(2008). Nature Biotechnology, 26(9) , 1035-1040. doi: 10.1038/nbt.1493. Article Pubmed
To generate RSFPs with different switching properties, we used rsFastLime (Dronpa-V157G) as a template because of its favorable switching behavior, and its large dynamic range (67:1) in the fluorescence signal between the on- and the off-state.
At 37 °C, Padron* behaves as a true monomer, although it has a slight tendency for dimerization at lower temperatures (∼15% dimer at 4 °C). The dimerization tendency was removed by exchanging hydrophobic amino acid residues that are located in potential dimer interfaces, namely Ile94, Ile100 and Leu141...The variant obtained [ Padron*(star) ] exhibits a slightly reduced dynamic range in the switchable signal (off-state fluorescence is 2% instead of 0.7% of the on-state signal), and its bleaching per cycle amounts to 3% instead of 2%. Hence Padron*(star) is particularly useful for applications at low temperatures; for most in vivo applications that require elevated temperatures, Padron* is superior.
As its switching behavior is reverted to that of Dronpa, we termed this variant Padron*.
At 37 °C, Padron* behaves as a true monomer, although it has a slight tendency for dimerization at lower temperatures (∼15% dimer at 4 °C). The dimerization tendency was removed by exchanging hydrophobic amino acid residues that are located in potential dimer interfaces, namely Ile94, Ile100 and Leu141...The variant obtained [ Padron*(star) ] exhibits a slightly reduced dynamic range in the switchable signal (off-state fluorescence is 2% instead of 0.7% of the on-state signal), and its bleaching per cycle amounts to 3% instead of 2%. Hence Padron*(star) is particularly useful for applications at low temperatures; for most in vivo applications that require elevated temperatures, Padron* is superior.
During the screen for Padron*, we identified a mutant with a cysteine residue at position 159 that was negatively switchable but exhibited a broad asymmetric absorption spectrum that was shifted to shorter wavelengths. After further improvements, we obtained a bright green fluorescent Dronpa variant with eight mutations, displaying an absorption maximum at 460 nm in the on-state. Termed bsDronpa (for broad-absorption-spectrum Dronpa), this RSFP combines a relatively large Stokes shift of 44 nm with a broad on-state absorption spectrum. It is efficiently excited with UV light, while maintaining good switching characteristics.