Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer

The biochemical and biophysical properties of a red fluorescent protein fro m a Discosoma species (DsRed) were investigated. The recombinant DsRed expr essed in E. coli showed a complex absorption spectrum that peaked at 277, 3 35, 487, 530, and 558 nm. Excitation at each of the absorption peaks produc ed a main emission peak at 583 nm, whereas a subsidiary emission peak at 50 0 nm appeared with excitation only at 277 or 487 nm. Incubation of E. coli or the protein at 37 degreesC facilitated the maturation of DsRed, resultin g in the loss of the 500-nm peak and the enhancement of the 583-nm peak. In contrast, the 500-nm peak predominated in a mutant DsRed containing two am ino acid substitutions (Y120H/ K168R). Light-scattering analysis revealed t hat DsRed proteins expressed in E. coli and HeLa cells form a stable tetram er complex. DsRed in HeLa cells grown at 37 degreesC emitted predominantly at 583 nm. The red fluorescence was imaged using a two-photon laser (Nd:YLF , 1047 nm) as well as a one-photon laser (He:Ne, 543.5 nm). When fused to c almodulin, the red fluorescence produced an aggregation pattern only in the cytosol, which does not reflect the distribution of calmodulin. Despite th e above spectral and structural complexity, fluorescence resonance energy t ransfer (FRET) between Aequorea green fluorescent protein (GFP) variants an d DsRed was achieved. Dynamic changes in cytosolic free Ca2+ concentrations were observed with red cameleons containing yellow fluorescent protein (YF P), cyan fluorescent protein (CFP), or Sapphire as the donor and RFP as the acceptor. using conventional microscopy and one- or two-photon excitation laser scanning microscopy, Particularly, the use of the Sapphire-DsRed pair rendered the red cameleon tolerant of acidosis occurring in hippocampal ne urons, because both Sapphire and DsRed are extremely pH-resistant.