Spectroscopic studies of model chromophores of green and red fluorescent proteins.

摘要

The green fluorescent protein (GFP) from Aequorea victoria has emerged as one of the most useful tools in molecular and cell biology. Due to it's internal fluorescent chromophore, which is formed from the Ser65-Tyr66-Gly67 tripeptide sequence post-translationally in the protein without involving any cofactor other than molecular oxygen, GFP can be introduced into living cells/organisms as a fusion partner to follow the trafficking and processing of the fusion partner in vivo. To further investigate the optical properties of GFP and elucidate protein-chromophore interactions, we have conducted extensive model studies on GFP and RFP model chromophores.; We have obtained Raman spectra of the model chromophores, 4′ -hydroxybenzylidene-1-acetate-2-methyl-imidazolinone (HBAMI), 4 ′-hydroxybenzylidene-2,3-dimethyl-imidazolinone (HBDI), wild-type GFP and the S65T mutant. To understand the complex coupling of the vibrational coordinates in the GFP chromophore, we unambiguously assigned the vibrational bands in HBDI with the help of isotopic labeling and ab initio normal mode calculations. It was found that the ground state structure of the anionic form of the chromophore is strongly dependent to the protein environment while the neutral form is not. A linear correlation between the absorption properties and the ground state structure was also demonstrated.; We also investigated the ability of the chromophore to isomerize about the exocyclic ethylene bond. Ionization/isomerization studies reveal that the cationic form of HBDI is present in 80% cis and 20% trans isomers about the bridging C=C bond at RT whereas there is less than 1% trans isomer in the neutral and anionic forms of the chromophore. This suggests that the protein may modulate the ability of the chromophore to isomerize via altering the protonation states of the chromophore.; Finally we synthesized and characterized two compounds designed to model the chromophore in DsRed, a red fluorescent protein. Comparison with model green fluorescent protein (GFP) chromophores indicates that the additional conjugation in the DsRed models can account, in part, for the red shifted absorption and emission properties of DsRed compared to GFP. In contrast to the GFP models, the DsRed models are fluorescent with quantum yields of 0.002–0.01 in CHCl₃.