Bacteriorhodopsin Molecules Research Articles

Imaging purple membranes in aqueous solutions at sub-nanometer resolution by atomic force microscopy

Purple membranes adsorbed to mica were imaged in buffer solution using the atomic force microscope. The hexagonal diffraction patterns of topographs from the cytoplasmic and the extracellular surface showed a resolution of 0.7 and 1.2 nm, respectively. On the cytoplasmic surface, individual bacteriorhodopsin molecules consistently exhibited a distinct substructure. Depending on the pH value of the buffer solution, the height of the purple membranes decreased from 5.6 nm (pH 10.5) to 5.1 nm (pH 4). The results are discussed with respect to the structure determined by cryo-electron microscopy.

Cooperativity-regulated parallel pathways of the bacteriorhodopsin photocycle

The paper demonstrates that the actinic light density dependence of the millisecond part of the bacteriorhodopsin (BR) photocycle at high pH predicts a model, which is the same in the sequence of the intermediates as concluded previously on the basis of double flash experiments [1992, FEBS Lett. 311, 267–270]. This model consists of the M f→N→BR and M s→BR parallel pathways, the relative yields of which are regulated by cooperative interaction of the BR molecules. The decay of M s is always slower than the decay of M f and described as a direct reprotonation of the Schiff-base from the bulk, and the recovery of the ground-state nearly at the same time. M s is decomposed into M′ f and M′ s. The first does not reprotonate, and similarly to M f, it is suggested to be before the conformational change (switch), which latter process would be just before the decay of M f. A simple way for the determination of the kinetics is also used. This confirms that the amount of N decreases with increasing fraction cycling and shows that the decay rate of N is independent of the fraction cycling. The differences in the kinetics are compared to each other, and they seem to allow a new way of kinetic evaluation at least under special conditions. The aim of this paper was briefly explained in my poster presented on the VIth International Conference on Retinal Proteins (see [14]).

Force-induced conformational change of bacteriorhodopsin

The cytoplasmic surface topography of purple membranes imaged by the atomic force microscope depends mainly on the force applied to the stylus. Imaged at forces of 300 pN, individual bacteriorhodopsin molecules reveal two domains. The resulting donut-shaped trimers reversibly transform into structures exhibiting three prominent protrusions when scanned at 100 pN. In parallel, the height of the protein moiety above the lipid layer increases from 4 A to 6 A. From the known structure of bacteriorhodopsin it appears that this change may be related to a bending of the most prominent cytoplasmic loop.

Photoelectrical Responses of Dried Purple Membrane Film in Blue Form and Acid Purple Form

The modification of photoelectrical property of dried purple membrane (PM) film by acid treatment and deionization method is reported. By acid treatment and deionization, blue and acid purple form of bacteriorhodopsin, which have been obtained only in suspension, are able to be obtained in dried PM films. Using these films, the photoelectrical responses for blue and acid purple form of bacteriorhodopsin in dried films are measured. The disappearance of the normal photocur rent and appearance of a photocurrent with an opposite polarity is observed for blue and acid purple form of dried PM. The photoelectrical property of bR in dried PM is able to be modified by acid treatment and deionization. These treatments are able to control the polarity of bacteriorhodopsin photoresponse without change of the orientation of the bacteriorhodopsin molecules.

Hyper-Rayleigh light scattering from an aqueous suspension of purple membrane

Here we report the first observation of hyper-Rayleigh light scattering from bacteriorhodopsin in the form of an aqueous suspension of unoriented purple membranes. A typical purple membrane suspension used in our experiments contains approximately 10(8) randomly oriented purple membranes. Each purple membrane contains approximately 10(5) bacteriorhodopsin molecules in a two-dimensional crystallinearray. Hyper-Rayleigh light scattering is observed when the purple membrane suspension is illuminated with light that has a wavelength of 1064 nm. We propose that the 532-nm scattered light from each of the bacteriorhodopsin molecules in a single purple membrane is coherent, and that the scattered light from different purple membranes is incoherent. This proposal is supported by the following experimental observations: (a) the 532-nm light intensity is proportional to the square of the incident power, (b) the intensity of the 532-nm signal is linearly proportional to the concentration of purple membrane in solution, (c) the scattered 532-nm light is incoherent, (d) the scattered 532-nm light intensity decreases if the size of the purple membranes is reduced while the bacteriorhodopsin concentration is kept constant, and (e) the 532-nm light is due to the retinal chromophore of the bacteriorhodopsin molecule. The ratio of horizontal polarized hyper-Rayleigh scattered light to vertically polarized hyper-Rayleigh scattered light gives the angle (23 ± 4°) of the retinal axis with respect to the plane of the purple membrane. The hyperpolarizability of the bacteriorhodopsin molecule is found to be 5 ± 0.4 × 10(-27) esu.

Molecular organization and dynamics in bacteriorhodopsin-rich reconstituted membranes: discrimination of lipid environments by the oxygen transport parameter using a pulse ESR spin-labeling technique.

Molecular organization and dynamics in protein-rich membranes have been studied by investigating transport (diffusion-concentration product) of molecular oxygen at various locations in reconstituted membranes of bacteriorhodopsin (BR) and L-alpha-dimyristoylphosphatidylcholine. Oxygen transport was evaluated by monitoring the bimolecular collision of molecular oxygen with four types of nitroxide lipid spin labels placed at various locations in the membrane. The collision rate was estimated from the spin-lattice relaxation times (T1's) measured at various oxygen partial pressures by analyzing the short-pulse saturation recovery ESR signals. CD spectra and decay of polarized flash-induced photodichroism of bacteriorhodopsin indicated that BR molecules are monomers in reconstituted membranes with a lipid/BR molar ratio of 80 (80-rec) and are 25% monomers and 75% trimers plus oligomers of trimers when the lipid/BR ratio is 40 (40-rec). In the 80-rec, the lipid environment is homogeneous on a microsecond scale (T1), probably because the exchange rate of lipids between the bulk and the boundary regions is greater than the T1 relaxation rate (approximately 10(6) s-1). The oxygen collision rate in the hydrophobic region of the 80-rec membrane is smaller by a factor of 1.6 than in that of the lipid membrane without BR, and the effect of BR in decreasing the collision rate is independent of the "depth" in the hydrophobic region. In the 40-rec, two collision rates were observed, one of which is close to those for purple membrane (or the gel-phase membrane), while the other is about the same as was measured in the 80-rec.(ABSTRACT TRUNCATED AT 250 WORDS)

A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin.

Ten mutants of bacteriorhodopsin, each containing a single cysteine residue regularly spaced along helix D and facing the lipid bilayer, were derivatized with a nitroxide spin label. Collision rates of the nitroxide with apolar oxygen increased with distance from the membrane/solution interface. Collision rates with polar metal ion complexes decreased over the same distance. Although the collision rates depend on steric constraints imposed by the local protein structure and on the depth in the membrane, the ratio of the collision rate of oxygen to those of a polar metal ion complex is independent of structural features of the protein. The logarithm of the ratio is a linear function of depth within the membrane. Calibration of this ratio parameter with spin-labeled phospholipids allows localization of the individual nitroxides, and hence the bacteriorhodopsin molecule, relative to the plane of the phosphate groups of the bilayer. The spacing between residues is consistent with the pitch of an alpha-helix. These results provide a general strategy for determining the immersion depth of nitroxides in bilayers.

Direct transmembraneous reconstitution of bacteriorhodopsin into planar phospholipid bilayers

A new method of incorporating bacteriorhodopsin molecules into planar lipid bilayers was developed and compared with a conventional system where purple membranes were adsorbed onto planar bilayers. By the new method, purple membrane sheets were first solubilized with a detergent and the solubilized fraction was directly added to an aqueous phase of a preformed planar bilayer membranes. Then, the solubilized bacteriorhodopsin molecules were spontaneously incorporated into the planar bilayers. Upon illumination, a steady state electric current was generated and the magnitude of the current was hardly affected by the presence of an uncoupler, FCCP. Furthermore, when bacteriorhodopsin molecules were incorporated into planar bilayers in high temperature range, a transient capacitive peak current was mostly eliminated by successive perfusions with a buffer at room temperature. These properties are in contrast with those of conventional method and strongly indicate that the bacteriorhodopsin molecules are transmembraneously reconstituted into planar bilayer membranes by the new method. The new method will prove useful in quantitative investigation of the properties of active ion transport.

Orthorhombic Crystal Form of Bacteriorhodopsin Nucleated on Benzamidine Diffracting to 3·6 Å Resolution

Freshly formed benzamidine crystals were found to provide a suitable nucleation surface for crystallisation of bacteriorhodopsin. At 20°C and 1% octylglucoside pseudohexagonal needles of bacteriorhodopsin nucleated on the benzamidine surface. At 4°C and reduced detergent concentration (0·5%) a new, better-ordered orthorhombic crystal form of bacteriorhodopsin was formed by heterogeneous nucleation on benzamidine. Polarised absorption spectroscopy and flash photolysis experiments were used to show that the crystalline bacteriorhodopsin is photoactive in both forms. The M-intermediate absorbs maximally at 405nm and formation of M does not disturb the crystal lattice. The plate-shaped crystals diffract to a resolution of 3·6 Å along the a and b directions and to 4·2 Å in the c direction. The most likely space group of the crystals in C 222 ( a = 107·5 Å, b = 117·0 Å, c = 69·5 Å). The crystals are built from layers of bacteriorhodopsin molecules that are tilted from the c axis by about 45°. In addition, as a background to the discovery of the new crystal form, the influence of different detergents, additives and precipitants on the formation of pseudohexagonal needles is presented.

Dimeric-like kinetic cooperativity of the bacteriorhodopsin molecules in purple membranes

The kinetics of the absorption changes accompanying the photocycle of bacteriorhodopsin (BR) strongly depend on the intensity of the exciting short laser pulse. The decrease in the flash intensity dependence of the M kinetics after different extents of bleaching of the purple membranes by hydroxylamine proves the existence of a cooperative interaction between the photocycling BR molecules. The yield of the slow component of the M decay (M(s)) is a quadratic function of the extent of the fraction cycling. The slope of the relative weight of M(s) versus the fraction cycling is 0.5. This slope indicates a dimeric-like cooperative interaction, although the structural units of the purple membranes are the trimers of the BR molecules. For the most probable cooperative mechanism an asymmetric trimeric interaction is suggested, which accounts for the apparently dimeric features. A photocycling molecule may influence only one of its two neighbors in the trimer. From this asymmetric feature a deformative interaction is expected to be the cooperative mechanism, which would be an allosteric regulating mechanism in the purple membrane.

Actinic light density dependence of the bacteriorhodopsin protocycle

The photocycle of bacteriorhodopsin (BR) was studied in the 0.3 microsecond to 10 s time interval after excitation, using a wide range of actinic light intensities (10 ns half-duration, 0.06-60 mJ/cm2), at neutral and alkaline pH values. The relative weights of the rapidly and the slowly decaying components of the M intermediate (Mf and M(s), respectively) and the yield of the third millisecond component, N(R,P), are the function of the exciting light intensity (density), while their lifetimes are not. The relative weight of M(s) is found to be a linear function of the portion of the BR molecules undergoing the photocycle. This suggests the existence of a cooperative interaction of the BR molecules arranged in the crystalline purple membrane sheets. Another source of M(s) is also found, which results a nonvanishing relative weight of M(s) even at very weak actinic light density values. The explanation for this may be a branching, or the heterogeneity of BR itself or with its environment. It is shown that the relative weights of the rising and decaying components of the M form(s) do not correlate directly with each other.

Fourier transform infrared analysis of bacteriorhodopsin secondary structure.

Fourier transform infrared spectroscopy at a resolution of 1 cm-1 has been used to study the conformation of dark-adapted bacteriorhodopsin in the native purple membrane, in H2O and D2O suspensions. A detailed analysis of the amide I bands was made using derivative and deconvolution techniques. Curve-fitting results of four independent experiments indicate, after estimation of the methodological errors, that native bacteriorhodopsin contains 52-73% alpha-helices, 13-19% reverse turns, 11-16% beta-sheets, and 3-7% unordered segments. Our analysis has enabled the identification of several components corresponding to alpha-helices, beta-sheets, and reverse turns. Besides the alpha I- and alpha II-helices (peaking at 1658 and 1665 cm-1), we propose that two more infrared bands arise from alpha-helical structures: one at 1650 cm-1 from alpha I and another one at 1642 cm-1 in H2O suspension, which could originate from type III beta-turns (i.e., one turn of 3(10)-helix). The relatively high content of reverse turns suggests the presence of one reverse turn per loop, plus another one in the C-terminal segment. On the other hand, several reasons argue that the calculated mean beta-sheet content of around 14% should be decreased somewhat. These beta-sheets could be located in the noncytoplasmatic links of the bacteriorhodopsin molecule.

Mutated Bacteriortiodopsins: Competitive Materials for Optical Information Processing?

In 1971, the first halobacterial retinal protein was discovered and named bacteriorhodopsin. Bacteriorhodopsin (BR) is a protein of 248 amino acids that is integrated into the bacterial membrane. It is a light driven pump, moving protons from the inside to the outside of the cell. The resulting gradient drives the production of ATP (adenosine triphosphate) for the organism. In the 1970s, rapidly growing research activities focused on the biochemical and biophysical properties of BR and resulted in a linearly increasing number of publications per year. The constant level of about 200 publications/year in the last decade signifies the steadily growing knowledge about the structure and bioenergetic function of BR and related molecules. Nowadays, BR is one of the best-investigated membrane proteins (e.g., References 3–10).Only five years after the unusual chemical and photophysical properties of BR were reported, several articles appeared suggesting technical applications of BR for sunlight energy conversion. Although no commercial applications of BR have resulted from these studies, new ideas for application have arisen, covering about 10% of the total publications on BR during the last two years. Is this an indicator of real potential for BR as a biological material in technology?

THE INWARD H+ PATHWAY IN BACTERIORHODOPSIN: THE ROLE OF M412 AND P(N)560 INTERMEDIATES*

Abstract— In purple bacteriorhodopsin sheets adsorbed onto the phospholipid‐impregnated collodion film, electrogenic stages are identified correlating with decays of the M and N(P)‐type intermediates. It is concluded that both M → N and N → bR transitions are electrogenic.The M decay is shown to be of a complex kinetics. In purple sheets, the lower the light intensity, the higher the rate of “slow M” decay. Such a dependence, which is absent from monomeric bacteriorhodopsin in proteoliposomes and from Triton X‐100‐solubilized protein, may be explained by the inhibiting effect of a light‐induced conformation change in a bacteriorhodopsin molecule upon the M decay in some other bacteriorhodopsin molecules within the same sheet.The light intensity‐independent “slow M” decay in solubilized bacteriorhodopsin is shown to correlate with the decay of the N intermediate and H+ uptake after the flash. In contrast to “fast M”, “slow M” is pH dependent, closely resembling in this respect the N intermediate. It is suggested that there is a fast light‐independent equilibration between M and N so that “slow M” represents the portion of the M pool that monitors the N concentration. The M → N equilibrium is assumed to be involved in the effect of the light‐induced electric field on the M decay. No direct effect of light on the equilibrium was found.

Photoresponse of Langmuir-Blodgett film containing bacteriorhodopsin

The photoresponse of the bacteriorhodopsin (BR) molecule in a purple membrane (PM) fragment LB film was investigated by pulsed laser excitation method. The initial results of laser flash photolysis carried out at room temperature indicated that the intermediate of the photochemical reaction is much the same as that of BR in the aqueous suspension of PM fragments. However the span of the appearance and dissappearance of the intermediates for multilayers is different from that for the aqueous suspension.

Molecular computing for edge-enhanced laser imaging

In order to illustrate the self-assembly capability, we consider a laser imaging experiment on a wet film that is made of bacteriorhodopsin (BR) molecules suspended in a diffusion-limited viscous medium. BR wet film is similar to a wet photograph film but having a finer resolution and adaptive pixel locations due to laser-induced thermal diffusion. The synergism between thermal diffusion of BR molecules (induced externally by a write-laser) and molecular photochromism (generated internally by a read-laser) is exploited naturally for edge-enhanced image applications.

Denaturation of purple membranes at the air/water interface studied by SEM

Fragments of purple membrane were spread at the air/water interface. The interfacial films were transferred onto a silicon wafer with time and initial coverage taken as parameters. The structure of the transferred films was examined with a scanning electron microscope. It was shown that both the size and the packing density of the membrane fragments decreased rapidly with time when the initial coverage was low, and that the intermembrane region was occupied by some substances. An assay by SDS polyacrylamide gel electrophoresis of a built-up film showed that bacteriorhodopsin molecules existed also in the intermembrane region. These results indicated that the denaturation of the purple membrane or the unfolding of bacteriorhodopsin occurred from the periphery of a membrane fragment at the air/water interface.

Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.

The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.

Imaging purple membranes dry and in water with the atomic force microscope

Purple membranes were imaged dry and in water with the atomic force microscope. Slowly air-dried purple membranes showed large cracks on one side and narrow cracks on the other side. Purple membranes that were imaged in water adsorbed to mica showed no cracks. The hexagonal arrangement of the bacteriorhodopsin molecules could be seen in the two-dimensional Fourier transform from images obtained in water. Often spots to the fifth order corresponding to about 1.1 nm resolution were observed.

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.