Abstract
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.
Highlights
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
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.”
In 2012, a gene encoding a eubacterial proton pumping rhodopsin was identified in the genome of an extreme thermophile, Thermus thermophilus JL-18 strain, which was isolated in the Great Boiling Spring in the United States Great Basin at ϳ75 °C [6]
Summary
In 2012, a gene encoding a eubacterial proton pumping rhodopsin was identified in the genome of an extreme thermophile, Thermus thermophilus JL-18 strain, which was isolated in the Great Boiling Spring in the United States Great Basin at ϳ75 °C [6] Since that time, this protein, named thermophilic rhodopsin (TR), has been investigated to elucidate its molecular properties, including its light-driven proton pump activity, thermal stability, and photoreaction [7, 8]. We determined its x-ray crystal structure and elucidated possible mechanisms for its thermal stability together with the results of molecular dynamics (MD) simulations These findings, which advance the understanding of ion transport mechanisms and the thermal stability of membrane proteins, should lead to the rational design of rhodopsin-based optogenetic tools
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