Rotational dynamics of protein and boundary lipid in sarcoplasmic reticulum membrane

We have used spin labels and electron paramagnetic resonance (EPR) to study the correlation between the rotational dynamics of protein and lipid in sarcoplasmic reticulum (SR) membranes. A short-chain maleimide spin label was used to monitor the submillisecond rotational mobility of the Ca-ATPase enzyme (using saturation transfer EPR); a free fatty acid spin label was used to monitor the submicrosecond rotational mobility of the bulk lipid hydrocarbon chains (using conventional EPR); and a fatty acid spin label derivative (long-chain maleimide) attached to the enzyme was used to monitor the mobility of hydrocarbon chains adjacent to the protein (i.e., boundary lipid). In the native SR membranes, the protein was highly mobile (effective correlation time 50 microseconds). The spectra of the hydrocarbon probes both contained at least two components. For the unattached probe, the major component indicated nearly as much mobility as in the absence of protein (effective rotational correlation time 3 ns), while a minor component, corresponding to 25-30% of the total signal, indicated strong immobilization (effective correlation time greater than or equal to 10 ns). For the attached hydrocarbon probe, the major component (approximately 70% of the total) was strongly immobilized, and the mobile component was less mobile than that of the unattached probe. When the lipid-to-protein ratio was reduced 55% by treatment with deoxycholate, protein mobility decreased considerably, suggesting protein aggregation. A concomitant increase was observed in the fraction of immobilized spin labels for both the free and attached hydrocarbon probes. The observed hydrocarbon immobilization probably arises in part from immobilization at the protein-lipid boundary, but protein-protein interactions that trap hydrocarbon chains may also contribute. When protein aggregation was induced by glutaraldehyde crosslinking, submillisecond protein mobility was eliminated, but there was no effect on either hydrocarbon probe. Thus protein aggregation does not necessarily cause hydrocarbon chain immobilization.     

Rotational dynamics of lipid and the Ca-ATPase in sarcoplasmic reticulum. The molecular basis of activation by diethyl ether

We have investigated the role of lipid and protein dynamics in the activation of the Ca2+-dependent ATPase in sarcoplasmic reticulum (SR) by diethyl ether. Conventional and saturation-transfer electron paramagnetic resonance (EPR) were used to probe rotational motions of spin labels attached either to fatty acid hydrocarbon chains or to the Ca-ATPase in SR. We confirm previous studies (Salama, G., and Scarpa, A. (1980) J. Biol. Chem. 255, 6525-6528; Salama, G., and Scarpa, A. (1983) Biochem. Pharmacol. 32, 3465-3477; Kidd, A., Scales, D., and Inesi, G. (1981) Biochem. Biophys. Acta 65, 124-131) reporting that addition of diethyl ether to SR results in an approximately 2-fold enzymatic activation, without loss of coupling. Diethyl ether progressively fluidizes the SR membrane with respect to lipid hydrocarbon chain dynamics probed at several depths in the bilayer. Digital substractions, used to analyze two-component lipid spin label spectra, reveal that a 2-fold mobilization occurs in the population of lipid probes motionally restricted by the protein, while the remaining more mobile population is less affected. The microwave saturation properties of lipid probes also indicate that restricted motions of these probes are mobilized in maximally activated SR membranes. Saturation-transfer EPR, applied to maleimide spin-labeled Ca-ATPase, demonstrates that a 2-fold increase in microsecond rotational motion of the Ca-ATPase correlates with the maximal enzymatic activation. Effects of diethyl ether on both the enzymatic activity and molecular dynamics are completely reversible by dilution with buffer. We propose that ether activates by selectively mobilizing lipid chains adjacent to the enzyme, thus facilitating protein motions that are essential for calcium transport.     

Electron spin resonance analysis of irreversible changes induced by calcium perturbation of erythrocyte membranes.

Introduction of calcium during hemolysis of erythrocytes causes irreversible membrane changes, including protein aggregation. These changes have been investigated by incorporation of one protein and three fatty acid spin label probes into washed membranes from erythrocytes hemolyzed with a range of Ca2+ concentrations. Electron spin resonance spectra of the lipid probes were analyzed for changes in the order parameters, isotropic coupling constants and mean angular deviations of the lipid hydrocarbon chains. The results generally indicated an increased freedom of mobility of the probes with increased Ca2+ concentration during hemolysis, but the response of each probe showed a different concentration dependence. The maximal response was obtained with the I(5, 10) probe. Variations in the responses were interpreted to reflect different modes of protein-lipid or protein-probe interactions arising from Ca2+ -induced membrane protein alterations. Spectra from membranes treated with the protein spin label showed an increased ratio of immobilized to mobile label with increased Ca2+ concentrations at hemolysis. This is consistent with the membrane protein aggregation phenomena previously observed. It is suggested that the increased protein-protein interactions formed as a result of calcium treatment permit an increased lipid mobility in the membrane regions monitored by the fatty acid probes.     

The influence of protein-lipid interactions on the order-disorder conformational transitions of the hydrocarbon chain.

The phases of simple systems involving one type of protein (lysozyme or cytochrome c) and one type of lipid (phosphatidic acid) have been characterized by X-ray crystallography, chemical analysis and spin-labeling technique as a function of temperature. They are of the lamellar type with alternative protein monolayers and lipid bilayers. According to the pH, two types of lamellar phases are obtained, one where the lipid-protein interactions are mainly hydrophobic, the other where they are electrostatic. In both cases, a phase transition occurs as temperature is lowered, between a high temperature phase, where all the lipids are in the liquid-like state, and another phase where some lipid chains are rigid. In the case of the phases with electrostatic interaction, it is shown that the onset of the order-disorder transition is shifted towards low temperature as compared with the homologous lipid-water phase and that the protein content of the phase decreases as the ratio of the liquid to rigid hydrocarbon chains decreases. This leads us to suggest that in the systems studied in this work the proteins interact only with lipid in the liquid-like state. In the case of the phases with hydrophobic interaction, it is shown that the extent of hydrophobic interaction between protein and lipid increases as the unsaturation of the hydrocarbon chains increases. The onset of the order-disorder transition shows a greater shift towards low temperature than the one observed in the case of the phase with electrostatic interaction.     

The influence of protein-lipid interactions on the order-disorder conformational transitions of the hydrocarbon chain

The phases of simple systems involving one type of protein (lysozyme or cytochrome $\text{c}$) and one type of lipid (phosphatidic acid) have been characterized by X-ray crystallography, chemical analysis and spin-labeling technique as a function of temperature. They are of the lamellar type with alternative protein monolayers and lipid bilayers. According to the pH, two types of lamellar phases are obtained, one where the lipid-protein interactions are mainly hydrophobic, the other where they are electrostatic. In both cases, a phase transition occurs as temperature is lowered, between a high temperature phase, where all the lipids are in the liquid-like state, and another phase where some lipid chains are rigid. In the case of the phases with electrostatic interaction, it is shown that the onset of the order-disorder transition is shifted towards low temperature as compared with the homologous lipid-water phase and that the protein content of the phase decreases as the ratio of the liquid to rigid hydrocarbon chains decreases. This leads us to suggest that in the systems studied in this work the proteins interact only with lipid in the liquid-like state. In the case of the phases with hydrophobic interaction, it is shown that the extent of hydrophobic interaction between protein and lipid increases as the unsaturation of the hydrocarbon chains increases. The onset of the order-disorder transition shows a greater shift towards low temperture than the one observed in the case of the phase with electrostatic interaction.     

Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.     

Relay model of lipid peroxidation in biological membranes

The present study examines the evidence for the important role of free radicals, localized on carbon atoms of the hydrocarbon chains, in lipid peroxidation. These radicals show a great inter- and intramolecular mobility in membranes by the way of relay-transfer (isomerisation). The sequence of intermediate steps of shift of free radicals in membranes with correction for molecular organization of the hydrocarbon zone of membranes, the intramembrane localization of unsaturated links and the gradient of mobility of the hydrocarbon chains are described. The effect of inhibitors in lipid peroxidation are interpreted in terms of decay of hydrocarbon free radicals as a result of its interaction with the antioxidant molecules. The natural antioxidants having a side chain (such as tocopherols) may be regarded as a some kind of "channel" through which free radicals leave the hydrocarbon moiety of the membrane. The processes of lipid peroxidation in membranes are subjected to a great extent to the requirements of the theory of oxidation of solid polymers and hydrocarbons.     

The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity

Lipid composition and Ca(2+)-ATPase activity both change with age and disease in many tissues. We explored relationships between lipid composition/structure and plasma membrane Ca(2+)-ATPase (PMCA) activity. PMCA was purified from human erythrocytes and was reconstituted into liposomes prepared from human ocular lens membrane lipids and synthetic lipids. Lens lipids were used in this study as a model for naturally ordered lipids, but the influence of lens lipids on PMCA function is especially relevant to the lens since calcium homeostasis is vital to lens clarity. Compared to fiber cell lipids, epithelial lipids exhibited an ordered to disordered phase transition temperature that was 12 degrees C lower. Reconstitution of PMCA into lipids was essential for maximal activity. PMCA activity was two to three times higher when the surrounding phosphatidylcholine molecules contained acyl chains that were ordered (stiff) compared to disordered (fluid) acyl chains. In a completely ordered lipid hydrocarbon chain environment, PMCA associates more strongly with the acidic lipid phosphatidylserine in comparison to phosphatidylcholine. PMCA associates much more strongly with phosphatidylcholine containing disordered hydrocarbon chains than ordered hydrocarbon chains. PMCA activity is influenced by membrane lipid composition and structure. The naturally high degree of lipid order in plasma membranes such as those found in the human lens may serve to support PMCA activity. The absence of PMCA activity in the cortical region of human lenses is apparently not due to a different lipid environment. Changes in lipid composition such as those observed with age or disease could potentially influence PMCA function.     

Lipid fluidity directly modulates the overall protein rotational mobility of the Ca-ATPase in sarcoplasmic reticulum

We have developed a quantitative and relatively model-independent measure of lipid fluidity using EPR and have applied this method to compare the temperature dependence of lipid hydrocarbon chain fluidity, overall protein rotational mobility, and the calcium-dependent enzymatic activity of the Ca-ATPase in sarcoplasmic reticulum. We define membrane lipid fluidity to be T/eta, where eta is the viscosity of a long chain hydrocarbon reference solvent in which a fatty acid spin label gives the same EPR spectrum (quantitated by the order parameter S) as observed for the same probe in the membrane. This measure is independent of the reference solvent used as long as the spectral line shapes in the membrane and the solvent match precisely, indicating that the same type of anisotropic probe motion occurs in the two systems. We argue that this empirical measurement of fluidity, defined in analogy to the macroscopic fluidity (T/eta) of a bulk solvent, should be more directly related to protein rotational mobility (and thus to protein function) than are more conventional measures of fluidity, such as the rate or amplitude of rotational motion of the lipid hydrocarbon chains themselves. This new definition thus offers a fluidity measure that is more directly relevant to the protein's behavior. The direct relationship between this measure of membrane fluidity and protein rotational mobility is supported by measurements in sarcoplasmic reticulum. The overall rotational motion of the spin-labeled Ca-ATPase protein was measured by saturation-transfer EPR. The Arrhenius activation energy for protein rotational mobility (11-12 kcal/mol/degree) agrees well with the activation energy for lipid fluidity, if defined as in this study, but not if more conventional definitions of lipid fluidity are used. This agreement, which extends over the entire temperature range from 0 to 40 degrees C, suggests that protein mobility depends directly on lipid fluidity in this system, as predicted from hydrodynamic theory. The same activation energy is observed for the calcium-dependent ATPase activity under physiological conditions, suggesting that protein rotational mobility (dependent on lipid fluidity) is involved in the rate-limiting step of active calcium transport.     

The crystal structure of cholesteryl dodecanoate: co-packing of steroid skeleta and hydrocarbon chains

The crystal structure of cholesteryl dodecanoate has been determined. The compound shows a co-packing of cholesterol skeleta and hydrocarbon chains. There are two molecules in the asymmetric unit both almost fully extended. The hydrocarbon chain axes are however somewhat bent in order to get a good close-packing side by side with the rigid cholesterol skeleta. The two non-symmetry related skeleta show different packing surroundings. One skeleton packs with both hydrocarbon chains and other skeleta while the other skeleton is completely surrounded by hydrocarbon chains. The latter packing is of particular interest as it is considered to indicate important packing principles in biological lipid bilayers.     

The role of the lipid matrix for structure and function of the GPCR rhodopsin

Photoactivation of rhodopsin in lipid bilayers results within milliseconds in a metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium that is very sensitive to the lipid composition. It has been well established that lipid bilayers that are under negative curvature elastic stress from incorporation of lipids like phosphatidylethanolamines (PE) favor formation of MII, the rhodopsin photointermediate that is capable of activating G protein. Furthermore, formation of the MII state is favored by negatively charged lipids like phosphatidylserine and by lipids with longer hydrocarbon chains that yield bilayers with larger membrane hydrophobic thickness. Cholesterol and rhodopsin-rhodopsin interactions from crowding of rhodopsin molecules in lipid bilayers shift the MI-MII equilibrium towards MI. A variety of mechanisms seems to be responsible for the large, lipid-induced shifts between MI and MII: adjustment of the thickness of lipid bilayers to rhodopsin and adjustment of rhodopsin helicity to the thickness of bilayers, curvature elastic deformations in the lipid matrix surrounding the protein, direct interactions of PE headgroups and polyunsaturated hydrocarbon chains with rhodopsin, and direct or lipid-mediated interactions between rhodopsin molecules. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Membrane thickness and acyl chain length

It appears reasonable to expect that the primary result of a change in the length of the acyl chains within a lipid bilayer is a similar change in the bilayer thickness. In the present communication we draw attention to the somewhat more complicated effects which are found experimentally for phosphatidylcholine bilayers as the hydrocarbon chain is varied from twelve to eighteen carbons in length. The major change in dimension which occurs with variation in acyl chain length is the area occupied per molecule rather than the bilayer thickness. The same effect is seen with solute hydrocarbon such as hexane which partition into the membrane and cause only a small variation in membrane thickness but a large increase in the molecular area of the lipid. The origin of this effect arises from the almost isotropic distribution of the additional hydrocarbon to the lipid core of the membrane.     

Constant normal pressure, constant surface tension, and constant temperature molecular dynamics simulation of hydrated 1,2-dilignoceroylphosphatidylcholine monolayer

A constant normal pressure, constant surface tension, and constant temperature (NP(N)gammaT) molecular dynamics (MD) simulation of the liquid condensed phase of a 1,2-dilignoceroylphosphatidylcholine (DLGPC) monolayer has been performed at 293.15 K. A DLGPC molecule has two saturated 24-carbon acyl chains, giving the hydrocarbon core thickness of the monolayer approximately 28 A, which is close to the hydrocarbon core thickness of a membrane of a living system. NP(N)gammaT ensemble was used to reproduce the experimental observations, such as area/lipid, because surface tension is an essential factor in determining the monolayer structure. Data analysis on DLGPC/water monolayer shows that various liquid condensed-phase properties of the monolayer have been well reproduced from the simulation, indicating that surface tension 22.9 mN/M used in the simulation is an appropriate condition for the condensed-phase NP(N)gammaT simulation. The simulation results suggest that this long-chain phospholipid monolayer shares many structural characteristics with typical short-chain 1,2-diacylphosphatidylcholine systems, such as DPPC/water monolayer in the condensed phase and DPPC/water bilayer in the gel phase. Furthermore, it was found that DLGPC/water monolayer has almost completely rotationally disordered acyl chains, which have not been observed so far in short-chain 1,2-diacylphosphatidylcholine/water bilayers. This study indicates the good biological relevance of the DLGPC/water monolayer which might be useful in protein/lipid studies to reveal protein structure and protein/lipid interactions in a membrane environment.     

Membrane effects of lysozyme amyloid fibrils

The influence of mature lysozyme fibrils on the structural and physical properties of model membranes composed of phosphatidylcholine (PC) and its mixtures with cardiolipin (CL) (10 mol%) and cholesterol (Chol) (30 mol%) was studied using fluorescent probes DPH, pyrene, Laurdan and MBA. Analysis of pyrene fluorescence spectra along with the measurements of DPH fluorescence anisotropy revealed that the structure of hydrocarbon chains region of lipid bilayer is not affected by the fibrillar aggregates of lysozyme. In contrast, probing the membrane effects by Laurdan and MBA showed the rise of both the generalized polarization of Laurdan and the MBA fluorescence anisotropy, suggesting that amyloid protein induces reduction of bilayer hydration and increase of lipid packing in the interfacial region of model membranes.     

Thermodynamics of glycophorin in phospholipid bilayer membranes

We have developed a model of glycophorin in a phospholipid bilayer membrane in order to study the thermodynamics of this system and to understand the detailed behavior of recent calorimetric data. We assume that the larger glycophorin polar group can be considered as either adopting a pancakelike conformation at the bilayer interface (D state) or be directed generally away from the interface (U state) [Ruppel, D., Kapitza, H.G., Galla, H.J., Sixl, F., & Sackmann, E. (1982) Biochim. Biophys. Acta 692, 1-17]. Lipid hydrocarbon chains are described either as excited (e state) with high energy and relatively many gauche conformers or as generally extended (g state) with low energy. We performed a Monte-Carlo simulation using the Glauber and Kawasaki procedures on a triangular lattice which represents the plane of half of the bilayer. Lattice sites can be occupied either by lipid hydrocarbon chains or by model glycophorin alpha-helical hydrophobic cores. The states D and U are represented by hexagons of different sizes in the plane of the lattice, and the hard core repulsion between two such polar groups is accounted for by forbidding hexagon-hexagon overlap. We have studied the effect of having the glycophorin polar group interact in various ways with the lipid bilayer. We find that the protein polar group in its D state interacts, either directly or indirectly, with the lipid bilayer so as to reduce the effective lateral pressure acting on the lipid hydrocarbon chains by about 3 dyn/cm. Polar groups in their U states do not reduce this lateral pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of N-(1-methyldodecyl)-N,N-dimethylaminoxide on the conformation of hydrocarbon chains in phospholipid bilayers isolated from Escherichia coli

Using the method of ESR spectroscopy of stearic acid spin probes labeled by the doxyl group on the 12th or 16th carbon, it has been found that bactericidal surfactant N-(1-methyldodecyl)-N,N-dimethylamine oxide increases the effective energy difference between trans- and gauche conformers Eg and decreases the probability of gauche conformers formation pg in lipid hydrocarbon chains in multilamellar liposomes prepared from Escherichia coli-isolated phospholipids, at low surfactant concentrations. Above the surfactant: phospholipid molar ratio of 1:14 to 1:17, the value of Eg decreases and that of pg increases. The results are interpreted using the cluster model of lipid bilayer. At low concentrations the surfactant molecules are inserted into the dynamical defects between the clusters, thereby increasing the packing density of chains in the bilayer. At high concentrations the surfactant molecules penetrate into the clusters perturbing the dense packing of chains in clusters.     

Stability and phase separation in mixed monopolar lipid/bolalipid layers

The phase stability of a fluid lipid layer that is a mixture of conventional monopolar lipids and C20 bipolar bolalipids was studied using a mean field theory that explicitly includes molecular details and configurational properties of the lipid molecules. The effect of changing the fraction of bolalipids, as well as the length of the hydrocarbon chain of the monopolar lipids, was probed. A phase separation between two liquid lipid phases was found when a mismatch exists in the optimal hydrophobic thicknesses of the pure bolalipid and monopolar lipid layers. The lipid mixture phase separates into a thin bolalipid-rich layer and a thicker monopolar-rich layer. The thin membrane phase is mainly composed of transmembrane bolalipid molecules whose polar heads are positioned at opposite membrane-water interfaces. In the monopolar lipid-rich phase, bolalipids are the minor component and most of them assume a looping configuration where both headgroups are present at the same membrane-water interface. For mixed layers that form a single lipid phase across all bolalipid concentrations, the hairpin-transmembrane ratio strongly depends on the hydrocarbon chain length of the monopolar lipid and the bolalipid concentration. The C-D bond order parameters of the different species have been calculated. Our findings suggest that the concentration-dependent phase transition should be experimentally observable by measuring of the order parameters through quadrupolar splitting experiments. The driving force for the phase separation in the monopolar lipid/bolalipid mixture is the packing mismatch between hydrophobic regions of the monopolar lipid hydrocarbon chains and the membrane-spanning bolalipid chains. The results from the molecular theory may be useful in the design of stable lipid layers for integral membrane protein sensing.     

Temperature dependence of rotational dynamics of protein and lipid in sarcoplasmic reticulum membranes

We have investigated the relationship between function and molecular dynamics of both the lipid and the Ca-ATPase protein in sarcoplasmic reticulum (SR), using temperature as a means of altering both activity and rotational dynamics. Conventional and saturation-transfer electron paramagnetic resonance (EPR) was used to probe rotational motions of spin-labels attached either to fatty acid hydrocarbon chains or to the Ca-ATPase sulfhydryl groups in SR. EPR studies were also performed on aqueous dispersions of extracted SR lipids, in order to study intrinsic lipid properties independent of the protein. While an Arrhenius plot of the Ca-ATPase activity exhibits a clear change in slope at 20 degrees C, Arrhenius plots of lipid hydrocarbon chain mobility are linear, indicating that an abrupt thermotropic change in the lipid hydrocarbon phase is not responsible for the Arrhenius break in enzymatic activity. The presence of protein was found to decrease the average hydrocarbon chain mobility, but linear Arrhenius plots were observed both in the intact SR and in extracted lipids. Lipid EPR spectra were analyzed by procedures that prevent the production of artifactual breaks in the Arrhenius plots. Similarly, using sample preparations and spectral analysis methods that minimize the temperature-dependent contribution of local probe mobility to the spectra of spin-labeled Ca-ATPase, we find that Arrhenius plots of overall protein rotational mobility also exhibit no change in slope. The activation energy for protein mobility is the same as that of ATPase activity above 20 degrees C; we discuss the possibility that overall protein mobility may be essential to the rate-limiting step above 20 degrees C.