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PubMed1997

Reducing the flexibility of retinal restores a wild-type-like photocycle in bacteriorhodopsin mutants defective in protein-retinal coupling.

The thermal re-isomerization of retinal from the 13-cis to the all-trans state is a key step in the final stages of the photocycle of the light-driven proton pump, bacteriorhodopsin. This step is greatly slowed upon replacement of Leu-93, a residue in van der Waals contact with retinal. The most likely role of this key interaction is that it restricts the flexibility of retinal. To test this hypothesis, we have exchanged native retinal in Leu-93 mutants with bridged retinal analogs that render retinal less flexible by restricting free rotation around either the C10-C11 (9,11-bridged retinal) or C12-C13 (11,13-bridged retinal) single bonds. The effect of the analogs on the photocycle was then determined spectroscopically by taking advantage of the previous finding that the decay of the O intermediate in the Leu-93 mutants provides a convenient marker for retinal re-isomerization. Time-resolved spectroscopic studies showed that both retinal analogs resulted in a dramatic acceleration of the photocycling time by increasing the rate of decay of the O intermediate. In particular, exchange of native retinal in the Leu-93 --> Ala mutant with the 9,11-bridged retinal resulted in an acceleration of the decay of the O intermediate to a rate similar to that seen in wild-type bacteriorhodopsin. We conclude that the protein-induced restriction of conformational flexibility in retinal is a key structural requirement for efficient protein-retinal coupling in the bacteriorhodopsin photocycle.

Alanine↗
PubMed1997

A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin.

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (i) the pKa values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (ii) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (iii) preventing rotation of the C13-C14 double bond by replacing the retinal with an all-trans locked analogue makes little difference to the Schiff base pKa. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.

Bacteriorhodopsins↗
PubMed1997

The C9 methyl group of retinal interacts with glycine-121 in rhodopsin.

The visual pigment rhodopsin is a prototypical G protein-coupled receptor. These receptors have seven transmembrane helices and are activated by specific receptor-ligand interactions. Rhodopsin is unusual in that its retinal prosthetic group serves as an antagonist in the dark in the 11-cis conformation but is rapidly converted to an agonist on photochemical cis to trans isomerization. Receptor-ligand interactions in rhodopsin were studied in the light and dark by regenerating site-directed opsin mutants with synthetic retinal analogues. A progressive decrease in light-dependent transducin activity was observed when a mutant opsin with a replacement of Gly121 was regenerated with 11-cis-retinal analogues bearing progressively larger R groups (methyl, ethyl, propyl) at the C9 position of the polyene chain. A progressive decrease in light activity was also observed as a function of increasing size of the residue at position 121 for both the 11-cis-9-ethyl- and the 11-cis-9-propylretinal pigments. In contrast, a striking increase of receptor activity in the dark-i.e., without chromophore isomerization-was observed when the molecular volume at either position 121 of opsin or C9 of retinal was increased. The ability of bulky replacements at either position to hinder ligand incorporation and to activate rhodopsin in the dark suggests a direct interaction between these two sites. A molecular model of the retinal-binding site of rhodopsin is proposed that illustrates the specific interaction between Gly121 and the C9 methyl group of 11-cis-retinal. Steric interactions in this region of rhodopsin are consistent with the proposal that movement of transmembrane helices 3 and 6 is concomitant with receptor activation.

Animals↗
PubMed1997

Synthesis and characterization of a novel retinylamine analog inhibitor of constitutively active rhodopsin mutants found in patients with autosomal dominant retinitis pigmentosa.

Two different mutations of the active-site Lys-296 in rhodopsin, K296E and K296M, have been found to cause autosomal dominant retinitis pigmentosa (ADRP). In vitro studies have shown that both mutations result in constitutive activation of the protein, suggesting that the activated state of the receptor may be responsible for retinal degeneration in patients with these mutations. Previous work has highlighted the potential of retinylamine analogs as active-site directed inactivators of constitutively active mutants of rhodopsin with the idea that these or related compounds might be used therapeutically for cases of ADRP involving mutations of the active-site Lys. Unfortunately, however, amine derivatives of 11-cis-retinal, although highly effective against a K296G mutant of rhodopsin, were without affect on the two naturally occurring ADRP mutants, presumably because of the greater steric bulk of Glu and Met side chains in comparison to Gly. For this reason we synthesized a retinylamine analog one carbon shorter than the parent 11-cis-retinal and show that this compound is indeed an effective inhibitor of both the K296E and K296M mutants. The 11-cis C19 retinylamine analog 1 inhibits constitutive activation of transducin by these mutants and their constitutive phosphorylation by rhodopsin kinase, and it does so in the presence of continuous illumination from room lights.

Animals↗
PubMed1997

Single amino acid residue as a functional determinant of rod and cone visual pigments.

The visual transduction processes in rod and cone photoreceptor cells begin with photon absorption by the different types of visual pigments. Cone visual pigments exhibit faster regeneration from 11-cis-retinal and opsin and faster decay of physiologically active intermediate (meta II) than does the rod visual pigment, rhodopsin, as expected, due to the functional difference between rod and cone photoreceptor cells. To identify the amino acid residue(s) responsible for the difference in molecular properties between rod and cone visual pigments, we selected three amino acid positions (64, 122, and 150), where cone visual pigments have amino acid residues electrically different from those of rhodopsin, and prepared mutants of rhodopsin and chicken green-sensitive cone visual pigment. The results showed that the replacement of Glu-122 of rhodopsin by the residue containing green- or red-sensitive cone pigment converted rhodopsin's rates of regeneration and meta II decay into those of the respective cone pigments, whereas the introduction of Glu-122 into green-sensitive cone visual pigment changed the rates of these processes into rates similar to those of rhodopsin. Furthermore, exchange of the residue at position 122 between rhodopsin and chicken green-sensitive cone pigment interchanges their efficiencies in activating retinal G protein transducin. Thus, the amino acid residue at position 122 is a functional determinant of rod and cone visual pigments.

Animals↗
PubMed1998

Effect of vitamin A supplementation on rhodopsin mutants threonine-17 --> methionine and proline-347 --> serine in transgenic mice and in cell cultures.

A therapeutic effect of vitamin A supplementation on the course of photoreceptor degeneration, previously reported for patients with retinitis pigmentosa, was tested in two transgenic mouse models of this disease, each carrying a dominant rhodopsin mutation. The threonine-17 --> methionine (T17M) mutation is a class II rhodopsin mutation, characterized by a thermal instability/folding defect and minimal regeneration with the chromophore. The proline-347 --> serine (P347S) mutation belongs to class I, comprised of a smaller number of mutations that exhibit no recognized biochemical abnormality in vitro. In the present study, each of the two mouse models was fed a diet containing 2.5 mg of vitamin A palmitate (control) or 102.5 mg of vitamin A palmitate (high vitamin A) per kilogram of diet. Dark-adapted, full-field electroretinograms showed that the high vitamin A diet significantly reduced the rate of decline of a-wave and b-wave amplitudes in the T17M mice but had no significant effect on the decline of electroretinogram amplitude in the P347S mice. Correspondingly, histologic evaluation revealed that the treatment was associated with significantly longer photoreceptor inner and outer segments and a thicker outer nuclear layer in the T17M mice but had no effect on photoreceptor morphology in the P347S mice. In a separate series of experiments, the instability defect of the T17M mutant opsin expressed in vitro was partially alleviated by inclusion of 11-cis-retinal in the culture media. These results show that vitamin A supplementation slows the rate of photoreceptor degeneration caused by a class II rhodopsin mutation. Vitamin A supplementation may confer therapeutic benefit by stabilizing mutant opsins through increased availability of the chromophore.

Animals↗
PubMed1999

The nop-1 gene of Neurospora crassa encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins.

Opsins are a class of retinal-binding, seven transmembrane helix proteins that function as light-responsive ion pumps or sensory receptors. Previously, genes encoding opsins had been identified in animals and the Archaea but not in fungi or other eukaryotic microorganisms. Here, we report the identification and mutational analysis of an opsin gene, nop-1, from the eukaryotic filamentous fungus Neurospora crassa. The nop-1 amino acid sequence predicts a protein that shares up to 81.8% amino acid identity with archaeal opsins in the 22 retinal binding pocket residues, including the conserved lysine residue that forms a Schiff base linkage with retinal. Evolutionary analysis revealed relatedness not only between NOP-1 and archaeal opsins but also between NOP-1 and several fungal opsin-related proteins that lack the Schiff base lysine residue. The results provide evidence for a eukaryotic opsin family homologous to the archaeal opsins, providing a plausible link between archaeal and visual opsins. Extensive analysis of Deltanop-1 strains did not reveal obvious defects in light-regulated processes under normal laboratory conditions. However, results from Northern analysis support light and conidiation-based regulation of nop-1 gene expression, and NOP-1 protein heterologously expressed in Pichia pastoris is labeled by using all-trans [3H]retinal, suggesting that NOP-1 functions as a rhodopsin in N. crassa photobiology.

Amino Acid Sequence↗
PubMed1999

Femtochemistry.

The topic of femtochemistry is surveyed from both theoretical and experimental points of view. A time-dependent wave packet description of the photodissociation of the O---C---S molecule reveals vibrational motion in the transition-state region and suggests targets for direct experimental observation. Theoretical approaches for treating femtosecond chemical phenomena in condensed phases are featured along with prospects for laser-controlled chemical reactions by using tailored ultrashort chirped pulses. An experimental study of the photoisomerization of retinal in the protein bacteriorhodopsin is discussed with an aim to gain insight into the potential energy surfaces on which this remarkably efficient and selective reactions proceeds. Finally, a prospective view of new frontiers in femtochemistry is given.

Bacteriorhodopsins↗
PubMed1999

Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin: expression of 15N-lysine- and 13C-glycine-labeled opsin in a stable cell line.

The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6-15N-lysine and 2-13C-glycine. Typical yields were 1.5-1.8 mg/liter. Incorporation of 6-15N-lysine was quantitative, whereas that of 2-13C-glycine was about 60%. The rhodopsin pigment formed by binding of 11-cis retinal was spectrally indistinguishable from native bovine rhodopsin. Magic angle spinning (MAS) NMR spectra of labeled rhodopsin were obtained after its incorporation into liposomes. The 15N resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-15N-lysine-labeled rhodopsin. This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 A, consistent with structural water in the binding site hydrogen bonded with the Schiff base nitrogen and the Glu-113 counterion. The present study demonstrates that structural studies of rhodopsin and other G protein-coupled receptors by using MAS NMR are feasible.

Amino Acid Sequence↗
PubMed1999

Structure and function in rhodopsin: kinetic studies of retinal binding to purified opsin mutants in defined phospholipid-detergent mixtures serve as probes of the retinal binding pocket.

In the current standard procedure for preparation of mammalian rhodopsin mutants, transfected COS-1 cells expressing the mutant opsin genes are treated with 5 microM 11-cis-retinal before detergent solubilization for purification. We found that binding of 11-cis-retinal to opsin mutants with single amino acid changes at Trp-265 (W265F,Y,A) and a retinitis pigmentosa mutant (A164V) was far from complete and required much higher concentrations of 11-cis-retinal. By isolation of the expressed opsins in a stable form, kinetic studies of retinal binding to the opsins in vitro have been carried out by using defined phospholipid-detergent mixtures. The results show wide variation in the rates of 11-cis-retinal binding. Thus, the in vitro reconstitution procedure serves as a probe of the retinal-binding pocket in the opsins. Further, a method is described for purification and characterization of the rhodopsin mutants after retinal binding to the opsins in vitro.

Amino Acid Sequence↗
PubMed1995

Transducin activation by the bovine opsin apoprotein.

The interaction of the bovine opsin apoprotein with transducin in rod outer segment membranes was investigated using a guanyl nucleotide exchange assay. In exhaustive binding experiments, opsin activates transducin, with half-maximal exchange activity occurring at 0.8 mol of opsin/mol of transducin. The opsin activity was light-insensitive, hydroxylamine-resistant, unaffected by stoichiometric concentrations of retinaloxime, and more heat-labile than rhodopsin. The t1/2 of transducin activation in the presence of excess opsin was 8.5 min, compared with 0.7 min for metarhodopsin (II). The second-order rate constants were determined to be 0.012 pmol of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) bound per min/nM opsin and 0.35 pmol of GTP gamma S bound per min/nM metarhodopsin (II). Opsin was able to activate more than one transducin, although there appeared to be a turnover-dependent inactivation of the apoprotein. Opsin showed a broad pH range (5.8-7.4) for optimal activity, with no activity in buffers of pH > 9, whereas metarhodopsin (II) exhibited activity at pH > 9. Regulation of opsin activity by stoichiometric amounts of retinal was observed, with inhibition by 11-cis-retinal and stimulation by all-trans-retinal. A model for opsin activity is proposed.

Animals↗
PubMed1995

Reduced light-dependent phosphorylation of an analog visual pigment containing 9-demethylretinal as its chromophore.

9-Demethyl rhodopsin (9dR), an analog of vertebrate rhodopsin, consists of opsin and a covalently attached chromophore of 11-cis 9-demethylretinal. Electrophysiological evidence that photoactivated 9dR (9dR*) undergoes abnormally slow deactivation in salamander rods (Corson, D. W., Cornwall, M. C., and Pepperberg, D. R. (1994) Visual Neurosci. 11, 91-98) raises the possibility that opsin phosphorylation, a reaction involved in visual pigment deactivation, operates abnormally on 9dR*. This possibility was tested by measuring the light-dependent phosphorylation of 9dR in preparations obtained from bovine rod outer segments. Outer segment membranes containing 9dR or regenerated rhodopsin were flash-illuminated in the presence of [gamma-32P]ATP and rhodopsin kinase, further incubated in darkness, and then analyzed for opsin-bound [32P]Pi. [32P]Pi incorporation by 9dR* increased with both incubation period and bleaching extent but, under all conditions tested, was less than that measured in rhodopsin controls. Results obtained with 30-s incubation periods indicated that the maximal initial rate of incorporation by 9dR* is about 25% of that by photoactivated rhodopsin. The results imply that the low incorporation of Pi by 9dR* results from a reduced rate of phosphorylation by rhodopsin kinase and are consistent with the prolonged lifetime of 9dR* determined electrophysiologically.

Animals↗
PubMed1995

The role of water in retinal complexation to bacterio-opsin.

A system is described that allows for the delineation of the factors that effect the complexation of retinal to the apoprotein of bacteriorhodopsin. This complexation is investigated in various states of hydration, in H2O and D2O, at a variety of pH levels, with mutant membranes and labeled retinals. The complexation reaction was also investigated using absorption spectroscopy and vibrational spectra using difference Fourier transform infrared spectroscopy. The results demonstrate the crucial role of water in controlling the protein conformations that lead to protein/ligand binding reactions and begin to shed new light on the protein control of a reaction that normally cannot take place in an aqueous medium.

Bacteriorhodopsins↗
PubMed1995

Alteration in the retinoid specificity of retinoic acid receptor-beta by site-directed mutagenesis of Arg269 and Lys220.

Retinoic acid receptor-beta (RAR-beta) specifically binds retinoic acid (RA) and functions as a RA-inducible transcriptional regulatory factor. Simultaneous mutation of Arg269 and Lys220 of RAR-beta to Ala results in a dramatic reduction in both transactivation and affinity for RA along with creating a RA concentration-dependent dominant negative mutant. In this report, we found that mutation of these two amino acid residues singly and simultaneously to Gln results in mutant RAR-beta s, each displaying a more dramatic reduction in transactivation and affinity for RA than their corresponding Ala mutant, with the R269Q more profoundly affected than K220Q. Furthermore, we examined both the Ala and Gln mutants for their ability to transactivate and bind two other retinoids with different functional end groups (all-trans-retinol and all-trans-retinal). Mutation of Lys220 to either an Ala or a Gln favors transactivation and binding of retinal, while mutation of either Lys220 or Arg269 to Gln favors retinol transactivation and binding. Taken together, these results suggest that Arg269 and Lys220 lie within the ligand binding pocket of RAR-beta and Lys220 lie within the ligand binding pocket of RAR-beta and that these two amino acid residues play an important role in determining retinoid specificity most likely by directly interacting with the carboxylate group of RA.

Animals↗
PubMed1995

Spectral tuning in bacteriorhodopsin in the absence of counterion and coplanarization effects.

The basis for wavelength regulation in bacteriorhodopsin (BR) and retinylidene proteins in general has been studied for decades but is still only partially understood. Here we report the preparation and spectroscopic characterization of BR analogs aimed at investigating the existence of spectral tuning mechanisms other than the two widely accepted mechanisms, weakened counterion interactions and ring/chain coplanarization. We synthesized two novel retinal analogs containing a saturated 13-14 bond, which interrupts the interaction of the protein counterions with the chromophore conjugation system. Furthermore, one of the analogs has a planar polyene system so that the contribution to the red shift of BR by retinal ring/chain coplanarization is also absent. We incorporated these analogs into bacterioopsin and discovered a sizable amount of red shift, which can be accounted for by interactions between the polar or polarizable groups of the protein and the retinal polyene chain. Our results suggest that the wavelength regulation in BR is achieved by synergistic chromophore/protein interactions including ring/chain coplanarization, excited state stabilization by polar or polarizable protein side chains located along the polyene chain, and weakened counterion interactions near the Schiff base positive charge.

Bacteriorhodopsins↗
PubMed1996

Auto-regulation of retinoic acid biosynthesis through regulation of retinol esterification in human keratinocytes.

In this report, we describe an auto-regulatory loop in human keratinocytes, whereby all-trans retinoic acid (retinoic acid) regulates its own biosynthesis from all-trans retinol (retinol) through regulation of retinol esterification. Retinol esterification activity was low in normal proliferating human keratinocytes, cultured in retinoid-free media. Treatment of keratinocytes with retinoic acid induced retinol esterifying activity (8-fold). Induction of retinol esterifying activity was blocked by either actinomycin D or cycloheximide. Based on substrate specificity and inhibitor sensitivity, lecithin:retinol acyltransferase (LRAT) was identified as the retinoic acid-inducible retinol esterifying enzyme. Induction of LRAT by retinoic acid reduced conversion of retinol to retinoic acid by 50%. This reduction in retinoic acid synthesis resulted from sequestration of retinol as retinyl esters, since inhibition of LRAT restored retinoic acid synthesis to control levels. In normal human skin, undifferentiated keratinocytes, in the lowest cell layer, esterified retinol 4 times greater, than differentiating keratinocytes, in upper cell layers, reflecting an induced state, under conditions of retinol sufficiency. Regulation of LRAT activity by retinoic acid provides a novel mechanism through which retinoic acid can regulate its own level by controlling availability of retinol for conversion to retinoic acid. In human skin in vivo, retinyl esters synthesized in basal keratinocytes could undergo hydrolysis during differentiation and thus serve as a source of retinol for keratinocytes in upper layers of skin.

Acyltransferases↗
PubMed1996

Cloning of a cDNA encoding an aldehyde dehydrogenase and its expression in Escherichia coli. Recognition of retinal as substrate.

The biosynthesis of the hormone retinoic acid from retinol (vitamin A) involves two sequential steps, catalyzed by retinol dehydrogenases and retinal dehydrogenases, respectively. This report describes the cloning of a cDNA encoding a heretofore unknown aldehyde dehydrogenase from a rat testis library and its expression in Escherichia coli. This enzyme has been designated retinal dehydrogenase, type II, RalDH(II). The deduced amino acid sequence of RalDH(II) had the highest identity with mammalian aldehyde dehydrogenases that feature low Km values (microM) for retinal: human ALDH1 (72.2%), rat retinal dehydrogenase, type I (71.5%), bovine retina (72.7%), and mouse AHD-2 (71.5%). RalDH(II) expressed in E. coli recognizes as substrates free retinal, with a Km of approximately 0.7 microM, and cellular retinol-binding protein-bound retinal, with a Km of approximately 0.2 microM. RalDH(II) also can utilize as substrate retinal generated in situ by microsomal retinol dehydrogenases, from the physiologically most abundant substrate: retinol bound to cellular retinol-binding protein. Rat testis expresses RalDH(II) mRNA most abundantly, followed by (relative to testis): lung (6.7%), brain (6.3%), heart (5.2%), liver (4.4%), and kidney (2.7%). RalDH(II) does not recognize citral, benzaldehyde, acetaldehyde, and propanal efficiently as substrates, but does metabolize octanal and decanal efficiently. These data support a function for RalDH(II) in the pathway of retinoic acid biogenesis.

Alcohol Oxidoreductases↗
PubMed1996

Docosahexaenoic acid modulates the interactions of the interphotoreceptor retinoid-binding protein with 11-cis-retinal.

Rapid transport of retinoids across the interphotoreceptor matrix is a critical part of the visual cycle, since it serves to replenish bleached rhodopsin with its chromophore 11-cis-retinal. The transport of retinoids in the interphotoreceptor matrix is believed to be mediated by the interphotoreceptor retinoid-binding protein (IRBP), a protein that, in addition to possessing two retinoid-binding sites, associates in vivo with long chain fatty acids. Here, the interrelationships between binding of the two types of ligands to IRBP were studied. The composition of fatty acids associated with IRBP in bovine retina was determined, and it was found that polyunsaturated fatty acids constitute a significant fraction of those. It was further found that docosahexaenoic acid, but not palmitic acid, induced a rapid and specific release of 11-cis-retinal from one of the protein's retinoid-binding sites. Based on these results and on the additional observation that a steep concentration gradient of docosahexaenoic acid exists between photoreceptor and pigment epithelium cells, a model for the mechanism by which IRBP may target 11-cis-retinal to photoreceptor cells is proposed.

Allosteric Regulation↗