Structural changes of pharaonis phoborhodopsin upon photoisomerization of the retinal chromophore: infrared spectral comparison with bacteriorhodopsin

Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins. In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K. Spectral comparison of the C--C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR. This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta-ionone ring and the Schiff base regions. In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR. Similar frequency changes upon photoisomerization are observed for the C==N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR. Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR. In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations. In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes. Such structure of the K intermediate of ppR is probably correlated with its high thermal stability. In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR. Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.     

Changes in the protonation state of bacterio-opsin during reconstitution of bacteriorhodopsin

Protonation changes of the protein occur during the reconstitution of bacteriorhodopsin from bacterio-opsin and all-trans retinal in the purple membrane of Halobacterium halobium. The protonation changes are conveniently determined from measures of the pH changes after photoisomerisation of 9-cis retinal in apomembrane preparations, which induces the reconstitution. In addition, to the omega-amino group of the lysine which is involved in the condensation of retinal and bacterio-opsin, the dissociation equilibria of at least two other amino acid residues are changed during the reconstitution. The results are consistent with a proposed model of chromophore structure in which an interaction of the Schiff's base occurs with two protonable amino acid residues.     

Early steps of the intramolecular signal transduction in rhodopsin explored by molecular dynamics simulations

We present molecular dynamics simulations of bovine rhodopsin in a membrane mimetic environment based on the recently refined X-ray structure of the pigment. The interactions between the protonated Schiff base and the protein moiety are explored both with the chromophore in the dark-adapted 11-cis and in the photoisomerized all-trans form. Comparison of simulations with Glu181 in different protonation states strongly suggests that this loop residue located close to the 11-cis bond bears a negative charge. Restrained molecular dynamics simulations also provide evidence that the protein tightly confines the absolute conformation of the retinal around the C12-C13 bond to a positive helicity. 11-cis to all-trans isomerization leads to an internally strained chromophore, which relaxes after a few nanoseconds by a switching of the ionone ring to an essentially planar all-trans conformation. This structural transition of the retinal induces in turn significant conformational changes of the protein backbone, especially in helix VI. Our results suggest a possible molecular mechanism for the early steps of intramolecular signal transduction in a prototypical G-protein-coupled receptor.     

Trans-cis isomerization of retinal and a mechanism for ion translocation in halorhodopsin

The chromophore in halorhodopsin (HR) which acts as a light-driven chloride pump in halobacteria shares many properties with its counterpart in bacteriorhodopsin (BR): (i) a similar retinal protein interaction, (ii) trans to cis isomerization and (iii) similar intermediates of its photocycle. One major difference between the two chromoproteins is that the HR chromophore does not become deprotonated during its photocycle. A mechanism for the photocycle of HR is presented, which, in close analogy to an earlier proposed mechanism for BR, involves the sequence of all-trans 13-cis, 14s-cis 13-cis all-trans isomerizations of the chromophore, a Schiff base of retinal. In contrast to the situation in BR the 13-cis, 14s-cis13-cis isomerization is induced not by deprotonation of the retinal Schiff base chromophore but rather by the movement of an anion (Cl^-) towards the protonated nitrogen of the Schiff's base. The suggested mechanism involves the Schiff base directly in the chloride translocation in halorhodopsin.     

Correlation between absorption maxima and thermal isomerization rates in bacteriorhodopsin

The reported rates of thermal 13-cis to all-trans isomerization of the protonated Schiff base of retinal (PSBR) in solution and in bacteriorhodopsin (BR) are shown to be correlated with the red shift in the absorption maximum of the chromophore, though the linear fit is different for BR and for a model PSBR in solution. Because the red shift in the absorption has been previously shown to be correlated with π-electron delocalization in the chromophore, this suggests that the thermal isomerization rate is largely regulated by the amount of double bond character in the chromophore. Because the linear fit of isomerization rates with absorption maxima is different for BR and the model PSBR, specific interactions of the protein with the chromophore must also be a factor in determining thermal isomerization rates in BR. A model of the later steps in the photocycle of BR is presented in which the 13-cis to all-trans thermal isomerization occurs during the O intermediate.     

Deactivation of rhodopsin in the transition from the signaling state meta II to meta III involves a thermal isomerization of the retinal chromophore C[double bond]D

Light-induced isomerization of rhodopsin's retinal chromophore to the activating all-trans geometry initializes the formation of the active receptor state, Meta II. In the absence of peripheral regulatory proteins, the activity of Meta II is switched off spontaneously by two independent pathways: either by hydrolysis of the retinal Schiff base and dissociation of the light receptor into apoprotein opsin plus free retinal or by formation of Meta III, an inactive species with intact retinal protonated Schiff base absorbing at 470 nm. By FTIR spectroscopy on rhodopsin reconstituted with isotopically labeled chromophores in combination with quantum mechanical DFT calculations, we show that the deactivating step during formation of Meta III involves a thermal isomerization of the chromophore C[double bond]N, such that the chromophore in Meta III is all-trans-15-syn. This isomerization step is catalyzed by the protein environment and proceeds via Meta I, as suggested by its dependence on pH and on properties of the lipid/detergent environment of the protein. In the long term, Meta III decays likewise to opsin and free retinal by slow hydrolysis of the Schiff base.     

The interaction of Triton X-100 with purple membranes. Detergent binding, spectral changes and membrane solubilization

The interaction of the non-ionic surfactant Triton X-100 with Halobacterium purple membranes has been examined at sublytic and lytic surfactant concentrations. These membranes present a number of important peculiarities in their behaviour towards the surfactant. Although solubilization is a very slow process, with a half-time of the order of hours, detergent binding appears to occur at the same fast rate as that found in other membranes. Lipids are solubilized more easily than proteins, so that hardly any protein is solubilized at surfactant concentrations at which about 75% of the lipid is in the form of detergent-mixed micelles; once started, protein solubilization takes place within a narrow range of surfactant concentrations. Retinal provides a built-in probe to monitor detergent-induced conformational changes by spectroscopy in the visible range. No spectral variation is detected at the prelytic stage, i.e. when detergent is incorporated into the membrane in monomeric form. Membrane disruption is accompanied by a blue shift in the absorption maximum, retinal isomerization (from all-trans to 13-cis), and a decrease in specific absorbance (bleaching). Increasing detergent concentrations after solubilization is completed do not produce further shifts in the spectral maximum, but the specific absorbance is progressively decreased. It is shown that Triton X-100 has a complex effect on the retinal chromophore, modifying its configuration and microenvironment (changes in maximum wavelength) and promoting hydrolysis of the retinal-bacteriorhopsin Schiff's base (bleaching).     

Interaction of aromatic retinal analogues with apopurple membranes of Halobacterium halobium

Absorption spectral properties of aromatic analogues of retinal with apopurple membrane of Halobacterium halobium were studied. The spectra of the all-trans forms were composed of two or more absorption bands. During incubation at 20 degrees C, an absorption band above 500 nm increased in intensity gradually at the expense of an absorption band in the shorter wavelength region with no isomerization of the chromophore. The longer wavelength species was shown to be the protonated form of the shorter wavelength species by changing the pH of the medium. Upon irradiation with blue light, the bandwidth of the spectrum became smaller with isomerization of the chromophore to its 13-cis form. Irreversible binding of protons on the membrane occurred during this process. The rate of the increase in the longer wavelength absorption band was especially low in the reaction with the all-trans form of retinal analogues having a bulky substituent at the para or meta positions of the phenyl ring. In contrast, the 13-cis isomer of aromatic retinal analogues gave a single absorption peak. The extent of the spectral shift upon binding to apopurple membranes was compared over a series of aromatic retinals, and the results were explained in terms of steric interactions of the chromophore with the protein.     

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)     

Chromophore configuration of pharaonis phoborhodopsin and its isomerization on photon absorption.

The configuration of the retinylidene chromophore in pharaonis phoborhodopsin (ppR) and its changes during the photoreaction cycle were investigated by means of a chromophore extraction method followed by HPLC analysis. The ppR has an all-trans chromophore, and unlike bacteriorhodopsin, it exhibits no dark isomerization of the chromophore. Irradiation of a ppR sample in the presence of 10 mM hydroxylamine, at which concentration a negligible amount of ppR was bleached, caused the formation of 90% 13-cis- and 10% all-trans-retinal oximes. Because the ppR sample under the continuous irradiation was a mixture containing original ppR, ppRM, and a small amount of ppRO, the above results showed that the chromophores of ppRM and ppRO are in a 13-cis form and an all-trans form, respectively. Therefore, the all-trans chromophore of ppR is isomerized to the 13-cis form on photon absorption, and it is thermally reisomerized to the all-trans form on the conversion process from ppRM to ppRO. The extracted retinal oximes from ppR and ppRO were mainly the 15-syn form, while that from ppRM was mainly the 15-anti form. This fact indicated that the attack of hydroxylamine on the chromophore is stereoselective owing to the unique structure of the chromophore binding site near the Schiff base region of the chromophore.     

Effects of light adaptation on the purple membrane structure of Halobacterium halobium.

Absorption, circular dichroism and optical rotatory dispersion of the bacteriorhodopsin containing purple membrane form Halobacterium halobium were studied in regard to the structural stability of this membrane during the photoisomerization of the retinal of the bacteriorhodopsin from the 13-cis to the all-trans configuration. The following conclusions were reached: (a) the macromolecular structure (protein-protein interaction which may result in the possible exciton interaction of the retinal pi-pi* (NV1) transition moments and protein-lipid interaction) are not significantly altered, (b) possibilities of delocalized conformation changes of the apoprotein involving secondary and/or tertiary structure can be ruled out, (c) localized secondary structure conformation changes of the apoprotein must be limited to the involvement of no more than one or two amino acid residues and localized tertiary structure conformation changes of the apoprotein must be limited to a very short segment of the protein chain containing only a few aromatic amino acid residues, and (d) the interaction between the apoprotein and retinal seems to be relatively more pronounced when the retinal is in the all-trans form than the 13-cis from and also the apoprotein seems to impose a more pronounced dissymmetric constraint on the retinal in the all-trans form than in the 13-cis form.     

Chromophore equilibria in bacteriorhodopsin.

An investigation of the dark equilibria between different chromophores of bacteriorhodopsin (BR) and studies of the kinetics of their interconversion and photochemical activity have led to the following conclusions. (a) A component of the 605-nm chromophore of BR decays in the millisecond range and is likely to be identical to the intermediate O of the photochemical cycle of BR and is assumed to be formed from the purple complex (PC) by the binding of one proton to BR. (b) An acidic form the PC, PCaL-, arises from the 605-nm chromophore by selective binding of anions L- (F- greater than Cl- greater than Br- greater than I- greater than Cl04-) to BR. (c) The isomeric equilibrium between 13-cis and all-trans retinal is approximately 0.15/0.85 in PCaCl-, 0.3/0.7 in the 605-nm chromophore as compared to 0.5/0.5 in the PC. (d) The 500-nm chromophore is formed from the PC by release of nearly one proton from BR. (e) The pH range in which the PC exists is reduced in a high-temperature structure of the purple membrane as compared to its low temperature structure. A model for the chromophore structure is proposed as a hypothesis, which allows a comprehensive interpretation of the results. In this model the absorption spectrum of the retinylidene lysine Schiff base is modulated by its protonation state and the interaction with an anionic group.     

FTIR study of the photoisomerization processes in the 13-cis and all-trans forms of Anabaena sensory rhodopsin at 77 K

Archaeal-type rhodopsins can accommodate either all-trans- or 13-cis,15-syn-retinal in their chromophore binding site in the dark, but only the former isomer is functionally important. In contrast, Anabaena sensory rhodopsin (ASR), an archaeal-type rhodopsin found in eubacteria, exhibits a photochromic interconversion of both forms, suggesting that ASR functions as a photosensor which interacts with its 14 kDa soluble transducer differently in the all-trans and 13-cis,15-syn forms. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the 13-cis,15-syn form of ASR (13C-ASR) at 77 K and compared the local structure around the chromophore and its structural changes upon retinal photoisomerization with those of the all-trans form (AT-ASR) [Furutani, Y., Kawanabe, A., Jung, K. H., and Kandori, H. (2005) Biochemistry 44, 12287-12296]. By use of [zeta-15N]lysine-labeled ASR, we identified the N-D stretching vibrations of the Schiff base (in D2O) at 2165 cm(-1) for 13C-ASR and at 2163 and 2125 cm(-1) for AT-ASR. The frequencies indicate strong hydrogen bonds of the Schiff base with a water molecule for both 13C-ASR and AT-ASR. In contrast, the N-D stretching vibration appears at 2351 cm(-1) and at 2483 cm(-1) for the K states of 13C-ASR (13C-ASR(K)) and AT-ASR (AT-ASR(K)), respectively, indicating that the Schiff base still forms a hydrogen bond in 13C-ASR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller for 13C-ASR than for AT-ASR, the latter altering hydrogen bonding of the Schiff base similar to bacteriorhodopsin (BR), a light-driven proton pump. Appearance of several hydrogen-out-of-plane vibrations and amide I vibrations in 13C-ASR(K), but not in AT-ASR(K), suggests that structural changes are distributed widely along the polyene chain for 13C-ASR. On the other hand, retinal photoisomerization in AT-ASR breaks the hydrogen bond of the Schiff base, and localized structural changes in the Schiff base region are induced.     

Retinal analog restoration of photophobic responses in a blind Chlamydomonas reinhardtii mutant. Evidence for an archaebacterial like chromophore in a eukaryotic rhodopsin.

The strain CC-2359 of the unicellular eukaryotic alga Chlamydomonas reinhardtii originally described as a low pigmentation mutant is found to be devoid of photophobic stop responses to photostimuli over a wide range of light intensities. Photophobic responses of the mutant are restored by exogenous addition of all-trans retinal. We have combined computer-based cell-tracking and motion analysis with retinal isomer and retinal analog reconstitution of CC-2359 to investigate properties of the photophobic response receptor. Most rapid and most complete reconstitution is obtained with all-trans retinal compared to 13-cis, 11-cis, and 9-cis retinal. An analog locked by a carbon bridge in a 6-s-trans conformation reconstitutes whereas the corresponding 6-s-cis locked analog does not. Retinal analogs prevented from isomerization around the 13-14 double bond by a five-membered ring in the polyene chain (locked in either the 13-trans or 13-cis configuration) do not restore the response, but enter the chromophore binding pocket as evidenced by their inhibition of all-trans retinal regeneration of the response. Results of competition experiments between all-trans and each of the 13-locked analogs fit a model in which each chromophore exhibits reversible binding to the photoreceptor apoprotein. A competitive inhibition scheme closely fits the data and permits calculation of apparent dissociation constants for the in vivo reconstitution process of 2.5 x 10(-11) M, 5.2 x 10(-10) M, and 5.4 x 10(-9) M, for all-trans, 13-trans-locked and 13-cis-locked analogs, respectively. The chromophore requirement for the trans configuration and 6-s-trans conformation, and the lack of signaling function from analogs locked at the 13 position, are characteristic of archaebacterial rhodopsins, rather than the previously studied eukaryotic rhodopsins (i.e., visual pigments).     

细菌视紫红质的质子传输机理

细菌视紫红质（bR）是嗜盐菌紫膜中的唯一蛋白质成分, 具有质子泵、电荷分离和光致变色功能. bR分子中的发色团视黄醛通过质子化席夫碱以共价键与Lys216相连. bR分子受可见光照射后, 视黄醛发生从全-反到13-顺式构型的异构化, 导致席夫碱的去质子化,继之以可极化基团位置的改变. 力场的变化引起包括蛋白质三级结构在内的诸多变化, 这些变化促进并保证了质子从细胞质侧向细胞外侧的定向传输.     

Hydration switch model for the proton transfer in the Schiff base region of bacteriorhodopsin

In a light-driven proton-pump protein, bacteriorhodopsin (BR), protonated Schiff base of the retinal chromophore and Asp85 form ion-pair state, which is stabilized by a bridged water molecule. After light absorption, all-trans to 13-cis photoisomerization takes place, followed by the primary proton transfer from the Schiff base to Asp85 that triggers sequential proton transfer reactions for the pump. Fourier transform infrared (FTIR) spectroscopy first observed O-H stretching vibrations of water during the photocycle of BR, and accurate spectral acquisition has extended the water stretching frequencies into the entire stretching frequency region in D(2)O. This enabled to capture the water molecules hydrating with negative charges, and we have identified the water O-D stretch at 2171 cm(-1) as the bridged water interacting with Asp85. We found that retinal isomerization weakens the hydrogen bond in the K intermediate, but not in the later intermediates such as L, M, and N. On the basis of the observation particularly on the M intermediate, we proposed a model for the mechanism of proton transfer from the Schiff base to Asp85. In the "hydration switch model", hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have raised the pK(a) of the proton acceptor, and the proton transfer is from the Schiff base to Asp85.     

Proton translocation by bacteriorhodopsin in the absence of substantial conformational changes

Unlike wild-type bacteriorhodopsin (BR), the BR triple mutant D96G/F171C/F219L has been shown to undergo only minor structural rearrangements during its photocycle. Nonetheless, the mutant is capable of transporting protons at a rate of 125(+/-40) H+/BR per minute under light-saturating conditions. Light adaptation of the triple mutant's retinal proceeds in a pH-dependent manner up to a maximum of 63% all-trans. These two findings imply that the transport activity of the triple mutant comprises 66% of the wild-type activity. Time-resolved spectroscopy reveals that the identity and sequence of intermediates in the photocycle of the triple mutant in the all-trans configuration correspond to that of wild-type BR. The only differences relate to a slower rise and decay of the M and O intermediates, and a significant spectral contribution from a 13-cis component. No indication for accumulation of the N intermediate is found under a variety of conditions that normally favor the formation of this species in wild-type BR. The Fourier transform infrared (FTIR) spectrum of the M intermediate in the triple mutant resembles that of wild type. Minor changes in the amide I region during the photocycle suggest that only small movements of the protein backbone occur. Electron microscopy reveals large differences in conformation between the unilluminated state of the mutant protein and wild-type but no light-induced changes in time-resolved measurements. Evidently, proton transport by the triple mutant does not require the major conformational rearrangements that occur on the same time-scale with wild-type. Thus, we conclude that large conformational changes observed in the photocycle of the wild-type and many BR mutants are not a prerequisite for the change in accessibility of the Schiff base nitrogen atom that must occur during vectorial catalysis to allow proton transport.     

A comparison of the second harmonic generation from light-adapted, dark-adapted, blue, and acid purple membrane.

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.     

A bacteriorhodopsin analog reconstituted with a nonisomerizable 13-trans retinal derivative displays light insensitivity.

With the aim of preparing a light-insensitive bacteriorhodopsin-like pigment, bacterio-opsin expressed in Escherichia coli was treated in phospholipid-detergent micelles with the retinal analog II, in which the C13-C14 trans-double bond cannot isomerize due to inclusion in a cyclopentene ring. The formation of a complex with a fine structure (lambda max, 439 nm) was first observed. This partially converted over a period of 12 days to a bacteriorhodopsin-like chromophore (ebR-II) with lambda max, 555 nm. An identical behavior has been observed previously upon reconstitution of bleached purple membrane with the analog II. Purification by gel filtration gave pure ebR-II with lambda max, 558 nm, similar to that of light-adapted bacterio-opsin reconstituted with all-trans retinal (ebR-I). Spectrophotometric titration of ebR-II as a function of pH showed that the purple to blue transition of bacteriorhodopsin at acidic pH was altered, and the apparent pKa of Schiff base deprotonation at alkaline pH was lowered by 2.4 units, relative to that of ebR-I. ebR-II showed no light-dark adaptation, no proton pumping, and no intermediates characteristic of the bacteriorhodopsin photocycle. In addition, the rates of reaction with hydroxylamine in the dark and in the light were similar. These results show, as expected, that isomerization of the C13-C14 double bond is required for bacteriorhodopsin function and that prevention of this isomerization confers light insensitivity.     

Synthetic pigment analogues of the purple membrane protein.

Nonphysiological analogues of retinal have been shown to form pigments in reactions with the apoprotein of the purple membrane of Halobacterium halobium. Both the all-trans and 13-cis isomers of a retinal analogue, having an elongated chain with an extra double bond, formed pigments. Unlike the native all-trans and 13-cis retinal1-based pigments, the new pigments were not interconvertible with each other and were unstable against hydroxylamine. When incorporated into phospholipid vesicles, they showed no proton pumping activity upon illumination. The ability of the extended-length retinal to form pigments contrasts with its nonreactivity with opsin (apoprotein of rhodopsin), suggesting a less stringent binding site for the purple membrane chromophore. All-trans retinal2 also combined with bleached purple membrane to form a blue pigment absorbing at ca. 590 nm. Like the native purple membrane, the blu membrane showed proton pumping activity upon illumination in phospholipid vesicles.