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PubMed2006

Assignment of the hydrogen-out-of-plane and -in-plane vibrations of the retinal chromophore in the K intermediate of pharaonis phoborhodopsin.

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor protein for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all-trans to 13-cis initiates conformational changes of the protein leading to activation of the cognate transducer protein (pHtrII). Elucidation of the initial photoreaction, formation of the K intermediate of ppR, is important for understanding the mechanism of storage of photon energy. We have reported the K minus ppR Fourier transform infrared (FTIR) spectra, including several vibrational bands of the retinal, the protein, and internal water molecules. It is interesting that more vibrational bands were observed in the hydrogen-out-of-plane (HOOP) region than for the light-driven proton pump, bacteriorhodopsin. This result implied that the steric constraints on the retinal chromophore in the binding pocket of ppR are distributed more widely upon formation of the initial intermediate. In this study, we assigned the HOOP and hydrogen-in-plane vibrations by means of low-temperature FTIR spectroscopy applied to ppR reconstituted with retinal deuterated at C7, C8, C10-C12, C14, and C15. As a result, the 966 (+)/971 (-) and 958 (+)/961 (-) cm(-1) bands were assigned to the C7=C8 and C11=C12 Au HOOP modes, respectively, suggesting that the structural changes spread to the middle part of the retinal. The positive bands at 1001, 994, 987, and 979 cm(-1) were assigned to the C15-HOOP vibrations of the K intermediate, whose frequencies are similar to those of the K(L) intermediate of bacteriorhodopsin trapped at 135 K. Another positive band at 864 cm(-1) was assigned to the C14-HOOP vibration. Relatively many positive bands of hydrogen-in-plane vibrations supported the wide distribution of structural changes of the retinal as well. These results imply that the light energy was stored mainly in the distortions around the Schiff base region while some part of the energy was transferred to the distal part of the retinal.

Deuterium↗
PubMed2006

Induced chirality of the light-harvesting carotenoid salinixanthin and its interaction with the retinal of xanthorhodopsin.

In xanthorhodopsin, a retinal protein-carotenoid complex of Salinibacter ruber, the carotenoid salinixanthin functions as a light-harvesting antenna in supplying additional excitation energy for retinal isomerization and proton transport. Another retinal protein, archaerhodopsin, has been shown to contain a carotenoid, bacterioruberin, but without an antenna function. We report here that the binding site confers a chiral geometry on salinixanthin in xanthorhodopsin and confirm that the same is true for bacterioruberin in archaerhodopsin. Cell membranes containing these rhodopsins exhibit CD spectra with sharp positive bands in the visible region where the carotenoids absorb, and in the case of xanthorhodopsin a negative band at 536 nm, as well as bands in the UV region. The carotenoid in ethanol has very weak optical activity in the visible region of the spectrum. Denaturation of the opsin upon deprotonation of the Schiff base at pH 12.5 eliminates the induced CD bands in both proteins. In one of these proteins, but not in the other, the carotenoid binding site depends entirely on the retinal. Hydrolysis of the retinal Schiff base of xanthorhodopsin with hydroxylamine eliminates the induced CD bands of salinixanthin. In contrast, hydrolysis of the Schiff base in archaerhodopsin does not abolish the CD bands of bacterioruberin. Thus, consistent with its antenna function, the carotenoid binding site interacts closely with the retinal only in xanthorhodopsin, and this interaction is the major source of the CD bands. In this protein, protonation of the counterion with a decrease in pH from 8 to 5 causes significant changes in the CD spectrum. The observed spectral features suggest that binding of salinixanthin in xanthorhodopsin involves the cyclohexenone ring of the carotenoid and its conformational heterogeneity is restricted.

Archaeal Proteins↗
PubMed2006

Propagating structural perturbation inside bacteriorhodopsin: crystal structures of the M state and the D96A and T46V mutants.

The X-ray diffraction structure of the non-illuminated D96A bacteriorhodopsin mutant reveals structural changes as far away as 15 A from residue 96, at the retinal, Trp-182, Ala-215, and waters 501, 402, and 401. The Asp-to-Ala side-chain replacement breaks its hydrogen bond with Thr-46, and the resulting separation of the cytoplasmic ends of helices B and C is communicated to the retinal region through a chain of covalent and hydrogen bonds. The unexpected long-range consequences of the D96A mutation include breaking the hydrogen bond between O of Ala-215 and water 501 and the formation of a new hydrogen bond between water molecules 401 and 402 in the extracellular region. Because in the T46V mutant a new water molecule appears at Asp-96 and its hydrogen-bond to Ile-45 replaces Thr-46 as its link to helix B, the separation of helices B and C is smaller than that in D96A, and there are no atomic displacements elsewhere in the protein. Propagation of conformational changes along the chain between the retinal and Thr-46 had been observed earlier in the crystal structures of the D96N and E204Q mutants but in the trapped M state. Consistent with the perturbation of the retinal region in D96A, little change of the Thr-46 region occurs between the non-illuminated and M states of this mutant. It appears that a local perturbation can propagate along a track in both directions between the retinal and the Asp-96/Thr-46 pair, either from photoisomerization of the retinal in the wild-type protein in one case or from the D96A mutation in the other.

Amino Acid Substitution↗
PubMed2006

Conformational coupling between the cytoplasmic carboxylic acid and the retinal in a fungal light-driven proton pump.

Many fungal rhodopsins, eukaryotic structural homologues of the archaeal light-driven proton pump bacteriorhodopsin, have been discovered in the course of genome sequencing projects. Recently, two fungal rhodopsins were characterized in vitro and exhibited very different photochemical behavior. Neurospora rhodopsin possesses a slow photocycle and shows no ion transport, reminiscent of sensory rhodopsins, while Leptosphaeria rhodopsin has a fast bacteriorhodopsin-like photocycle and pumps protons light-dependently. Such a dramatic difference is surprising considering the very high degree of sequence homology of the two proteins. In this paper, we investigate whether the chemical structure of a cytoplasmic carboxylic acid, the homologue of Asp-96 of bacteriorhodopsin serving as a proton donor for the retinal Schiff base, can define the photochemical properties of fungal rhodopsins. We studied mutants of Leptosphaeria rhodopsin in which this aspartic acid was replaced with Glu or Asn using spectroscopy in the infrared and visible ranges. We show that Glu at this position is inefficient as a proton donor similar to a nonprotonatable Asn. Moreover, this replacement induces long-range structural perturbations of the retinal environment, as evidenced by changes in the vibrational bands of retinal (especially, hydrogen-out-of-plane modes) and neighboring aspartic acids and water molecules. The conformational coupling of the mutation site to the retinal may be mediated by helical rearrangements as suggested by the changes in amide and proline vibrational bands. We conclude that the difference in the photochemical behavior of fungal rhodopsins from Leptosphaeria and Neurospora may be ascribed, to some extent, to the replacement of the cytoplasmic proton donor Asp with Glu.

Amino Acid Substitution↗
PubMed1996

Structure around C6-C7 bond of the chromophore in bathorhodopsin: low-temperature spectroscopy of 6s-cis-locked bicyclic rhodopsin analogs.

To elucidate the structural changes near the beta-ionone ring region of the chromophore during the photobleaching process of rhodopsin, the photochemical and subsequent thermal reactions of rhodopsin analogs, whose retinylidene chromophores were fixed in a 6s-cis form with a five-membered ring (6,5-rhodopsin) and a seven-membered ring (6,7-rhodopsin), respectively, were investigated by low-temperature spectroscopy. Like rhodopsin, both the rhodopsin analogs convert to the respective batho-intermediates upon absorption of light at -190 degrees C. The extinction coefficient of batho-intermediate of 6,5-rhodopsin is similar to that of bathorhodopsin, while that of 6,7-rhodopsin is considerably smaller than that of bathorhodopsin. Like bathorhodopsin, the batho-intermediate of 6,5-rhodopsin directly converts to lumi-intermediate, while that of 6,7-rhodopsin first converts to a blue-shifted intermediate and then to lumi-intermediate. These results strongly suggest that the structure around the beta-ionone ring region of the bathorhodopsin chromophore resembles 6,5-retinal rather than 6,7-retinal. From the comparison of the structural features among retinal, 6,5-retinal, and 6,7-retinal, a possible conformation around C6-C7 bond of the bathorhodopsin chromophore is discussed.

Animals↗
PubMed1996

Kinetics and specificity of human liver aldehyde dehydrogenases toward aliphatic, aromatic, and fused polycyclic aldehydes.

Human mitochondrial aldehyde dehydrogenase (ALDH-2) has a Km for acetaldehyde that is 900-fold lower than that for the cytosolic isozyme, ALDH-1. An increase in aliphatic aldehyde chain length decreases the ALDH-2 Km by up to 10-fold but decreases that of ALDH-1 by 5 orders of magnitude. As a consequence, the Km of ALDH-1 for decanal is 8 times lower than that of ALDH-2, i.e. 2.9 +/- 0.4 and 22 +/- 3 nM, respectively. Determination of these low Km values required kinetic analysis of the simultaneous enzymatic conversion of two aldehyde substrates, an approach also applied to aromatic and fused polycyclic aldehydes. For most of these substrates, maximum velocities are 5-100 times lower than those for acetaldehyde. Addition of one of these tight-binding, slow-turnover substrates to a reaction mixture containing ALDH, NAD+, and a "reference" aldehyde substrate (e.g. acetaldehyde) blocks the principal (reference) enzymatic reaction temporarily and reversibly. Once the first substrate is converted to product, the enzyme can act on the reference substrate. In terms of apparent affinity and blocking capacity, naphthalene and phenanthrene aldehydes were the most potent effectors. Other aromatic and fused polycyclic and heterocyclic aldehydes, as well as derivatives of coumarin, quinoline, indole, and pyridine, are tight-binding, slow-turnover substrates for ALDH-2 and relatively weak inhibitors of ALDH-1. The hydrophobicity of substituents of benzaldehydes, and particularly of naphthaldehydes, correlates with their binding constants toward ALDH-2. Vitamin A1 aldehydes are specific natural substrates for ALDH-1; at pH 7.5, for all-trans- and 13-cis-retinal, Km = 1.1 and 0.37 micromolar, respectively, and kcat/Km is 50-100 times higher than that for acetaldehyde. At the same time, the retinals are inhibitors of ALDH-2, all-trans-retinal being a particularly potent inhibitor (competitive Ki = 43 nM, noncompetitive Ki = 316 nM). These properties suggest that all-trans-retinal is a possible regulatory compound for ALDH-2 in vivo. The data in general point to specialized roles for both major human liver ALDH isozymes in the oxidation of bulky/hydrophobic natural compounds, with Km values in the low nanomolar range.

Acetaldehyde↗
PubMed1996

Opsin/all-trans-retinal complex activates transducin by different mechanisms than photolyzed rhodopsin.

In rhodopsin, the 11-cis-retinal chromophore forms a complex with Lys296 of opsin via a protonated Schiff base. Absorption of light initiates the activation of rhodopsin by cis/trans photoisomerization of retinal. Thermal relaxation through different intermediates leads into the metarhodopsin states which bind and activate transducin (Gt) and rhodopsin kinase (RK). all-trans-Retinal also recombines with opsin independent of light, forming activating species of the receptor. In this study, we examined the mechanism by which all-trans-retinal activates opsin. To exclude other amines except active site Lys296 from formation of Schiff bases, we reductively methylated rhodopsin (PM-rhodopsin), which we then bleached to generate PM-opsin. Using spectroscopic methods and a Gt activation assay, we found that all-trans-retinal interacted with PM-opsin, producing a noncovalent complex that activated Gt. The residual nucleotide exchange in Gt catalyzed by opsin was approximately 1/250 lower relative to that of photoactivated rhodopsin (pH 8.0, 23 degrees C). Addition of equimolar all-trans-retinal led to an occupancy of one-tenth of the putative retinal binding site(s) of opsin and enhanced the Gt activation rate 2-fold. When the concentration of all-trans-retinal was increased to saturation, the Gt activation rate of the opsin/all-trans-retinal complex was approximately 1/33 lower compared to that of photoactivated rhodopsin. We conclude that all-trans-retinal can form a noncovalent complex with opsin that activates Gt by different mechanisms than photolyzed rhodopsin.

Animals↗
PubMed1996

Retinal binding during folding and assembly of the membrane protein bacteriorhodopsin.

The factors driving folding and assembly of integral membrane proteins are largely unknown. In order to determine the role that the retinal chromophore plays in assembly of bacteriorhodopsin, we have determined the kinetics and thermodynamics of retinal binding during regeneration of bacteriorhodopsin, from denatured apoprotein, in vitro. Regeneration is initiated by rapid, stopped-flow, mixing of the denatured apoprotein bacterioopsin in sodium dodecyl sulfate micelles with mixed detergent/lipid micelles containing retinal. Regeneration kinetics are measured by time-resolving changes in protein fluorescence. The dependence of each kinetic component on retinal concentration is determined. Only one experimentally observed rate constant is dependent on retinal concentration, leading to identification of only one second-order reaction involving retinal and bacterioopsin. This reaction occurs after a rate-limiting step in bacterioopsin folding, and results in formation of a noncovalent retinal/protein complex. The free energy change of this retinal binding step is determined, showing that thermodynamic information can be obtained on transient intermediates involved in membrane protein regeneration.

Bacteriorhodopsins↗
PubMed1996

Conformation and dynamics of [3-13C]Ala- labeled bacteriorhodopsin and bacterioopsin, induced by interaction with retinal and its analogs, as studied by 13C nuclear magnetic resonance.

13C nuclear magnetic resonance (NMR) spectra of [3-13C]Ala-labeled bacteriorhodopsin (bR), bacterioopsin (bO), and regenerated bR with retinal or bO complex with retinal analogs were recorded in order to gain insights into how the conformation and dynamics of apoprotein (bO) vary with or without retinal or its analogs. First, we assigned the 13C NMR peak resonating at 16.3 ppm to Ala 53 of both bR and bO, which appears to contact the side chain of Lys 216 at the site of the Schiff base in the former, utilizing the 13C NMR peaks of A53V and A53G proteins in comparison with those of wild-type bR and bO. Characteristic spectral differences between the apoprotein and bR were observed upon removal of the retinal: the changes of the peak intensities at 16.4, 15.9, and 16.9 ppm are notable. We found that the loops (17.4 ppm) and transmembrane alpha II helical region (15.9 ppm) acquired motional freedom with a correlation time of 10(-5)s when the retinal was removed, as detected by proton spin-lattice relaxation times in the rotating frame. A 13C NMR spectrum very similar to that of native bR was recorded when bR was regenerated by addition of retinal to bO. On the other hand, the addition of the retinal analogs retinol or beta-ionone, which are bound in the retinal binding site but are incapable of forming a Schiff base to the apoprotein, caused distinct spectral changes different from those of bR, as manifested from the displacements of 13C chemical shifts. These spectral changes must be ascribed to significant conformational changes of apoprotein at various locations in the protein, including the site of Ala 53 induced by modified interaction between the apoprotein and chromophore.

Alanine↗
PubMed1996

Steric interaction between the 9-methyl group of the retinal and tryptophan 182 controls 13-cis to all-trans reisomerization and proton uptake in the bacteriorhodopsin photocycle.

The hypothesis was tested whether in bacteriorhodopsin (BR) the reduction of the steric interaction between the 9-methyl group of the chromophore all-trans-retinal and the tryptophan at position 182 causes the same changes as observed in the photocycle of 9-demethyl-BR. For this, the photocycle of the mutant W182F was investigated by time-resolved UV-vis and pH measurements and by static and time-resolved FT-IR difference spectroscopy. We found that the second half of the photocycle was similarly distorted in the two modified systems: based on the amide-I band, the protonation state of D96, and the kinetics of proton uptake, four N intermediates could be identified, the last one having a lifetime of several seconds; no O intermediate could be detected; the proton uptake showed a pronounced biphasic time course; and the pKa of group(s) on the cytoplasmic side in N was reduced from 11 in wild type BR to around 7.5. In contrast to 9-demethyl-BR, in the W182F mutant the first part of the photocycle does not drastically deviate from that of wild type BR. The results demonstrate the importance of the steric interaction between W182 and the 9-methyl group of the retinal in providing tight coupling between chromophore isomerization and the late proton transfer steps.

Halobacterium↗
PubMed1996

Involvement of alcohol dehydrogenase, short-chain dehydrogenase/reductase, aldehyde dehydrogenase, and cytochrome P450 in the control of retinoid signaling by activation of retinoic acid synthesis.

The effects of vitamin A (retinol) on growth and development are mediated by the active metabolite retinoic acid which controls a nuclear receptor signaling pathway. While elegant work on the retinoic acid receptor family has focused attention upon how the receptor controls this pathway, there now exists a relatively large gap in our understanding of how retinol is activated to form the ligand. During vertebrate embryogenesis and in adult organs retinoic acid is detected in a distinct spatiotemporal pattern, suggesting that it is produced from retinol in a regulated fashion. Enzymes involved in retinol and retinal metabolism are likely candidates for regulators of tissue retinoic acid levels. Members of the alcohol dehydrogenase and short-chain dehydrogenase/reductase enzyme families catalyze the reversible interconversion of retinol and retinal, the rate-limiting step, whereas members of the aldehyde dehydrogenase and cytochrome P450 enzyme families catalyze the irreversible oxidation of retinal to retinoic acid. The identification of enzymes likely to catalyze retinol oxidation in vivo has been particularly controversial, and this is made even more difficult by the reversible nature of this reaction. Taking into account enzymatic properties and coenzyme preferences, a case can be made that class IV alcohol dehydrogenase catalyzes retinol oxidation to provide retinal for retinoic acid synthesis, whereas microsomal retinol dehydrogenase (a short-chain dehydrogenase/reductase) catalyzes the reduction of retinal to retinol to promote retinoid storage. Further studies on these enzyme families will allow this layer of control in the retinoid signaling pathway to be understood.

Alcohol Dehydrogenase↗
PubMed1996

Relationship of retinal configuration and internal proton transfer at the end of the bacteriorhodopsin photocycle.

In the last step of the bacteriorhodopsin photocycle the initial state is regenerated from the O intermediate in an essentially unidirectional reaction. Comparison of the rate of this photocycle step and the rate of deprotonation of Asp-85 in pH jump experiments with various site-specific mutants indicates that recovery of the initial state is influenced by (1) residues such as Glu-204 that affect deprotonation of Asp-85 and (2) residues such as Leu-93 that contact the retinal and therefore must affect its thermal reisomerization from 13-cis to all-trans as suggested by Delaney, Schweiger, and Subramaniam (Proc. Natl. Acad. Sci. U.S.A. 92, 11120-11124, 1995). These results, together with FTIR spectra (Kandori, Hatanaka, Yamazaki, Needleman, Brown, Richter, Lanyi, & Maeda, manuscript in preparation) of the last intermediate in the photocycles of representatives of the two kinds of mutants, E204Q and L93M, suggest the following sequence of events: reisomerization of the retinal from 13-cis to an all-trans configuration that contains a twisted chain (with high amplitude hydrogen out-of-plane vibrational bands) triggers proton transfer from Asp-85 to Glu-204 or directly to the extracellular surface, and the proton transfer in turn triggers relaxation of the twist in the retinal. The involvement of the proton transfer in the kinetics of this sequence suggests the reason for the unidirectionality of the overall reaction: upon reisomerization of the retinal the very low pKa of Asp-85 in the unphotolyzed protein is reestablished and this residue thereby becomes a good proton donor.

Aspartic Acid↗
PubMed1996

Chromophore configuration of iodopsin and its photoproducts formed at low temperatures.

The photochemical reactions of iodopsin at low temperatures were investigated by a combination of absorption spectroscopy and chromophore extraction to show the formation of isomeric photoproducts other than the all-trans intermediates. We first confirmed that the chromophore in iodopsin is an 11-cis-retinal. Next, iodopsin samples were irradiated with light of different wavelengths at selected temperatures ranging from -190 to 0 degrees C, and their retinylidene chromophores were extracted as oximes after warming the sample to 0 degree C. The isomeric composition of the extracted chromophores was analyzed by high-performance liquid chromatography. It was confirmed that bathoiodopsin produced at -190 degrees C has an all-trans chromophore, but a considerable amount of its chromophore thermally reisomerizes to the 11-cis form upon warming. Photoproducts formerly assigned as lumi- and metaiodopsins [Yoshizawa, T., & Wald, G. (1967) Nature 214, 566-571; Hubbard, R., & Kropf, A. (1959) Nature 183, 448-450] were produced by extensive irradiation of iodopsin at -80 and -40 degrees C, respectively, with red light, but their chromophores were identified to be 7-cis-retinals instead of all-trans-retinals. Thus these photoproducts are artificial byproducts formed as a result of photon absorption by all-trans intermediates. The absorption spectrum of the 7-cis product formed from bovine rhodopsin shows no spectral shift when it is warmed from -80 to 0 degrees C, but the spectrum of 7-cis species formed from iodopsin shifted about 40 nm to the blue at a transition temperature of -60 degrees C. This result indicates a unique chromophore-opsin interaction in iodopsin. Four all-trans intermediates of iodopsin were identified above -80 degrees C under irradiation conditions in which no 7-cis products accumulated. Their absorption maxima were estimated to be approximately 570, approximately 530, approximately 470, and approximately 380 nm. These species should correspond to BL-iodopsin, lumiiodopsin, metaiodopsin I, and metaiodopsin II, respectively, assigned by room temperature laser photolysis [Shichida, Y., Okada, T., Kandori, H., Fukada, Y., & Yoshizawa, T. (1993) Biochemistry 32, 10832-10838].

Animals↗
PubMed1996

Retinal analog study of the role of steric interactions in the excited state isomerization dynamics of rhodopsin.

The role of intramolecular steric interactions in the isomerization of the 11-cis-retinal chromophore in the photoreceptor protein rhodopsin is examined with resonance Raman and CD spectroscopy combined with quantum yield experiments. The resonance Raman spectra and CD spectra of 13-demethylrhodopsin indicate that its chromophore, an analog in which the nonbonded interaction between the 10-H and the 13-CH3 groups is removed, is less distorted in the C10...C13 region than the native chromophore. The reduced torsional and hydrogen-out-of-plane resonance Raman intensities further indicate that the excited state potential energy surface has a much shallower slope along the isomerization coordinate. This is consistent with the decrease in quantum yield from 0.67 in rhodopsin to 0.47 in 13-demethylrhodopsin. The resonance Raman intensities show that the steric twist is reintroduced by addition of a methyl group at the C10 position. However, the quantum yield of 10-methyl-13-demethylrhodopsin is found to be only 0.35. This is attributed to nonisomorphous protein-analog interactions. The nonbonded interaction between the 10-hydrogen and the 13-methyl group in 11-cis-retinal makes this isomer particularly effective as the light-sensing chromophore in all visual pigments.

Animals↗
PubMed1997

General concept for ion translocation by halobacterial retinal proteins: the isomerization/switch/transfer (IST) model.

Bacteriorhodopsin (BR), which transports protons out of the cell in a light-driven process, is one of the best-studied energy-transducing proteins. However, a consensus on the exact molecular mechanism has not been reached. Matters are complicated by two experimental facts. First, recent results using BR mutants (BR-D85T) and the homologous protein sensory rhodopsin I demonstrate that the vectoriality of active proton transport may be reversed under appropriate conditions. Second, in BR-D85T as well as in the homologous halorhodopsin, protons and chloride ions compete for transport; e.g. the same molecule may transport either a positive or a negative ion. To rationalize these results, we propose a general model for ion translocation by bacterial rhodopsins which is mainly based on two assumptions. First, the isomerization state of the retinylidene moiety governs the accessibility of the Schiff base in the protein; e.g. all-trans, 15-anti, and 13-cis-15-anti direct the Schiff base to extracellular and cytoplasmic accessibility, respectively, but change in accessibility (called the "switch") is a time-dependent process in the millisecond time range. A light-induced change of the isomerization state induces not only a change in accessibility but also an ion transfer reaction. Second, we propose that these two processes are kinetically independent, e.g. that relative rate constants in a given molecule determine which process occurs first, ultimately defining the vectoriality of active transport.

Bacterial Proteins↗
PubMed1997

Evidence that bilayer bending rigidity affects membrane protein folding.

The regeneration kinetics of the integral membrane protein bacteriorhodopsin have been investigated in a lipid-based refolding system. Previous studies on bacteriorhodopsin regeneration have involved detergent-based systems, and in particular mixed dimyristoylphosphatidylcholine (DMPC)/CHAPS micelles. Here, we show that the short chain lipid dihexanoylphosphatidylcholine (DHPC) can be substituted for the detergent CHAPS and that bacteriorhodopsin can be regenerated to high yield in mixed DMPC/DHPC micelles. Bacteriorhodopsin refolding kinetics are measured in the mixed DMPC/DHPC micelles. Rapid, stopped flow mixing is employed to initiate refolding of denatured bacterioopsin in SDS micelles with mixed DMPC/DHPC micelles and time-resolved fluorescence spectroscopy to follow changes in protein fluorescence during folding. Essentially identical refolding kinetics are observed for mixed DMPC/CHAPS and mixed DMPC/DHPC micelles. Only one second-order retinal/apoprotein reaction is identified, in which retinal binds to a partially folded apoprotein intermediate, and the free energy of this retinal binding reaction is found to be the same in both types of mixed micelles. Formation of the partially folded apoprotein intermediate is a rate-limiting step in protein folding and appears to be biexponential. Both apparent rate constants are found to be dependent on the relative proportion of DMPC present in the mixed DMPC/DHPC micelles as well as on the pH of the aqueous phase. Increasing the DMPC concentration should increase the bending rigidity of the amphiphilic bilayer, and this is found to slow the rate of formation of the partially folded apoprotein intermediate. Increasing the mole fraction of DMPC from 0.3 to 0.6 slows the two apparent rate constants associated with formation of this intermediate from 0.29 and 0.031 to 0.11 and 0.013 s-1, respectively. Formation of the intermediate also slows with increasing pH, from 0.11 and 0.013 s-1 at pH 6 to 0.033 and 0.0053 s-1 at pH 8. Since this pH change has no known effect on the phase behavior of lecithins, this is more likely to represent a direct effect on the protein itself. Thus, it appears to be possible to control the rate-limiting process in bacterioopsin folding through both bilayer bending rigidity and pH.

Bacteriorhodopsins↗
PubMed1997

Interaction between Asp-85 and the proton-releasing group in bacteriorhodopsin. A study of an O-like photocycle intermediate.

Upon light adaptation by continuous (or pulsed) illumination, the artificial bacteriorhodopsin (bR) pigments, I and II, derived from synthetic 14F retinal and a short polyenal, respectively produce a long-lived red-shifted species denoted O1. An analogous phenomenon was observed by Sonar, S., et al. [(1993) Biochemistry 32, 2263-2271], in the case of the Y185F mutant (pigment III). The nature of these O1 species was investigated by studying a series of effects, primarily their red light photoreversibility, the associated proton uptake and release processes, and the effects of pH on their relative amounts, which are interpreted in terms of pH-dependent acid-base equilibria. Experiments were also carried out with pigments I and II derived from the mutants D96A, E204Q, R82Q, and D85N. The O1 species of pigments I and II (and possibly also that of pigment III) are identified as an unusually long-lived (all-trans) intermediate of the photocycle of their 13-cis isomer. It is concluded that in O1, Asp-85 is protonated, a process associated with proton uptake from the extracellular side. Subsequent proton release (to the same side of the membrane) occurs from Glu-204 (or from a group closely interacting with it) prior to the decay of O1. At high pH (>9), O1 reversibly converts to a purple form, due to deprotonation of Asp-85, while at still higher pH (> 11), a blue-shifted species characterized by a deprotonated Schiff base is generated. These transitions constitute the first demonstration of the titration of a photocycle intermediate of a retinal protein. The respective pKa values are determined and discussed in relation to those pertaining to the unphotolyzed (dark-adapted) pigments. It appears that the pKa values are controlled by a hydrogen bond network involving water molecules, which binds the protonated Schiff base with Asp-85 and Glu-204. The disruption of this network in pigments I-III may also be responsible for the long lifetime of the O1 species, due to the inhibition of thermal trans-13-cis isomerization. The results are relevant to the molecular mechanism of the photocycles of both 13-cis- and all-trans-bR, primarily to the nature and to the deprotonation mechanism of the proton-releasing group.

Arylsulfonates↗
PubMed1997

Reconstitution of bacteriorhodopsin from the apoprotein and retinal studied by Fourier-transform infrared spectroscopy.

The reconstitution of the retinal-containing protein bacteriorhodopsin (BR) from the apoprotein and retinal has been studied by Fourier-transform infrared (FTIR) difference spectroscopy. 9-cis-Retinal which occupies the binding site but does not reconstitute the chromophore was used as "caged retinal". Photoisomerization to the all-trans isomer triggers the reconstitution reaction. Absorption bands in the FTIR difference spectra of the educt and product of the reaction could be assigned by comparison with a 9-cis-retinal FTIR spectrum or an FT-Raman spectrum of BR and due to band shifts observed upon deuterium exchange. Specific difference bands were assigned to the protonated carboxyl groups of D96 and D115 by use of the mutants D115N and D96N. Both aspartic acids are protonated also in the apoprotein with pKa values above 10 and undergo a frequency shift toward higher wavenumbers indicating a more hydrophobic environment in the reconstituted protein. No indication was found for protonation changes of carboxyl groups or other protonatable residues when carrying out the reaction at pH values between 4 and 10. The pH-dependent protonation changes reported earlier [Fischer & Oesterhelt (1980) Biophys. J. 31, 139-146] therefore may be caused by protons in a hydrogen-bonded network. Mutations of E204, but not of D38 or E9, cancel proton uptake during reconstitution at high pH as well as proton release at low pH. It is concluded, that E204, without changing its protonation state itself, is part of a protonatable hydrogen-bonded network which changes its pKa during reconstitution thereby causing the observed protonation changes.

Bacteriorhodopsins↗