Salt bridge in the conserved His–Asp cluster in <i>Gloeobacter</i> rhodopsin contributes to trimer formation

Tsukamoto, Takashi; Kikukawa, Takashi; Kurata, Takuro; Jung, Kwang Hwan; Kamo, Naoki; Demura, Makoto

doi:10.1016/j.febslet.2012.12.022

Cited by 47 publications

(115 citation statements)

References 33 publications

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“…Therefore, it is assumed that the trimer/monomer transition can occur within a few milliseconds during the photoreaction because the primary counterion Asp121 is protonated upon formation of the Mintermediate that results in breakage of the salt bridge. 26 On the other hand, recent X-ray crystallographic investigations have revealed that a marine eubacterial proton pump, blue light absorbing PR (BPR), can form pentamers and hexamers, 29 which was also proposed by previous studies on green light absorbing PR (GPR; Figure 1C). 30,31 In the crystallographic structure of BPR, the conserved His75 residue, corresponding to His87 in GR, forms a hydrogen bond not only to a counterion Asp97 but also to Trp34 in a neighboring protomer.…”

Section: ■ Introductionmentioning

confidence: 67%

“…26,35,36 After purification, the sample media were replaced by the appropriate buffer solution by centrifugation for several times using an Amicon Ultra filter (30000 MW cutoff; Merck Millipore, Bedford, MA, U.S.A.).…”

Section: ■ Methodsmentioning

confidence: 99%

“…16−24 For BR and HR, it has been shown that some amino acids on the interprotomer interaction surface contribute to the trimer formation and that inter-and intratrimer interactions are crucial for maintaining appropriate photoreactions. 22,25 A nonmarine eubacterial proton pump, Gloeobacter rhodopsin (GR), forms a trimer as well as BR and HR, 26 however, its molecular mechanism is different from BR and HR because the sign of the visible CD spectrum of GR is opposite from those of BR and HR ( Figure 1C). 20,22,27,28 The trimeric assembly of GR is maintained by an His87−Asp121 salt bridge, which is completely conserved among the eubacterial proton pumps.…”

Section: ■ Introductionmentioning

confidence: 99%

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Irreversible Trimer to Monomer Transition of Thermophilic Rhodopsin upon Thermal Stimulation

2014

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Assembly is one of the keys to understand biological molecules, and it takes place in spatial and temporal domains upon stimulation. Microbial rhodopsin (also called retinal protein) is a membrane-embedded protein that has a retinal chromophore within seven-transmembrane α-helices and shows homo-, di-, tri-, penta-, and hexameric assemblies. Those assemblies are closely related to critical physiological properties such as stabilizing the protein structure and regulating their photoreaction dynamics. Here we investigated the assembly and disassembly of thermophilic rhodopsin (TR), which is a novel proton-pumping rhodopsin derived from a thermophile living at 75 °C. TR was characterized using size-exclusion chromatography and circular dichroism spectroscopy, and formed a trimer at 25 °C, but irreversibly dissociated into monomers upon thermal stimulation. The transition temperature was estimated to be 68 °C. The irreversible nature made it possible to investigate the photochemical properties of both the trimer and the monomer independently. Compared with the trimer, the absorption maximum of the monomer is blue-shifted by 6 nm without any changes in the retinal composition, pKa value for the counterion or the sequence of the proton movement. The photocycling rate of the monomeric TR was similar to that of the trimeric TR. A similar trimer-monomer transition upon thermal stimulation was observed for another eubacterial rhodopsin GR but not for the archaeal rhodopsins AR3 and HwBR, suggesting that the transition is conserved in bacterial rhodopsins. Thus, the thermal stimulation of TR induces the irreversible disassembly of the trimer.

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Section: ■ Introductionmentioning

confidence: 67%

Section: ■ Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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Irreversible Trimer to Monomer Transition of Thermophilic Rhodopsin upon Thermal Stimulation

2014

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“…However, because the cells do not synthesize retinal, an alternative role for the actinorhodopsin apoprotein cannot be ruled out. Many microbial rhodopsins oligomerize in the membrane and form large aggregates (55)(56)(57)(58)(59)(60). Without a cofactor, actinorhodopsin might still aggregate in the membrane, providing structural stability against membrane stresses.…”

Section: Discussionmentioning

confidence: 99%

Characterization of an Unconventional Rhodopsin from the Freshwater Actinobacterium Rhodoluna lacicola

2015

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Rhodopsin-encoding microorganisms are common in many environments. However, knowing that rhodopsin genes are present provides little insight into how the host cells utilize light. The genome of the freshwater actinobacterium Rhodoluna lacicola encodes a rhodopsin of the uncharacterized actinorhodopsin family. We hypothesized that actinorhodopsin was a light-activated proton pump and confirmed this by heterologously expressing R. lacicola actinorhodopsin in retinal-producing Escherichia coli. However, cultures of R. lacicola did not pump protons, even though actinorhodopsin mRNA and protein were both detected. Proton pumping in R. lacicola was induced by providing exogenous retinal, suggesting that the cells lacked the retinal cofactor. We used high-performance liquid chromatography (HPLC) and oxidation of accessory pigments to confirm that R. lacicola does not synthesize retinal. These results suggest that in some organisms, the actinorhodopsin gene is constitutively expressed, but rhodopsin-based light capture may require cofactors obtained from the environment. IMPORTANCEUp to 70% of microbial genomes in some environments are predicted to encode rhodopsins. Because most microbial rhodopsins are light-activated proton pumps, the prevalence of this gene suggests that in some environments, most microorganisms respond to or utilize light energy. Actinorhodopsins were discovered in an analysis of freshwater metagenomic data and subsequently identified in freshwater actinobacterial cultures. We hypothesized that actinorhodopsin from the freshwater actinobacterium Rhodoluna lacicola was a light-activated proton pump and confirmed this by expressing actinorhodopsin in retinalproducing Escherichia coli. Proton pumping in R. lacicola was induced only after both light and retinal were provided, suggesting that the cells lacked the retinal cofactor. These results indicate that photoheterotrophy in this organism and others may require cofactors obtained from the environment. Rhodopsin-containing photoheterotrophic microbes are common inhabitants of marine, terrestrial, and freshwater environments, where they have been identified by cultivation (1-3), metagenomic sequencing (4-6), targeted amplicon sequencing (7-9), and quantitative PCR (9, 10). The cosmopolitan distribution of rhodopsin-containing microbes in diverse habitats is reflected in the variety of effects the rhodopsins have on their hosts. For instance, certain rhodopsins transport protons or other ions, while others affect gene expression through signaling networks (11). Within a single organism, multiple rhodopsins can be present and perform different roles (12, 13). Closely related rhodopsin-containing organisms have been shown to react to light differently: in Dokdonia spp., light exposure has been shown to provide a growth advantage for one species while offering no measurable benefit to another (14-16). In addition, rhodopsins may differ in their maximum absorption peaks (480 to 560 nm [17]) or their abilities to bind additional carotenoids (18,19) ...

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“…During the reaction, His 61 and Arg 92 are thought to be involved in the maintenance of both the pK a values of the charged residues and the proper structural changes of the peptide backbone analogous to the other eubacterial proton pumps including XR (18,(43)(44)(45)(46). Unfortunately, we cannot discuss the structural details of TR at the atomic level by precisely comparing them with XR or other microbial rhodopsins because of the present modest resolution (2.8 Å).…”

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confidence: 99%

X-ray Crystallographic Structure of Thermophilic Rhodopsin

Tsukamoto

Mizutani

Hasegawa

et al. 2016

Journal of Biological Chemistry

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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.

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Salt bridge in the conserved His–Asp cluster in Gloeobacter rhodopsin contributes to trimer formation

Cited by 47 publications

References 33 publications

Irreversible Trimer to Monomer Transition of Thermophilic Rhodopsin upon Thermal Stimulation

Irreversible Trimer to Monomer Transition of Thermophilic Rhodopsin upon Thermal Stimulation

Characterization of an Unconventional Rhodopsin from the Freshwater Actinobacterium Rhodoluna lacicola

X-ray Crystallographic Structure of Thermophilic Rhodopsin

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