Purple-to-blue transition of bacteriorhodopsin in a neutral lipid environment.

The red shift in the absorption maximum of native purple membrane suspensions caused by deionization is missing in lipid-depleted purple membrane, and the pK of the acid-induced transition is down-shifted to pH approximately 1.4 and has become independent of cation concentration (Szundi, I., and W. Stoeckenius. 1987. Proc. Natl. Acad. Sci. USA. 84:3681-3684). However, the proton pumping function cannot be demonstrated in these membranes. When native acidic lipids of purple membrane are exchanged for egg phosphatidylcholine or digalactosyldiglyceride, bacteriorhodopsin is functionally active in the modified membrane. It shows spectral shifts upon light-dark adaptation, a photocycle with M-intermediate and complex decay kinetics; when reconstituted into vesicles with the same neutral lipids, it pumps protons. Unlike native purple membrane, lipid-substituted modified membranes do not show a shift of the absorption maximum to longer wavelength upon deionization. A partial shift can be induced by titration with HCl; it has a pK near 1.5 and no significant salt dependence. Titration with HNO3 and H2SO4, which causes a complete transition in the lipid-depleted membranes, i.e., it changes their colors from purple to blue, does not cause the complete transition in the lipid-substituted preparations. These results show that the purple color of bacteriorhodopsin is independent of cations and their role in the purple-to-blue transition of native membranes is indirect. The purple and blue colors of bacteriorhodopsin are interpreted as two conformational states of the protein, rather than different protonation states of a counterion to the protonated Schiff base.     

Structure and thermal stability of monomeric bacteriorhodopsin in mixed phospholipid/detergent micelles

Thermal unfolding experiments on bacteriorhodopsin in mixed phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 +/- 13 A in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near-UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field induced conformational changes of bacteriorhodopsin were studied in six types of dried film (randomly and electrically oriented membranes of purple as well as cation-depleted blue bacteriorhodopsin) by measuring the frequency dependence of the optical absorbance change and the dielectric dispersion and absorption. For the purple bacteriorhodopsin the optical absorbance change induced by alternating rectangular electric fields of ±300 kV/cm altered the sign twice in the frequency range from 0.001 Hz to 100 kHz (around 0.03 Hz and 100 kHz), indicating that the electric field induced conformational change in these samples consists of, at least, three steps. Similarly, it was found for the blue bacteriorhodopsin that at least two steps are involved. In accord with optical measurements, the dielectric behaviour due to alternating sinusoidal electric fields of±6kV/cm in the frequency range from 10 Hz to 10 MHz showed two broad dispersion/absorption regions, one below 1 kHz and the other around 10–100 kHz. This suggests that the conformational change of bacteriorhodopsin is also reflected by its dielectrical properties and that it is partially induced at 6 kV/cm. Including previous results obtained by analysis of the action of DC fields on purple membrane films, a model for a field-induced cyclic reaction for purple as well as blue bacteriorhodopsin is proposed. In addition it was found that there are electrical interactions among purple membrane fragments in dried films.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field-induced absorption changes of bacteriorhodopsin were studied with different samples of purple membranes which were prepared as randomly oriented and electrically oriented films of purple as well as cation-depleted blue bacteriorhodopsin. The absorption changes were proportional to the square of the field strength up to 300 kV/cm. The electric field from the intracellular side to the extracellular side of the purple bacteriorhodopsin induces a spectrum change, resulting in a spectrum similar to that of the cation-depleted blue bacteriorhodopsin. When the field was removed, the purple state was regenerated. The blue state was mainly affected by an electric field in the opposite direction, suggesting a reversible interaction with the Schiff's base bond of the retinal. Since the field-induced reaction of bacteriorhodopsin was observed in the presence of a concomitant steady ion flux, it is assumed that the generation of a local diffusion potential may play an important role in these spectral reactions. Although the fragments were fixed in the dried film, electric dichroism was observed. The dichroic contribution of the total absorbance change was about 15%. The angular displacement of the retinal transition moment was calculated to be 1.5° toward the membrane normal.     

Electrooptical measurements on purple membrane containing bacteriorhodopsin mutants.

Electrooptical measurements on purple membrane containing the wild-type and 10 different bacteriorhodopsin mutants have shown that the direction of the permanent electric dipole moment of all these membranes reverses at different pH values in the range 3.2-6.4. The induced dipole moment and the retinal angle exhibit an increased value at these pHs. The results demonstrate that the bacteriorhodopsin protein makes an important contribution to the electrooptical properties of the purple membrane.     

Fourier-transform infrared studies on cation binding to native and modified purple membranes

Fourier-transform infrared spectroscopy has been used to examine the structural differences in the protein moiety between the native purple and the deionized blue membranes, both at pH 5.0. The spectra demonstrate that deionization of purple membrane decreases the content of the distorted alpha II-helices in favor of the more common alpha I-helices. Changes in the signals from beta-turns are also observed. The changes corresponding to the carboxyl groups suggest that deionization leads to a decrease in the strength of the hydrogen bonds involving carboxyl groups. Most of these effects are reversed progressively upon binding of one to five Mn2+ per bacteriorhodopsin to the deionized membrane. Binding of Hg2+ to the deionized membranes does not restore the purple color but induces global changes similar to, but less intense than, those brought about by Mn2+ binding. However, the effects attributed to the carboxyl groups are opposite to those found for Mn2+. Schiff base reduction or bleaching induces a decrease of the content of the alpha II-helix in favor of the alpha I-helix and a decrease in the strength of hydrogen bonds to carboxyl groups. Deionization of these modified membranes leads to a further loss in the alpha II content. These results indicate a conformational rearrangement of the protein structure between the native purple membrane and the deionized membrane, which could arise from surface potential changes elicited by bound cations. The changes observed in the carboxyl groups suggest that some of them are located structurally close to the retinal environment and may be involved in cation binding.     

脱脂菌紫质与天然紫膜水合变化的比较

本实验通过不同水合度下天然紫膜、脱脂菌紫质吸附等温线分析、红外光谱对比,讨论了天然紫膜小磷脂、蛋白质、水三者作用关系,认为磷脂对天然紫膜中蛋白质表而一些极性基团的分布及水合有重要作用,这些位点的水合对蛋白质进一步水合变化起重要作用.     

Influence of cations on the blue to purple transition of bacteriorhodopsin. Comparison of Ca2+ and Hg2+ binding and their effect on the surface potential

We have investigated the effect of Ca2+ and Hg2+ binding on various properties of the blue membrane prepared by deionization of the Halobacterium halobium purple membrane. Binding of radioactive 45Ca2+ and 203Hg2+ was monitored by a filtration technique. Five high and medium affinity sites for Ca2+ and seven low affinity sites for Hg2+ were found per bacteriorhodopsin. Competitive binding was observed only for three Ca2+ and three Hg2+. Visible absorption studies indicated that Ca2+ binding could restore the purple color of bacteriorhodopsin while Hg2+ was inefficient. Hg2- could partially reverse to blue the Ca2+-regenerated purple membrane in parallel with the displacement of three Ca2+. Effects of cation binding on the surface potential of the membrane were measured by Electron Spin Resonance spectroscopy using a cationic spin-labeled amphiphile. Cations such as La3+, Ca2+, Mg2+, or Na+ strongly increased (i.e. rendered less negative) the surface potential. An univocal correlation was found between the cation-induced variation of surface potential and the extent of regeneration of the purple color. Hg2+ induced a smaller increase in surface potential than that corresponding to the effective divalent cations. This lower effect appears to be due to binding to sites not related to those of other cations.     

Bacteriorhodopsin thermal stability: influence of bound cations and lipids on the intrinsic protein fluorescence

Temperature-induced changes in protein intrinsic fluorescence of native, delipidated and deionized purple membranes are investigated. It is found that the removal of cations most strongly affects the protein and its thermal stability. The denaturation of dei-BR completes at 70 degrees C, while delipidated and native BR still maintain their native structure at this temperature. Both the quantum yield and the fluorescence maximum suggest correlation between the Trp-retinal coupling and protein structural stability. The low red shift of the fluorescence maximum caused by increasing of temperature indicates limited unfolding of bacteriorhodopsin upon denaturation.     

Role of carboxyl residues and membrane lipids in cation binding to bacteriorhodopsin

To investigate the site specificity of cation binding to bacteriorhodopsin, carboxyl groups were chemically modified in purple membrane preparations from Halobacterium halobium. Cation binding followed by EPR and visible spectroscopy has led us to the conclusion that two cations bind to the surface regions and that at least one cation binds to carboxyl groups in the protein interior. Conformational freedom is necessary for the cooperative conversion of deionized blue species to cation-reconstituted purple species. Studies of white membranes from the JW-5 strain showed that a higher content of charged lipids results in the binding of approximately 100 more color-regulating cations and in negative cooperativity in the blue-to-purple species conversion. A greater dependence of protein structure on these bound cations suggests a role for cations in the modulation of opsin-lipid interaction.     

Protein-chromophore interactions in bacteriorhodopsin: the effects of a change in surface potential.

The chromophore retinal is bound to bacteriorhodopsin via a protonated Schiff base linkage. The retinal binding site is reported to be buried in the transmembrane portion of the protein, distant from the membrane surfaces. When bound to bacteriorhodopsin, the absorption maximum of retinal is red-shifted from 366 nm to 568 nm producing a purple color. This color persists across a wide pH range. However, when the pH is raised above 12.0, the membranes become pink in color, while at pH values of 3.0 or below, a blue color is produced. The blue color can also be obtained by removing the divalent cations bound to the surface of the protein. In this study, bacteriorhodopsin was examined by circular dichroism and absorption spectroscopy to determine if protein conformational changes were associated with the color shifts. It was found that although the retinal chromophore can be completely removed by bleaching with hydroxylamine with no significant influence on the secondary structure of the protein, a change in the surface charge of bacteriorhodopsin results in measurable conformational change in the protein, which apparently affects the nature of the retinal binding site.     

Proteoliposomes with right-side-out oriented purple membrane/bacteriorhodopsin require cations inside for proton pumping

Proteoliposomes were prepared by sonication of phospholipids and blue membranes (cation-free purple membranes carrying little activity of light-driven proton pumping) in an acidic medium of very low ionic strength. The majority of the bacteriorhodopsin population in these proteoliposomes was in the right-side-out (as in living cells) orientation as judged from the resultant polypeptides after papain digestion. By raising the pH of sonication, the population of right-side-out oriented bacteriorhodopsin decreased, and consequently that of the inversely oriented one increased. In KCl and NaCl up to certain concentrations or in choline chloride even at high concentrations, in the light, the proteoliposomes with right-side-out bacteriorhodopsin did not pump protons, whereas those with inversely oriented bacteriorhodopsin did. The former began to pump only after cations were likely incorporated/permeated into the proteoliposome and reached the carboxyl terminal (cytosol) side of bacteriorhodopsin/purple membrane.     

Eu3+ binding to europium-regenerated bacteriorhodopsin upon delipidation and monomerization

We have studied the effect of monomerization of the purple membrane lattice, as well as removal of 75% of the lipids, on the binding properties of Eu(3+) ions. We found that delipidation and monomerization do not cause the cations to lose their binding ability to the protein. This suggests that the three most strongly bound Eu(3+) cations do not bind to the lipids, but directly bind to the protein. Furthermore, we found that delipidation actually causes a slight increase in the binding affinity. This is likely a result of reduced aggregation of europium-regenerated bacteriorhodopsin (bR) upon lipid removal causing more exposure of the binding sites to the Eu(3+) cations. These results, taken with those from our previous publication [Heyes and El-Sayed, Biophys. J. 85 (2003) 426-434], might suggest that the cations remain bound upon delipidation of bR, but have no effect on the function. This is discussed with respect to the role of cations in the function of native bR.     

Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis

Summary Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated³H leucine from the digested membranes.During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.     

Mechanism and Role of Divalent Cation Binding of Bacteriorhodopsin

Several observations have already suggested that the carboxyl groups are involved in the association of divalent cations with bacteriorhodopsin (Chang et al., 1985). Here we show that at least part of the protons released from deionized purple membrane (`blue membrane') samples when salt is added are from carboxyl groups. We find that the apparent pK of magnesium binding to purple membrane in the presence of 0.5 mM buffer is 5.85. We suggest this is the pK of the carboxyl groups shifted from their usual pK because of the proton concentrating effect of the large negative surface potential of the purple membrane. Divalent cations may interact with negatively charged sites on the surface of purple membrane through the surface potential and/or through binding either by individual ligands or by conformation-dependent chelation. We find that divalent cations can be released from purple membrane by raising the temperature. Moreover, purple membrane binds only about half as many divalent cations after bleaching. Neither of these operations is expected to decrease the surface potential and thus these experiments suggest that some specific conformation in purple membrane is essential for the binding of a substantial fraction of the divalent cations. Divalent cations in purple membrane can be replaced by monovalent, (Na⁺ and K⁺), or trivalent, (La⁺⁺⁺) cations. Flash photolysis measurements show that the amplitude of the photointermediate, O, is affected by the replacement of the divalent cations by other ions, especially by La⁺⁺⁺. The kinetics of the M photointermediate and light-induced H⁺ uptake are not affected by Na⁺ and K⁺, but they are drastically lengthened by La⁺⁺⁺ substitution, especially at alkaline pHs. We suggest that the surface charge density and thus the surface potential is controlled by divalent cation binding. Removal of the cations (to make deionized blue membrane) or replacement of them (e.g. La⁺⁺⁺-purple membrane) changes the surface potential and hence the proton concentration near the membrane surface. An increase in local proton concentration could cause the protonation of critical carboxyl groups, for example the counter-ion to the protonated Schiff's base, causing the red shift associated with the formation of both deionized and acid blue membrane. Similar explanations based on regulation of the surface proton concentration can explain many other effects associated with the association of different cations with bacteriorhodopsin.     

Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base.

The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.     

Location of a cation-binding site in the loop between helices F and G of bacteriorhodopsin as studied by 13C NMR

The high-affinity cation-binding sites of bacteriorhodopsin (bR) were examined by solid-state 13C NMR of samples labeled with [3-13C]Ala and [1-13C]Val. We found that the 13C NMR spectra of two kinds of blue membranes, deionized (pH 4) and acid blue at pH 1.2, were very similar and different from that of the native purple membrane. This suggested that when the surface pH is lowered, either by removal of cations or by lowering the bulk pH, substantial change is induced in the secondary structure of the protein. Partial replacement of the bound cations with Na+, Ca2+, or Mn2+ produced additional spectral changes in the 13C NMR spectra. The following conclusions were made. First, there are high-affinity cation-binding sites in both the extracellular and the cytoplasmic regions, presumably near the surface, and one of the preferred cation-binding sites is located at the loop between the helix F and G (F-G loop) near Ala196, consistent with the 3D structure of bR from x-ray diffraction and cryoelectron microscopy. Second, the bound cations undergo rather rapid exchange (with a lifetime shorter than 3 ms) among various types of cation-binding sites. As expected from the location of one of the binding sites, cation binding induced conformational alteration of the F-G interhelical loop.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

The purple to blue transition of bacteriorhodopsin is accompanied by a loss of the hexagonal lattice and a conformational change

X-ray diffraction measurements show that in contrast to the purple membrane, the bacteriorhodopsin molecules are not organized in a hexagonal lattice in the deionized blue membrane. Addition of Ca2+ restores both the purple color and the normal (63 A) hexagonal protein lattice. In the blue state, the circular dichroism spectrum in the visible has the typical exciton features indicating that a trimeric structure is retained. Time-resolved linear dichroism measurements show that the blue patch rotates in aqueous suspension with a mean correlation time of 11 ms and provide no evidence for rotational mobility of bacteriorhodopsin within the membrane. The circular dichroism spectra of the blue and the Ca2+-regenerated purple state in the far-UV are different, indicating a small change in secondary structure. The thermal stability of the blue membrane is much smaller than that of the purple membrane. At pH 5.0, the irreversible denaturation transition of the blue form has a midpoint at 61 degrees C. The photocycle of the blue membrane (lambda ex 590 nm) has an L intermediate around 540 nm whose decay is slowed down into the millisecond time range (5 ms). Light-dark adaptation in the blue membrane is rapid with an exponential decay time of 38 s at 25 degrees C. The purple to blue transition apparently involves a conformational change in the protein leading to a change in the aggregation state from a highly ordered and stable hexagonal lattice to a disordered array of thermally more labile trimers.(ABSTRACT TRUNCATED AT 250 WORDS)