RESUMO
Microbial pump rhodopsins are highly versatile light-driven membrane proteins that couple protein conformational dynamics with ion translocation across the cell membranes. Understanding how microbial pump rhodopsins use specific amino acid residues at key functional sites to control ion selectivity and ion pumping direction is of general interest for membrane transporters, and could guide site-directed mutagenesis for optogenetics applications. To enable direct comparisons between proteins with different sequences we implement, for the first time, a unique numbering scheme for the microbial pump rhodopsin residues, NS-mrho. We use NS-mrho to show that distinct microbial pump rhodopsins typically have hydrogen-bond networks that are less conserved than anticipated from the amino acid residue conservation, whereas their hydrophobic interaction networks are largely conserved. To illustrate the role of the hydrogen-bond networks as structural elements that determine the functionality of microbial pump rhodopsins, we performed experiments, atomic-level simulations, and hydrogen bond network analyses on GR, the outward proton pump from Gloeobacter violaceus, and KR2, the outward sodium pump from Krokinobacter eikastus. The experiments indicate that multiple mutations that recover KR2 amino acid residues in GR not only fail to convert it into a sodium pump, but completely inactivate GR by abolishing photoisomerization of the retinal chromophore. This observation could be attributed to the drastically altered hydrogen-bond interaction network identified with simulations and network analyses. Taken together, our findings suggest that functional specificity could be encoded in the collective hydrogen-bond network of microbial pump rhodopsins.
Assuntos
Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Rodopsinas Microbianas , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/genética , Simulação de Dinâmica Molecular , Flavobacteriaceae/metabolismo , Flavobacteriaceae/química , Bombas de Próton/metabolismo , Bombas de Próton/química , Cianobactérias/metabolismoRESUMO
Proton (H+) motive force (PMF) serves as the energy source for the flagellar motor rotation, crucial for microbial motility. Here, to control PMF using light, we introduced light-driven inward and outward proton pump rhodopsins, RmXeR and AR3, into Escherichia coli. The motility of E. coli cells expressing RmXeR and AR3 significantly decreased and increased upon illumination, respectively. Tethered cell experiments revealed that, upon illumination, the torque of the flagellar motor decreased to nearly zero (28 pN nm) with RmXeR, while it increased to 1170 pN nm with AR3. These alterations in PMF correspond to +146 mV (RmXeR) and -140 mV (AR3), respectively. Thus, bidirectional optical control of PMF in E. coli was successfully achieved by using proton pump rhodopsins. This system holds a potential for enhancing our understanding of the roles of PMF in various biological functions.
Assuntos
Escherichia coli , Força Próton-Motriz , Rodopsinas Microbianas , Escherichia coli/metabolismo , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Luz , Bombas de Próton/metabolismo , Bombas de Próton/químicaRESUMO
Exiguobacterium sibiricum rhodopsin (ESR) functions as a light-driven proton pump utilizing Lys96 for proton uptake and maintaining its activity over a wide pH range. Using a combination of methodologies including the linear Poisson-Boltzmann equation and a quantum mechanical/molecular mechanical approach with a polarizable continuum model, we explore the microscopic mechanisms underlying its pumping activity. Lys96, the primary proton uptake site, remains deprotonated owing to the loss of solvation in the ESR protein environment. Asp85, serving as a proton acceptor group for Lys96, does not form a low-barrier H-bond with His57. Instead, deprotonated Asp85 forms a salt-bridge with protonated His57, and the proton is predominantly located at the His57 moiety. Glu214, the only acidic residue at the end of the H-bond network exhibits a pKa value of â¼6, slightly elevated due to solvation loss. It seems likely that the H-bond network [Asp85···His57···H2O···Glu214] serves as a proton-conducting pathway toward the protein bulk surface.
Assuntos
Exiguobacterium , Ligação de Hidrogênio , Exiguobacterium/metabolismo , Exiguobacterium/química , Prótons , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bombas de Próton/metabolismo , Bombas de Próton/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genéticaRESUMO
Common proton pumps, e.g. HsBR and PR, transport protons out of the cell. Xenorhodopsins (XeR) were the first discovered microbial rhodopsins which come as natural inward proton pumps. In this work we combine steady-state (cryo-)FTIR and Raman spectroscopy with time-resolved IR and UV/Vis measurements to roadmap the inward proton transport of NsXeR and pinpoint the most important mechanistic features. Through the assignment of characteristic bands of the protein backbone, the retinal chromophore, the retinal Schiff base and D220, we could follow the switching processes for proton accessibility in accordance with the isomerization / switch / transfer model. The corresponding transient IR signatures suggest that the initial assignment of D220 as the proton acceptor needs to be questioned due to the temporal mismatch of the Schiff base and D220 protonation steps. The switching events in the K-L and MCP-MEC transitions are finely tuned by changes of the protein backbone and rearrangements of the Schiff base. This finely tuned mechanism is disrupted at cryogenic temperatures, being reflected in the replacement of the previously reported long-lived intermediate GS* by an actual redshifted (O-like) intermediate.
Assuntos
Bombas de Próton , Rodopsina , Luz , Bombas de Próton/química , Prótons , Rodopsina/química , Bases de Schiff/química , Espectroscopia de Infravermelho com Transformada de Fourier , Vibração , Análise Espectral RamanRESUMO
The creation of unidirectional ion transporters across membranes represents one of the greatest challenges in chemistry. Proton-pumping rhodopsins are composed of seven transmembrane helices with a retinal chromophore bound to a lysine side chain via a Schiff base linkage and provide valuable insights for designing such transporters. What makes these transporters particularly intriguing is the discovery of both outward and inward proton-pumping rhodopsins. Surprisingly, despite sharing identical overall structures and membrane topologies, these proteins facilitate proton transport in opposite directions, implying an underlying rational mechanism that can transport protons in different directions within similar protein structures. In this study, we unraveled this mechanism by examining the chromophore structures of deprotonated intermediates in schizorhodopsins, a recently discovered subfamily of inward proton-pumping rhodopsins, using time-resolved resonance Raman spectroscopy. The photocycle of schizorhodopsins revealed the cis-trans thermal isomerization that precedes reprotonation at the Schiff base of the retinal chromophore. Notably, this order has not been observed in other proton-pumping rhodopsins, but here, it was observed in all seven schizorhodopsins studied across the archaeal domain, strongly suggesting that cis-trans thermal isomerization preceding reprotonation is a universal feature of the schizorhodopsin family. Based on these findings, we propose a structural basis for the remarkable order of events crucial for facilitating inward proton transport. The mechanism underlying inward proton transport by schizorhodopsins is straightforward and rational. The insights obtained from this study hold great promise for the design of transmembrane unidirectional ion transporters.
Assuntos
Bacteriorodopsinas , Bombas de Próton , Bombas de Próton/química , Prótons , Bacteriorodopsinas/química , Bases de Schiff/química , Transporte de Íons , LuzRESUMO
Retinal-containing light-sensitive proteins - rhodopsins - are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein of Exiguobacterium sibiricum. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct electrometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins.
Assuntos
Bacteriorodopsinas , Prótons , Transporte de Íons , Bombas de Próton/química , Bombas de Próton/metabolismo , Rodopsinas Microbianas/metabolismo , Bacteriorodopsinas/químicaRESUMO
Bacteriorhodopsin (bR) is a transmembrane protein that functions as a light-driven proton pump in halophilic archaea. The bR photocycle has been well-characterized; however, these measurements almost exclusively measured purified bR, outside of its native membrane. To investigate what effect the cellular environment has on the bR photocycle, we have developed a Raman-based assay that can monitor the activity of the bR in a variety of conditions, including in its native membrane. The assay uses two continuous-wave lasers, one to initiate photochemistry and one to monitor bR activity. The excitation leads to the steady-state depletion of ground-state bR, which directly relates to the population of photocycle intermediate states. We have used this assay to monitor bR activity both in vitro and in vivo. Our in vitro measurements confirm that our assay is sensitive to bulk environmental changes reported in the literature. Our in vivo measurements show a decrease in bR activity with increasing extracellular pH for bR in its native membrane. The difference in activity with increasing pH indicates that the native membrane environment affects the function of bR. This assay opens the door to future measurements into understanding how the local environment of this transmembrane protein affects function.
Assuntos
Bacteriorodopsinas , Bacteriorodopsinas/química , Bombas de Próton/química , Fotoquímica , CinéticaRESUMO
Cytochrome c oxidase (CcO) reduces O2 in the O2-reduction site by sequential four-electron donations through the low-potential metal sites (CuA and Fea). Redox-coupled X-ray crystal structural changes have been identified at five distinct sites including Asp51, Arg438, Glu198, the hydroxyfarnesyl ethyl group of heme a, and Ser382, respectively. These sites interact with the putative proton-pumping H-pathway. However, the metal sites responsible for each structural change have not been identified, since these changes were detected as structural differences between the fully reduced and fully oxidized CcOs. Thus, the roles of these structural changes in the CcO function are yet to be revealed. X-ray crystal structures of cyanide-bound CcOs under various oxidation states showed that the O2-reduction site controlled only the Ser382-including site, while the low-potential metal sites induced the other changes. This finding indicates that these low-potential site-inducible structural changes are triggered by sequential electron-extraction from the low-potential sites by the O2-reduction site and that each structural change is insensitive to the oxidation and ligand-binding states of the O2-reduction site. Because the proton/electron coupling efficiency is constant (1:1), regardless of the reaction progress in the O2-reduction site, the structural changes induced by the low-potential sites are assignable to those critically involved in the proton pumping, suggesting that the H-pathway, facilitating these low-potential site-inducible structural changes, pumps protons. Furthermore, a cyanide-bound CcO structure suggests that a hypoxia-inducible activator, Higd1a, activates the O2-reduction site without influencing the electron transfer mechanism through the low-potential sites, kinetically confirming that the low-potential sites facilitate proton pump.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Prótons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Cianetos , Bombas de Próton/química , Oxirredução , Metais , Cristalografia por Raios XRESUMO
Hydrogen bond formation and deformation are crucial for the structural construction and functional expression of biomolecules. However, direct observation of exchangeable hydrogens, especially for oxygen-bound hydrogens, relevant to hydrogen bonds is challenging for current structural analysis approaches. Using solution-state NMR spectroscopy, this study detected the functionally important exchangeable hydrogens (i.e., Y49-ηOH and Y178-ηOH) involved in the pentagonal hydrogen bond network in the active site of R. xylanophilus rhodopsin (RxR), which functions as a light-driven proton pump. Moreover, utilization of the original light-irradiation NMR approach allowed us to detect and characterize the late photointermediate state (i.e., O-state) of RxR and revealed that hydrogen bonds relevant to Y49 and Y178 are still maintained during the photointermediate state. In contrast, the hydrogen bond between W75-εNH and D205-γCOO- is strengthened and stabilizes the O-state.
Assuntos
Bombas de Próton , Rodopsina , Rodopsina/química , Bombas de Próton/química , Ligação de Hidrogênio , Espectroscopia de Ressonância MagnéticaRESUMO
Proton transport is indispensable for cell life. It is believed that molecular mechanisms of proton movement through different types of proton-conducting molecules have general universal features. However, elucidation of such mechanisms is a challenge. It requires true-atomic-resolution structures of all key proton-conducting states. Here we present a comprehensive function-structure study of a light-driven bacterial inward proton pump, xenorhodopsin, from Bacillus coahuilensis in all major proton-conducting states. The structures reveal that proton translocation is based on proton wires regulated by internal gates. The wires serve as both selectivity filters and translocation pathways for protons. The cumulative results suggest a general concept of proton translocation. We demonstrate the use of serial time-resolved crystallography at a synchrotron source with sub-millisecond resolution for rhodopsin studies, opening the door for principally new applications. The results might also be of interest for optogenetics since xenorhodopsins are the only alternative tools to fire neurons.
Assuntos
Bombas de Próton , Prótons , Bombas de Próton/química , Transporte de ÍonsRESUMO
Microbial rhodopsins are a large family of photoreceptive membrane proteins with diverse light-regulated functions. While the most ubiquitous microbial rhodopsins are light-driven outward proton (H+) pumps, new subfamilies of microbial rhodopsins transporting H+ inwardly, i.e., light-driven inward H+ pumps, have been discovered recently. Although structural and spectroscopic studies provide insights into their ion transport mechanisms, the minimum key element(s) that determine the direction of H+ transport have not yet been clarified. Here, we conducted the first functional conversion study by substituting key amino acids in a natural outward H+-pumping rhodopsin (PspR) with those in inward H+-pumping rhodopsins. Consequently, an artificial inward H+ pump was constructed by mutating only three residues of PspR. This result indicates that these residues govern the key processes that discriminate between outward and inward H+ pumps. Spectroscopic studies revealed the presence of an inward H+-accepting residue in the H+ transport pathway and direct H+ uptake from the extracellular solvent. This finding of the simple element for determining H+ transport would provide a new basis for understanding the concept of ion transport not only by microbial rhodopsins but also by other ion-pumping proteins.
Assuntos
Bombas de Próton , Rodopsina , Bombas de Próton/química , Rodopsina/química , Rodopsinas Microbianas/metabolismo , Transporte de Íons , Bombas de Íon/metabolismo , Prótons , LuzRESUMO
The K intermediate of proton pumping bacteriorhodopsin is the first intermediate generated after isomerization of retinal to the 13-cis form. Although various structures have been reported for the K intermediate until now, these differ from each other, especially in terms of the conformation of the retinal chromophore and its interaction with surrounding residues. We report here an accurate X-ray crystallographic analysis of the K structure. The polyene chain of 13-cis retinal is observed to be S-shaped. The side chain of Lys216, which is covalently bound to retinal via the Schiff-base linkage, interacts with residues, Asp85 and Thr89. In addition, the Nζ-H of the protonated Schiff-base linkage interacts with a residue, Asp212 and a water molecule, W402. Based on quantum chemical calculations for this K structure, we examine the stabilizing factors of distorted conformation of retinal and propose a relaxation manner to the next L intermediate.
Assuntos
Bacteriorodopsinas , Bacteriorodopsinas/química , Modelos Moleculares , Bombas de Próton/química , Conformação Molecular , Transporte de ÍonsRESUMO
Schizorhodopsins (SzRs) are light-driven inward proton pumping membrane proteins. A H+ is released to the cytoplasmic solvent from the chromophore, retinal Schiff base (RSB), after light absorption, and then another H+ is bound to the RSB at the end of photocyclic reaction. However, the mechanistic detail of H+ transfers in SzR is almost unknown. Here we studied the deuterium isotope effect and the temperature dependence of the reaction rate constants of elementary steps in the photocycles of SzRs. The former indicated that deprotonation and reprotonation of RSB is mainly accomplished by H+ hopping between heavy atoms with similar H+ affinity. Furthermore, the temperature dependence of the rate constants revealed that most of H+ transfer events have a high entropy barrier. In contrast, the activation enthalpy and entropy of extremely thermostable SzR (MsSzR) are significantly higher than other types of SzRs (SzR1 and MtSzR) suggesting that its highly thermostable structure is optimized with at the cost of slower reaction rates at ambient temperatures.
Assuntos
Bombas de Próton , Prótons , Cinética , Bombas de Próton/química , Bombas de Próton/metabolismo , Bases de Schiff/química , Bases de Schiff/metabolismo , TermodinâmicaRESUMO
Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. This two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum, ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively.
Assuntos
Bacteriorodopsinas , Prótons , Bacteriorodopsinas/química , Exiguobacterium , Concentração de Íons de Hidrogênio , Bombas de Próton/química , Bombas de Próton/metabolismo , Bases de Schiff/químicaRESUMO
Recent discoveries of light-driven inward proton-pumping rhodopsins have opened new avenues to exploring the mechanism of unidirectional transport because these proteins transport protons in the opposite direction to conventional proton-pumping rhodopsins, despite their similar protein structure and membrane topology. Schizorhodopsin (SzR) is a newly discovered rhodopsin family of light-driven inward proton pumps. Here, we report time-resolved resonance Raman spectra showing that cis-trans thermal reisomerization precedes reprotonation at the Schiff base of the retinal chromophore in the photocycle of SzR AM_5_00977. This sequence has not been observed for the photocycles of conventional proton-pumping rhodopsins, in which reisomerization follows reprotonation, and thus provides insights into the mechanism of proton uptake to the chromophore during inward proton pumping. The present findings are expected to contribute to controlling the direction of proton transport in engineered proteins.
Assuntos
Bombas de Próton , Prótons , Transporte de Íons , Bombas de Próton/química , Rodopsina/química , Bases de SchiffRESUMO
In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.
Assuntos
Bombas de Próton , Rodopsina , Trifosfato de Adenosina/genética , Carbono/metabolismo , Luz , Optogenética , Bombas de Próton/química , Bombas de Próton/genética , Bombas de Próton/metabolismo , Prótons , Rodopsina/química , Rodopsina/genética , Rodopsina/metabolismo , Rodopsinas Microbianas/genéticaRESUMO
Water structuring on the outer surface of protein molecules called the hydration shell is essential as well as the internal water structures for higher-order structuring of protein molecules and their biological activities in vivo. We now show the molecular-scale hydration structure measurements of native purple membrane patches composed of proton pump proteins by a noninvasive three-dimensional force mapping technique based on frequency modulation atomic force microscopy. We successfully resolved the ordered water molecules localized near the proton uptake channels on the cytoplasmic side of the individual bacteriorhodopsin proteins in the purple membrane. We demonstrate that the three-dimensional force mapping can be widely applicable for molecular-scale investigations of the solid-liquid interfaces of various soft nanomaterials.
Assuntos
Bacteriorodopsinas , Água , Bacteriorodopsinas/química , Microscopia de Força Atômica/métodos , Proteínas/análise , Bombas de Próton/química , Membrana Purpúrea/química , Água/químicaRESUMO
DTG/DTS rhodopsin, which was named based on a three-residue motif (DTG or DTS) that is important for its function, is a light-driven proton-pumping microbial rhodopsin using a retinal chromophore. In contrast to other light-driven ion-pumping rhodopsins, DTG/DTS rhodopsin does not have a cytoplasmic proton donor residue, such as Asp, Glu, or Lys. Because of the lack of cytoplasmic proton donor residue, proton directly binds to the retinal chromophore from the cytoplasmic solvent. However, mutational experiments that showed the complicated effects of mutations were not able to clarify the roles played by each residue, and the detail of proton uptake pathway is unclear because of the lack of structural information. To understand the proton transport mechanism of DTG/DTS rhodopsin, here we report the three-dimensional structure of one of the DTG/DTS rhodopsins, PspR from Pseudomonas putida, by X-ray crystallography. We show that the structure of the cytoplasmic side of the protein is significantly different from that of bacteriorhodopsin, the best-characterized proton-pumping rhodopsin, and large cytoplasmic cavities were observed. We propose that these hydrophilic cytoplasmic cavities enable direct proton uptake from the cytoplasmic solvent without the need for a specialized cytoplasmic donor residue. The introduction of carboxylic residues homologous to the cytoplasmic donors in other proton-pumping rhodopsins resulted in higher pumping activity with less pH dependence, suggesting that DTG/DTS rhodopsins are advantageous for producing energy and avoiding intracellular alkalization in soil and plant-associated bacteria.
Assuntos
Bombas de Próton , Rodopsina , Cristalografia por Raios X , Luz , Bombas de Próton/química , Prótons , Rodopsina/metabolismo , Rodopsinas Microbianas/química , SolventesRESUMO
Apoptosis is a type of programmed cell death that commonly occurs in multicellular organisms including humans and that is essential to eliminate unnecessary cells to keep organisms healthy. Indeed, inappropriate apoptosis leads to various diseases such as cancer and autoimmune disease. Here, we developed an optical method to regulate apoptotic cell death by controlling the intracellular pH with outward or inward proton pump rhodopsins, Archaerhodopsin-3 (AR3) or Rubricoccus marinas xenorhodopsin (RmXeR), respectively. The alkalization-induced shrinking of human HeLa cells cultured at pH 9.0 was significantly accelerated or decelerated by light-activated AR3 or RmXeR, respectively, implying the contribution of intracellular alkalization to the cell death. The light-activated AR3 induced cell shrinking at a physiologically neutral pH 7.4 and biochemical analysis revealed that the intracellular alkalization caused by AR3 triggered the mitochondrial apoptotic signaling pathway, which resulted in cell death accompanied by morphological changes. Phototriggered apoptosis (PTA) was also observed for other human cell lines, SH-SY5Y and A549 cells, implying its general applicability. We then used the PTA method with the nematode Caenorhabditis elegans as a model for living animals. Irradiation of transgenic worms expressing AR3 in chemosensing amphid sensory neurons significantly decreased their chemotaxis responses, which suggests that AR3 induced the cell death of amphid sensory neurons and the depression of chemotaxis responses. Thus, the PTA method has a high applicability both in vivo and in vitro, which suggests its potential as an optogenetic tool to selectively eliminate target cells with a high spatiotemporal resolution.
Assuntos
Bombas de Próton , Rodopsina , Animais , Apoptose , Células HeLa , Humanos , Transporte de Íons , Bombas de Próton/química , Rodopsina/químicaRESUMO
We have developed a methodology for identifying further thermostabilizing mutations for an intrinsically thermostable membrane protein. The methodology comprises the following steps: (1) identifying thermostabilizing single mutations (TSSMs) for residues in the transmembrane region using our physics-based method; (2) identifying TSSMs for residues in the extracellular and intracellular regions, which are in aqueous environment, using an empirical force field FoldX; and (3) combining the TSSMs identified in steps (1) and (2) to construct multiple mutations. The methodology is illustrated for thermophilic rhodopsin whose apparent midpoint temperature of thermal denaturation Tm is â¼91.8 °C. The TSSMs previously identified in step (1) were F90K, F90R, and Y91I with ΔTm â¼5.6, â¼5.5, and â¼2.9 °C, respectively, and those in step (2) were V79K, T114D, A115P, and A116E with ΔTm â¼2.7, â¼4.2, â¼2.6, and â¼2.3 °C, respectively (ΔTm denotes the increase in Tm). In this study, we construct triple and quadruple mutants, F90K+Y91I+T114D and F90K+Y91I+V79K+T114D. The values of ΔTm for these multiple mutants are â¼11.4 and â¼13.5 °C, respectively. Tm of the quadruple mutant (â¼105.3 °C) establishes a new record in a class of outward proton pumping rhodopsins. It is higher than Tm of Rubrobacter xylanophilus rhodopsin (â¼100.8 °C) that was the most thermostable in the class before this study.