Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein.
Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, approximately 20 A from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated fluorophore. Caspase-3 cleavage of constructs with a caspase-3 sequence near the C-terminus in the sequence between the native fluorophore and the new cysteine, located C-terminal to the caspase site, destroyed the FRET, the emitted color reverting to that of unmodified GFP. This process was demonstrated in vitro with caspase-3 and lysates from cells undergoing apoptosis. Real-time emission changes for the Alexa Fluor 532 conjugate of this GFP, studied quantitatively in vivo for single HeLa cells using the ratios of fluorescence at the red and green maxima by confocal microscopy, showed that caspase-3 action in the cytosol preceded that in the nucleus.
Suzuki
Miho
M
Department of Functional Materials Science, Faculty of Engineering, Saitama University, 255 Shimo-okubo, Saitama, Saitama 338-8570, Japan. miho@fms.saitama-u.ac.jp
Ito
Yoichiro
Y
Sakata
Ichiro
I
Sakai
Takafumi
T
Husimi
Yuzuru
Y
Douglas
Kenneth T
KT
D000595:Amino Acid Sequence; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015398:Signal Transduction
D049452:Green Fluorescent Proteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases