Conformational fluctuations in a green fluorescent protein-like Akane family protein: a high-pressure fluorescence study at 0.1–700 MPa

We have investigated conformational fluctuations in a new green fluorescent protein(GFP)-like protein rb-Akane found in a red-brown-colored octocoral, Scleronephthya gracillima (Kuekenthal)), with high pressure fluorescence spectroscopy at 0.1-700MPa. Besides the green fluorescence at 510nm, two red fluorescence peaks are observed at 590 and 629nm, the relative intensity of which varies reversibly with pressure. The phenomenon is interpreted as representing the cis-trans isomerization of the chromophore accompanied by the conformational transition between two sub-states of the red fluorescence form of rb-Akane. The two sub-states are separated only marginally in free energy (G(0)=1.9 +/- 0.4kJmol(-1)), but significantly in partial molar volume (V-0=-19.8 +/- 1.4mlmol(-1)) at 0.1MPa (pH 7.5, 25 degrees C). Above 500MPa, the fluorescence at (max) 629nm undergoes another reversible change with pressure, showing the onset of unfolding.