Novel pH-Sensitive Microbial Rhodopsin from Sphingomonas paucimobilis.

This work provides the first characteristics of the rhodopsin SpaR from Sphingomonas paucimobilis, aerobic bacteria associated with opportunistic infections. The sequence analysis of SpaR has shown that this protein has unusual DTS motif which has never reported in rhodopsins from Proteobacteria. We report that SpaR operates as an outward proton pump at low pH; however, proton pumping is almost absent at neutral and alkaline pH. The photocycle of this rhodopsin in detergent micelles slows down with an increase in pH because of longer Schiff base reprotonation. Our results show that the novel microbial ion transporter SpaR of interest both as an object for basic research of membrane proteins and as a promising optogenetic tool.

Unusual features of the c-ring of F1FO ATP synthases.

Membrane integral ATP synthases produce adenosine triphosphate, the universal "energy currency" of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it.

Expression and purification of an engineered human endothelin receptor B in a monomeric form.

In humans, two endothelin receptors, ETa and ETb, are activated by three endogenous 21-mer cyclic peptides, ET-1, ET-2, and ET-3, which control various physiological processes, including vasoconstriction, vasodilation, and stimulation of cell proliferation. The first stage of this study it to produce a stable solubilized and purified receptor in a monodisperse state. This article is focused on the engineering, expression, purification, and characterization of the endothelin receptor B for subsequent structural and functional studies.

ESR - a retinal protein with unusual properties from Exiguobacterium sibiricum.

This review covers the properties of a retinal protein (ESR) from the psychrotrophic bacterium Exiguobacterium sibiricum that functions as a light-driven proton pump. The presence of a lysine residue at the position corresponding to intramolecular proton donor for the Schiff base represents a unique structural feature of ESR. We have shown that Lys96 successfully facilitates delivery of protons from the cytoplasmic surface to the Schiff base, thus acting as a proton donor in ESR. Since proton uptake during the photocycle precedes Schiff base reprotonation, we conclude that this residue is initially in the uncharged state and acquires a proton for a short time after Schiff base deprotonation and M intermediate formation. Involvement of Lys as a proton donor distinguishes ESR from the related retinal proteins - bacteriorhodopsin (BR), proteorhodopsin (PR), and xanthorhodopsin (XR), in which the donor function is performed by residues with a carboxyl side chain. Like other eubacterial proton pumps (PR and XR), ESR contains a histidine residue interacting with the proton acceptor Asp85. In contrast to PR, this interaction leads to shift of the acceptor's pKa to more acidic pH, thus providing its ability to function over a wide pH range. The presence of a strong H-bond between Asp85 and His57, the structure of the proton-conducting pathways from cytoplasmic surface to the Schiff base and to extracellular surface, and other properties of ESR were demonstrated by solving its three-dimensional structure, which revealed several differences from known structures of BR and XR. The structure of ESR, its photocycle, and proton transfer reactions are discussed in comparison with homologous retinal proteins.

Study of visual pigment rhodopsin supramolecular organization in photoreceptor membrane by small-angle neutron scattering method with contrast variation.

Supramolecular organization of rhodopsin in the photoreceptor membrane was investigated by small-angle neutron scattering method. The experiments, which were performed with mixtures of heavy/light water as solvent (contrast variation method), were aimed at obtaining information about the lipid and protein components of the photoreceptor disc membrane separately. It was shown that the packaging density of the rhodopsin molecules in the photoreceptor membrane was unusually high: the distance between the centers of the molecules was approximately 56 Å. The probability of the monomeric state of rhodopsin molecules in the photoreceptor membrane, according to the data obtained, is rather high.

Time-resolved microspectroscopy on a single crystal of bacteriorhodopsin reveals lattice-induced differences in the photocycle kinetics.

The determination of the intermediate state structures of the bacteriorhodopsin photocycle has lead to an unprecedented level of understanding of the catalytic process exerted by a membrane protein. However, the crystallographic structures of the intermediate states are only relevant if the working cycle is not impaired by the crystal lattice. Therefore, we applied visible and Fourier transform infrared spectroscopy (FTIR) microspectroscopy with microsecond time resolution to compare the photoreaction of a single bacteriorhodopsin crystal to that of bacteriorhodopsin residing in the native purple membrane. The analysis of the FTIR difference spectra of the resolved intermediate states reveals great similarity in structural changes taking place in the crystal and in PM. However, the kinetics of the photocycle are significantly altered in the three-dimensional crystal as compared to PM. Strikingly, the L state decay is accelerated in the crystal, whereas the M decay is delayed. The physical origin of this deviation and the implications for trapping of intermediate states are discussed. As a methodological advance, time-resolved step-scan FTIR spectroscopy on a single protein crystal is demonstrated for the first time which may be used in the future to gauge the functionality of other crystallized proteins with the molecular resolution of vibrational spectroscopy.

Strength of thermal undulations of phospholipid membranes.

The temperature dependence of intermembrane interactions in freely suspended multilamellar membranes of dimiristoylphosphatidylcholine in D2O was studied using small-angle neutron scattering (SANS) and high-resolution x-ray diffraction (HRXRD). The study reveals that the Helfrich's undulation force is the dominating repulsion force at temperatures above 48.6 degrees C and intermembrane distances larger than 20.5 A. At approximately 77 degrees C the onset of the unbinding transition in the multilamellar membranes is observed. This transition has a continuous behavior in agreement with theoretical predictions and proceeds in accordance with a two-state model. Complimentary analysis of SANS and HRXRD data permits accurate calculation of the fundamental undulation force constant cfl. The obtained value of cfl=0.111+/-0.005 is in good agreement with theoretical calculations. The results of this work demonstrate a key role of Helfrich's undulations in the balance of intermembrane interactions of lipid membranes under physiological temperatures and suggest that thermal undulations play an important part in the interactions of biological membranes. The agreement of the predictions with the experimental data confirms that lipid membranes can be considered as random fluctuating surfaces that can be described well by current theoretical models and that they can serve as a powerful tool for studying behavior of such surfaces.

Lipid membrane structure and interactions in dimethyl sulfoxide/water mixtures.

In this paper we have investigated via x-ray diffraction the influence of dimethyl sulfoxide (DMSO), known for its biological and therapeutic properties, on the structure of lipid membranes of dipalmitoylphosphatidylcholine (DPPC) in excess of the solvent (DMSO/water) at mole DMSO fractions XDMSO in (0.1) and under equilibrium conditions. At small XDMSO

Accuracy of determination of position and width of molecular groups in biological and lipid membranes via neutron diffraction.

Neutron diffraction combined with the deuterium-labelled molecular groups of biological and model membrane components allows one to detect with high accuracy the structure of these objects. Experiments of this kind are only possible at unique high-flux neutron sources, and the planning of neutron-diffraction experiments must take into account some special requirements primarily related to the duration of the experiment and the accuracy of estimation of membrane structure parameters as a result of finite time of the measurements. This paper deals with the question of statistical accuracy of the position x(0) and width v of the distribution of deuterium labels in membranes along the normal of their plane, which are determined in a neutron diffraction experiment. It is shown that the accuracy of x(0) and v estimation does not depend on membrane constitution. It is dependent only on the scattering amplitude of the deuterium label, the label position x(0) and the distribution width v. Analytic calculations show that the statistical errors Deltax(0) and Deltav are inversely proportional to the scattering amplitude of the label and, as usual, to the square root of measurement time. The question of Deltax0 and Deltav dependence on the number of structure factors used in the calculations of x(0) and v is also studied. It is shown that, the accuracy of x(0) estimation is approximately constant with down to four structure factors used, and, with the number of the factors below four, it deteriorates drastically. Analogous is the behaviour of Deltav(h(max)) relation with one exception: abrupt deterioration of the accuracy occurs beginning with five structure factors used. One does not have to measure the highest diffraction reflections which takes a much longer time compared with the first ones. It is an important result. All the problems mentioned above have also been considered for the case of two different deuterium labels in membranes.

Investigation of the structure of thylakoid membranes (spinach) by means of small-angle neutron scattering.

This paper presents experimental data on the determination of the thickness of thylakoid membranes by small-angle neutron scattering. The thylakoids were isolated from spinach chloroplasts. The partial volume of proteins and lipids in the "washed" and "unwashed" membranes was estimated. It is shown that the thickness of thylakoid membranes, measured with this techniques depends on the way the membranes were separated. When isolated thylakoids by the standard method, the membrane thickness amounted to 75 A but if the extracted thylakoids were additionally washed with the isolation medium, the measured thickness was 50 A. In this case a significant decrease in the protein partial volume of the membrane was observed. The obtained results make it possible to explain numerous data on X-ray and small-angle neutron scattering by thylakoid membranes of different origins, proceeding from the assumption that all these membranes have a unified structure and consist of a stable core with a thickness of about 50 A, and layers of peripheral, weakly bound proteins with a thickness which may depends on the nature of the object under investigation and extracting conditions.

Definition of lipid membrane structural parameters from neutronographic experiments with the help of the strip function model.

Neutron diffraction is an effective method for investigating model and biological membranes. Yet, to obtain accurate structural information it is necessary to use deuterium labels and much time is needed to acquire experimental data as there are a large number of diffraction reflections to register. This paper offers a way to define the hydrophobic boundary position in lipid membranes with high accuracy and for this purpose it is sufficient to take into consideration three structural factors. The method is based on modeling the density of the neutron diffraction amplitude rho(x) in the direction of the bilayer plane normal by means of a strip function, but it also takes into consideration the fact that the multiplication of the strip function amplitude rho i by the step width zi-zi-1 makes the sum of neutron scattering amplitudes of the atoms included in the step region. On the basis of the analysis of a large number of experimental data for different membranes, the effectiveness of this method in the determination of the position of hydrophilic/hydrophobic boundary is demonstrated, including the case of various rho(x) modifications in the region of polar heads and also the different phase states of membranes. However, it is shown in the present paper that the strip function model is not an adequate instrument for the determination of other structural parameters of membranes.

A neutron diffraction study of the influence of ions on phospholipid membrane interactions.

Neutron diffraction is used to examine the effects of Ca2+ and ClO4- ions on interactions and some structural features of dipalmitoylphosphatidylcholine membranes in both solid and fluid lamellar phases. The results are described within the framework of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory with reference to electrostatic, van der Waals, and hydration components of disjoining pressure. The Hamaker constants are evaluated under equilibrium conditions. Addition of 100 mM CaCl2 to the aqueous phase substantially increases the lamellar repeat spacing (d), which is interpreted in terms of adsorption of Ca2+ ions to bilayers followed by electrostatic repulsion between membranes. The rise of NaClO4 concentration in the presence of 100 mM CaCl2 leads to gradual decrease in d, evidently resulted from the diminution of Ca(2+)-induced positive surface potential by both electrostatic screening and binding of ClO4- ions. In the absence of CaCl2, elevation of NaClO4 concentration to 100-300 mM drastically enhances the repeat spacing and then dramatically decreases d at about 1 M NaClO4. Estimation of the hydration coefficients showed that the pronounced decrease of the repeat spacing at high NaClO4 concentrations was resulted mainly from the (partial) disruption of the structure of intermembrane bound water by chaotropic ClO4- ions and subsequent decrease in hydration repulsive pressure. Moreover, in the case of solid membranes (20 degrees C) high concentrations of ClO4- induced formation of interdigitated phase paralleled with marked reduction in bilayer thickness and corresponding increase in the effective cross-sectional area per lipid molecule.

Incorporation of phosphonic acid diesters into lipid model membranes. Part II. X-ray and neutron diffraction studies.

Mixtures of egg phosphatidylcholine and phosphonic acid diethyl or dibutyl esters of the general type RP(O)(OR')2 with R = hexane or dodecane were studied at room temperature in the fluid lamellar state by X-ray and by neutron diffraction. Generally a molar ratio of lipid and ester of 1:0.5 was used. Additionally an equimolar lipid/ester mixture of hexane phosphonic acid diethyl ester was studied. Depending on the ester used and its concentration a single L alpha-phase was observed above a certain water content which changes to an L alpha + water two phase system at high water concentration. Despite the large amounts of the amphiphilic ester molecules incorporated in the membrane and their high molecular asymmetry, the mixtures qualitatively show the typical hydration and swelling behaviour of non-charged lipid membranes. However, the incorporation of the esters induces a higher hydration capacity, a lateral extension and a decrease in membrane thickness. The position of the ester molecules and their orientation in the membrane were determined by neutron diffraction using partially deuterated esters. The esters were found to be located with their phosphonic moiety near or in the lipid/water interface. The lamellar structure contradicts this location of the cone-shaped ester molecules which should increase the tendency to form hexagonal structures. However, the experimental findings can be understood if one considers a partial interdigitation of the last hydrocarbon groups of the lipid chains accompanied by a larger disorder in the hydrophobic centre of the membrane. In the case of hexane phosphonic acid dibutyl ester, a vertical translocation of the ester takes place below a certain water content where it is distributed between two locations at the lipid water interface and the centre of the membrane.

Detection of structural defects in phosphatidylcholine membranes by small-angle neutron scattering. The cluster model of a lipid bilayer.

The oriented DPPC multilayers hydrated by D2O have been studied by a small-angle neutron scattering method in the Guinier range, and the gyration radius of the structural inhomogeneities has been estimated at about 29 A. They are interpreted as the annular defects between adjacent clusters uniting the all-trans chain 'segments' adjacent to the polar head group regions. The angle of the 'segment' tilt is determined by the hydrated polar group area (59.2 A2 for DPPC bilayers) and has been estimated to be about 44 degrees under the given experimental conditions. The hydrocarbon interior of a bilayer can be suggested as a 'sandwich' that is formed by two clustered layers (approx. 7 A of the thickness) and the central disordered (liquid) layer. The average cluster size along the bilayer surface is estimated to be approx. 24 A which correlates with the estimations of the short order region dimensions from the halfwidth of the X-ray 'packing' reflex (4.6 A)-1. The average interchain separation of approx. 5 A and the average cross-section area of a chain in a cluster (21.4 A2) were estimated from the reflex position and the chain cross-section geometry. The total volume of defects and the fraction of a bilayer surface occupied by them were estimated too.