The RoGFP protein contains two engineered cysteine thiols, as first described by Remington et al. (RoGFP2) 11 (link). The cDNA encoding the protein was created by introducing four mutations in the mammalian GFP expression vector (pEGFP-N1) (C48S, Q80R, S147C, and Q204C) using a QuikChange Multi Site-directed mutagenesis kit (Strategene). The RoGFP construct was ligated into the VQ Ad5CMV K-NpA adenoviral shuttle vector between the KpnI and NotI sites; after sequencing and amplification this plasmid was used to generate a recombinant adenovirus to permit widespread expression in our cells (ViraQuest Inc., North Liberty, IA). The resulting redox-sensitive protein has excitation maxima at 400 and 484 nm, with emission at 525 nm. In response to changes in redox conditions, RoGFP exhibits reciprocal changes in intensity at the two excitation maxima 12 (link), and its ratiometric characteristics render it insensitive to expression levels 13 (link)-15 (link). Although RoGFP’s fluorescence behavior is relatively independent of pH and it does not respond to authentic nitric oxide (NO), reduced NADH, or the antioxidant N-acetyl-L-cysteine (NAC), its spectrum is slightly affected by reduced glutathione (GSH) possibly due to thiol-disulfide exchange (Online Figures I and II ).
RoGFP was expressed in the mitochondrial matrix (Mito-RoGFP) by appending a 48 bp region encoding the mitochondrial targeting sequence from cytochrome oxidase subunit IV, at the 5′ end of the coding sequence. This construct was then ligated into the VQ Ad5CMV K-NpA plasmid between the KpnI and NotI sites, and used to generate an adenoviral vector. RoGFP was targeted to the mitochondrial inter-membrane space (IMS-RoGFP) by appending it to glycerol phosphate dehydrogenase (GPD). A cDNA construct encoding GPD, an integral protein of the inner mitochondrial membrane whose C-terminus protrudes into the inter-membrane space 17 (link), was ligated in-frame with cDNA encoding RoGFP 17 (link). The corresponding polypeptide includes amino acids 1–626 of GPD, with RoGFP at the carboxy terminus. This method has been used previously to express YFP in the inter-membrane space 18 (link). (SeeOnline Supplemental Material for characterization of the RoGFP sensors and experimental protocols).
RoGFP was expressed in the mitochondrial matrix (Mito-RoGFP) by appending a 48 bp region encoding the mitochondrial targeting sequence from cytochrome oxidase subunit IV, at the 5′ end of the coding sequence. This construct was then ligated into the VQ Ad5CMV K-NpA plasmid between the KpnI and NotI sites, and used to generate an adenoviral vector. RoGFP was targeted to the mitochondrial inter-membrane space (IMS-RoGFP) by appending it to glycerol phosphate dehydrogenase (GPD). A cDNA construct encoding GPD, an integral protein of the inner mitochondrial membrane whose C-terminus protrudes into the inter-membrane space 17 (link), was ligated in-frame with cDNA encoding RoGFP 17 (link). The corresponding polypeptide includes amino acids 1–626 of GPD, with RoGFP at the carboxy terminus. This method has been used previously to express YFP in the inter-membrane space 18 (link). (See
