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PubMed2002

Retinoid composition and retinal localization in the eggs of teleost fishes.

Retinoids in the eggs of four teleosts, chum salmon (Oncorhynchus keta), black porgy (Acanthopagrus schlegeli), marbled flounder (Pleuronectes yokohamae), and stingfish (Inimicus japonicus), were analyzed by high performance liquid chromatography. Retinal (RAL1) or both RAL1 and 3,4-didehydroretinal (RAL2) were major or exclusive retinoids in the eggs of every species examined. In O. keta eggs, both RAL1 and RAL2 were present at the ratio of approximately 3:4, whereas RAL1 was the only retinal in the eggs of the other three marine species. RAL1 was the exclusive retinoid in the eggs of P. yokohamae and I. japonicus, whose eggs lack lipid bodies. In the eggs of O. keta and A. schlegeli, which have lipid bodies, retinylesters were also detected, and retinals composed 69% and 93%, respectively, of total retinoids. In O. keta eggs, retinals were present mostly in the aqueous part and were bound to a protein homologous to lipovitellin 1, an amphibian yolk protein, and retinylesters were located in lipids. These results indicate that retinals are the essential mode of retinoid storage in eggs of teleosts and they are the precursors of functional retinoids, such as retinoic acid and visual pigment chromophores. Retinylesters are additional retinoids that accompany lipid accumulation.

Animals↗
PubMed2003

Storage of retinal in the eggs of the ascidian, Halocynthia roretzi.

Retinoids in the eggs of the solitary ascidian, Halocynthia roretzi, were analyzed by high performance liquid chromatography. Retinal was the almost exclusive retinoid (>>99%), and the concentration of retinal was 25.9-40.1 (30.6 on average) ng/mg of protein. The egg retinal consisted of four isomers: all-trans (50.9%), 9-cis (6.8%), 11-cis (20.4%) and 13-cis (21.9%). The presence of retinal in the eggs of this ascidian is a characteristic shared with the wide range of oviparous vertebrates, although the isomer composition differs between ascidian eggs and vertebrate eggs; in vertebrate eggs, almost all the retinal is in the all-trans form. The egg retinal was bound to a protein complex via a Schiff base linkage. The electrophoretic characteristics of the protein complex were similar to that of egg yolk proteins of oviparous vertebrates. The results presented in this study strongly suggest that, as is found with oviparous vertebrates, retinal in the ascidian eggs is the essential mode of retinoid storage, and is the precursor of photoreceptive pigment chromophores and retinoic acid during development.

Animals↗
PubMed1998

Release of retinol and denaturation of its plasma carrier, retinol-binding protein.

BACKGROUND: Retinol is tightly packed inside the structure of its plasma carrier (retinol-binding protein, RBP). It was found that retinol release from RBP to aqueous solutions is facilitated by either very low pH or very high temperatures (i.e. by non-physiological conditions that cause protein denaturation). It was also found that alcohols induce protein conformational transitions to denatured states. On this basis, it may be suggested that retinol release in vivo is facilitated by the partial unfolding of the carrier resulting from the concerted action of the moderate local decrease of pH and the moderate local decrease of dielectric constant in proximity to the target membranes. RESULTS: In vitro, at 37 degrees C, retinol is removed from its plasma carrier by the concerted action of the moderately low pH and the moderately low dielectric constant of solutions containing a low ionic strength buffer and methanol in variable proportions. Release of retinol is accompanied by a conformational transition of RBP from the native to the molten-globule state. CONCLUSIONS: The physiological function of RBP-targeted delivery of retinol-is mimicked in vitro by the facilitated release of retinol (associated with a partial unfolding of the protein carrier) in solutions exhibiting pH and dielectric constant values that are within the range of values expected in the in vivo microenvironment.

Circular Dichroism↗
PubMed1999

Interactions of retinoid binding proteins and enzymes in retinoid metabolism.

Naturally occurring retinoids (vitamin A or retinol and its active metabolites) are vital for vision, controlling the differentiation program of epithelial cells in the digestive tract and respiratory system, skin, bone, the nervous system, the immune system, and for hematopoiesis. Retinoids are essential for growth, reproduction (conception and embryonic development), and resistance to and recovery from infection. The functions of retinoids in the embryo begin soon after conception and continue throughout the lifespan of all vertebrates. Both naturally occurring and synthetic retinoids are used in the therapy of various skin diseases, especially acne, for augmenting the treatment of diabetes, and as cancer chemopreventive agents. Retinol metabolites serve as ligands that activate specific transcription factors in the superfamily of steroid/retinoid/thyroid/vitamin D/orphan receptors and thereby control gene expression. Additionally, retinoids may also function through non-genomic actions. Various retinoid binding proteins serve as partners in retinoid function. These binding proteins show high specificity and affinity for specific retinoids and seem to control retinoid metabolism in vivo qualitatively and quantitatively by reducing 'free' retinoid concentrations, protecting retinoids from non-specific interactions, and chaperoning access of metabolic enzymes to retinoids. Implementation of the physiological effects of retinoids depends on the spatial-temporal expressions of binding proteins, receptors and metabolic enzymes. This review will discuss current understanding of the enzymes that catalyze retinol and retinoic acid metabolism and their unique and integral relationship to retinoid binding proteins.

Alcohol Dehydrogenase↗
PubMed2001

Differential effects of retinoids on proliferation of bovine mammary epithelial cells in collagen gel culture.

The effects of increasing concentrations of retinol, retinal and retinoic acid on proliferation of bovine mammary epithelial cells were investigated in collagen gel cultures. All retinoids significantly inhibited proliferation of mammary epithelial cells. The relative inhibitory potency of the retinoids was: retinoic acid > retinal > retinol. Maximal inhibition at 10 microg/ml corresponded to a 75-95% inhibition of proliferation obtained in basal medium. Retinol, retinal and retinoic acid also inhibited proliferation of cells growth-stimulated with insulin-like growth factor-I (IGF-I). Retinoids in highest concentrations (10 microg/ml) inhibited 68-85% of proliferation of cells obtained in culture medium containing 25 ng IGF-I/ml. Retinol and retinoic acid also inhibited proliferation of cells growth-stimulated by insulin and other growth factors from the IGF growth factor family (des(1-3)IGF-I and IGF-II), as well as growth factors from the epidermal growth factor family (EGF and TGF-alpha), with retinoic acid being more effective than retinol. At a concentration of 100 ng/ml retinol and retinoic acid inhibited respectively 24-38 and 44-52% of mammary cell proliferation stimulated by growth factors of the IGF family, and at 10,000 ng/ml, 61-71% of cell proliferation was inhibited. The growth-stimulating effect of insulin, EGF and TGF-alpha was inhibited 42-64% by retinol and retinoic acid at 100 ng/ml, and 64-84% at 10,000 ng/ml. The present results show that retinol, retinal and retinoic acid are potent inhibitors of bovine mammary epithelial cell proliferation. It is suggested that retinoids may have concentration-dependent roles in regulation of pubertal mammary growth and development, indicating that the milk yield potential of heifers may be affected by vitamin A status.

Animals↗
PubMedDate not supplied

Movement of retinal along cone and rod photoreceptors.

Single isolated photoreceptors can be taken through a visual cycle of light adaptation by bleaching visual pigment, followed by dark adaptation when supplied with 11-cis retinal. Light adaptation after bleaching is manifested by faster response kinetics and a permanent reduction in sensitivity to light flashes, presumed to be due to the presence of bleached visual pigment. The recovery of flash sensitivity during dark adaptation is assumed to be due to regeneration of visual pigment to pre-bleach levels. In previous work, the outer segments of bleached, light-adapted cells were exposed to 11-cis retinal. In the present work, the cell bodies of bleached photoreceptors were exposed. We report a marked difference between rods and cones. Bleached cones recover sensitivity when their cell bodies are exposed to 11-cis retinal. Bleached rods do not. These results imply that retinal can move freely along the cone photoreceptor, but retinal either is not taken up by the rod cell body or retinal cannot move from the rod cell body to the rod outer segment. The free transfer of retinal along cone but not along rod photoreceptors could explain why, during dark adaptation in the retina, cones have access to a store of 11-cis retinal which is not available to rods. Additional experiments investigated the movement of retinal along bleached rod outer segments. The results indicate that retinal can move along the rod outer segment, but that this movement is slow, occurring at about the same rate as the regeneration of visual pigment.

Ambystoma↗
PubMedDate not supplied

Packaging of rhodopsin and porphyropsin in the compound eye of the crayfish.

The distribution of 3-dehydroretinal (Ral2) in dorsal, middle, and ventral slices of eyes of the crayfish Procambarus clarkii was examined by HPLC. No pronounced differences were found. Similar results were obtained when the eyes were cut into anterior, intermediate, and posterior portions. Dichroic difference spectra were measured in single halves of microvillar layers of isolated rhabdoms and the proportions of rhodopsin (P1) and porphyropsin (P2) estimated by comparison with computer-generated mixtures of these pigments, whose spectra are known from previous work. The fraction of visual pigment that is porphyropsin appears to be uniform throughout individual retinular cells and among the retinular cells of individual rhabdoms, but various substantially among different rhabdoms from the same eye. The interommatidial variation in the amount of P2 greatly exceeds the gross regional variation in Ral2. This means there is an intermingling of ommatidia with different levels of P2. The variability in P2 among ommatidia is not likely to have important implications for the vision of the crayfish but suggests that in the metabolism of retinoids, individual ommatidia are quasi-independent metabolic units. The results are compatible with a single opsin for both crayfish rhodopsin and porphyropsin.

Animals↗
PubMedDate not supplied

The retinoids of seven species of mantis shrimp.

Eyes of stomatopod crustaceans, or mantis shrimps, contain the greatest diversity of visual pigments yet described in any species, with as many as ten or more spectral classes present in a single retina. In this study, the eyes of seven species of mantis shrimp from three superfamilies of stomatopods were examined for their content of retinoids. Only retinal and retinol were found; neither hydroxyretinoids nor dehydroretinoids were detected. The principal isomers were 11-cis and all-trans. The eyes of most of these species contain stores of 11-cis retinol, principally as retinyl esters, and in amounts in excess of retinal. Squilla empusa is particularly noteworthy, with over 5000 pmoles of retinol per eye.

Animals↗
PubMedDate not supplied

Evidence for the prolonged photoactivated lifetime of an analogue visual pigment containing 11-cis 9-desmethylretinal.

Following bright flashes, rod photoreceptors exhibit a period of photocurrent saturation that increases linearly with the logarithm of flash intensity. In a recent report, Pepperberg et al. (1992) presented evidence that the slope of the function relating the saturation period (T) to the natural logarithm of flash intensity (ln If) represents the exponential lifetime (tau) of photoactivated visual pigment: tau = delta T/delta [ln If]. In salamander rods, 11-cis 9-desmethylretinal combines with opsin to form 9-desmethyl rhodopsin. Dim flash responses mediated by this analogue visual pigment exhibited slow recovery kinetics relative to those of native pigment (Corson et al., 1991). This observation raises the hypothesis that the physiological lifetime of photoactivated 9-desmethyl rhodopsin is substantially longer than that of native visual pigment. To test this hypothesis, we have examined the relation between the period of photocurrent saturation and flash intensity in salamander rods containing a mixture of the two pigments. Brief stimuli at two widely separated wavelengths (440 and 640 nm) elicited saturating photocurrent responses that were preferentially mediated by 9-desmethyl rhodopsin or residual native pigment, respectively. Plots of T vs. ln If revealed a linear increase in the period of response saturation over a large range of saturating intensities at both wavelengths. However, the slope of the relation between T and ln If with 440-nm flashes was more than twice as large (4.1 +/- 0.5 s, n = 5) as that measured with 640-nm flashes (1.7 +/- 0.4 s). For rods subjected only to bleaching of the native pigment, or to bleaching and resensitization with 11-cis retinal, the slope of the relation between T and ln If remained independent of wavelength and indistinguishable from that of native pigment in unbleached cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Spectral and polarization sensitivity of photocurrents of amphibian rods in the visible and ultraviolet.

Photocurrents from isolated rods of adults and sub-adults of three species of amphibians, Rana pipiens, Ambystoma tigrinum, and Xenopus laevis, were measured with suction pipette electrodes. The intensity for a half-maximal response was 0.91+/-0.48 photons microm(-2) flash(-1) (mean +/- S.D., 10-ms flashes) for Rana, 0.92+/-0.44 for Ambystoma, and 6.14+/-1.33 for Xenopus. The mean number of photoisomerizations at half-saturation was 22+/-12 for Rana, 50+/-24 for Ambystoma, and 221+/-48 for Xenopus. The photocurrent per photoisomerization is several times smaller in Xenopus rods than in the other two species. Spectral sensitivity was measured from 277-737 nm with light polarized both parallel and perpendicular to the planes of the membrane disks. Dichroism fell in the near UV and was absent in the region of absorption by tryptophan and tyrosine. Maximum sensitivity of Rana was at 503.9+/-2.6 nm (n = 86), and of Ambystoma, 505.8+/-1.8 nm (n = 24). Animals from these same batches that were sampled by HPLC had no 3-dehydroretinal (retinal2). Xenopus containing about 94% retinal2 and 6% retinal1 had lambda(max) at 519.3+/-2.7 nm (n = 11). Spectral position of the beta-band, estimated by the method of Stavenga et al. (1993), appears to be at longer wavelengths in amphibian photoreceptors than in other vertebrates. Fits of log sensitivity to a normalized-frequency template that tracks the long-wavelength tail of the alpha-band (Lamb, 1995) show that the rod pigments of Rana and Ambystoma are slightly narrower than those found in the photoreceptors of fish and mammals.

Ambystoma↗
PubMed1995

Interaction of tryptophan-182 with the retinal 9-methyl group in the L intermediate of bacteriorhodopsin.

An intense indole N-H stretching vibrational band at 3486 cm-1 in the difference Fourier transform infrared spectrum is one of the characteristic features of the L intermediate of bacteriorhodopsin [Maeda, Sasaki, Ohkita, Simpson, & Herzfeld (1992) Biochemistry 31, 12543]. This band is now assigned to tryptophan-182. The Trp182-->Phe (W182F) protein shows specific features in the difference spectrum in the visible region upon L formation, and exhibits great delay in the L-M conversion. Fourier transform infrared difference spectra further indicate that while the intensity of the C-methyl in-plane bending vibration at 1009 cm-1 is lost in the L intermediate of the wild type, its intensity remains high in the W182F protein. The intensity of the N-H stretching vibration upon L formation is diminished considerably in an artificial bacteriorhodopsin containing 9-desmethylretinal. It also exhibits delayed M formation. These results suggest that Trp182 interacts with the retinal side chain through the 9-methyl group, and thereby affects the L-to-M conversion.

Bacteriorhodopsins↗
PubMed1995

NMR constraints on the location of the retinal chromophore in rhodopsin and bathorhodopsin.

Rhodopsin is the photoreceptor in vertebrate rod cells responsible for vision at low light intensities. The photoreactive chromophore in rhodopsin is 11-cis-retinal bound to the protein via a protonated Schiff base with Glu113 as the counterion. We have used the observed 13C NMR chemical shifts of the conjugated retinal carbons in combination with semiempirical molecular orbital calculations to establish the major charge interactions in the retinal binding site of rhodopsin and its primary photoproduct, bathorhodopsin. In rhodopsin, the NMR data constrain one of the carboxylate oxygens (O1) of Glu113 to be approximately 3 A from the C12 position of the retinal with the second oxygen oriented away from the conjugated retinal chain. The O1-C12-H angle is constrained by taking into account the 500 nm absorption maximum of the protein-bound retinal as well as the chemical shift data. The bathorhodopsin retinal binding site structure is generated from the rhodopsin model by isomerization of the C11 = C12 bond and incorporation of C-C single bond twists from C8 to C15. The resulting structure yields a moderate fit to both the chemical shift data and the 543 nm absorption maximum of bathorhodopsin. In both the rhodopsin and bathorhodopsin models, we have included a structural water molecule hydrogen bonded with the Schiff base to account for the high C = N stretching vibrations previously observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed2000

Relocation of internal bound water in bacteriorhodopsin during the photoreaction of M at low temperatures: an FTIR study.

Changes in the FTIR difference spectra upon photoconversion of the M intermediate to its photoproduct(s) M' were studied in wild-type bacteriorhodopsin and several mutants at low temperatures. The studies aimed at examining whether internally bound water molecules interact with the chromophore and the key residues Asp85 and Asp96 in M, and whether these water molecules participate in the reprotonation of the Schiff base. We have found that three water molecules are perturbed by the isomerization of the chromophore in the M --> M' transition at 80 K. The perturbation of one water molecule, detected as a bilobe at 3567(+)/3550(-) cm(-)(1), relaxed in parallel with the relaxation of an Asp85 perturbation upon increasing temperature from 80 to 100 and 133 K (before the reprotonation of the Schiff base). Two water bands of M at 3588 and 3570 cm(-)(1) shift to 3640 cm(-)(1) upon photoconversion at 173 K. These bands were attributed to water molecules which are located in the vicinity of the Schiff base and Asp85 (Wat85). In the M to M' transition at 80 and 100 K, where the Schiff base remained unprotonated, the Wat85 pair stayed in similar states to those in M. The reprotonation of the Schiff base at 133 K occurred without the restoration of the Wat85 band around 3640 cm(-)(1). This band was restored at higher temperatures. Two water molecules in the region surrounded by Thr46, Asp96, and Phe219 (Wat219) were perturbed in the M to M' transition at 80 K and relaxed in parallel with the relaxation of the perturbation of Asp96 upon increasing the temperature. Mutant studies show that upon photoisomerization of the chromophore at 80 K one of the Wat219 water molecules moves closer to Val49 (located near the lysine side chain attached to retinal, and close to the Schiff base). These data along with our previous results indicate that the water molecules in the cytoplasmic domain participate in the connection of Asp96 with the Schiff base and undergo displacement during photoconversions, presumably shuttling between the Schiff base and a site close to Asp96 in the L to M to N transitions.

Aspartic Acid↗
PubMed1995

Long-range effects on the retinal chromophore of bacteriorhodopsin caused by surface carboxyl group modification.

Carboxyl groups of bacteriorhodopsin (bR) that are modified by 1-ethyl-3-[3-(trimethylamino)-propyl]carbodiimide (ETC) have been identified. Reaction of deionized purple membrane with a 400-fold molar excess of ETC or [14C]ETC for 1 h at 0 degree C incorporates about 3.5 mol of ETC/mol of bR. Proteinase K cleavage of ETC-modified bacterioopsin (bO) produced small 14C-labeled peptides. Amino acid sequence analysis showed three major ETC-modified residues: Glu 234, Asp 38, and Glu 74. Proteolysis of purple membrane with papain removes the ETC site at Glu 234. Treatment of ETC-modified, papain-cleaved purple membrane with hydroxylamine removes half of the remaining ETC label. Subsequent cleavage with chymotrypsin, followed by amino acid sequence analysis, revealed that most of the remaining label was at Glu 74. bR modified by ETC primarily at Glu 74 displays two alterations in the retinal chromophore, located in the membrane interior at a distance more than 2 nm away from the modified carboxyl group. (1) The acid-induced purple-to-blue transition undergoes a shift in apparent pK from 3.2 to 2.3. (2) The second-order rate constant for chromophore regeneration from bO and retinal is diminished from 3600 to 1700 M-1 s-1 in membrane sheets. Most of the shift in the pK of the purple-to-blue transition can be explained by the quaternary ammonium ion of ETC attached to Glu 74 overlapping the postulated location of the guanidinium group of Arg 82.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Photoregeneration of bovine rhodopsin from its signaling state.

In rhodopsin, 11-cis-retinal is bound by a protonated Schiff base and acts as a strong antagonist, which holds the receptor in its inactive ground state conformation. Light induces cis-/trans-retinal isomerization and a sequence of thermal transitions through intermediates. The active conformation that catalyzes GDP/GTP exchange in the G-protein (Gt) is generated from the metarhodopsin II intermediate (MII) and mediated by Schiff base proton translocation and proton uptake from the aqueous phase. In the stable nucleotide-free MII-Gt complex, any thermal transition of MII into other forms of rhodopsin is blocked. We have now studied how Gt affects flash-induced photochemical conversions of MII. Difference spectra from measured absorption changes show that MII photolyzes through two parallel pathways, with fast (1 ms) and slow (50 ms) kinetics (12 degrees C, pH 6). The slow pathway regenerates rhodopsin (9- or 11-cis) via Schiff base reprotonation and proton release. We infer a cis-isomerized early photoproduct (reverted meta, RM) preceding these thermal transitions. When MII is photolyzed in the MII-Gt complex, the slow absorption change is abolished, indicating that Gt blocks the completion of the regeneration process. This is due to the formation of a stable RM-Gt complex, as shown by successive photolysis of MII, RM, and ground state rhodopsin, and the application of GTP gamma S at different stages. The complex dissociates with GTP gamma S, and rhodopsin relaxes to the ground state. The results indicate that cis-retinal and Gt can bind to the receptor at the same time. We discuss the result that the protonations in the meta II state uncouple retinal geometry from Gt interaction.

Animals↗
PubMed1995

Test of the contribution of an amino-aromatic hydrogen bond to protein function.

Hydrogen bonds which form between a hydrogen bond donor and an aromatic ring as acceptor are thought to contribute to the stability and function of proteins. We have tested the function of such an interaction in a highly homologous pair of proteins, cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type II [CRBP(II)]. Both proteins bind the ligand all-trans-retinal with comparable affinities, but CRBP has an approximately 100-fold higher affinity for all-trans retinal. The greater affinity of CRBP for all-trans-retinol has been attributed to the presence of an amino-aromatic hydrogen bond, which is absent in CRBP(II). We have generated a pair of mutant proteins, in which the amino-aromatic interaction was removed from CRBP and introduced into CRBP(II). Spectral analyses of retinol when bound to the wild-type and mutant CRBP suggested that it adopted an identical conformation within both proteins, a conformation that was distinct from that of retinol bound to CRBP(II), both wild-type and mutant. Unexpectedly, the affinities of the mutant binding proteins for all-trans-retinol were indistinguishable from those of their corresponding wild-type proteins. Further, in ligand competition experiments, there were no observable differences between mutant and wild-type CRBP, or between mutant and wild-type CRBP(II), in their preferences for binding all-trans-retinol versus all-trans-retinal. The results of this direct test of the proposed function of an amino-aromatic hydrogen bond did not support a functional role for such bonds, at least in this system.

Animals↗
PubMed1995

pKa of the protonated Schiff base and aspartic 85 in the bacteriorhodopsin binding site is controlled by a specific geometry between the two residues.

The structure and function of the light-driven proton pump bacteriorhodopsin appear to be determined by the exact geometrical conformation of specific groups in the retinal binding site, including bound water molecules. This applies to the pKa values of the protonated Schiff base, which links the retinal chromophore to Lys216, and to Asp85. In the present work we show that the geometrical constraints imposed by the ring structures of several synthetic retinals can induce substantial changes in the pKa values of the Schiff base and of Asp85. Thus, the artificial pigments derived from 13-demethyl-11,14-epoxyretinal (2) and 13-demethyl-9,12-epoxyretinal (3) show protonated Schiff base pKa values of 8.2 +/- 0.1 and 9.1 +/- 0.1, respectively, as compared with 13.3 in the native (all-trans-retinal) pigment. We also suggest that in both systems the pKa of Asp85 increases from 3.2 in the native bR to above 9. Analogous, though smaller, effects are obtained for artificial bR pigments derived from 12,14-ethanoretinal (4), 11,13-propanoretinal (5), 11,13-ethanoretinal (6), and p-(CH3)2N-C6H4-HC = CH-C(CH3) = CH-CHO 7. The effects of geometry on the pKa values (those on Asp85 being more pronounced) are attributed to the disruption of the original, well-defined, structure in which the Schiff base and its Asp85 counterion are bridged by bound water molecules. These results are the first to show that it is possible to modify the pKa values of the Schiff base and Asp85 in appropriate artificial pigments, without inducing intrinsic pKa changes in the chromophore or introducing a mutation in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗
PubMed1995

The complex extracellular domain regulates the deprotonation and reprotonation of the retinal Schiff base during the bacteriorhodopsin photocycle.

During the L-->M reaction of the bacteriorhodopsin photocycle the proton of the retinal Schiff base is transferred to the anionic D85. This step, together with the subsequent reprotonation of the Schiff base from D96 in the M-->N reaction, results in the translocation of a proton across the membrane. The first of these critical proton transfers occurs in an extended hydrogen-bonded complex containing two negatively charged residues (D85 and D212), two positively charged groups (the Schiff base and R82), and coordinated water. We simplified this region by replacing D212 and R82 with neutral residues, leaving only the proton donor and acceptor as charged groups. The D212N/R82Q mutant shows essentially normal proton transport, but in the photocycle neither of this protein nor of the D212N/R82Q/D96N triple mutant does a deprotonated Schiff base (the M intermediate) accumulate. Instead, the photocycle contains only the K, L, and N intermediates. Infrared difference spectra of D212N/R82Q and D212N/R82Q/D96N demonstrate that although D96 becomes deprotonated in N, D85 remains unprotonated. On the other hand, M is produced at pH > 8, where according to independent evidence the L<==>M equilibrium should shift toward M. Likewise, M is restored in the photocycle when the retinal is replaced with the 14-fluoro analogue that lowers the pKa of the protonated Schiff base, and now D85 becomes protonated as in the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗