High Thermal Stability of Oligomeric Assemblies of Thermophilic

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment Tomomi Shionoya, Misao Mizuno, Takashi Tsukamoto, Kento Ikeda, Hayato Seki, Keiichi Kojima, Mikihiro Shibata, Izuru Kawamura, Yuki Sudo, and Yasuhisa Mizutani J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04894 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 16, 2018

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The Journal of Physical Chemistry

High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment Tomomi Shionoya,† Misao Mizuno,† Takashi Tsukamoto,‡¶ Kento Ikeda,♠ Hayato Seki,# Keiichi Kojima,‡ Mikihiro Shibata,§♦ Izuru Kawamura,# Yuki Sudo,‡ and Yasuhisa Mizutani†* †

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan

‡

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 11-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan ♠

Mathematical and Physical Sciences, Graduate School of Natural Science & Technology, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan

§

Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kakuma, Kanazawa, 9201192, Japan ♦

High-speed AFM for Biological Application Unit, Institute for Frontier Science Initiative, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan

#

Graduate School of Engineering, Yokohama National University, Hodogaya-ku, Yokohama 240-8501, Japan

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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 ºC). In the present study, we used resonance Raman (RR) spectroscopy, solid-state NMR spectroscopy, and highspeed atomic force microscopy (HS-AFM) 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 Thermus 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.


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INTRODUCTION Many microbial rhodopsins identified to date perform transmembrane light-driven ion translocations and consist of seven transmembrane helices that form an interior pocket for the all-trans-retinal chromophore.1-2 The retinal chromophore is covalently bound to the protein moiety through a protonated Schiff base (PSB) linkage. Light-induced trans-cis isomerization of the chromophore leads to a cyclic reaction called the photocycle that involves a series of intermediates.1-2 Each step in the photocycle is accompanied by a conformational reorganization of the surrounding protein moiety and results in ion transport. Ion pumping rhodopsins generally form oligomers in the cell membrane, suggesting that oligomeric assembly is associated with their efficient ion transport. Thermophilic rhodopsin (TR) was the first microbial rhodopsin to be identified in a thermophilic bacterium and functions as a light-driven proton pump.3-4 The first TR to be characterized was from the extreme thermophile Thermus thermophilus JL-18 strain, isolated from the Great Boiling Spring in the United States Great Basin, which is at a temperature of between 73−77 ºC.3-4 The molecular properties and structure of TR were studied, including its light-driven proton pump activity, thermal stability, and photoreaction.4-7 A previous study showed that this TR has the highest thermal stability of all known microbial rhodopsins.4 Retinal is readily degraded above 50 ºC8 and thus a robust protein structure is required to hold retinal inside the protein and retain its optimal function at the high temperature of the native habitat of Thermus thermophilus JL-18. Another interesting feature of TR is an irreversible change in its oligomeric assembly in the solubilized state,5 in which TR solubilized with detergent forms a trimer at room temperature which irreversibly dissociates into monomers when the protein is incubated at physiological temperature (around 75 ºC). It is likely that the monomeric TR generated by heat treatment has


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ion pumping activity because it exhibits a photocycle similar to that of trimeric TR.5 These findings suggest that TR functions in the monomeric form at physiological temperature in the extreme thermophile but the thermal stability and oligomeric assembly of TR in a lipid environment remain largely unknown. It is crucial to understand the behavior of TR in a lipid environment because previous studies on other microbial rhodopsins reported that solubilization with detergent affected both thermal stability and oligomerization.9-11 Therefore, understanding how the protein environment affects the thermal stability and structure of TR will help us understand the mechanisms of ion transport and the thermostabilization of microbial rhodopsins. In this study, we characterized the oligomeric assembly and protein structure of TR using resonance Raman (RR) spectroscopy, solid-state NMR spectroscopy, and high-speed atomic force microscopy (HS-AFM). For samples solubilized with detergent, the RR spectra of the retinal chromophore showed that the bond alternation of the polyene and the hydrogen bond of the PSB of the chromophore are different between the oligomeric and monomeric forms. The observed spectral differences served as markers to distinguish the oligomeric assembly from the monomeric form. In a lipid environment, TR exhibited the characteristic spectral features of the oligomer at 75 ºC, showing that the protein does not become monomeric upon heat treatment in a lipid environment. Furthermore, HS-AFM images of TR demonstrated that TR adopts a pentameric form in a lipid environment at room temperature with or without prior heat treatment. In addition, the NMR data indicated that the oligomeric assembly of TR in a lipid environment was stable towards heat treatment. Based on the obtained data, we discuss the distinct difference in thermal stabilities of the oligomeric assembly when incorporated in a micelle or in a lipid environment, and the physiological significance of these thermal stabilities for TR under high temperature conditions.


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MATERIALS AND METHODS Sample Preparation for Resonance Raman Measurements. The TR sample was prepared as described previously4 with some modifications. Briefly, Escherichia coli BL21(DE3) cells harboring a plasmid encoding TR with a C-terminal His tag were grown in 2×YT medium containing 50 μg/mL ampicillin. Isopropyl-β-D-thiogalactopyranoside and all-trans-retinal were added to the medium at final concentrations of 1 mM and 10 μM, respectively, in the middle of the logarithmic growth phase. The cells were harvested after 3 hours, disrupted by sonication, and the cell membranes were solubilized with 1.5% (w/v) n-dodecyl-β-D-maltoside (DDM, Dojindo Laboratories). The solubilized TR proteins were purified using a Ni-affinity column (GE Healthcare, HisTrap FF) and an ion-exchange chromatography column (GE Healthcare, HiTrap Q HP). The purities of the samples were checked by their absorption ratio at 280 nm and 530 nm. Samples with a ratio of less than 1.6 were collected and suspended in a buffer comprising 50 mM Tris-HCl (pH 7.0), 1 M NaCl, 300 mM Na2SO4, and 0.05% DDM. The H2O buffer was exchanged with a D2O buffer or a mixed buffer (H2O:D2O = 1:1) as described previously.12 The monomeric form of solubilized TR was prepared by heating a solution of TR at 85 ºC for 10 min in an incubator (Eppendorf, ThermoStat plus), then confirming the monomeric solubilized state by size exclusion chromatography and UV-vis absorption spectroscopy as described previously.5 Samples of TR embedded in egg PC (L-α-phosphatidylcholine; Avanti Polar Lipids, Inc.) were prepared by dialysis at a molar ratio of 1:20 TR:egg PC as described previously13 except that dialysis was conducted for about 1 month in this study. Following dialysis, the egg PCreconstituted sample was washed more than five times by centrifugation at 4 ºC using 50 mM


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Tris-HCl (pH 7.0) containing 1 M NaCl, then the sample was suspended in the same buffer for RR measurements. Resonance Raman Measurements. RR spectra were measured upon excitation with the 532nm line from a cw DPSS laser (Cobolt, Samba 04-01). The 90° scattering was detected using a spectrometer (HORIBA Jobin Ybon, iHR320) equipped with a cryogenically-cooled CCD camera (Roper Scientific, PyLoN:400B_eXelon VISAR). The spectrometer was calibrated using the Raman bands of acetone, cyclohexane, ethanol, 2-propanol, and toluene. The calibration error was within 1 cm−1 for defined RR bands. RR measurements of solubilized TR were conducted by placing the sample solution in a 10-mmϕ glass NMR tube used as a spinning cell which was spun at 1800 rpm at room temperature. A typical beam diameter (1/e2 width) at the sample point was about 0.19 mm. The mechanical width of the entrance slit of the spectrometer was set to 0.10 mm, which corresponded to the spectral slit width of 4.8 cm−1 at the center position of the CCD camera as judged by the spectral dispersion of the spectrometer. The spectral dispersion was about 0.4 cm−1/pixel on the CCD camera. For TR embedded in egg PC, the sample solution was circulated in a cylindrical glass tube. Time necessary for the one cycle of the circulation was about 45 seconds, which is long enough to ensure recovery of photolyzed protein to the unphotolyzed state. The spectrum at 75 ºC was obtained while continuously heating the sample solution in a reservoir at 92 ºC using a hot water bath. The unheated sample and the sample following the heat treatment were measured at room temperature. To avoid contamination of spectral contributions of the photolyzed species, we carefully examined dependence of the power of the probe laser on the RR spectra of TR. We found that the probe power of 0.2 mW at the sample position was weak enough to avoid the contamination based on the linearity of the RR intensities of TR with respect to the probe power and the absence of the


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additional bands due to the photolyzed protein (Figure S1 and S2 in the Supporting Information). Detailed descriptions of the experimental setup are described in elsewhere.12 Solid-state NMR Measurements. TR was overexpressed in E. coli BL21 (DE3) cells grown in M9 medium with 10 μM all-trans-retinal and 1.0 g of

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NH4Cl (Cambridge Isotope

Laboratories) per liter at 37 ºC until the OD reached 0.8. After cell lysis by ultra-sonication, the recombinant protein was solubilized with 1.5% DDM and purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose. The U-15N-labeled TR was reconstituted into egg PC membranes at a molar ratio of 1:30 and the samples were suspended in 10 mM Tris-HCl (pH 7.0) buffer containing 100 mM NaCl. The reconstituted sample was concentrated by centrifugation and center-packed into a 4.0 mm diameter zirconia rotor.

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N cross-polarization magic-angle spinning (CP-MAS) NMR

measurements were performed on a 600 MHz Bruker Avance III spectrometer equipped with an E-free probe at 5 ºC. 15N NMR spectra were obtained of U-15N-labeled TR without heating and following heat treatment at 75 ºC for 2 hours. An MAS frequency of 10.0 kHz and a Spinal-64 proton high power decoupling frequency of 78 kHz were employed during each acquisition. The 15

N chemical shift was referenced to glycine powder at 11.59 ppm (NH4Cl at 0.0 ppm). High-speed AFM Measurements.

HS-AFM measurements of TR embedded in lipid

membranes were obtained by reconstituting TR into lipids, followed by the nanodisc protocol provided by the manufacturer (Sigma-Aldrich) with minor modifications. Briefly, the reconstituted lipids comprised a mixture of phospholipids (asolectin) from soybean (SigmaAldrich, No. 11145). Asolectin (240 μg) was suspended in 50 μL buffer (20 mM HEPES-KOH (pH 7.4), 100 mM NaCl, and 4% DDM) and sonicated for about 1 min, then solubilized TR protein (1 nmol) and MPS (50 μL, 1 mg/mL) (MSP1E3D1, Sigma-Aldrich, No. M7074) were


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added and mixed well for at least 1 hour. Finally, we added 60 mg Bio-beads SM-2 (Bio-Rad, No. 1523920) and dialyzed the samples in detergent overnight. We did not purify the reconstituted samples using a column but obtained flat membranes

High Thermal Stability of Oligomeric Assemblies of Thermophilic

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