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Pressure and temperature dependence of fluorescence anisotropy of Green Fluorescent Protein

We have studied the effect of high hydrostatic pressure and temperature on the steady state fluorescence anisotropy of Green Fluorescent Protein (GFP). We find that the fluorescence anisotropy of GFP at a constant temperature decreases with increasing pressure. At atmospheric pressure, anisotropy decreases with increasing temperature but exhibits a maximum with temperature for pressure larger than 20 MPa. The temperature corresponding to the maximum of anisotropy increases with increasing pressure. By taking into account of the rotational correlation time changes of GFP with the pressure-temperature dependent viscosity of the solvent, we argue that viscosity increase with pressure is not a major contributing factor to the decrease in anisotropy with pressure. Our results suggest that the decrease of fluorescence anisotropy with pressure may result from changes in H-bonding environment around the chromophore.