X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION.

Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study， we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin， including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR， including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions， were also clarified. Based on the crystal structure， the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain， the movement of extracellular domains， the formation of a hydrogen bond， and the tilting of transmembrane helices were observed. From the computational and mutational analysis， we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability， acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.