Bacteriorhodopsin Molecules Research Articles

A new approach for structure analysis of two‐dimensional membrane protein crystals using X‐ray powder diffraction data

The application of powder diffraction methods to problems in structural biology is generally regarded as intractable because of the large number of unresolved, overlapping X-ray reflections. Here, we use information about unit cell lattice parameters, space group transformations, and chemical composition as a priori information in a bootstrap process that resolves the ambiguities associated with overlapping reflections. The measured ratios of reflections that can be resolved experimentally are used to refine the position, the shape, and the orientation of low-resolution molecular structures within the unit cell, in leading to the resolution of the overlapping reflections. The molecular model is then made progressively more sophisticated as additional diffraction information is included in the analysis. We apply our method to the recovery of the structure of the bacteriorhodopsin molecule (bR) to a resolution of 7 Å using experimental data obtained from two-dimensional purple membrane crystals. The approach can be used to determine the structure factors directly or to provide reliable low-resolution phase information that can be refined further by the conventional methods of protein crystallography.

Amphiphilic block copolymers significantly influence functions of bacteriorhodopsin in water

This paper investigates the effects of a macromolecular amphiphile poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO–PPO–PEO) on a photoresponsive membrane protein, bacteriorhodopsin (BR). After incubation of BR in EO23–PO65–EO23 (P123) solution, BR maintained its function as a light-driven proton pump; however, the rate of proton uptake and lifetime of the M intermediate in the photocycle of BR upon illumination were, under appropriate conditions, prolonged by about three orders of magnitude compared with that of native BR, even at neutral pH. Measurements using circular dichroism spectroscopy and dynamic light scattering indicated that BR molecules were still in a trimer state after treatment with the copolymers. This is quite different to BR, which showed a much slower photoresponse during drying. The BR–P123 assemblies did not exhibit significantly different photoresponsive behavior with changes to the water content, which implied that in the case of dried BR films or dried BR–polymer films, the elongation of the M decay may be caused not by lack of water molecules necessary for proton transfer, but by protein immobilization. Determination of the critical micelle concentration of P123 with and without BR revealed that this prolongation effect is closely related to the formation of micelles. The above phenomenon was also observed with 6 other Pluronic copolymers. In solutions of small molecular detergents, such as Triton X-100, the photoresponse of BR was prolonged as well; the extent of prolongation was, however, much less than in solutions of macromolecular amphiphiles. The formation of a local polymer coating due to self assembly of the copolymer and protein molecules might be responsible for this very significant prolongation effect, which is beneficial for the potential application of BR as an information material.

Curvature of Purple Membranes Comprising Permanently Wedge-Shaped Bacteriorhodopsin Molecules Is Regulated by Lipid Content

Purple membrane (PM) from Halobacterium salinarum has been studied by many groups and is commonly described as a flat 2-D crystalline membrane microdomain which contains a hexagonal crystalline lattice of bacteriorhodopsin (BR) trimers in a stoichiometric ratio of 10:1 between lipids and BR. BR is the key protein in the halobacterial photosynthetic system and acts as a light-driven proton pump. Upon absorption of a photon, BR undergoes a cyclic series of intramolecular changes, among them a transient "wedge-like" geometrical change of the protein due to a tilt in helix F, one of the seven alpha-helical domains of BR. Due to the strong coupling between the BRs in the crystalline lattice, this may affect membrane topography. In nature, only low light levels occur and the total number of BRs in the "wedge-shaped" state is negligible. For mutated PMs like PM-D85T and PM-D85N (PM-D85X, X = neutral residue), the change of the membrane topography can be triggered in a pH-dependent manner. PMs containing BR-D85X look like "cups" at certain pH values. How does nature deal with a mutated PM like PM-D96G/F171C/F219L (PM-Tri) which comprises permanently "wedge-shaped" BRs and how does this influence membrane assembly? Astonishingly, we observed that PM-Tri is flat. Obviously, the morphology of Halobacterium salinarum is highly conserved and requires flat PMs to be assembled. We found that the lipid content of PM-Tri is specifically altered to assemble a hexagonal crystalline PM-Tri lattice of flat topography.

Layer by layer composite film of dimyristoyl-phosphatidylcoline and bacteriorhodopsin fabricated by multilayer molecular thin film method using fatty acid and lipid

A composite film of dimyristoyl-phosphatidylcoline (DMPC) and bacteriorhodopsin (BR) was fabricated by multilayer molecular thin film method using fatty acid and lipid on a quartz substrate or a hydrogenated amorphous silicon thin film. FTIR reflection absorption spectrums and UV absorption spectrums of the films were characterized on the detail of surface structure of the films. The spectroscopic data exhibited a specific layer by layer interaction of BR and environmental molecule DMPC above fatty acid. Especially, 4 layer composite LB films DMPC and BR exhibited an entirely different feature of IR reflection absorption spectrum depend on fatty acid species.

Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic force microscopy

We have used high-speed atomic force microscopy to study the dynamics of bacteriorhodopsin (bR) molecules at the free interface of the crystalline phase that occurs naturally in purple membrane. Our results reveal temporal fluctuations at the crystal edges arising from the association and dissociation of bR molecules, most predominantly pre-formed trimers. Analysis of the dissociation kinetics yields an estimate of the inter-trimer single-bond energy of -0.9kcal/mol. Rotational motion of individual bound trimers indicates that the inter-trimer bond involves W10-W12 tryptophan residues.

Coulomb blockade effect and negative differential resistance in the electronic transport of bacteriorhodopsin

A functional protein of bacteriorhodopsin has been employed for an approach to develop molecular single-electron transistors. A purple-membrane nanofragment shows a characteristic, cyclic, and reproducible I-V curve having negative differential resistance and an on-off peak-to-valley ratio of 660. The conductance increases exponentially with temperature increase with an activation energy of 47 meV, comparable to the charging energy of a bacteriorhodopsin molecule. This with observed I-V scaling relationship indicates that the Coulomb blockade is the primary conductance-limiting feature and that charges are carried by arrayed Coulomb islands of bacteriorhodopsin.

Optical Characteristics of Polymer Films Based on Bacteriorhodopsin for Irreversible Recording of Optical Information†

Bacteriorhodopsin (BR)-containing polymer films have been developed in which photoinduced transformations of BR molecules take place during the process of photoinduced hydroxylaminolysis (PHA). The routine simplified scheme of the phototransformation is B <--> M(412) in the case of polymer films based on nonmodified BR. In the case of polymer films based on BR modified by hydroxylamine (HA), this scheme is changed to [formula in text] where retinal oxime (RO) is a final product of chemical trapping of retinal by the HA molecule. Under certain conditions, the rate of RO to B regeneration is infinitely low. So, the irreversibility of RO formation allows one to use PHA for the preparation of films for irreversible write once recording. Some optical sensitometric characteristics are compared for polymer-BR films and polymer-BR-HA films. It has been shown that the photosensitivity level for polymer-BR-HA films depends on the time after film preparation. A method is offered to increase the photosensitivity of polymer-BR-HA films and to avoid the photosensitivity decrease with the time after film preparation.

Application of bacteriorhodopsin film for polarization phase-shifting interferometry

Photoinduced anisotropy in bacteriorhodopsin (BR) film is based on photoanisotropic selective bleaching of BR molecules under linearly polarized excitation light. It is modulated by the polarization orientation of the linearly polarized light. The anisotropic information recorded in the BR film is read by a circularly polarized light, which is in turn converted into an elliptical polarized light by the BR film. The rotation angle and the ellipticity of the elliptical polarized light are dependent on the polarization orientation of the linearly polarized excitation light. A phase-shifting interferometer based on the photoinduced anisotropy of BR film is presented theoretically and experimentally. Phase shift is controlled by the polarization orientation of the external excitation light, thus, the phase shift can be controlled without moving parts inside the interferometer, which contributes to the mechanical stability of the system.

Kinetics of photoinduced anisotropy in bacteriorhodopsin film under two pumping beams

Photoinduced anisotropy in bacteriorhodopsin (BR) film arises from the selective bleaching of BR molecules to linearly polarized light. The kinetics of photoinduced anisotropy excited by single and two pumping beams are investigated theoretically and experimentally. Compared with a single pumping beam (650 nm), which produces comparatively small photoinduced anisotropy, dual-wavelength linearly polarized pumping beams (650 and 405 nm) can obviously change the photoinduced anisotropy. When the polarization orientation of the 405 nm pumping beam is perpendicular to that of the 650 nm pumping beam, the peak and steady values of the photoinduced anisotropy kinetic curves are remarkably enhanced. But when the two pumping beams have parallel polarization orientation, the peak and steady values are restrained. At a fixed intensity of the 650 nm pumping beam, there exists an optimal intensity for the 405 nm pumping beam to maximize the value of the photoinduced anisotropy. The photoinduced transmittance of the polarizer-BR-analyzer system is modulated by the polarization angle of the 405 nm pumping beam in an approximate-cosine form.

Optical trigger based on the molecules of bacteriorhodopsin adsorbed on AgBr nanocrystals

AbstractIt is well known that light absorption triggers transformation chain of bacteriorhodopsin (bR). Protein goes through the intermediate states which usually are denoted in alphabetic order: K,L,M,N, and O. These transformations and electric potential changes correlated with them occur in 10 ps. The value of electric potential photoinduced by bR molecules on single membrane is 100 mV. The energy is stored electrostatically in accordance with the mechanisms of molecular condenser appearing under flash action. To obtain high photopotentials (up to 10–12 V) it is necessary to use packs of oriented membranes of bR molecules.We propose to use the molecules of bR adsorbed on silver halide crystals to overcome the threshold of spontaneous nucleation from small silver specks to colloidal ones in very short‐time interval. Earlier we demonstrated the kinetic curves of the chemical development process for ultrafine‐grained emulsion. Colloidal silver particles appeared to lead to effective scattering and absorption of actinic light. Such systems can be considered in the framework of integrated chemical systems. Copyright © 2008 John Wiley &amp; Sons, Ltd.

Real-Time Simulation of Isolated Biomolecule Characterization with Frequency and Force Modulation Atomic Force Microscopy

Recently a new AFM technique, frequency and force modulation AFM (FFM-AFM), was proposed for the characterization of delicate samples and applied within numerical simulations to calculate the cross-sectional scan of a single bacteriorhodopsin molecule with a carbon nanotube probe in air. A real-time imaging simulation of the same molecule and probe is presented here, illustrating the effect of horizontal scan speed, cantilever stiffness, effective frequency setpoint, and quality factor on the images obtained. Controls parameters are proposed for scan speeds of ∼150 and ∼25 nm/s in air and water, respectively. The results indicate that the most common causes leading to distorted images are excessive sample compression and denting by the tip when it approaches the sample horizontally, and loss of contact with the surface as the tip continues traveling horizontally past the sample, both of which can occur for high scan speeds and large cantilever force constants. The calculations also show that imaging conditions can be found for which the tip−sample repulsive forces and sample deformation are small and depend only weakly on imaging parameters other than scan speed.

Single Biomolecule Imaging with Frequency and Force Modulation in Tapping-Mode Atomic Force Microscopy

A new intermittent-contact atomic force microscopy (AFM) mode (frequency and force modulation AFM, FFM-AFM) has been recently proposed to characterize soft samples. This method uses excitation force frequency and amplitude modulation to eliminate bistability and reduce the tip-sample forces. This letter describes theoretical modeling of FFM-AFM applied to a single bacteriorhodopsin molecule on a substrate, showing that its cross section can be measured without damage, in contrast to conventional tapping-mode AFM. Speculations are made regarding nonideal conditions and the ability of FFM-AFM to perform quantitative nanoelasticity measurements.

Influence of the Membrane Potential on the Protonation of Bacteriorhodopsin: Insights from Electrostatic Calculations into the Regulation of Proton Pumping

Proton binding and release are elementary steps for the transfer of protons within proteins, which is a process that is crucial in biochemical catalysis and biological energy transduction. Local electric fields in proteins affect the proton binding energy compared to aqueous solution. In membrane proteins, also the membrane potential affects the local electrostatics and can thus be crucial for protein function. In this paper, we introduce a procedure to calculate the protonation probability of titratable sites of a membrane protein in the presence of a membrane potential. In the framework of continuum electrostatics, we use a modified Poisson-Boltzmann equation to include the influence of the membrane potential. Our method considers that in a transmembrane protein each titratable site is accessible for protons from only one side of the membrane depending on the hydrogen bond pattern of the protein. We show that the protonation of sites receiving their protons from different sides of the membrane is differently influenced by the membrane potential. In addition, the effect of the membrane potential is combined with the effect of the pH gradient resulting from proton pumping. Our method is applied to bacteriorhodopsin, a light-activated proton pump. We find that the proton pumping of this protein might be regulated by Asp115, a conserved residue for which no function has been identified yet. According to our calculations, the interaction of Asp115 with Asp85 leads to the protonation of the latter if the pH gradient or the membrane potential becomes too large. Since Asp85 is the primary proton acceptor in the photocycle, bacteriorhodopsin molecules in which Asp85 is protonated cannot pump protons. Furthermore, we estimate how the membrane potential affects the energetics of the individual proton-transfer reactions of the photocycle. Most reactions, except the initial proton transfer from the Schiff base to Asp85, are influenced. Our calculations give new insights into the mechanism with which bacteriorhodopsin senses the membrane potential and the pH gradient and how the proton pumping is regulated by these parameters.

Molecular Mechanics of Single Molecules

The mechanics of single molecules of bacteriorhodopsin interacting with purple membrane have been investigated from both sides of the membrane by Kessler and Gaub (2006) in this issue of Structure. Remarkably, barriers can be associated with specific amino acid sequences to an accuracy of ±3 amino acids.

Characterizing Molecular Interactions in Different Bacteriorhodopsin Assemblies by Single-molecule Force Spectroscopy

Using single-molecule force spectroscopy we characterized inter- and intramolecular interactions stabilizing structural segments of individual bacteriorhodopsin (BR) molecules assembled into trimers and dimers, and monomers. While the assembly of BR did not vary the location of these structural segments, their intrinsic stability could change up to 70% increasing from monomer to dimer to trimer. Since each stable structural segment established one unfolding barrier, we conclude that the locations of unfolding barriers were determined by intramolecular interactions but that their strengths were strongly influenced by intermolecular interactions. Subtracting the unfolding forces of the BR trimer from that of monomer allowed us to calculate the contribution of inter- and intramolecular interactions to the membrane protein stabilization. Statistical analyses showed that the unfolding pathways of differently assembled BR molecules did not differ in their appearance but in their population. This suggests that in our experiments the membrane protein assembly does not necessarily change the location of unfolding barriers within the protein, but certainly their strengths, and thus alters the probability of a protein to choose certain unfolding pathways.

Lipid composition of integral purple membrane by 1H and 31P NMR

In the purple membrane (PM) of halobacteria, lipids stabilize the trimeric arrangement of bacteriorhodopsin (BR) molecules and mediate the packing of the trimers in a regular crystalline arrangement. To date, the identification and quantification of these lipids has been based either on lipid extraction procedures or structural models. By directly solubilizing PMs from Halobacterium salinarum in aqueous detergent solutions (SDS or Triton X-100), we avoided any separation or modification steps that might modify the lipid composition or even the lipid molecules themselves. Our analysis of integral PM preparations should resolve partially conflicting literature data on the lipid composition of the PM. Using 31P and 1H NMR of detergent-solubilized but otherwise untreated samples, we found two glycolipids and 6.4 +/- 0.1 phospholipids per BR molecule, 4.4 +/- 0.1 of the latter being the phosphatidylglycerophosphate methyl ester. The only glycolipid detected was S-TGD-1. For an additional glycolipid, glycocardiolipin, that was recently identified in lipid extracts, we show that it was produced mainly during the lipid extraction procedure but also was partially dependent on the preparation of the PM suspensions.

Single Molecule Studies of Antibody–Antigen Interaction Strength Versus Intra-molecular Antigen Stability

We investigated molecular recognition of antibodies to membrane-antigens and extraction of the antigens out of membranes at the single molecule level. Using dynamic force microscopy imaging and enzyme immunoassay, binding of anti-sendai antibodies to sendai-epitopes genetically fused into bacteriorhodopsin molecules from purple membranes were detected under physiological conditions. The antibody/antigen interaction strength of 70-170 pN at loading rates of 2-50 nN/second yielded a barrier width of x = 0.12 nm and a kinetic off-rate (corresponding to the barrier height) of k(off) = 6s(-1), respectively. Bacteriorhodopsin unfolding revealed a characteristic intra-molecular force pattern, in which wild-type and sendai-bacteriorhodopsin molecules were clearly distinguishable in their length distributions, originating from the additional 13 amino acid residues epitope in sendai purple membranes. The inter-molecular antibody/antigen unbinding force was significantly lower than the force required to mechanically extract the binding epitope-containing helix pair out of the membrane and unfold it (126 pN compared to 204 pN at the same loading rate), meeting the expectation that inter-molecular unbinding forces are weaker than intra-molecular unfolding forces responsible for stabilizing native conformations of proteins.

Unraveling Bacteriorhodopsin

Unraveling Bacteriorhodopsin

The photochemical conversion in the chemically modified bacteriorhodopsin using polyvinyl alcohol as the protectant

In recent years, it has been shown that chemically modified bacteriorhodopsin (BR) containing large organic cations has a long lifetime of the O state. So it can be used to study the photochemical conversion between the O state and the P state. Chemically modified BR was obtained by adding cations to the deionized blue membrane (dIbR). However, BR molecules were easy to aggregate because the large organic cations were bound to dIbR. In this paper, a water-soluble polymer, polyvinyl alcohol (PVA), was used as a protectant and added to dIbR before the addition of large organic cations. It can prevent the aggregation of the molecules of chemically modified BR. The surface binding of PVA not only affected the stability properties but also played a very important role in controlling the photochemical properties of chemically modified BR.

Probing the Energy Landscape of the Membrane Protein Bacteriorhodopsin

The folding and stability of transmembrane proteins is a fundamental and unsolved biological problem. Here, single bacteriorhodopsin molecules were mechanically unfolded from native purple membranes using atomic force microscopy and force spectroscopy. The energy landscape of individual transmembrane α helices and polypeptide loops was mapped by monitoring the pulling speed dependence of the unfolding forces and applying Monte Carlo simulations. Single helices formed independently stable units stabilized by a single potential barrier. Mechanical unfolding of the helices was triggered by 3.9–7.7 Å extension, while natural unfolding rates were of the order of 10 −3 s −1. Besides acting as individually stable units, helices associated pairwise, establishing a collective potential barrier. The unfolding pathways of individual proteins reflect distinct pulling speed-dependent unfolding routes in their energy landscapes. These observations support the two-stage model of membrane protein folding in which α helices insert into the membrane as stable units and then assemble into the functional protein.