X-ray Crystallographic Structure of Thermophilic Rhodopsin

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.

X-ray Crystallographic Structure and Oligomerization of Gloeobacter Rhodopsin

“…As a result, the tip of the B-C loop is facing towards the periphery of the GR molecule and the central ion-releasing cavity at the extracellular surface is exposed. This change in orientation of the β-sheet was first found in the crystal structure of XR and then successively identified in sodium-pumping rhodopsin (KR2) 14,28 , thermophilic rhodopsin (TR) 29 and eubacterial light-driven chloride-pumping rhodopsin (ClR) 29,30 . In case of KR2 and ClR, an additional N-terminal helix is accommodated close to the extracellular cavity to “cap” the ion-releasing/uptaking region.…”

“…Since GR in our crystals does not have any additional carotenoids, the movement of the extracellular end of helix E is likely to reflect the carotenoid-free structure of GR. It should be noted that other XR-type sodium/chloride pumps, KR2 14 and ClR 30 have the same helix E position as GR in the absence of carotenoid, and molecular dynamics simulations on TR showed the inward movement of helix E at higher temperature 29 , suggesting the flexibility of this region.…”

Quantitation of the neural silencing activity of anion channelrhodopsins in Caenorhabditis elegans and their applicability for long-term illumination

“…4B, in transgenic worms expressing GtACR1, GtACR2 or Arch, elongation of the body length was induced by approximately 5% immediately by the illumination. That elongation was likely to be mediated by the silencing of motor neurons governing muscular contraction 16,30 . After turning off the light, the body length rapidly recovered to the initial level within 1.0 sec.…”

“…elegans ) 9–14 . The neural inhibition can be evoked through hyperpolarization of the membrane potential and light-driven outward proton pumping rhodopsins such as archaerhodopsin-3 (Arch) and thermophilic rhodopsin (TR) have been used for neural inhibition in animals 14–16 . A light-driven inward chloride pumping rhodopsin, Natronomonas pharaonis halorhodopsin ( Np HR), can be also applied for neural inhibition 17 .…”

Molecular mechanism for thermal denaturation of thermophilic rhodopsin

“…For example, a recently discovered outward proton pump, thermophilic rhodopsin (TR) that shows 74% similarity in amino acid sequence to gloebacter rhodopsin (GR), exhibits much higher thermal stability than GR 6. TR, prepared from the genome of an extreme thermophile Thermus thermophilus bacterium living in a hot spring in the United States, showed high resistance to temperature change 6–8. The excited state photophysics of TR is reported to be independent of temperature up to about 70 °C 9.…”