High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment

Abstract

Thermophilic rhodopsin (TR) is a light-driven proton pump from the extreme thermophile Thermus thermophilus JL-18. Previous studies on TR solubilized with detergent showed that the protein exhibits high thermal stability and forms a trimer at room temperature but irreversibly dissociates into monomers when incubated at physiological temperature (75 °C). In the present study, we used resonance Raman (RR) spectroscopy, solid-state NMR spectroscopy, and high-speed atomic force microscopy to analyze the oligomeric structure of TR in a lipid environment. The obtained spectra and microscopic images demonstrate that TR adopts a pentameric form in a lipid environment and that this assembly is stable at the physiological temperature, in contrast to the behavior of the protein in the solubilized state. These results indicate that the thermal stability of the oligomeric assembly of TR is higher in a lipid environment than in detergent micelles. The observed RR spectra also showed that the retinal chromophore is strongly hydrogen bonded to an internal water molecule via a protonated Schiff base, which is characteristic of proton-pumping rhodopsins. The obtained data strongly suggest that TR functions in the pentameric form at physiological temperature in the extreme thermophile T. thermophilus JL-18. We utilized the high thermal stability of the monomeric form of solubilized TR and here report the first RR spectra of the monomeric form of a microbial rhodopsin. The observed RR spectra revealed that the monomerization of TR alters the chromophore structure: there are changes in the bond alternation of the polyene chain and in the hydrogen-bond strength of the protonated Schiff base. The present study revealed the high thermal stability of oligomeric assemblies of TR in the lipid environment and suggested the importance of using TR embedded in lipid membrane for elucidation of its functional mechanism.

Structure of a retinal chromophore of dark-adapted middle rhodopsin as studied by solid-state nuclear magnetic resonance spectroscopy

“…Specifically, the λ max values corresponding to the three retinal configurations of MR were distinguished as 485 nm (all- trans ), 479 nm (13- cis , 15- syn ), and 495 nm (11- cis ) by the extraction of single component UV-Vis spectra [ 19 ]. As a reference for the maximum MR data, the value for MR was plotted together with those for BR [ 48 ], Krokinobacter rhodopsin 2 (KR2) [ 40 ], ASR [ 49 ], Thermophilic rhodopsin (TR) [ 50 ], and Np SRII [ 51 ] as shown in Figure 5 (a) and (b). MR with the all- trans form (485 nm/184.5 ppm) agreed well with the trend of the linear relationships shown in the model compound ( Fig.…”

“…Specifically, the  max values corresponding to the three retinal configurations of MR were distinguished as 485 nm (all-trans), nm (13-cis, 15-syn), and 495 nm (11-cis) by the extraction of single component UV-Vis spectra [19]. As a reference for the maximum MR data, the value for MR was plotted together with those for BR [48], Krokinobacter rhodopsin 2 (KR2) [40], ASR [49], Thermophilic rhodopsin (TR) [50], and NpSRII [51] as shown in Figure 5 nm is induced by a displacement of helix G, resulting in the observed distance between PSB and Asp201 [52]. Thus, our 15 N NMR result suggested that the strong electrostatic interaction between PSB and Asp84 in helix C or Asp213 in helix G is significantly linked to the shorter  max of the all-trans form in MR.…”

“…The linear relationships were as follows: for all- trans model, (λ max (nm)= –5.0872 δ (ppm)+1415.1); 13- cis model, (λ max (nm)= –4.6709 δ (ppm)+1341.3) [ 46 ]. 15 N PSB chemical shift values of BR [ 48 ], KR2 [ 40 ], TR [ 50 ], and Np SRII [ 51 ] are recalibrated to the reference signal of 15 NH 4 Cl.…”

Discovery of a microbial rhodopsin that is the most stable in extreme environments

“…The stability of microbial rhodopsins was tested by considering several factors, including temperature, pH, detergent, high salt concentration, and dehydration. This stable protein was extracted from microorganisms living in extreme environments ( Ece et al., 2015 ; Ji et al., 2017 ; Otzen, 2002 ; Shionoya et al., 2018 ), and the results led to continued research on various applications such as biochips and biosensors, as well as optogenetic tools ( Li et al., 2018 ). In this study, we investigated Tara76 rhodopsin, which exhibits stability against temperature, detergent, pH, salt, and dehydration.…”

“…These TRs have been reported to be stable and functional at high temperatures, and they may be valuable optogenetic tools as light sensors. Recently, an oligomeric state was reported for the lipid state of TR, which is monomeric at high temperatures and has strong stability ( Shionoya et al., 2018 ; Tsukamoto et al, 2013 , 2016 ). In addition, RxR found in Rubrobacter xylanophilus DSM 9941 T exhibits high thermal stability (85°C for 10 h) ( Kanehara et al., 2017 ).…”