Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
"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.
Zapata-Hommer
Otilia
O
Max-Planck-Institut für Neurobiologie Am Klopferspitz 18a 82152 Martinsried, Germany. zapata@neuro.mpg.de
Griesbeck
Oliver
O
D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D017510:Protein Folding
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D010450:Endopeptidases