Prokaryotes and eukaryotes are equipped with a variety of membrane-embedded proteins necessary for events such as, but not limited to, solute transport, catalytic activity, adherence to other cells or abiotic structures and signal transduction. In the case of prokaryotes specifically, multiple proteins are, in many cases, independently required for both passive and active transport, which may differ entirely in terms of structural homology from those of eukaryotic organisms. Bacteriorhodopsin, for instance, is a membrane-embedded structure in salt-loving, or halophilic archaea, commonly Halobacterium salinarum, that harvests light energy to ultimately generate a proton gradient. It is unique in the sense that it is exclusive to the archaeal domain and is much less complex in nature than the light harvesting complexes of photosynthetic organisms, specifically eukaryotes. Energy in the form of light is crucial for the isomerization of a bound retinaldehyde to 13-cis retinal in a photocycle and upon this event, hydrogen cations (H+) are delivered to the extracellular environment. Given that bacteriorhodopsin is not scarce in the membranes of Haloarchaea, the proton gradient acquired from retinaldehyde conversion and resulting H+ export can provide energy for other biochemical processes. Examining the rhodopsin family in which bacteriorhodopsin and related proteins belong may provide for a better understanding of the evolutionary relationships among organisms of all three recognized domains of life and a plausible path of phylogenetic division from the Last Universal Common Ancestor, or LUCA. Many studies have also suggested practical implementations for bacteriorhodopsin in many biotechnological fields.
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Bacteriorhodopsin is a retinaldehyde-containing, light-driven H+ pump located within the plasma membranes of Haloarchaea belonging to the Euryarchaeal phylum. It is a microbial opsin, or type I opsin, that consists of a chromophore (retinaldehyde, colloquially retinal) (“Opsin,” 2018). This protein is natively purple in color, thus providing a reddish-purplish hue to cell membranes in its presence (“Bacteriorhodopsin,” 2018). In fact, many halobacterium may possess even upwards of 40% bacteriorhodopsin within their cell membranes (“Bacteriorhodopsin,” 2018). The structure of bacteriorhodopsin consists of a trimer spanning the transmembrane region of the cell membrane, as confirmed by high-speed atomic force microscopy (Shibata, Yamashita, Uchihashi, Kandori & Ando, 2010). Each monomer constituent of the trimer contains exactly one retinaldehyde, or retinal molecule enclosed within seven longitudinally spanning alpha-helices appropriately termed A, B, C, D, E, F, and G helices and suppled with interconnecting loops (“Bacteriorhodopsin,” 2018). These inter-helical loops combine to create an anti-parallel beta-pleated sheet that is located opposite to the extracellular leaflet and is hydrogen-bond stabilized (Griffith & Kunka, n.d.). Lysine and aspartate residues are imperative to the proper construction of bacteriorhodopsin. A conserved lysine residue on the final helix (Helix G) is bound covalently to the inner retinaldehyde molecule in the cis configuration by a centralized Schiff base, while specific aspartate residues are essential for bacteriorhodopsin-mediated H+ movement at both intracellular and extracellular regions of the cell membrane (Pandey, 2006). These aspartic acid residues localized in opposite regions of the membrane, Asp-96 and Asp-85, the latter functioning as a proton acceptor (upon photoactivation) contained within a half channel able to permit the flow of protons into extracellular space, must be oriented and accessed in a specific manner to allow for electrostatic interactions and subsequent modifications in protonation of the Schiff base during photocycle events (Pandey, 2006). The photocycle refers to the series of conformational and configurational changes of a molecule, in this case, retinaldehyde, in the presence of light. Upon light absorption, retinaldehyde achieves an excited state, known as K, and then proceeds to L, M1, M2, M2’, N, N’, O, and finally, to its resting state, BR (light is absent) (Layni, 2006). The conversion of M1 to M2 is irreversible (Réat, Patzel, Ferrand, Pfister, Oesterhelt & Zaccai, 1998). The involvement of certain amino acid residues with bacteriorhodopsin’s retinal molecules is key in the absorption and conversion of light energy into usable energy in Haloarchaea. This molecule, as mentioned previously, is surrounded by seven alpha-helices attaching at interconnecting loops in an arc-like structure at each end or pole, giving much of bacteriorhodopsin an ellipse shape. Bacteriorhodopsin is afforded a total molecular weight of roughly between 27,000 and 28,000 Daltons and less than 250 amino acid residues (Subramaniam & Henderson, 2000). The retinaldehyde molecule positioned within the protein only accounts for a small portion of the overall mass when bound- just under 300 grams per mole and is connected to bacteriorhodopsin via an alpha-helical lysine residue (Lys-216) (Subramaniam & Henderson, 2000). This twenty-carbon chromophore exists in the all-trans-retinal state prior to the initiation of photocycle events. However, upon photoactivation (absorption of a photon by the aldehyde), the retinaldehyde undergoes isomerization and is converted to 13-cis retinal. Of course, in the absence of light, the default, or ground state (all-trans-retinal) is achieved (Subramaniam & Henderson, 2000). Light absorption by retinaldehyde cues proton export across the plasma membrane generating a proton gradient which can be used as an energy source for other cellular processes, including secondary active transport and can even be coupled with phosphorylation reactions to generate high-energy intermediates such as adenosine triphosphate (ATP). The generated proton-motive force is also notable for reducing the negative effects of environmental stressors such as pH shifts, as low pH conditions permit irreversible structural adjustments within bacteriorhodopsin, and this opsin itself may have some role in archaeal phototaxis (Griffith & Kunka, n.d. and Kouyama, Kinosita, & Ikegami, 1988). In the study conducted by Subramanian and Henderson (2000) titled, “Molecular mechanism of vectorial proton translocation by bacteriorhodopsin,” (referenced above), amino acid residues of alpha-helices B, C, F and G were key components in limiting proton flow from intracellular spaces to Asp-96, the residue associated with proton movement from the cytosolic end. These residues, Phe-42, Leu-100, Thr-170, Phe-171, and Leu-223, in dark-state settings, are unable to reach this specific aspartate residue due to the congestion of lipophilic residues spanning from Asp-96 to the Schiff base, and the situation is further deemed unfavorable due to the low Ka value of Asp-96 prior to light exposure (Subramaniam & Henderson, 2000). The study provides evidence of a “switch” apparatus at work to direct proton flow from the cytoplasm to the extracellular space following retinaldehyde conversion. Upon photon collision and absorption by a retinaldehyde molecule, a proton is lost in the change in configuration from trans to cis. The unidirectional movement of protons to ensure the proper functioning of bacteriorhodopsin as a light driven proton pump is dictated mainly by the positioning of phenylalanine residues within the sixth and seventh alpha-helices, F and G (Subramaniam & Henderson, 2000). The artificial construction of D96G, F171C, F219L triple mutant bacteriorhodopsin proteins in this study exhibited a gaping, or “open” region at the cytoplasmic end where Asp-96 would be in wild-type bacteriorhodopsin due to the significant tilting of helix F away from the center of the protein and the intermediate sloping of helix G in the same direction. Cytosolic rearrangements of F and G helices not extending to the extracellular region of bacteriorhodopsin allow for increased access to the designated channel region and proton movement to the Schiff base from the cytoplasm (Subramaniam & Henderson, 2000). The swapping of specific tryptophan residues within the seven alpha-helices may also contribute to the altered utility of available light energy by purple bacteriorhodopsin. Mogi, Marti, and Khorana (1989) in their work, “Structure-function studies on bacteriorhodopsin. IX. Substitutions of tryptophan residues affect protein-retinal interactions in bacteriorhodopsin,” made note of the effects of switching eight tryptophan residues within the transmembrane helices of the opsin. Likely, the existence of these tryptophan residues may be tied to optimal retinaldehyde functioning. For example, the replacement of Trp-86, Trp-182 and Trp-189 in the third and sixth alpha-helix with phenylalanine residues have suggested changes in the absorption spectra of retinal (Mogi, Marti & Khorana, 1989). This study demonstrated the presence of a retinal-specific binding pocket that is absent in altered residues. Furthermore, helix E’s Trp-137 modification may have also encouraged the result of blue shifts caused by the substitution of previous tryptophan residues, however, the swapping of residues 137 and 138 with cysteine and the substitution of the tryptophan residues of alpha-helices A, C and E with phenylalanine did not affect overall light absorption via chromophore or consequent proton pumping (Mogi, Marti & Khorana, 1989). Thus, every aspect of protein morphology is important in bacteriorhodopsin’s involvement in cell physiology and even minute changes in the primary structure can alter its function. While bacteriorhodopsin maintains its characteristic of acting as a primary active transporter in the generation of a proton gradient via a light-dependent reaction, it is starkly different from its eukaryotic counterparts, specifically plants, and even the rhodopsins of other prokaryotes that may serve comparable function. Plants use reaction centers and light-harvesting complexes which contain photopigments such as chlorophyll-a and chlorophyll-b, as well as accessory pigments including carotenoids (utilized by xanthorodopsin, a retinal-based photosensitive proton pump of Salinobacter strains) and phycobilins; the latter in the case of cyanobacteria (Speer, 1997 and Balashov, Imasheva, Boichenko, Antón, Wang & Layni, 2005). Purple photosynthetic bacteria, not to be confused with Haloarchaea of similar color, also contain two light-harvesting complexes containing bacteriochlorophyll within their cellular membranes and use light-dependent mechanisms to fix carbon in the presence of light and may form sulfur (Hu, Ritz, Damjanović, Autenrieth & Schulten, 2002). Bacteriorhodopsin is unmatched in that it utilizes light energy to systematically pump hydrogen ions across the cell membrane of haloarchaea, thereby generating a proton-motive force, in a manner untraditional to higher-level organisms. This rhodopsin, naturally purple in hue, largely absorbs green light at a wavelength range between 500 to 650 nanometers (maximum 568 nm) (“Bacteriorhodopsin,” 2018 and Max Planck Institute of Biochemistry, n.d.). The truly unique aspect of bacteriorhodopsin is that it is able to harvest energy from light sources (naturally, solar energy) without the need for chlorophyll and accessory pigments, thus, it does not participate in photosynthesis. Bacteriorhodopsin uses rhodopsin-based phototropy (photocycle) in hypoxic conditions, much like archaerhodopsin, and proteorhodopsin in bacteria, and interestingly, does not generate oxygen in the process (Bruslind, n.d.). In fact, oxygen-generating photosynthesis is not known to occur in archaea. Haloarchaea are also not known to fix carbon while employing bacteriorhodopsin in light-dependent reactions, therefore, they are considered photoheterotrophs (“Haloarchaea,” 2018).
Within microbial rhodopsins, including bacteriorhodopsin, many differences can be identified. These rhodopsins can be characterized by the presence of seven transmembrane alpha-helices encasing a retinaldehyde in each monomeric unit with a conserved chromophore molecule (Ernst, Lodowski, Elstner, Hegemann, Brown & Kandori, 2013). Channelrhodopsins, important components in algal phototaxis, differ from bacteriorhodopsin in amino acid composition at residue 185, where the tyrosine is replaced with a phenylalanine, indicating the lack of hydrogen-bond-related stabilization at that region (Ernst, Lodowski, Elstner, Hegemann, Brown & Kandori, 2013 and “Channelrhodopsin,” 2018). Archaerhodopsins, prevalent in both halophilic and non-halophilic archaea, and xanthorhodopsins both function as proton pumps in their respective cells, but may differ in terms of water molecule localization, side chain order, and secondary structures (Ernst, Lodowski, Elstner, Hegemann, Brown & Kandori, 2013). Halorhodopsin and sensory rhodopsin function as a light-sensitive chloride pump and chemotactic mediator, respectively, and belong in the same protein family as bacteriorhodopsin, the archaeal/bacterial/fungal rhodopsin family (Kurihara & Sudo, 2015). Proteins of this family are widely represented in all domains of life and viruses. Comparing prokaryotic or microbial rhodopsins (type 1) with metazoan rhodopsins (type 2) revealed similarities in the overall structure of the proteins, with seven conserved transmembrane helices A-G, as well as a conserved WXXY motif in helix F that is likely involved in the formation of the retinaldehyde binding pocket (Shen, Chen, Zheng & Jin, 2013). It is interesting to note that the lysine residue involved in binding the centralized retinaldehyde molecule via a Schiff base in the seventh transmembrane helix in bacteriorhodopsin is resides closer to the N-terminus in studied metazoan species (Shen, Chen, Zheng & Jin, 2013). From an evolutionary perspective, the results of Shen, Chen, Zheng and Jin’s study (2013), “The Evolutionary Relationship between Microbial Rhodopsins and Metazoan Rhodopsins,” indicate a stronger-than-moderate likelihood of residue conservation as a result of common origin between microbial and metazoan rhodopsins, given the presence of the tyrosine residue. Multiple rhodopsin genes were found to coexist in simpler prokaryotes than in their metazoan counterparts, and due to the absence of rhodopsin genes in many metazoans, the authors of the aforementioned study suggested rhodopsin’s nonessential role. Sequence identity of tested microbial and metazoan organisms remained under 30%, indicating that statistically, homology between type 1 and type 2 rhodopsins remains in question and cannot be entirely elucidated (Shen, Chen, Zheng & Jin, 2013). Thus, convergent evolution or extremely dated divergence from a pre-metazoan common ancestor is possible. Chen, Chen, Zheng, and Jin (2013) did propose that this phylogenetic speciation event may have occurred as recent as 250 million years ago. Most studies have not found any statistically significant evidence indicating any homology between the two types of rhodopsins. However, all retinal-based rhodopsins do feature some level of structural homology and pose a major advantage in prokaryote survival. In bacteriorhodopsin alone, phylogenetic tree constructions taking into account both 16S rRNA genes and Maximum Likelihood models attribute the existence of multiple copies of bacteriorhodopsin genes to duplication events and prospective mutations rather than an initial horizontal gene transfer event (Jani, 2012). Comparisons with sensory rhodopsin out-group trees could not trace definitive horizontal gene transfer events in the Haloferax genus, however, such phenomenon cannot be overlooked in divergent species of Haloarchaea (Jani, 2012). Also, the distribution of bacteriorhodopsin alone cannot gather much support for the emergent endosymbiotic theory, however, G-protein coupled receptors and microbial rhodopsins, which are exceedingly similar morphologically rather than by sequence, when compared to sodium-translocating microbial rhodopsins via sequence alignment, match more closely to each other than to their rather primitive counterpart individually (Shalaeva, Galperin & Mulkidjanian, 2015). While non-opsin G protein coupled receptors are more closely related to microbial receptors at the sodium-binding-site level than animal receptors are to these G proteins coupled receptors, sequence alignments were unable to confirm this, thus, differences may lie within the evolutionarily determined helical interactions with the retinaldehyde chromophore (Shalaeva, Galperin & Mulkidjanian, 2015). Also, certain classes of G protein coupled receptors, namely class C, likely evolved separately from other G protein coupled receptors, and this is reinforced by the aforementioned alignments (Shalaeva, Galperin & Mulkidjanian, 2015). Thus, multiple factors could be associated with the general divergence of metazoan and microbial retinal-based rhodopsins and Na+-translocating rhodopsins, which are even further removed.
The practical applications of bacteriorhodopsin in biotechnological fields is quite numerous. As mentioned previously, bacteriorhodopsin can be used in vitro in conjunction with ATP synthase to convert light to energy via an electrochemical gradient to assist in ATP synthesis via chemiosmotic coupling mechanisms (“Bacteriorhodopsin,” 2018). This event can also be emulated in laboratory settings via expression of bacteriorhodopsin in non-native bacterium in the synthesis of high energy nucleotides (ATP) through liposomal structures (Freisleben, Zwicker, Jezek, John, Bettin-Bogutzki, Ring & Nawroth, 1995). The inclusion of a light-sensitive proton pump may provide for accelerated rates of antibiotic metabolism in antibiotic-resistant pathogens and their rapid growth and division. Likewise, the energy generation by bacteriorhodopsin could be mitigated to other biochemical processes such as gas vesicle function. The function of gas vesicles is multifaceted in halophilic archaea; potential upward-floating activity may be attributed to a need for oxygen in hypoxic and anoxic environments, reduction of overall cell volume, and increased bacteriorhodopsin activity, which may provide energy for buoyancy regulation (Oren, 2013). Expression of bacteriorhodopsin and genes coding for the structure and regulation of gas vesicles in pathogens may also pose some benefit in medicine due to selective targeting techniques, although this needs more research and may not be especially advantageous in vivo. Due to the structural similarities of microbial rhodopsins such as bacteriorhodopsin and sensory rhodopsins, there is a possibility that artificial human retinas can be utilized in the near future (Hathaway, 2002). Of course, this will take some time as more information would need to be gathered on how exactly this microbial rhodopsin can mimic sensory rhodopsin function, specifically in relaying visual information to mammalian neural cells. Bacteriorhodopsins also have potential use in data storage and other bioelectronics that are currently in use, including biofilms and biosensors (Li, Tian, Tian, Tu, Gou, Wang, Qiao, Yang & Ren, 2018). The utilization of rhodopsins in long-term data storage is quite interesting, requiring conversions from intermediate photoactivated states of bacteriorhodopsin for read-write capabilities (Li et al, 2018). One area of particular interest includes energy storage technology. Photovoltaic cells constructed using metal oxide nanofibers, such as titanium (IV) oxide, in conjunction with bacteriorhodopsin-driven H+ output is not entirely new and may be improved upon the substitution of substrate and improved pH control (Allam, Yen, Near & El-Sayed, 2011). Further improvements to this area of bacteriorhodopsin-based biotechnology seem highly promising and may drive down the costs of wireless electronic gadgets in the future if graphene technology does not take over sooner. Such improvements in fuel cell technology may also provide for a reduced battery waste, as prokaryotic components are virtually expendable, and solar energy is technically everlasting.
Bacteriorhodpsin is an extensively researched archaeal protein that features a retinaldehyde chromophore and seven transmembrane helices per monomer. The trimeric arrangement of this rhodopsin allows for the efficient absorption of light energy in the generation of a proton-motive force that is likely nonessential but assists in the maintenance of cell homeostasis to a large degree. Evolutionarily, the protein family of which bacteriorhodopsin is a member of is extremely diverse and includes some non-opsin proteins as well. Due to its simplicity and availability, bacteriorhodopsin has become of increasing research interest, with potential roles in photovoltaic cells and artificial retinas. Further research outlook may involve more in-depth studies of bacterorhodpsin and related photosensitive proton pumps to determine whether or not it can effectively replace or assist sensory rhodopsin in light processing. The creation and modification of hybrid electrodes using bacteriorhodopsin and other substrates may revolutionize modern technology and thus, our dependence. Further phylogenetic analyses is still required to provide more insight into the splitting of the domains of life from the primordial LUCA.
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