Fluorescence properties of fluorophores are known to be affected by various environmental factors (e.g. pH, temperature and permittivity of solvent). By utilizing these properties, we think various fluorescent probes been developed and utilized to monitor the intracellular environment of living cells. The properties of fluorescent proteins (FPs) are also affected by the environment but in a more complicated manner due to their unique chromophore structures and the surrounding b-barrel region. In this study, we investigated how the fluorescent properties of FPs are affected by solvents, and explored the possibility of using them as specific probes to characterize the local environment of a target protein.When purified FPs (BFP, ECFP, EGFP, Venus, mCherry and mRFP) were exposed to various alchols (methanol, 1-propanol, 2-propanol, ethanol, 2,2,2trifluoroethanol (TFE)), fluorescent intensities were significantly affected. The fluorescence intensities of ECFP, EGFP and BFP, but not Venus, mRFP and mCherry, were dramatically decreased in a solution of more than 30% TFE, whereas a lower concentration of alcohol had almost no effect. The circular dichroism (CD) spectrum of ECFP demonstrated that b-sheets are significantly collapsed in 30% TFE. Molecular dynamics (MD) simulations also demonstrated significant changes in the b-barrel structure and in the accessibility of solvents to the chromophore. The fluorescence life-time measurements demonstrated that the life-time of ECFP in 30% TFE was drastically decreased, suggesting that solvent relaxation occurs. These results suggest that hydrophobic solvent such as TFE first attacks the b-barrel structure of FPs, which then allows solvent molecules to access the chromophore. By combining environment-sensitive and -intensive FPs, we successfully developed FP probes to monitor the local environment of target proteins.
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