The hydroxylamine reaction of sensory rhodopsin II: light-induced conformational alterations with C13=C14 nonisomerizable pigment

Sensory rhodopsin II, a repellent phototaxis receptor from Natronomonas (Natronobacterium) pharaonis (NpSRII), forms a complex with its cognate transducer (NpHtrII). In micelles the two proteins form a 1:1 heterodimer, whereas in membranes they assemble to a 2:2 complex. Similarly to other retinal proteins, sensory rhodopsin II undergoes a bleaching reaction with hydroxylamine in the dark which is markedly catalyzed by light. The reaction involves cleavage of the protonated Schiff base bond which covalently connects the retinal chromophore to the protein. The light acceleration reflects protein conformation alterations, at least in the retinal binding site, and thus allows for detection of these changes in various conditions. In this work we have followed the hydroxylamine reaction at different temperatures with and without the cognate transducer. We have found that light irradiation reduces the activation energy of the hydroxylamine reaction as well as the frequency factor. A similar effect was found previously for bacteriorhodopsin. The interaction with the transducer altered the light effect both in detergent and membranes. The transducer interaction decreased the apparent light effect on the energy of activation and the frequency factor in detergent but increased it in membranes. In addition, we have employed an artificial pigment derived from a retinal analog in which the critical C13=C14 double bond is locked by a rigid ring structure preventing its isomerization. We have observed light enhancement of the reaction rate and reduction of the energy of activation as well as the frequency factor, despite the fact that this pigment does not experience C13=C14 double bond isomerization. It is suggested that retinal excited state polarization caused by light absorption of the "locked" pigment polarizes the protein and triggers relatively long-lived protein conformational alterations.     

Rhodopsin with 11-cis-locked chromophore is capable of forming an active state photoproduct

The visual pigment rhodopsin is characterized by an 11-cis retinal chromophore bound to Lys-296 via a protonated Schiff base. Following light absorption the C(11)=C(12) double bond isomerizes to trans configuration and triggers protein conformational alterations. These alterations lead to the formation of an active intermediate (Meta II), which binds and activates the visual G protein, transducin. We have examined by UV-visible and Fourier transform IR spectroscopy the photochemistry of a rhodopsin analogue with an 11-cis-locked chromophore, where cis to trans isomerization around the C(11)=C(12) double bond is prevented by a 6-member ring structure (Rh(6.10)). Despite this lock, the pigment was found capable of forming an active photoproduct with a characteristic protein conformation similar to that of native Meta II. This intermediate is further characterized by a protonated Schiff base and protonated Glu-113, as well as by its ability to bind a transducin-derived peptide previously shown to interact efficiently with native Meta II. The yield of this active photointermediate is pH-dependent and decreases with increasing pH. This study shows that with the C(11)=C(12) double bond being locked, isomerization around the C(9)=C(10) or the C(13)=C(14) double bonds may well lead to an activation of the receptor. Additionally, prolonged illumination at pH 7.5 produces a new photoproduct absorbing at 385 nm, which, however, does not exhibit the characteristic active protein conformation.     

Effective light-induced hydroxylamine reactions occur with C13 = C14 nonisomerizable bacteriorhodopsin pigments.

The light-driven proton pump bacteriorhodopsin (bR) undergoes a bleaching reaction with hydroxylamine in the dark, which is markedly catalyzed by light. The reaction involves cleavage of the (protonated) Schiff base bond, which links the retinyl chromophore to the protein. The catalytic light effect is currently attributed to the conformational changes associated with the photocycle of all-trans bR, which is responsible for its proton pump mechanism and is initiated by the all-trans --> 13-cis isomerization. This hypothesis is now being tested in a series of experiments, at various temperatures, using three artificial bR molecules in which the essential C13==C14 bond is locked by a rigid ring structure into an all-trans or 13-cis configuration. In all three cases we observe an enhancement of the reaction by light despite the fact that, because of locking of the C13==C14 bond, these molecules do not exhibit a photocycle, or any proton-pump activity. An analysis of the rate parameters excludes the possibility that the light-catalyzed reaction takes place during the approximately 20-ps excited state lifetimes of the locked pigments. It is concluded that the reaction is associated with a relatively long-lived (micros-ms) light-induced conformational change that is not reflected by changes in the optical spectrum of the retinyl chromophore. It is plausible that analogous changes (coupled to those of the photocycle) are also operative in the cases of native bR and visual pigments. These conclusions are discussed in view of the light-induced conformational changes recently detected in native and artificial bR with an atomic force sensor.     

Light-induced hydrolysis and rebinding of nonisomerizable bacteriorhodopsin pigment

Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13-cis locked bR pigment (bR5.13; lambda(max) = 550 nm) in which isomerization around the critical C13==C14 double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (lambda(max) = 450 nm) and H430 (lambda(max) = 430 nm), which are characterized by a 13-cis configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process.     

A nonbleachable rhodopsin analogue with a slow photocycle

Archaeal rhodopsins, e.g. bacteriorhodopsin, all have cyclic photoreactions. Such cycles are achieved by a light-induced isomerization step of their retinal chromophores, which thermally re-isomerize in the dark. Visual pigment rhodopsins, which contain in the dark state an 11-cis retinal Schiff base, do not share such a mechanism, and following light absorption, they experience a bleaching process and a subsequent release of the photo-isomerized all-trans chromophore from the binding pocket. The pigment is eventually regenerated by the rebinding of a new 11-cis retinal. In the artificial visual pigment, Rh(6.10), in which the retinal chromophore is locked in an 11-cis geometry by the introduction of a six-member ring structure, an activated receptor may be formed by light-induced isomerization around other double bonds. We have examined this activation of Rh(6.10) by UV-visible and FTIR spectroscopy and have revealed that Rh(6.10) is a nonbleachable pigment. We could further show that the activated receptor consists of two different subspecies corresponding to 9-trans and 9-cis isomers of the chromophore. Both subspecies relax in the dark via separate pathways back to their respective inactive states by thermal isomerization presumably around the C(13)=C(14) double bond. This nonbleachable pigment can be repeatedly photolyzed to undergo identical activation-relaxation cycles. The rate constants of these photocycles are pH-dependent, and the half-times vary between several hours at acidic pH and about 1.5 min at neutral to alkaline pH, which is several orders of magnitude longer than for bacteriorhodopsin.     

Crystal structure of the 13-cis isomer of bacteriorhodopsin in the dark-adapted state

The atomic structure of the trans isomer of bacteriorhodopsin was determined previously by using a 3D crystal belonging to the space group P622. Here, a structure is reported for another isomer with the 13-cis, 15-syn retinal in a dark-adapted crystal. Structural comparison of the two isomers indicates that retinal isomerization around the C13[double bond]C14 and the C15[double bond]N bonds is accompanied by noticeable displacements of a few residues in the vicinity of the retinal Schiff base and small re-arrangement of the hydrogen-bonding network in the proton release channel. On the other hand, aromatic residues surrounding the retinal polyene chain were found to scarcely move during the dark/light adaptation. This result suggests that variation in the structural rigidity within the retinal-binding pocket is one of the important factors ensuring the stereospecific isomerization of retinal.     

Studies on structure and function of rhodopsin by use of cyclopentatrienylidene 11-cis-locked-rhodopsin

The photochemical reaction of cyclopentatrienylidene 11-cis-locked-rhodopsin derived from cyclopentatrienylidene 11-cis-locked-retinal and cattle opsin was spectrophotometrically studied. The difference absorption spectrum between the cyclopentatrienylidene 11-cis-locked-rhodopsin and its retinal oxime had its maximum at 495 nm (P-495). Irradiation of P-495 at -196 degrees C with either blue light or orange light caused no spectral change, supporting the cis-trans isomerization hypothesis for formation of bathorhodopsin. Upon irradiation of P-495 at 0 degree C with orange light, however, its absorption spectrum shifted to a shorter wavelength owing to formation of a hypsochromic product. The difference absorption spectrum between this product (P-466) and its retinal oxime showed its maximum at 466 nm. Analysis of retinal isomers by high-performance liquid chromatography showed that this spectral shift was not accompanied by photoisomerization of the chromophore. P-466 could almost completely be photoconverted to the original pigment (P-495) by irradiation at 0 degree C with blue light with little formation of the other isomeric form of its chromophore. The alpha-band of the circular dichroism spectrum of P-495 was very small in comparison with that of rhodopsin, while that of P-466 was comparable to it. These facts suggest that P-495 has a planar conformation in the side chain of the chromophore and that P-466 has a twisted one, probably at the C8-C9 single bond. Cyclic-GMP phosphodiesterase in frog rod outer segment was activated by neither P-495 nor P-466. This result suggests that the isomerization of the retinylidene chromophore of rhodopsin is indispensable in the phototransduction process.     

Molecular dynamics study of the nature and origin of retinal's twisted structure in bacteriorhodopsin

The planarity of the polyene chain of the retinal chromophore in bacteriorhodopsin is studied using molecular dynamics simulation techniques and applying different force-field parameters and starting crystal structures. The largest deviations from a planar structure are observed for the C(13)==C(14) and C(15)==N(16) double bonds in the retinal Schiff base structure. The other dihedral angles along the polyene chain of the chromophore, although having lower torsional barriers in some cases, do not significantly deviate from the planar structure. The results of the simulations of different mutants of the pigment show that, among the studied amino acids of the binding pocket, the side chain of Trp-86 has the largest impact on the planarity of retinal, and the mutation of this amino acid to alanine leads to chromophore planarity. Deletion of the methyl C(20), removal of a water molecule hydrogen-bonded to H(15), or mutation of other amino acids to alanine did not show any significant influence on the distortion of the chromophore. The results from the present study suggest the importance of the bulky residue of Trp-86 in the isomerization process, in both ground and excited states of the chromophore, and in fine-tuning of the pK(a) of the retinal protonated Schiff base in bacteriorhodopsin. The dark adaptation of the pigment and the last step of the bacteriorhodopsin photocycle imply low barriers against the rotation of the double bonds in the Schiff base region. The twisted double bonds found in the present study are consistent with the proposed mechanism of these ground state isomerization events.     

Non-Isomerizable Artificial Pigments: Implications for the Primary Light-Induced Events in Bacteriorhodopsin

The primary events in the photosynthetic retinal protein bacteriorhodopsin (bR) are reviewed in light of photophysical and photochemical experiments with artificial bR in which the native retinal polyene is replaced by a variety of chromophores. Focus is on retinals in which the critical C₁₃=C₁₄ bond is locked with respect to isomerization by a rigid ring structure. Other systems include retinal oxime and non-isomerizable dyes noncovalently residing in the binding site. The early photophysical events are analyzed in view of recent pump–probe experiments with sub-picosecond time resolution comparing the behavior of bR pigments with those of model protonated Schiff bases in solution. An additional approach is based on the light-induced cleavage of the protonated Schiff base bond that links retinal to the protein by reacting with hydroxylamine. Also described are EPR experiments monitoring reduction and oxidation reactions of a spin label covalently attached to various protein sites. It is concluded that in bR the initial relaxation out of the Franck–Condon (FC) state does not involve sub-stantial C₁₃=C₁₄ torsional motion and is considerably catalyzed by the protein matrix. Prior to the decay of the relaxed fluorescent state (FS or I state), the protein is activated via a mechanism that does not require double bond isomerization. Most plausibly, it is a result of charge delocalization in the excited state of the polyene (or other) chromophores. More generally, it is concluded that proteins and other macromolecules may undergo structural changes (that may affect their chemical reactivity) following optical excitation of an appropriately (covalently or non-covalently) bound chromophore. Possible relations between the light-induced changes due to charge delocalization, and those associated with C₁₃=C₁₄ isomerization (that are at the basis of the bR photocycle), are discussed. It is suggested that the two effects may couple at a certain stage of the photocycle, and it is the combination of the two that drives the cross-membrane proton pump mechanism.     

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.     

Formation of a long-lived photoproduct with a deprotonated Schiff base in proteorhodopsin, and its enhancement by mutation of Asp227

Proteorhodopsin, a retinal protein of marine proteobacteria similar to bacteriorhodopsin of the archaea, is a light-driven proton pump. Absorption of a light quantum initiates a reaction cycle (turnover time of ca. 50 ms), which includes photoisomerization of the retinal from the all-trans to the 13-cis form and transient deprotonation of the retinal Schiff base, followed by recovery of the initial state. We report here that in addition to this fast cyclic conversion, illumination at high pH results in accumulation of a long-lived photoproduct absorbing at 362 nm. This photoconversion is much more efficient in the D227N mutant in which the anionic Asp227, which together with Asp97 constitutes the Schiff base counterion, is replaced with a neutral residue. Upon illumination at pH 8.5, most of the D227N pigment is converted to the 362 nm species, with a quantum efficiency of ca. 0.2. The pK(a) for this transition in the wild type is 9.6, but decreased to 7.5 after mutation of Asp227. The short wavelength of the absorption maximum of the photoproduct indicates that it has a deprotonated Schiff base. In the dark, this photoproduct is converted back to the initial pigment with a time constant of 30 min (in D227N, at pH 8.5), but it can be reconverted more rapidly by illumination with near-UV light. Experiments with "locked" retinal analogues which selectively exclude rotation around either the C9=C10, C11=C12, or C13=C14 bond show that formation of the 362 nm species involves isomerization around the C13=C14 bond. In agreement with this, retinal extraction indicates that the 362 nm photoproduct is 13-cis whereas the initial state is predominantly all-trans. A rapid shift of the pH from 8.5 to 4 greatly accelerates thermal reconversion of the 362 nm species to the initial pigment, suggesting that its recovery involving the thermal isomerization of the chromophore is controlled by ionizable residues, primarily the Schiff base and Asp97. The transformation to the long-lived 362 nm photoproduct is apparently a side reaction of the photocycle, a response to high pH, caused by alteration of the normal reprotonation and reisomerization pathway of the Schiff base.     

The Effect of Protonation and Electrical Interactions on the Stereochemistry of Retinal Schiff Bases

Based on quantumchemical MNDOC calculations it is shown that the ground-state properties of a retinal Schiff base depend sensitively on its protonation state and charge environment. This is exemplified for the equilibrium geometry, for the distribution of partial charges and, in particular, for the thermal isomerization barriers around the π-bonds. It is demonstrated that a protein, by protonating the retinal Schiff base and by providing one or two negative ions in its environment, can reduce double-bond isomerization barriers from 50 kcal/mol for the unprotonated compound to ~ 5 kcal/mol and can increase single bond barriers from 5 kcal/mol to ~ 20 kcal/mol. Thereby, the specific location of the ions relative to the polyene chain of the protonated retinal Schiff base determines the barrier heights. The results explain the ground-state isomerization reactions of retinal observed in bacteriorhodopsin and in squid retinochrome.     

Photoreactions of metarhodopsin III

Meta III is an inactive intermediate thermally formed following light activation of the visual pigment rhodopsin. It is produced from the Meta I/Meta II photoproduct equilibrium of rhodopsin by a thermal isomerization of the protonated Schiff base C=N bond of Meta I, and its chromophore configuration is therefore all-trans 15-syn. In contrast to the dark state of rhodopsin, which catalyzes exclusively the cis to trans isomerization of the C11=C12 bond of its 11-cis 15-anti chromophore, Meta III does not acquire this photoreaction specificity. Instead, it allows for light-dependent syn to anti isomerization of the C15=N bond of the protonated Schiff base, yielding Meta II, and for trans to cis isomerizations of C11=C12 and C9=C10 of the retinal polyene, as shown by FTIR spectroscopy. The 11-cis and 9-cis 15-syn isomers produced by the latter two reactions are not stable, decaying on the time scale of few seconds to dark state rhodopsin and isorhodopsin by thermal C15=N isomerization, as indicated by time-resolved FTIR methods. Flash photolysis of Meta III produces therefore Meta II, dark state rhodopsin, and isorhodopsin. Under continuous illumination, the latter two (or its unstable precursors) are converted as well to Meta II by presumably two different mechanisms.     

Structural changes in the L photointermediate of bacteriorhodopsin

The L to M reaction of the bacteriorhodopsin photocycle includes the crucial proton transfer from the retinal Schiff base to Asp85. In spite of the importance of the L state in deciding central issues of the transport mechanism in this pump, the serious disagreements among the three published crystallographic structures of L have remained unresolved. Here, we report on the X-ray diffraction structure of the L state, to 1.53-1.73 A resolutions, from replicate data sets collected from six independent crystals. Unlike earlier studies, the partial occupancy refinement uses diffraction intensities from the same crystals before and after the illumination to produce the trapped L state. The high reproducibility of inter-atomic distances, and bond angles and torsions of the retinal, lends credibility to the structural model. The photoisomerized 13-cis retinal in L is twisted at the C(13)=C(14) and C(15)=NZ double-bonds, and the Schiff base does not lose its connection to Wat402 and, therefore, to the proton acceptor Asp85. The protonation of Asp85 by the Schiff base in the L-->M reaction is likely to occur, therefore, via Wat402. It is evident from the structure of the L state that various conformational changes involving hydrogen-bonding residues and bound water molecules begin to propagate from the retinal to the protein at this stage already, and in both extracellular and cytoplasmic directions. Their rationales in the transport can be deduced from the way their amplitudes increase in the intermediates that follow L in the reaction cycle, and from the proton transfer reactions with which they are associated.     

Retinal-Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis

Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of the retinal protein group and serves as a light-driven chloride pump in which chloride ions are transported through the membrane following light absorption by the retinal chromophore. In this study, we examined two main issues: (1) factors controlling the binding of the retinal chromophore to the NpHR opsin and (2) the ability of the NpHR opsin to catalyze the thermal isomerization of retinal isomers. We have revealed that the reconstitution process of pharaonis HR (NpHR) pigment from its apoprotein and all-trans retinal depends on the pH, and the process has a pK_a of 5.8 ± 0.1. It was proposed that this pK_a is associated with the pK_a of the lysine residue that binds the retinal chromophore (Lys256). The pigment formation is regulated by the concentration of sodium chloride, and the maximum yield was observed at 3.7 M NaCl. The low yield of pigment in a lower concentration of NaCl (< 3 M) may be due to an altered conformation adopted by the apomembrane, which is not capable of forming the pigment. Unexpectedly and unlike the apomembrane of bacteriorhodopsin, NpHR opsin produces pigments with 11-cis retinal and 9-cis retinal owing to the thermal isomerization of these retinal isomers to all-trans retinal. The isomerization rate depends on the pH, and it is faster at a higher pH. The pK_a value of the isomerization process is similar to the pK_a of the binding process of these retinals, which suggests that Lys256 is also involved in the isomerization process. The isomerization is independent of the sodium chloride concentration. However, in the absence of sodium chloride, the apoprotein adopts such a conformation, which does not prevent the isomerization of retinal, but it prevents a covalent bond formation with the lysine residue. The rate and the thermodynamic parameter analysis of the retinal isomerization by NpHR apoprotein led to the conclusion that the apomembrane catalyzes the isomerization via a triplet mechanism.     

Transition of rhodopsin into the active metarhodopsin II state opens a new light-induced pathway linked to Schiff base isomerization

Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage. 11-cis/all-trans isomerization, induced by absorption of green light, leads to active metarhodopsin II, in which the Schiff base is intact but deprotonated. The subsequent metabolic retinoid cycle starts with Schiff base hydrolysis and release of photolyzed all-trans-retinal from the active site and ends with the uptake of fresh 11-cis-retinal. To probe chromophore-protein interaction in the active state, we have studied the effects of blue light absorption on metarhodopsin II using infrared and time-resolved UV-visible spectroscopy. A light-induced shortcut of the retinoid cycle, as it occurs in other retinal proteins, is not observed. The predominantly formed illumination product contains all-trans-retinal, although the spectra reflect Schiff base reprotonation and protein deactivation. By its kinetics of formation and decay, its low temperature photointermediates, and its interaction with transducin, this illumination product is identified as metarhodopsin III. This species is known to bind all-trans-retinal via a reprotonated Schiff base and forms normally in parallel to retinal release. We find that its generation by light absorption is only achieved when starting from active metarhodopsin II and is not found with any of its precursors, including metarhodopsin I. Based on the finding of others that metarhodopsin III binds retinal in all-trans-C(15)-syn configuration, we can now conclude that light-induced formation of metarhodopsin III operates by Schiff base isomerization ("second switch"). Our reaction model assumes steric hindrance of the retinal polyene chain in the active conformation, thus preventing central double bond isomerization.     

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.     

Trans/13-cis isomerization is essential for both the photocycle and proton pumping of bacteriorhodopsin.

We studied an analogue of bacteriorhodopsin whose chromophore is based on all-trans retinal. A five-membered ring was built around the 13-14 double bond so as to prohibit trans to 13-cis isomerization. No light-induced photochemical changes were seen, other than those due to a small amount (approximately 5%) of unbleached bacteriorhodopsin remaining in the apomembrane used for regeneration. The techniques used included flash photolysis at room and liquid nitrogen temperatures and Fourier-transform infrared difference spectroscopy. When the trans-fixed pigment was incorporated into phospholipid vesicles, no evidence of light-initiated proton pumping could be found. The results indicate that trans to 13-cis isomerization is essential for the photochemical transformation and function of bacteriorhodopsin.     

Conformational change in bacterio-opsin on binding to retinal.

The detailed mechanism of retinal binding to bacterio-opsin is important to understanding retinal pigment formation as well as to the process of membrane protein folding. We have measured the temperature dependence of bacteriorhodopsin formation from bacterio-opsin and all-trans retinal. An Arrhenius plot of the apparent second-order rate constants gives an activation energy of 11.6 +/- 0.7 kcal/mol and an activation entropy of -4 +/- 2 cal/mol deg. Comparison of the activation entropy to model compound reactions suggests that chromophore formation in bacteriorhodopsin involves a substantial protein conformational change. Cleavage of the polypeptide chain between residues 71 and 72 has little effect on the activation energy or entropy, indicating that the connecting loop between helices B and C is not involved in this conformational change.     

Bioorganic chemistry of the purple membrane ofHalobacterium halobium — Chromophore and apoprotein modified bacteriorhodopsins

Iodophenyl and anthryl retinal analogues have been synthesized. Thetrans-isomers have been isolated and purified by high pressure liquid chromatography. The purified isomers have been further characterized by nuclear magnetic resonance and ultraviolet-visible spectroscopy. Incubation of these retinal analogues with apoprotein (bacterioopsin), isolated from the purple membrane ofHalobacterium halobium gave new bacteriorhodopsin analogues. These analogues have been investigated for their absorption properties and stability. The iodophenyl analogue has been found to bind to bacterioopsin rapidly. The pigment obtained from this analogue showed a dramatically altered opsin shift of 1343 cm^-1. The anthryl analogue based bacteriorhodopsin, however, showed an opsin shift of 3849 cm^-1. It has been found that bacteriorhodopsin is quite unrestrictive in the ionone ring site. The apoprotein seems to prefer chromophores that have the ring portion co-planar with the polyene side chain. The purple membrane has also been modified by treatment with fluorescamine, a surface active reagent specific for amino groups. Reaction under controlled stoichiometric conditions resulted in the formation of a modified pigment. The new pigment showed a band at 390 nm—indicative of fluorescamine reaction with amino group (s) of apoprotein-besides retaining its original absorption band at 560 nm. Analysis of the fluorescamine modified bacteriorhodopsin resulted in the identification of lysine 129 as the modified amino acid residue. Fluorescamine-modified-bacteriorhodopsin suspension did not release protons under photolytic conditions. However, proteoliposomes of fluorescamine-modified-bacteriorhodopsin were found to show proton uptake, though at a reduced rate. Presented at the 3rd National Symposium on Bioorganic Chemistry, 1987, Hyderabad.