Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

Abstract

Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein-based biosensors for Ca 21 and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)-based biosensors by providing a means for varying the critical dipole-dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th b-strand of mCherry. These new variants may ultimately prove useful for the creation of single FP-based Ca 21 biosensors.

Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

“…Subsequently, we circularly permuted the red FP mCherry (Shaner et al, 2004) and replaced cpGFP in GCaMP3 with cp-mCherry; this protein was not fluorescent either (data not shown), in agreement with the absence of chromophore formation in cp-mCherry described previously (Carlson et al, 2010). We therefore selected another protein, mRuby (Kredel et al, 2009), as a template for a red calcium indicator.…”

Circular Permutation of Red Fluorescent Proteins

“…The excitation and emission maxima of the cp193g7, with 6 amino acid mutations, are 580 nm and 602 nm, respectively [19].bExtinction coefficients were measured by alkali-denatured chromophore method.cQuantum yields were measured using mCherry as the reference standard.dRelative brightness of chromophore (extinction coefficient × quantum yield) was compared with mCherry (91,000×0.22).eFluorescence of cp-mCherry relative to mCherry with fixed protein concentration (BCA assay).fOur data; the published data are 72,000 [7], 78,000 [9].gPublished values [18], [19], which were based on the protein quantification (absorption at 280 nm).iNot determined.…”

“…The excitation and emission maxima of the cp193g7, with 6 amino acid mutations, are 580 nm and 602 nm, respectively [19].…”

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

“…Circularly permutated FPs are generated by genetically linking the original N-and C-termini with a short polypeptide linker and introducing new N-and C-termini at a position elsewhere in the protein. 91,92 For FP-based biosensor construction, the new N-and C-termini are introduced close to the chromophore such that conformational changes in the extrinsic recognition element cause alterations in the chromophore environment and, correspondingly, in the fluorescence intensity or hue of the FP.…”