. Acceptor channel emission will be contaminated with donor emission even under conditions in which no FRET occurs, depending on transfection efficiencies and expression levels of the constructs that may vary significantly. Therefore alternative fluorescent labels with reduced overlap are desirable.
Sapphire, also termed H9-40 , is a mutant of GFP in which the T203I mutation abolishes the second excitation peak at 475 nm that can be found in wildtype GFP. As a result the mutant protein exhibits a huge Stoke's shift, with an excitation peak at 399 nm and an emission peak at 511 nm [3, 4]. Sapphire so far has not been much considered for intensity-based FRET (fluorescence resonance energy transfer) applications because its emission was too much overlapping with that of other GFP mutants used as acceptors. Also, so far no attempts have been made to improve its folding and expression properties inside cells, a prerequisite to allow precise targetings and successful fusions to proteins of interest. We therefore examined the effects of a series of folding mutations on Sapphire expression and characterized the resulting proteins spectroscopically. We also assessed the folding properties of one resulting variant when expressed in the endoplasmic reticulum of HEK293 cells and explored possibilities of using Sapphire as a donor protein in FRET-based genetically encoded indicators.
Results and Discussion
Sapphire variants with improved folding properties and circular permutations
Spectroskopic properties and pKa of Sapphire mutants.
Targeted expression in HEK293 cells
Long emission wavelength genetic indicators of protease activity
In general, red-shifted indicators are useful because excitation occurs at longer wavelengths, emission is further removed from auto-fluorescence and detection instruments often are more effective at red wavelengths. They are complementary to indicators based on the CFP-YFP pair and can therefore be used for double-labelings or imaging of several parameters within the same cell. It has to be mentioned that first generation DsRed has several disadvantages for use in FRET experiments. It is a tetramer, forms aggregates within cells and matures slowly and incompletely . However, most of these problems have been overcome recently and even monomeric versions have been generated [14, 15] so that that the combination Sapphire/DsRed should become the pair of choice for FRET experiments in the future. Therefore the development of an efficiently folding variant of Sapphire is complementary to the current evolution of red fluorescent proteins.
By directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP and permutations thereof with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag. Its absorption spectrum is pH-stable and the pKa of its emission is 4.9, making it very resistant to pH perturbation inside cells. We believe T-Sapphire will be useful in a number of applications ranging from labelling of cellular structures to FRET where the combination of T-Sapphire as donor and a red fluorescent protein such as DsRed as acceptor allows the complete separation of donor and acceptor emissions in intensity-based FRET experiments.
Gene clonings were done into the bacterial expression plasmid pRSETB (Invitrogen). Circular permutations with new N-termini were made by two separate PCRs and corresponding 5' and 3' fragments were linked with the peptide sequence GGTGGS that included a KpnI site. Site-directed mutagenesis was performed using the QuickChange kit (Stratagene). Fusion proteins between different GFP mutants and DsRed were done by first cloning DsRed into the BamHI-site of pRSETB, then inserting the corresponding GFP mutant in frame into the Nhe-site after the polyhistidine tag, thereby creating a short linker with an enterokinase cleavage site between the two fluorescent proteins.
Protein expression and spectroscopy
All constructs of interests were cloned into pRSETB (Invitrogen) and transformed into the bacterial strain E. coli BL-21. Purification of the His-tagged proteins followed via Nickel-chelate column chromatography according to previous procedures . Spectroscopy of purified protein was usually performed in 100 mM KCl, 10 mM K-MOPS, pH 7.25, in a fluorescence spectrometer (Cary Eclipse, Varian). pH-titrations were performed as described . Extinction coefficients were determined according to the "base denatured chromophore" method . Proteolysis of fusion constructs was done at room temperature with recombinant enterokinase (Invitrogen).
Cell culture and microscopy
In order to evaluate targeted expression of Sapphire mutants, identical amounts of DNA (30 μg) of ER-Sapphire or ER-T-Sapphire in pcDNA3 were transfected into HEK293 cells (500,000 per 50 mm dish). After 2 days of expression cells were suspended in Hanks-buffered saline solution (HBSS), normalized at OD 600 and measured in the fluorescence spectrometer. Pictures of 293 cells were taken with a charge-coupled device camera (CoolSnap, Roper Scientific). Ilumination was done with a DeltaRam monochromator (Photon Technology International) at 400 nm or 488 nm, emission filter was 535/25.
We would like to thank Alexander Borst for his support.
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