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 substantial 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.

Retinal isomerization in bacteriorhodopsin is controlled by specific chromophore-protein interactions. A study with noncovalent artificial pigments.

It has previously been shown that, in mutants lacking the Lys-216 residue, protonated Schiff bases of retinal occupy noncovalently the bacteriorhodopsin (bR) binding site. Moreover, the retinal-Lys-216 covalent bond is not a prerequisite for initiating the photochemical and proton pump activity of the pigment. In the present work, various Schiff bases of aromatic polyene chromophores were incubated with bacterioopsin to give noncovalent pigments that retain the Lys-216 residue in the binding site. It was observed that the pigment's absorption was considerably red-shifted relative to the corresponding protonated Schiff bases (PSB) in solution and was sensitive to Schiff base linkage substitution. Their PSB pK(a) is considerably elevated, similarly to those of related covalently bound pigments. However, the characteristic low-pH purple to blue transition is not observed, but rather a chromophore release from the binding site takes place that is characterized by a pK(a) of approximately 6 (sensitive to the specific complex). It is suggested that, in variance with native bR, in these complexes Asp-85 is protonated and Asp-212 serves as the sole negatively charged counterion. In contrast to the bound analogues, no photocycle could be detected. It is suggested that a specific retinal-protein geometrical arrangement in the binding site is a prerequisite for achieving the selective retinal photoisomerization.

Nonisomerizable non-retinal chromophores initiate light-induced conformational alterations in bacterioopsin.

The photoactivation of retinal proteins is usually interpreted in terms of C=C photoisomerization of the retinal moiety, which triggers appropriate conformational changes in the protein. In this work several dye molecules, characterized by a completely rigid structure in which no double-bond isomerization is possible, were incorporated into the binding site of bacteriorhodopsin (bR). Using a light-induced chemical reaction of a labeled EPR probe, it was observed that specific conformational alterations in the protein are induced following light absorption by the dye molecules occupying the binding site. The exact nature of these changes and their relationship to those occurring in the bR photocycle are still unclear. Nevertheless, their occurrence proves that C=C or C=NH(+) isomerization is not a prerequisite for protein conformational changes in a retinal protein. More generally, we show that conformational changes, leading to changes in reactivity, may be induced in proteins by optical excitation of simple nonisomerizable dyes located in the macromolecular matrix.

Specific binding sites for cations in bacteriorhodopsin.

The Asp-85 residue, located in the vicinity of the retinal chromophore, plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment the protonation of Asp-85 is responsible for the transition from the purple form (lambda(max) = 570 nm) to the blue form (lambda(max) = 605 nm) of bR. This transition can also be induced by deionization (cation removal). It was previously proposed that the cations bind to the bR surface and raise the surface pH, or bind to a specific site in the protein, probably in the retinal vicinity. We have reexamined these possibilities by evaluating the interaction between Mn(2+) and a nitroxyl radical probe covalently bound to several mutants in which protein residues were substituted by cystein. We have found that Mn(2+), which binds to the highest-affinity binding site, significantly affects the EPR spectrum of a spin label attached to residue 74C. Therefore, it is concluded that the highest-affinity binding site is located in the extracellular side of the protein and its distance from the spin label at 74C is estimated to be approximately 9.8 +/- 0.7 A. At least part of the three to four low-affinity cation binding sites are located in the cytoplasmic side, because Mn(2+) bound to these binding sites affects spin labels attached to residues 103C and 163C located in the cytoplasmic side of the protein. The results indicate specific binding sites for the color-controlling cations, and suggest that the binding sites involve negatively charged lipids located on the exterior of the bR trimer structure.

Bacteriorhodpsin experiences light-induced conformational alterations in nonisomerizable C(13)=C(14) pigments. A study with EPR.

The mechanism by which bacteriorhodopsin is activated following light absorption is not completely clear. We have detected protein conformational alterations following light absorption by retinal-based chromophores in the bacteriorhodopsin binding site by monitoring the rate of reduction-oxidation reactions of covalently attached spin labels, using EPR spectroscopy. It was found that the reduction reaction with hydroxylamine is light-catalyzed in the A103C-labeled pigment but not in E74C or M163C. The reaction is light-catalyzed even when isomerization of the C(13)=C(14) bond of the retinal chromophore is prevented. The reverse oxidation reaction with molecular oxygen is effective only in apomembrane derived from the mutant A103C. This reaction is light-accelerated following light absorption of the retinal oxime, which occupies the binding site. The light-induced acceleration is evident also in "locked" bacteriorhodopsin in which isomerization around the C(13)=C(14) bond is prevented. It is evident that the chromophore-protein covalent bond is not a prerequisite for protein response. In contrast to the case of the retinal oxime, a reduced C=N bond A103C-labeled pigment did not exhibit acceleration of the oxidation reaction following light absorption. Acceleration was observed, however, following substitution of the polyene by groups that modify the excited state charge delocalization. It is suggested that protein conformational alterations are induced by charge redistribution along the retinal polyene following light absorption.

On the protein residues that control the yield and kinetics of O(630) in the photocycle of bacteriorhodopsin.

The effects of pH on the yield (phi(r)), and on the apparent rise and decay constants (k(r), k(d)), of the O(630) intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in k(r), k(d), and phi(r) at high pH [pK(a)(1) approximately 8]; 2) A rise in phi(r) at low pH [pK(a)(2) approximately 4.5]; and 3) A drop in k(r) and k(d) at low pH [pK(a)(3) approximately 4. 5]. (pK(a) values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of phi(r), k(r), and k(d) in native and acetylated bR, and in its D96N and R82Q mutants. The D96N experiments were carried out in the presence of small amounts of the weak acids, azide, nitrite, and thiocyanate. Analysis of the mutant's data leads to the identification of the protein residue (R(1)) whose state of protonation controls the magnitude of phi(r), k(r), and k(d) at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pK(a) of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O(630) parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R(2) and R(3). R(2) affects the rise of phi(r) at low pH, whereas the state of protonation of R(3) affects both k(r) and k(d). Our data confirm a previous suggestion that R(3) should be identified as the proton release moiety (XH). A clear identification of R(2), including its possible identity with R(3), remains open.

The titrations of Asp-85 and of the cation binding residues in bacteriorhodopsin are not coupled.

An outstanding problem relating to the structure and function of bacteriorhodopsin (bR), which is the only protein in the purple membrane of the photosynthetic microorganism Halobacterium salinarium, is the relation between the titration of Asp-85 and the binding/unbinding of metal cations. An extensively accepted working hypothesis has been that the two titrations are coupled, namely, protonation of Asp-85 (located in the vicinity of the retinal chromophore) and cation unbinding occur concurrently. We have carried out a series of experiments in which the purple blue equilibrium and the binding of Mn2+ ions (monitored by electron spin resonance) were followed as a function of pH for several (1-4) R = [Mn2+]/[bR] molar ratios. Data were obtained for native bR, bR mutants, artificial bR and chemically modified bR. We find that in the native pigment the two titrations are separated by more than a pKa unit [delta pKa = pKa(P/B)-pKa(Mn2+) = (4.2-2.8) = 1.4]. In the non-native systems, delta pKa values as high as 5 units, as well as negative delta pKas, are observed. We conclude that the pH titration of cation binding residues in bR is not directly related to the titration of Asp-85. This conclusion is relevant to the nature of the high affinity cation sites in bR and to their role in the photosynthetic function of the pigment.

Generation of the O630 photointermediate of bacteriorhodopsin is controlled by the state of protonation of several protein residues.

The last stages of the photocycle of the photosynthetic pigment all-trans bacteriorhodopsin (bR570), as well as its proton pump mechanism, are markedly pH dependent. We have measured the relative amount of the accumulated O630 intermediate (Phir), as well as its rise and decay rate constants (kr and kd, respectively), over a wide pH range. The experiments were carried out in deionized membrane suspensions to which varying concentrations of metal cations and of large organic cations were added. The observed pH dependencies, s-shaped curves in the case of Phir and bell-shaped curves for kr and kd, are interpreted in terms of the titration of three protein residues denoted as R1, R2, and R3. The R1 titration is responsible for the increase in Phir, kr, and kd upon lowering the pH from pH approximately 9.5 to 7. At low pH Phir exhibits a secondary rise which is attributed to the titration of a low pKa group, R2. After reaching a maximum at pH approximately 7, kr and kd undergo a decrease upon decreasing the pH, which is attributed to the titration of R3. All three titrations exhibit pKa values which decrease upon increasing the salt concentration. As in the case of the Purple (bR570) if Blue (bR605) equilibrium, divalent cations are substantially more effective than monovalent cations in shifting the pKa values. Moreover, bulky organic cations are as effective as small metal cations. It is concluded that analogously to the Purple if Blue equilibrium, the salt binding sites which control the pKa values of R1, R2, and R3 are located on, or close to, the membrane surface. Possible identifications of the three protein residues are considered. Experiments with the E204Q mutant show that the mutation has markedly affected the R2 (Phir) titration, suggesting that R2 should be identified with Glu-204 or with a group whose pKa is affected by Glu-204. The relation between the R1, R2 and R3 titrations and the proton pump mechanism is discussed. It is evident that the pH dependence of Phir is unrelated to the measured pKa of the group (XH) which releases the proton to the extracellular medium during the photocycle. However, since the same residue may exhibit different pKa values at different stages of the photocycle, it cannot be excluded that R2 or R3 may be identified with XH.

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.

Titration kinetics of Asp-85 in bacteriorhodopsin: exclusion of the retinal pocket as the color-controlling cation binding site.

The spectrum (the purple blue transition) and function of the light-driven proton pump bacteriorhodopsin are determined by the state of protonation of the Asp-85 residue located in the vicinity of the retinal chromophore. The titration of Asp-85 is controlled by the binding/unbinding of one or two divalent metal cations (Ca2+ or Mg2+). The location of such metal binding site(s) is approached by studying the kinetics of the cation-induced titration of Asp-85 using metal ions and large molecular cations, such as quaternary ammonium ions, R4N+ (R = Et, Pr, a divalent 'bolaform ion' [Et3N+-(CH2)4-N+Et3] and the 1:3 molecular complex formed between Fe2+ and 1,10-phenanthroline (OP). The basic multi-component kinetic features of the titration, extending from 10(-2) to 10(4) s, are unaffected by the charge and size of the cation. This indicates that cation binding to bR triggers the blue --> purple titration in a fast step, which is not rate-determining. In view of the size of the cations involved, these observations indicate that the cation binding site is in an exposed location on, or close to, the membrane surface. This excludes previous models, which placed the color-controlling Ca2+ ion in the retinal binding pocket.

Probing the Sol-Gel Conversion in the Tetraethoxysilane/Alcohol/Water System with the Aid of Diffusion-Controlled Fluorescence Quenching

The sol-gel conversion in the tetraethoxysilane/alcohol/water system can be probed by means of the diffusion-controlled fluorescence quenching of Py* by Cu2+ ions. In the course of tetraethoxysilane hydrolysis, the viscosity of the system increases, thereby causing the quenching rate to decrease and the mean lifetime of the excited state to elongate dramatically as the system approaches the gel-point. Incorporating the equation proposed by Vogelsberger et al. (1992) for the time-dependence of the viscosity of gelating systems into the Smoluchowsky kinetic equation, an equation has been proposed to describe the observed time-dependence of the Py* lifetime. Copyright 1997 Academic Press. Copyright 1997Academic Press

Time-resolved titrations of ASP-85 in bacteriorhodopsin: the multicomponent kinetic mechanism.

The Asp-85 residue, located in the vicinity of the retinal chromophore, plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment the protonation of Asp-85 is responsible for the transition from the purple form (lambdamax = 570 nm) to the blue form (lambdamax = 605 nm) of bR (pKa = 3.5 in 20 mM NaCl). The Purple <=> Blue transition can also be induced by deionization (cation removal). These color changes offer a unique opportunity for time resolving the titration of a protein residue using conventional stopped-flow methodologies. We have studied the Purple <=> Blue equilibration kinetics in bR by exposing the system to pH and to cation jumps. Independently of the equilibration direction (Purple-->Blue or Blue-->Purple) and of the inducing concentration jump ([H+] or [cation]), the kinetics are found to exhibit analogous multicomponent features. Analysis of the data over a range of cation concentrations and pH values leads to the conclusion that the rate-determining step in the overall titration of Asp-85 is proton translocation through a specific proton channel. The multicomponent kinetics, extending over a wide time range (10(-2)-10(4) s), are accounted for in terms of a pH-dependent heterogeneity of proton channels. A model is presented in which the relative weight of four proton channels is determined by the state of protonation of two interacting, channel-controlling, protein residues A1 and A2. These findings bear on the mechanism of the vectorial proton translocation associated with the photocycle of bR.

Microsecond atomic force sensing of protein conformational dynamics: implications for the primary light-induced events in bacteriorhodopsin.

In this paper a new atomic force sensing technique is presented for dynamically probing conformational changes in proteins. The method is applied to the light-induced changes in the membrane-bound proton pump bacteriorhodopsin (bR). The microsecond time-resolution of the method, as presently implemented, covers many of the intermediates of the bR photocycle which is well characterized by spectroscopical methods. In addition to the native pigment, we have studied bR proteins substituted with chemically modified retinal chromophores. These synthetic chromophores were designed to restrict their ability to isomerize, while maintaining the basic characteristic of a large light-induced charge redistribution in the vertically excited Franck-Condon state. An analysis of the atomic force sensing signals lead us to conclude that protein conformational changes in bR can be initiated as a result of a light-triggered redistribution of electronic charge in the retinal chromophore, even when isomerization cannot take place. Although the coupling mechanism of such changes to the light-induced proton pump is still not established, our data question the current working hypothesis which attributes all primary events in retinal proteins to an initial trans<==>cis isomerization.

Atomic Force Sensing of Light-Induced Protein Dynamics with Microsecond Time Resolution in Bacteriorhodopsin and Photosynthetic Reaction Centers

This paper reports on experiments that have monitored protein microsecond dynamics with a cantilevered near-field optical glass fiber. In these experiments two photoactive proteins, bacteriorhodopsin (bR) and the photosynthetic reaction center (PS I), are used to demonstrate that such probes can measure light-induced microsecond protein dynamics even though the resonance frequencies of the glass cantilevers used are on the order of a few hundred kilohertz. In the case of the light-driven proton pump, bR, the light-induced atomic force sensing (AFS) signal is negative (indicating contraction) in the microsecond time domain of the L photointermediate and becomes positive (corresponding to expansion) in the subsequent M intermediate that lives for milliseconds. Double pulse experiments from M to bR show that the latter process reverses the AFS signal. Thus, the AFS structural changes are coupled with the (optical) photocycle intermediates. Light-induced contraction and expansion phenomena are also observed in the case of PS I. In both systems the time regime of the dynamic phenomena that have been measured with AFS is five orders of magnitude faster than the fastest previously recorded atomic force detection of dynamic phenomena. This advance portends a new era in dynamic imaging of protein conformational changes.

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.

An optical submicrometer calcium sensor with conductance sensing capability.

The identification of chemical species and the measurement of their concentrations with high (submicrometer) spatial resolution are of considerable importance in cell biology. In this article we report the first successful development of a > or = 0.1-micron Ca2+ sensor based on a pulled micropipet, filled with a conducting porous sol-gel glass which was doped with the fluorescent calcium green 1 Ca2+ indicator. Such sensors are potentially capable of measuring Ca2+ concentrations as low as 10(-8) M, in confined volumes, with a three-dimensional resolution which exceeds approximately 0.1 micron. A major advantage of the sensor is its capability to be integrated into a multifunctional probe which will measure chemical analyte concentrations and ion conductance.

Lateral organization of pyrene-labeled lipids in bilayers as determined from the deviation from equilibrium between pyrene monomers and excimers.

In lipid bilayers, pyrene and pyrene-labeled lipids form excimers in a concentration-dependent manner. The aromatic amine N, N-diethylaniline (DEA), which has a high membrane-to-medium partition coefficient, quenches the monomers only, and therefore it is expected that under conditions in which the monomers are in equilibrium with the excimers due to the mass law, the Stern-Volmer coefficient (Ksv) for monomers (M), defined as KM, should be identical to that of the excimer (E), defined as KE, and KE/KM = 1. 0. This is indeed the case for pyrene and pyrene valerate in egg phosphatidylcholine small unilamellar vesicles. However, for pyrene decanoate and pyrene dodecanoate in these vesicles, and for N-[12-(1-pyrenyl)dodecanoyl]sphingosylphosphocholine in a matrix of either N-stearoyl sphingosylphosphocholine or 1-palmitoyl-2-oleoyl phosphatidylcholine, KE < KM. This can be explained either by the existence of (a) two subpopulations of excimers, one in fast equilibrium with the monomers and the other, related to ground-state protoaggregates of pyrene lipids; (b) two monomer subpopulations where part of M cannot be quenched by DEA; or (c) two monomer subpopulations, both quenched by DEA, but only one of which produces excimers. The good agreement between the photophysical processes determined by steady state and time-resolved measurements supports the third explanation for the bilayers containing pyrene phospholipids. It also suggests that the main factors determining the immiscibility of pyrene lipids in phospholipid bilayers are the temperature, the difference in the gel-to-liquid-crystalline phase transition temperature (deltaTm) between the matrix and the pyrene lipid, and the structural differences between the matrix lipid and the pyrene-labeled lipid. These results indicate that the KE/KM ratio can serve as a very sensitive tool to quantify isothermal microscopic immiscibility in membranes. This novel approach has the following advantages: applicability to fluid phase immiscibility, requirement of a relatively low mol fraction of pyrene lipids, and conceivably, applicability to biological membranes.

Time-resolved titrations of the Schiff base and of the Asp85 residue in artificial bacteriorhodopsins.

Deprotonation/protonation processes involving the retinal Schiff base and the Asp85 residue play dominant roles in the light-induced proton pump of bacteriorhodopsin (bR). Although the pKa values of these two moieties in unphotolyzed bR are well established, the kinetics of the respective titrations in the native pigment are difficult to interpret, primarily due to the extreme (nonphysiological) pKa values of the two moieties (12.2 +/- 0.2 and 2.7, in 0.1 M NaCl, for the Schiff base and for Asp85, respectively). These difficulties are circumvented by applying stopped-flow techniques, time resolving the titrations of several artificial bRs in which the pKa values of the above two residues are substantially modified: 13-CF3 bR, pKa (Schiff base) = 8.2 +/- 0.2; 13-demethyl-11,14-epoxy bR, pKa (Schiff base) = 8.2 +/- 0.1 (in 0.1 M NaCl); aromatic bR, pKa (Asp85) = 5.2 +/- 0.1 (in water). The R82Q bR mutant, pKa (Asp85) congruent to 7.2 was also employed. A major objective was to verify whether the basic relationships of homogeneous kinetics obeyed by elementary acid/base systems in solution (primarily, the possibility to express the equilibrium constant as the ratio of the forward and back rate constants) are also obeyed by the Schiff base and Asp85 moieties. We found that this is the case for the Schiff base in the pH range between 7 and 9 but not at lower pH. These observations led to the conclusion that the Schiff base is titrable from the outside medium via a proton channel, which becomes saturated, and thus rate determining, below pH approximately equal to 7.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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

Low-temperature trapping of early photointermediates of alpha-isorhodopsin.

Alpha-Isorhodopsin, an artificial visual pigment with a 9-cis-4,5-dehydro-5,6-dihydro(alpha)retinal chromophore, was photolyzed at low temperatures and absorption difference spectra were collected as the sample was warmed. A bathorhodopsin (Batho)-like intermediate absorbing at ca 495 nm was detected below 55 K,a blue-shifted intermediate (BSI)-like intermediate absorbing at ca 453 nm was observed when the temperature was raised to 60 K and a lumirhodopsin (Lumi)-like intermediate absorbing at ca 470 nm was found when the sample was warmed to 115 K. Photointermediates from this pigment were compared to those of native rhodopsin and 5,6-dihydroisorhodopsin. As in native rhodopsin, Batho is the first intermediate detected in alpha-isorhodopsin, though unlike native rhodopsin at low temperatures BSI is observed prior to Lumi formation. Alpha-Isohodopsin behaves similarly to 5,6-dihydroisorhodopsin, with the same early intermediates observed in both artificial visual pigments lacking the C5-C6 double bond. The transition temperature for BSI formation is higher in alpha-isorhodopsin, suggesting an interaction involving the chromophore ring in BSI formation. The transition temperature for Lumi formation is similar for these two pigments as well as for native rhodopsin, suggesting comparable changes in the protein environment in that transition.