Bacteriorhodopsin Research Articles

Impact of the lipid environment on the protonation dynamics of bacteriorhodopsin studied with time-resolved step-scan FTIR spectroscopy

The membrane plays an important role in the structure and function of membrane proteins. We studied the influence of the lipid environment on the photocycle of the membrane protein bacteriorhodopsin (BR) with time-resolved step-scan FTIR spectroscopy. Proton transfer dynamics was monitored with mic rosecond time resolution for BR embedded in the native purple membrane as well as reconstituted into DOPC liposomes. We observed altered protonation dynamics of the Schiff base and the primary proton acceptor Asp85, revealing a faster rise as well as decay of the M state for BR surrounded by DOPC lipids. The purple membrane consists of a lipid composition that adapts better to the protein shape resulting in a stronger protein-membrane interaction as compared to the uniform DOPC lipid environment. Conformational dynamics and the correlated protonation dynamics are affected by the altered protein-membrane interaction explaining the faster photoreaction of BR in DOPC liposomes. Here we demonstrate the high sensitivity of the proton transfer dynamics to the lipid environment of BR.

Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH.

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H(+)-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as Ma), which is different from M, with a pKa of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pKa value of ca. 9.5 was estimated for this inversion and was in good agreement with the pKa value of the formation of Ma (∼ 9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed.

Photo-Current and TEM Imaging Characterization of Light-Gated Ion Pump Proteins in Lipid Membranes

We report our study on TEM imaging, and photocurrent characterization of two photosynthetic proteins, Bacteriorhodopsin (BR), and Halorhodopsin (HR), which act as light-gated ion pumps. Bacteriorhodopsin acts as a proton (H+) pump, and Halorhodopsin as a chloride ion (Cl-) pump. Both proteins are reconstituted in nano-bilayer membranes supported by Teflon and silicon-based substrates. When a beam of green or blue light shines on the membranes, it induces ion (H+ or Cl-) transport, which is measurable as a photocurrent. We measure how the photocurrent generated by BR and HR changes as a function of their concentration in the membranes and cross-membrane potential. Furthermore, we take TEM images of the membranes with BR and HR reconstituted, to characterize the structure and the density of the proteins in the lipid membranes. The relationship between these structures and the generated photocurrent will also be discussed.

Membrane Protein Analyses Using Alkylated Trihydroxyacetophenone (ATHAP) as a MALDI Matrix.

Membrane proteins containing hydrophobic regions have been difficult to analyze using MALDI-MS, probably due to the use of conventional matrices with a low affinity for hydrophobic peptides. Recently, we reported 1-(2,4,6-trihydroxyphenyl)octan-1-one (alkylated trihydroxyacetophenone (ATHAP)) as a matrix for hydrophobic peptides. In this study, ATHAP was applied to analyze membrane proteins containing transmembrane domains. As a result, we detected intact molecular ions for bacteriorhodopsin (BR) containing seven transmembrane domains that are difficult to detect using 2,4,6-trihydroxyacetophenone or sinapinic acid, by using ATHAP. In addition, we detected digest ions containing all seven transmembrane domains that are difficult to detect using α-cyano-4-hydroxycinnamic acid (CHCA), by using ATHAP. Moreover, ions for hydrophobic digests containing a single transmembrane domain for cadherin 1 (CDH1), fibroblast growth factor receptor 4 (FGFR4), epithelial cell adhesion molecule (EPCAM) recombinant proteins, and human epidermal growth factor receptor type 2 (HER2) were detected with higher sensitivity using ATHAP than with CHCA, confirming that ATHAP improved the membrane protein analyses, especially for hydrophobic regions such as transmembrane domains.

Silver Nanoparticles Strongly Affect the Properties of Bacteriorhodopsin, a Photosensitive Protein of Halobacterium Salinarium Purple Membranes

Interaction of silver nanoparticles (AgNPs) with purple membranes (PM) has been studied using atomic force microscopy and optical spectroscopy. It was shown that AgNPs alter the photocycle of the bacteriorhodopsin (BRh) molecules located in SERS-active spots. AgNPs suppresses the photocycle by “freezing” the state of retinal in which BRh has bound AgNPs. Outside SERS-active spots, BRh molecules retain the capacity for carrying out the photocycle but with shorter lifetimes of its photointermediate states. Thus, the AgNPs may affect the BRh photocycle through its acceleration in the regions of weak AgNPs-PM interaction or by the photocycle freezing in the SERS-active spots.

Diversity and functional analysis of light-driven pumping rhodopsins in marine Flavobacteria.

The aims of this study are the description of diversity for proteorhodopsin (PR)‐containing flavobacteria in marine environments, the finding of novel photoreceptive membrane proteins, and the elucidation of the effect of light on the growth of three rhodopsin genes containing flavobacterium. We investigated novel sodium ion rhodopsin (NaR) and halorhodopsin (HR) genes from PR‐containing flavobacteria that were previously isolated from diverse aquatic sites, mainly from tidal flat sediment (62.5%). In 16 PR‐containing isolates, three new types of genes were found. Among these three isolates, one (Nonlabens sp. YIK11 isolated from sediment) contained both the NaR and chloride ion rhodopsin (ClR) ‐ HR type of gene. The sequences showed that the DTE (proton pump), NDQ (sodium ion pump) and NTQ (chloride ion pump) motifs corresponding to the D85, T89, and D96 positions in bacteriorhodopsin (BR) were well conserved. Phylogenetic analysis indicated that three NaR and one ClR grouped within the same clade, as previously reported. Illumination of cell suspensions showed the change in proton pump activity, supporting that one or more rhodopsins are functional. The qRT‐PCR study revealed that three rhodopsin genes, especially NaR, are highly induced when they are incubated in the presence of light or in the absence of sufficient nutrients. The expression levels of the DTE, NDQ, and NTQ motif‐containing rhodopsin genes in YIK11 correlate positively with illumination, but negatively with nutrient levels. Based on those results, we concluded that light has a positive impact on the relative expression levels of the three rhodopsin genes in the flavobacterium, Nonlabens sp. YIK11, but with no apparent positive impact on growth. Consequently, light did not stimulate the growth of YIK11 as determined by cell numbers in a nutrient‐limited or ‐enriched medium, although it contains and induces three rhodopsins.

Structural and Functional Studies of a Newly Grouped Haloquadratum walsbyi Bacteriorhodopsin Reveal the Acid-resistant Light-driven Proton Pumping Activity

Retinal bound light-driven proton pumps are widespread in eukaryotic and prokaryotic organisms. Among these pumps, bacteriorhodopsin (BR) proteins cooperate with ATP synthase to convert captured solar energy into a biologically consumable form, ATP. In an acidic environment or when pumped-out protons accumulate in the extracellular region, the maximum absorbance of BR proteins shifts markedly to the longer wavelengths. These conditions affect the light-driven proton pumping functional exertion as well. In this study, wild-type crystal structure of a BR with optical stability under wide pH range from a square halophilic archaeon, Haloquadratum walsbyi (HwBR), was solved in two crystal forms. One crystal form, refined to 1.85 Å resolution, contains a trimer in the asymmetric unit, whereas another contains an antiparallel dimer was refined at 2.58 Å. HwBR could not be classified into any existing subgroup of archaeal BR proteins based on the protein sequence phylogenetic tree, and it showed unique absorption spectral stability when exposed to low pH values. All structures showed a unique hydrogen-bonding network between Arg(82) and Thr(201), linking the BC and FG loops to shield the retinal-binding pocket in the interior from the extracellular environment. This result was supported by R82E mutation that attenuated the optical stability. The negatively charged cytoplasmic side and the Arg(82)-Thr(201) hydrogen bond may play an important role in the proton translocation trend in HwBR under acidic conditions. Our findings have unveiled a strategy adopted by BR proteins to solidify their defenses against unfavorable environments and maintain their optical properties associated with proton pumping.

Structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin I from the marine plant Acetabularia acetabulum.

Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 Å) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pKa of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pKa of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.

Ion-pumping microbial rhodopsins.

Rhodopsins are light-sensing proteins used in optogenetics. The word “rhodopsin” originates from the Greek words “rhodo” and “opsis,” indicating rose and sight, respectively. Although the classical meaning of rhodopsin is the red-colored pigment in our eyes, the modern meaning of rhodopsin encompasses photoactive proteins containing a retinal chromophore in animals and microbes. Animal and microbial rhodopsins possess 11-cis and all-trans retinal, respectively, to capture light in seven transmembrane α-helices, and photoisomerizations into all-trans and 13-cis forms, respectively, initiate each function. Ion-transporting proteins can be found in microbial rhodopsins, such as light-gated channels and light-driven pumps, which are the main tools in optogenetics. Light-driven pumps, such as archaeal H+ pump bacteriorhodopsin (BR) and Cl− pump halorhodopsin (HR), were discovered in the 1970s, and their mechanism has been extensively studied. On the other hand, different kinds of H+ and Cl− pumps have been found in marine bacteria, such as proteorhodopsin (PR) and Fulvimarina pelagi rhodopsin (FR), respectively. In addition, a light-driven Na+ pump was found, Krokinobacter eikastus rhodopsin 2 (KR2). These light-driven ion-pumping microbial rhodopsins are classified as DTD, TSA, DTE, NTQ, and NDQ rhodopsins for BR, HR, PR, FR, and KR2, respectively. Recent understanding of ion-pumping microbial rhodopsins is reviewed in this paper.

Assay of bacteriorhodopsin stability on polycarbonate surface by using of FTIR-ATR: a model of disk-based bioassays.

Bacteriorhodopsin (BR) is a transmembrane protein which able to transport protons through cell membrane and thus converting solar energy to electrical energy. Up to now different strategies have been used to immobilize BR. In the present study the BR has been immobilized on polycarbonate surface with two different methods. The functional groups of polycarbonate were modified in two ways (sulfuric acid, PDAC and HNO3) and then the BR was immobilized on two different modified polycarbonate surfaces. The modified surfaces were characterized by ATR-FTIR and AFM techniques. Afterward the activity of bounded BR to two different modified polycarbonate surfaces was measured. Our results show that BR bounded to modified polycarbonate surface with HNO3 (nitrated polycarbonate) has higher activity in comparison to modified with sulfuric acid (electrostatically bounded BR). Also the activities of both types of Bounded BR after 10 days were measured. The results showed that unlike electrostatically bounded BR, bounding BR to nitrated polycarbonate keeps its activity after 10 days. In conclusion, nitrated polycarbonate surface is a suitable candidate due to immobilizing BR in order to manufacture of BioCDs.

Improved protease digestion conditions for membrane protein detection.

This work presents improved protease digestion conditions for membrane protein detection. The enzymatic digest of bacteriorhodopsin (BR), a model membrane protein with seven transmembrane domains (TMDs) was investigated. An initial in-gel digestion identified 17% BR sequence coverage, including part of the seventh TMD. To improve sequence coverage, BR digest was tested with different concentrations of RapiGest, methanol (MeOH) and SDS using either trypsin or chymotrypsin. Two improved conditions, 0.01% SDS or the combination of 10% MeOH and 0.01% RapiGest, were chosen. Trypsin digestions in both conditions achieved more than 40% BR sequence coverage compared to 0% using standard digestion conditions. Peptides detected from trypsin and chymotrypsin digestions in the same condition were combined to maximize sequence coverage. The same conditions were applied to a different membrane protein with one TMD, Selenoprotein S, and proteins from Escherichia coli. For Selenoprotein S, a higher sequence coverage, including a peptide from the TMD, was detected from the improved condition compared to the typical condition. The application of both improved conditions to a membrane protein fraction of Escherichia coli resulted in the identification of 309 (SDS) and 329 (MeOH/RapiGest) unique proteins of which 140/309 and 148/329 were membrane proteins.

ESR - a retinal protein with unusual properties from Exiguobacterium sibiricum.

This review covers the properties of a retinal protein (ESR) from the psychrotrophic bacterium Exiguobacterium sibiricum that functions as a light-driven proton pump. The presence of a lysine residue at the position corresponding to intramolecular proton donor for the Schiff base represents a unique structural feature of ESR. We have shown that Lys96 successfully facilitates delivery of protons from the cytoplasmic surface to the Schiff base, thus acting as a proton donor in ESR. Since proton uptake during the photocycle precedes Schiff base reprotonation, we conclude that this residue is initially in the uncharged state and acquires a proton for a short time after Schiff base deprotonation andM intermediate formation. Involvement of Lys as a proton donor distinguishes ESR from the related retinal proteins- bacteriorhodopsin (BR), proteorhodopsin (PR), and xanthorhodopsin (XR), in which the donor function is performed by residues with a carboxyl side chain. Like other eubacterial proton pumps (PR and XR), ESR contains a histidine residue interacting with the proton acceptor Asp85. In contrast to PR, this interaction leads to shift of the acceptor's pKa to more acidic pH, thus providing its ability to function over a wide pH range. The presence of a strong H-bond between Asp85 and His57, the structure of the proton-conducting pathways from cytoplasmic surface to the Schiff base and to extracellular surface, and other properties of ESR were demonstrated by solving its three-dimensional structure, which revealed several differences from known structures of BR and XR. The structure of ESR, its photocycle, and proton transfer reactions are discussed in comparison with homologous retinal proteins.

Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis

Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.

Exploration of a haloarchaeon, Halostagnicola larsenii, isolated from rock pit sea water, West Coast of Maharashtra, India, for the production of bacteriorhodopsin.

The aim of the present investigation was to isolate haloarchaea from rock pit sea water, West Coast of India and to explore their potential in the production of bacteriorhodopsin (BR) which converts light energy into electrical energy. Haloarchaeal strains were isolated from rock pit sea water samples collected from Rock garden, Malvan, West Coast of India. Based on morphological, physiological and biochemical characteristics, and 16S rRNA gene sequencing, all the 11 strains were identified as Halostagnicola larsenii. All the strains require at least 1·5moll(-1) NaCl for growth; grow optimally in the range of 3·5-5·2moll(-1) NaCl. BR was detected in all the strains ranging from 0·035 to 0·258gl(-1) . All 11 strains showed conversion of light energy into electrical energy in the range of 0·7-44·2mV, when exposed to sunlight. A haloarchaeon, Hst.larsenii is isolated from rock pit sea water and demonstrated to have BR that converted light energy into electrical energy. The present investigation is presumably the first report of the isolation of Hst.larsenii from low salinity environment and its potential in production of BR. The haloarchaeon could be explored for the generation of electrical energy.

Novel expression and characterization of a light driven proton pump archaerhodopsin 4 in a Halobacterium salinarum strain

Archaerhodopsin 4 (AR4), a new member of the microbial rhodopsin family, is isolated from Halobacterium species xz515 in a Tibetan salt lake. AR4 functions as a proton pump similar to bacteriorhodopsin (BR) but with an opposite temporal order of proton uptake and release at neutral pH. However, further studies to elucidate the mechanism of the proton pump and photocycle of AR4 have been inhibited due to the difficulty of establishing a suitable system in which to express recombinant AR4 mutants. In this paper, we report a reliable method for expressing recombinant AR4 in Halobacterium salinarum L33 with a high yield of up to 20mg/l. Experimental results show that the recombinant AR4 retains the light-driven proton pump characteristics and photo-cycling kinetics, similar to that in the native membrane. The functional role of bacterioruberin in AR4 and the trimeric packing of AR4 in its native and recombinant forms are investigated through light-induced kinetic measurements, two-dimensional solid-state NMR experiments, dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). Such approaches provide new insights into structure–function relationships of AR4, and form a basis for other archaeal rhodopsins.

F-state based permanent optical data storage in bacteriorhodopsin film

Upon excitation of high intensity linearly polarized femtosecond laser, a blue-shifted two-photon induced photoproduct named F540 state with permanent photo-induced anisotropy is observed in bacteriorhodopsin (BR) films. Based on the F-state, permanent optical data storage can be realized with spatial field modulation of femtosecond laser. By using a phase-only spatial light modulator to modulate the spatial phase distribution of the incident femtosecond laser beam, we generate spot-patterns in the focal plane of the microscopic objective lens, and the patterns are recorded in the BR film. At the same time, by varying the polarization direction of the excitation laser beam, polarization-multiplexed optical data storage in the BR film is demonstrated, which is applicable to high-density optical data storage and optical information encryption.

Effect of Complex Nutrients and Repeated-Batch Cultivation of Halobacterium salinarum on enhancing Bacteriorhodopsin Production

The extreme halophilic bacteria Halobacterium salinarum is known to produce bacteriorhodopsin (BR) protein in its purple membrane (PM) as a light-driven pump for the synthesis ATP. Its growth does not utilize simple carbon sources such as glucose, but relies on the complex carbon/nitrogen sources. The production yield of BR in the culture of H. salinarum also strongly depends on the complex nitrogen sources employed. From the various complex carbon/nitrogen sources employed in this work, tryptone rather than commonly used peptone was found to be the best complex nutrient for the growth of H. salinarum and its BR production. Bubble column photobioreactor operated in a repeated-batch mode was also employed to enhance the growth of H. salinarum by intermittent removing growth inhibitory metabolites. By employing 0.5% tryptone as carbon/nitrogen source in the culture medium, 201.8 mg/L of BR was obtained after 210 h repeated-batch cultivation which is about 50% higher than that obtained in shaker flask cultivation.

Vibrational Studies of Channelrhodopsin-1 from Chlamydomonas Augustae: Protonation Changes during the Early Photocycle

Channelrhodopsins (ChRs) from green algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs in order to bioengineer their properties for specific optogenetic applications. Most studies have focused so far on channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Here, low-temperature and time-resolved rapid scan FTIR-difference spectroscopy, along with kinetic UV-Visible transient absorbance spectroscopy, are applied to study protein conformational changes occurring during the photocycle of the red-shifted channelrhodopsin-1 from Chlamydomonas augustae (CaChR1). Substitutions with isotope-labeled retinals or the retinal analog A2, site-directed mutagenesis, hydrogen-deuterium exchange and H218O substitution are used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to CrChR2, almost exclusively all-trans to 13-cis isomerization of the retinal chromophore, similar to the primary phototransition of bacteriorhodopsin (BR). A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1 which, along with amide I bands, reflects alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2 indicating a more heterogeneous environment. Based on the data from mutations involving the residues Glu169 and Asp299 (homologs of the Asp85 and Asp212 Schiff base counterions in BR), as well as Phe139 (Lys132 in the C1C2 chimera located within hydrogen bonding distance of the homologs of Asp85 and Asp212), we propose a model for protonation changes occurring near the Schiff base during the P1 and P2 steps of the photocycle.

Using Cell-Free Expression to Create Light-Activated Proteins In Situ in Droplet Interface Bilayer Networks

ABSTRACTDroplet interface bilayers (DIBs) are physical lipid bilayers that mimic real membranes in living cells, and they are formed quickly using droplets of water and lipids in oil (Fig. 1A). DIBs allow biomolecular sensing and direct detection of transmembrane proteins or peptides and small molecules such as drugs, anesthetics, or even ions. Cell-free expression systems allow in vitro protein synthesis using actual natural machinery extracted from organisms (Fig. 1B). Previous attempts to combine DIBs with cell-free extracts (CFE) encountered bilayer destabilization due to components in the expression system. This study evaluates incorporation of Promega’s T7 S30 High Yield (HY) Expression system with DIBs to pave the way for future in situ expression of light-activated bacteriorhodopsin (BR) and other complex transmembrane proteins in DIBs. A secondary output includes establishing a method for real-time monitoring and modeling of CF expression reactions using minimal volume. The ability to quantify CF output in such small volumes reduces cost per reaction from $20 to around $0.40, and synthesized protein levels reach tens to hundreds of micrograms per milliliter in less than 1 hour at 37°C.

Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas algae.

Channelrhodopsins (ChRs) from green flagellate algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs to bioengineer their properties for specific optogenetic applications such as elucidating the function of specific neurons in brain circuits. While most studies have used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), in this work low-temperature Fourier transform infrared-difference spectroscopy is applied to study the conformational changes occurring during the primary phototransition of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1). Substitution with isotope-labeled retinals or the retinal analogue A2, site-directed mutagenesis, hydrogen–deuterium exchange, and H218O exchange were used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to that of CrChR2, almost exclusively an all-trans to 13-cis isomerization of the retinal chromophore, as in the primary phototransition of bacteriorhodopsin (BR). In addition, significant differences are found for structural changes of the protein and internal water(s) compared to those of CrChR2, including the response of several Asp/Glu residues to retinal isomerization. A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1, which reflects along with amide I bands alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2, indicating a more heterogeneous environment. Mutations involving residues Glu169 and Asp299 (homologues of the Asp85 and Asp212 Schiff base counterions, respectively, in BR) lead to the conclusion that Asp299 is protonated during P1 formation and suggest that these residues interact through a strong hydrogen bond that facilitates the transfer of a proton from Glu169.