Circular dichroism of heterochromophoric and partially regenerated purple membrane: search for exciton coupling

In order to determine the origin of the bisignate CD spectra of native purple membrane, heterochromophoric analogues containing bacteriorhodopsin regenerated with native all-trans-retinal and retinal analogues were investigated. The data collected for the purple membrane samples containing two different chromophores suggest the additive character of the CD spectra. This conclusion was supported by a series of spectra using 5,6-dihydroretinal and 3-dehydroretinal and by using 33% regenerated PM in buffer and in presence of osmolytes. Our results support the idea of conformational heterogeneity of the chromophores in the bR in the trimer, suggesting that the three bR subunits in the trimer are not conformationally equal, and therefore, the bisignate CD spectrum of bR in the purple membrane occurs rather due to a superposition of the CD spectra from variously distorted bR subunits in the trimer than interchromophoric exciton-coupling interactions.     

Circular dichroism study of bacteriorhodopsin-lipid interaction

Delipidated bacteriorhodopsin purified from purple membrane of H. halobium was reconstituted with the circular dichroism active phospholipid. The observed circular dichroism spectra in the 450-700 nm region characteristic of bacteriorhodopsin showed the temperature dependence characterized by a midpoint at ca. 45 degrees C and this spectral change showed the disaggregation of bacteriorhodopsin trimer to monomer. The circular dichroism spectra in the 250-400 nm region characteristic of the azo chromophore of phospholipid exhibited a remarkable temperature dependence synchronized with the disaggregation of bacteriorhodopsin, suggesting that a large proportion of the phospholipid is present as boundary lipid.     

温度对脂质囊泡中单体菌紫质分子结构和光电响应的影响

本实验用人工双分子平板膜系统(BLM)测量了紫膜碎片和在DMPC脂质襄泡膜中的单体菌紫质分子的光电响应以及与温度的关系(处理温度17℃至31℃).温度对紫膜碎片的光电响应影响不大,但对单体菌紫质分子的光电响应有明显影响.用园二色(CD)方法相应地观察了温度对紫膜碎片和单体菌紫质分子在可见波长范围内的CD谱的影响 同样观察到温度对单体菌紫质分子的CD谱有明显影响.两者的影响很可能与脂质襄泡中DMPC的相变温度有关.     

Reorientations in the bacteriorhodopsin photoscycle are pH dependent.

Chromophore reorientations during the bacteriorhodopsin photocycle in the purple membrane of Halobacterium salinarium have been detected by time-resolved linear dichroism measurements of the optical anisotropy over the pH range from 4 to 10 and at ionic strengths from 10 mM to 1 M. The results show that reorientations in the L and M states of bacteriorhodopsin are pH dependent, reaching their largest amplitude when the membrane is at pH 6-8. Reorientations on the millisecond time scale of unexcited spectator proteins in the native purple membrane also depend on pH, consistent with the suggestion that spectator reorientations are triggered by reorientation of the photoexcited protein. The results imply that a group with a PK(a) of 5 to 6 enables reorientations, and that the deprotonation of a site at pH values above 9 restricts reorientational motion. This suggests that reorientations in M may be correlated with proton release.     

Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity.

Specific lipids of the purple membrane of Halobacteria are required for normal bacteriorhodopsin structure, function, and photocycle kinetics [Hendler, R.W. & Dracheva, S. (2001) Biochemistry (Moscow)66, 1623-1627]. The decay of the M-fast intermediate through a path including the O intermediate requires the presence of a hydrophobic environment near four charged aspartic acid residues within the cytoplasmic loop region of the protein (R. W. Hendler & S. Bose, unpublished results). On the basis of the unique ability of squalene, the most hydrophobic purple membrane lipid, to induce recovery of M-fast activity in Triton-treated purple membrane, we proposed that this uncharged lipid modulates an electrostatic repulsion between the membrane surface of the inner trimer space and the nearby charged aspartic acids of the cytoplasmic loop region to promote transmembrane alpha-helical mobility with a concomitant increase in the speed of the photocycle. We examined Triton-treated purple membranes in various stages of reconstitution with native lipid suspensions using infrared spectroscopic techniques. We demonstrate a correlation between the vibrational half-width parameter of the protein alpha-helical amide I mode at 1660 cm-1, reflecting the motional characteristics of the transmembrane helices, and the lipid-induced recovery of native bacteriorhodopsin properties in terms of the visible absorbance maxima of ground state bacteriorhodopsin and the mean decay times of the photocycle M-state intermediates.     

Restricted motion of photoexcited bacteriorhodopsin in purple membrane containing ethanol.

The molecular motion of retinal within the purple membrane was investigated by flash-induced absorption anisotropies with or without ethanol. In the absence of ethanol, the measured anisotropies at several wavelengths exhibited almost the same slow decay. This slow decay was attributed to only the rotation of purple membrane sheet itself in the aqueous suspension. In the presence of ethanol, however, we observed the wavelength-dependent anisotropies. The fluidity of the purple membrane, investigated with a fluorescence anisotropy method, was increased by the addition of ethanol. These facts indicated that the characteristic motion of bacteriorhodopsin is induced in perturbed purple membrane with ethanol. The data analysis was performed, taking account of the overlapping of absorption from ground-state bacteriorhodopsin and photointermediates. The results showed that the rotational motion of photointermediates within the membrane was more restricted than that of nonexcited bacteriorhodopsin. The addition of ethanol facilitated the rotation of nonexcited protein, whereas it did not significantly affect the motion of photointermediates. The restricted motion of photointermediates is probably caused by a conformational change in them, which may hinder the rotation of monomer protein and/or induce the interaction between photointermediate and neighboring proteins.     

Characterization of the binding of valinomycin to bacteriorhodopsin

Circular dichroism spectroscopy has been used to investigate the binding of valinomycin to bacteriorhodopsin in purple membrane suspensions. Addition of valinomycin to purple membrane suspensions obtained from Halobacterium halobium causes the circular dichroism spectrum to shift from an aggregate spectrum to one resembling a monomer spectrum, indicating a loss of chromophore-chromophore interactions. By observing the spectral change upon titration of valinomycin, an apparent dissociation constant of 30–40 M for valinomycin binding was determined. Kinetics of dark adaptation for valinomycin-treated purple membrane are comparable to those for monomeric bacteriorhodopsin. Centrifugation studies demonstrate that valinomycin-treated purple membrane sediments the same as untreated purple membrane suspensions. These results are consistent with a model in which valinomycin binds specifically to bacteriorhodopsin without disrupting the purple membrane fragments.Abbreviations BR bacteriorhodopsin - CD circular dichroism - Tricine N-[tris-(hydroxymethyl) methyl] glycine     

Actinic light-energy dependence of proton release from bacteriorhodopsin

Measuring the light-density (fluence) dependence of proton release from flash excited bacteriorhodopsin with two independent methods we found that the lifetime of proton release increases and the proton pumping activity, defined as a number of protons per number of photocycle, decreases with increasing fluence. An interpretation of these results, based on bending of purple membrane and electrical interaction among the proton release groups of bacteriorhodopsin trimer, is presented.     

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)     

Chromophore reorientations in the early photolysis intermediates of bacteriorhodopsin.

The photoselection-induced time-resolved linear dichroism of a bacteriorhodopsin suspension of purple membrane from 350 to 750 nm is measured by a new pseudo-null measurement technique. In combination with time-resolved absorption measurements, these linear dichroism measurements are used to determine the reorientation of the retinal chromophore of bacteriorhodopsin from 50 ns to 50 microseconds after photolysis. This time range covers the times when the K photointermediate decays to form L, as well as the early times during the formation of the M intermediate in the photocycle. An analysis of the photoselection-induced linear dichroism measured directly, along with the absorbance changes polarized parallel to the linearly polarized excitation, shows that the anisotropy is invariant over this time period, implying that the photolyzed chromophore rotates less than 8 degrees C with respect to unphotolyzed chromophores during this part of the photocycle.     

Transient and linear dichroism studies on bacteriorhodopsin: determination of the orientation of the 568 nm all-trans retinal chromophore

The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways.In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a time-independent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78 ° ± 3 ° is determined.In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of the light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient dichroism method, linear dichroism measurements on oriented samples lead to an angle of 71 ° ± 4 °.No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substantially smaller than the recently suggested value of 20 nm (Ebrey et al., 1977).The orientation of the 568 nm transition dipole moment, which coincides with the direction of the all-trans polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane.     

Electric field effects in bacteriorhodopsin.

Exposure of aqueous suspensions of fragments of the purple membrane of Halobacterium halobium to electric field pulses leads to transient linear dichroism phenomena. The effects are interpreted in terms of field-induced alignments of the bacteriorhodopsin chromophore. Two observed relaxation times (tau) are attributed to rotation of the whole membrane fragments (tau s approximately 100 ms), and to a much faster reorientation of the chromophore within membrane (tau f approximately 260 microns).     

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)     

Biogenesis of the purple membrane of Halobacterium halobium

A protein closely resembling the purple membrane protein pre-exists in the cell membrane of H. halobium prior to the appearance of functional bacteriorhodopsin. It is associated with a differentiated membranous structure which has been isolated on a sucrose gradient and appears to be a precursor of the purple membrane. The identity of the precursor protein as a form of the purple membrane protein was established in different ways: (1) The cell proteins were labelled in vivo with ¹⁴C-proline during dark aerobic growth, the label was chased, and the cells transferred to the illuminated near-anaerobic conditions under which purple membrane is optimally synthesised (induction conditions). Cell lysates were fractionated on sucrose gradients at different times after induction. Label first found in the precursor fraction appeared within 24 h in the purple membrane fraction. (2) SDS-urea-acrylamide gel electrophoresis of the purple membrane protein and the precursor showed only one protein band whose migration coincided with that of the purple membrane band. (3) The amino-acid analysis of the purified precursor was very similar to that of the purple membrane.The absorption spectrum of the precursor showed little of the characteristic absorption of bacteriorhodopsin at 570 nm. A major band appears at 412 nm, the exact nature of which is not known. The difference spectrum (reduced versus oxidised) of a purified fraction showed only traces of cytochrome. Thin-layer chromatography of an acetone-soluble lipid extract indicated the presence of retinal and -carotene. Cells grown in the presence of nicotine did not develop purple membrane after induction: the species absorbing at 412 nm was much less abundant than in non-inhibited cells, but a new fraction was present with a sharp peak at 345 nm consisting mainly of lycopene.Abbreviations CTAB cetyltrimethyl ammonium bromide - SDS sodium dodecyl sulfate - CAP chloramphenicol - TLC thin layer chromatography - CD circular dichroism     

pH dependence of bacteriorhodopsin thermal unfolding

The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.     

Photoreactions of bacteriorhodopsin at acid pH.

It has been known that bacteriorhodopsin, the retinal protein in purple membrane which functions as a light-driven proton pump, undergoes reversible spectroscopic changes at acid pH. The absorption spectra of various bacteriorhodopsin species were estimated from measured spectra of the mixtures that form at low pH, in the presence of sulfate and chloride. The dependency of these on pH and the concentration of Cl- fit a model in which progressive protonation of purple membrane produces "blue membrane", which will bind, with increasing affinity as the pH is lowered, chloride ions to produce "acid purple membrane." Transient spectroscopy with a multichannel analyzer identified the intermediates of the photocycles of these altered pigments, and described their kinetics. Blue membrane produced red-shifted KL-like and L-like products, but no other photointermediates, consistent with earlier suggestions. Unlike others, however, we found that acid purple membrane exhibited a very different photocycle: its first detected intermediate was not like KL in that it was much more red-shifted, and the only other intermediate detectable resembled the O species of the bacteriorhodopsin photocycle. An M-like intermediate, with a deprotonated Schiff base, was not found in either of these photocycles. There are remarkable similarities between the photoreactions of the acid forms of bacteriorhodopsin and the chloride transport system halorhodopsin, where the Schiff base deprotonation seems to be prevented by lack of suitable aspartate residues, rather than by low pH.     

Control of bacteriorhodopsin color by chloride at low pH. Significance for the proton pump mechanism

The chromophore of bacteriorhodopsin undergoes a transition from purple (570 nm absorbance maximum) to blue (605 nm absorbance maximum) at low pH or when the membrane is deionized. The blue form was stable down to pH 0 in sulfuric acid, while 1 M NaCl at pH 0 completely converted the pigment to a purple form absorbing maximally at 565 Other acids were not as effective as sulfuric in maintaining the blue form, and chloride was the best anion for converting blue membrane to purple membrane at low pH. The apparent dissociation constant for Cl- was 35 mM at pH 0, 0.7 M at pH 1 and 1.5 M at pH 2. The pH dependence of apparent Cl- binding could be modeled by assuming two different types of chromophore-linked Cl- binding site, one pH-dependent. Chemical modification of bacteriorhodopsin carboxyl groups (probably Asp-96, -102 and/or -104) by 1-ethyl-3-dimethlyaminopropyl carbodiimide, Lys-41 by dansyl chloride, or surface arginines by cyclohexanedione had no effect on the conversion of blue to purple membrane at pH 1. Fourier transform infrared difference spectroscopy of chloride purple membrane minus acid blue membrane showed the protonation of a carboxyl group (trough at 1392 cm -1 and peak at 1731 cm -1). The latter peak shifted to 1723 cm -1 in D2O. Ultraviolet difference spectroscopy of chloride purple membrane minus acid blue membrane showed ionization of a phenolic group (peak at 243 nm and evidence for a 295 nm peak superimposed on a tryptophan perturbation trough). This suggests the possibility of chloride-induced proton transfer from a tyrosine phenolic group to a carboxylate side-chain. We propose a mechanism for the purple to acid blue to chloride purple transition based on these results and the proton pump model of Braiman et al. (Biochemistry 27 (1988) 8516-8520).     

Imaging the membrane protein bacteriorhodopsin with the atomic force microscope.

The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.     

Effect of fluorescamine modification of purple membranes on exciton coupling and light-to-dark adaptation

Purple membranes of $\text{Halobacterium}\text{,}\text{halobium}$ were modified with fluorescamine. At pH 8.8, with a molar ratio of fluorescamine to bacteriorhodopsin of 170, about 6 residues of lysine were modified while the arginines were not affected at all. Except for the appearance of the fluorescamine peak at 394 nm and some broadening of the chromophore peak at 570 nm, the absorption spectrum of bacteriorhodopsin was not significantly changed after modification. After fluorescamine modification, circular dichroism studies indicated loss of exciton coupling between bacteriorhodopsin molecules in the purple membrane. Rotational diffusion studies suggested enhanced mobility of the chromophore after modification. However, the spectral changes accompanying the light-to-dark adaptation of purple membranes were not prevented by fluorescamine modification. The implications of these findings are that exciton coupling between neighboring bacteriorhodopsin molecules in the purple membrane is not required for light-to-dark adaptation.     

Flash kinetic study of the last steps in the photoinduced reaction cycle of bacteriorhodopsin

The reaction cycle of light adapted bacteriorhodopsin (BR) in aqueous purple membrane suspensions was studied by laser flash photolysis at different temperatures (2–49°C) and pH values (3–10). The activation energy for several reaction steps was determined at pH 7.6. The kinetics of O-bacteriorhodopsin (one of the last intermediates in the cycle) were analyzed in some detail and it was found that the simple consecutive reaction scheme M-BR → O-BR → BR may explain the kinetics of O-bacteriorhodopsin as measured at 680 nm. Since the pH change in neutral aqueous suspensions of purple membrane follows a similar kinetics as O-bacteriorhodopsin it is suggested that protons are released during the reaction M-BR → O-BR and taken up again during the reaction O-BR → BR.Another long-lived intermediate, which absorbs to a greater extent than bacteriorhodopsin at 570 nm and less than bacteriorhodopsin at 420 nm, was identified with the strongly fluorescing species, pseudo- or P-bacteriorhodopsin. The decay of P-bacteriorhodopsin in bacteriorhodopsin had an activation energy of only approx. 1.2 kcal/mol, which suggests that the last step of the photocycle is a relaxation around a single bond.At pH 9–10, the simple first-order kinetics of all the intermediates were changed into a kinetics consisting of two first-order decays. This change of kinetics was accompanied by a drastic decrease in the rotational diffusion relaxation time.To explain the results obtained in this work and those of others, a model involving proton uptake and release by the Schiff base nitrogen combined with an isomerization reaction is finally proposed.