Multi-pixel spectral imaging of green fluorescent protein (GFP) in COS-7 cells: folding kinetics and chromophore formation

Spectrally resolved imaging of Green fluorescent protein (GFP) expressed in living COS-7 kidney cells distinguished the subcellular localization and demarcated the processes of protein folding and chromophore formation. COS-7 kidney cells were transfected by a plasmid pEGFP-N1 plasmid followed by incubation for 15 hrs for gene expression. At different intervals the cells were examined by fluorescence microscopy and analyzed by a spectral imaging system. After 7 hrs, GFP was detected in the cytoplasm, concentrated in a localized form. Spectra of the initial GFP evinced several components that belong both to the typical fluorescent signal as well as to unspecific fingerprints. At 10 and 15 hrs, GFP was seen spread in the cytoplasm as well as in the nucleus, and the specific spectra of the GFP were dominant at the later time. The typical GFP spectral fingerprint is the result of protein folding and chromophore formation following internal oxidation reactions. This folding and chromophore formation process, up to final conformation, was detected by spectral imaging as localized in the nucleus rather than in the cytosol. Thus, the method of fluorescence microscopy combined with multipixel spectral imaging demonstrates distinct biochemical pathways leading to GFP conformation.