Irreversible photobleaching of bacteriorhodopsin in a high-temperature intermediate state.

The photo-intermediate state of bacteriorhodopsin is a metastable state that spontaneously transforms to the ground state over the energy barrier of a local minimum. As the recovery of the photocycle to the ground state and irreversible photobleaching to the denatured state may occur from the same local energy minimum, depending on the temperature, the structural stability of bacteriorhodopsin under illumination at high temperature was measured in order to study the intra- and inter-molecular interactions that contribute to the recovery of the ground state. Visible CD spectra of bacteriorhodopsin began to change at 60 degrees C from a bilobed to positive type in accordance with an appearance of an absorption peak at 470 nm. Irreversible photobleaching, the light-induced denaturation, also started to occur at 60 degrees C, suggesting some correlation between irreversible photobleaching and the structural change to the high-temperature intermediate state. However, bacteriorhodopsin in the dark was stable up to 70 degrees C, suggesting that there is some additional factor that lends structural stability to bacteriorhodopsin in the dark. The contribution of protein-protein interactions to stability is discussed on the basis of the difference in the denaturation behaviors between light and dark conditions.