Leif Rilfors

Lipid Bilayer Stability in Biological Membranes

One of the most important problems in biophysics today is the self-assembly of membrane components, in particular lipids and proteins. It has long been known that many membrane lipids spontaneously form a bilayer when mixed with water (Luzzati, 1968). Recently, it was also indicated that even a membrane protein may form a bilayer structure (Carlsson, 1981). The Singer and Nicolson (1972) model of a biomembrane is based on the assumption that the lipids form a bilayer matrix in which the proteins are incorporated and are able to diffuse more or less freely in two dimensions. If the function of the lipids is only to form this fluid matrix, why does a biological membrane often contain more than 100 different lipid species? Furthermore, many lipids do not spontaneously form bilayers with water. Sometimes not even the major lipid in a membrane forms a bilayer, e.g., monogalactosyldiglyceride of chloroplasts (Shipley et al., 1973; Brentel et al., 1984c). However, the membrane lipids together with the proteins form a stable, functioning membrane, with the properties necessary for a living cell, i.e., to be both a barrier and a communicator to the surroundings. It can thus be expected that the membrane lipids are not working just as a fluid matrix, as is suggested by the Singer and Nicolson model. Most probably they play an important structural role in biological membranes as will be discussed in this chapter.

Hydrophobic molecules in lecithin-water systems. I. Formation of reversed hexagonal phases at high and low water contents

The system dioleoylphosphatidylcholine (DOPC)-n-dodecane-2H2O was investigated with different nuclear magnetic resonance (NMR) techniques: (a) a tentative phase diagram was determined by 2H- and 31P-NMR, (b) translational diffusion coefficients were determined for the three components with the pulsed magnetic field gradient NMR technique, and (c) order parameters for perdeuterated n-dodecane were obtained by 2H-NMR. n-Dodecane induces the formation of reversed hexagonal (HII) phases at low and high water concentrations, and cubic phases at low water contents. The translational diffusion coefficients of n-dodecane in a cubic phase with 6 mol water per mol DOPC, and in an HII phase with 48 mol water per mol DOPC, were just approximately 2.5 times lower than in pure dodecane. Perdeuterated dodecane gave large quadrupole splittings in a lamellar phase, much smaller in an HII phase at low water contents, and a narrow single peak in an HII phase at high water contents. This latter observation indicates that a large fraction of the dodecane molecules is located in separate regions between the water cylinders. Our results support the model given by Gruner concerning the aggregation of membrane lipids in the presence of hydrophobic molecules.

Regulation of lipid composition in biological membranes-biophysical studies of lipids and lipid synthesizing enzymes

The study of the role played by membrane lipids, functional lipidomics, has become increasingly important in membrane biology. The physico-chemical properties of the lipids in biological membranes are subject to some fundamental requirements. In general, the acyl chains shall be in a liquid-like state to keep the membrane proteins active, and the lipids must form a bilayer structure in order for the membrane to be an insulating barrier. However, a potential ability of the lipids to form nonbilayer structures seems to be a prequisite for several membrane-associated cell processes. Therefore, organisms exposed to changes in the environmental conditions, such as temperature and uptake of fatty acids, adjust their membrane lipid composition. Examples of prokaryotic organisms that have been studied in this respect are the cell wall-less bacterium Acholeplasma laidlawii, the Gram-negative bacterium Escherichia coli, and the Gram-positive bacteria Bacillus megaterium and Clostridium butyricum, and among eukaroytic organisms are found fungi, higher plants, and poikilothermic animals. By synthesizing a proper combination of acyl chain and polar head group structures, the organisms modify the phase transition temperatures of the membrane lipids so that they are maintained in a lamellar liquid crystalline phase, and the formation of a lamellar gel phase as well as reserved nonlamellar phases is avoided. It has been shown that A. laidlawii and E. coli maintain a balance between lamellar-formed and nonlamellar-forming lipids. A growing body of evidence shows that nonlamellar-forming membrane lipids play essential roles in many aspects of membrance functioning. Short-lived nonbilayer structures are probably formed in the processes of fusion and fission of lipid bilayers, and long-lived bilayer structures with a small radius of curvature occur in several types of biological membranes (e.g. smooth endoplasmic reticulum, inner mitochondrial membrane, and prolamellar bodies). The activity of membrane-associated proteins can be modulated by adding detergents or nonlamellar-forming lipids to bilayers. Some examples are the regeneration of denatured bacteriorphodopsin, and the activities of protein kinase C, some phospholipases, and some key lipid synthases involved in the lipid metabolism of eukaryotic cells, A. laidlawii, and E. coli. The physico-chemical properties of the lipid matrix can be a direct feed-back signal on the activity of the lipid synthases. Finally, nonlamellar-forming lipids are essential for a proper cell division, and an efficient translocation of proteins across the plasma membrane, of E. coli cells.

Lipid extracts from membranes of Acholeplasma laidlawii A grown with different fatty acids have a nearly constant spontaneous curvature

X-ray diffraction methods were used to explore the variation in the spontaneous curvature of lipid extracts from Acholeplasma laidlawii strain A-EF22 grown with different mixtures of palmitic acid and oleic acid. It was shown that the cells respond to the different growing conditions by altering the polar head group compositions in order to keep the phase transition between lamellar and nonlamellar structures within a narrow temperature range. This has been interpreted to mean that the membrane lipids are adjusted toward an optimal packing (Lindblom et al. (1986) Biochemistry 25, 7502). Here it is shown that for these extracts, the membrane curvature is kept within a narrow range (58-73 A), compared to the range in curvatures exhibited by pure lipids extracts from the membrane (17-123 A). These observations support the hypothesis (Gruner (1989) J. Phys. Chem. 93, 7562) that the spontaneous curvature is a functionally important membrane parameter which is regulated by the organism and is likely to be one of the constraints controlling the lipid composition of the bilayer.

Multicomponent spectra from 31P-NMR studies of the phase equilibria in the system dioleogylphosphatidylcholine-dioleoylphosphatidylthanolamine-water

Abstract The phase equilibria in mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and water were studied by 31P-NMR and 2H-NMR. The chemical shift anisotropy is greater for DOPC than for DOPE (6–9 ppm in the lamellar phase). This difference can most probably be ascribed to different order parameters for the two lipid head groups. 31P-NMR spectra recorded from a lamellar phase formed by DOPC-DOPE-water below maximum hydration exhibit two resolved, superimposed powder spectra. The chemical shift anisotropy for both phospholipids has greater values at excess water contents than below maximum hydration, and the spectral resolution between DOPC and DOPE in the lamellar phase is strikingly diminished at excess water contents. From 31P-NMR spectra it is possible to observe relative differences in composition between different lipid phase existing in equilibrium. The proportion of DOPE is decreased in the lamellar phase, and is increased in the reversed hexagonal phase, when these phases exist in equilibrium.

Interaction of Phosphatidylserine Synthase from E. coli with Lipid Bilayers: Coupled Plasmon-Waveguide Resonance Spectroscopy Studies

The interaction of phosphatidylserine (PS) synthase from Escherichia coli with lipid membranes was studied with a recently developed variant of the surface plasmon resonance technique, referred to as coupled plasmon-waveguide resonance spectroscopy. The features of the new technique are increased sensitivity and spectral resolution, and a unique ability to directly measure the structural anisotropy of lipid and proteolipid films. Solid-supported lipid bilayers with the following compositions were used: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) (80:20, mol/mol); POPC-POPA (60:40, mol/mol); and POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (75:25, mol/mol). Addition of either POPA or POPG to a POPC bilayer causes a considerable increase of both the bilayer thickness and its optical anisotropy. PS synthase exhibits a biphasic interaction with the bilayers. The first phase, occurring at low protein concentrations, involves both electrostatic and hydrophobic interactions, although it is dominated by the latter, and the enzyme causes a local decrease of the ordering of the lipid molecules. The second phase, occurring at high protein concentrations, is predominantly controlled by electrostatic interactions, and results in a cooperative binding of the enzyme to the membrane surface. Addition of the anionic lipids to a POPC bilayer causes a 5- to 15-fold decrease in the protein concentration at which the first binding phase occurs. The results reported herein lend experimental support to a previously suggested mechanism for the regulation of the polar head group composition in E. coli membranes.

Difference in packing properties between iso and anteiso methyl-branched fatty acids as revealed by incorporation into the membrane lipids of Acholeplasma laidlawii strain A

Abstract Acholeplasma laidlawii strain A was grown on the long-chain, branched, saturated fatty acids 12-methyltetradecanoic acid (anteiso-C 15 ), 14-methylhexadecanoic acid (anteiso-C 17 ), 13-methyltetradecanoic acid (iso-C 15 ) and 15-methylhexadecanoic acid (iso-C 17 ), and on the branched-chain fatty acid precursors 2-methylbutanoic acid (anteiso-C 5 ) and 3-methylbutanoic acid (iso-C 5 ). The membrane lipid composition as a function of the fatty acid supplement and the growth temperature was determined: (1) The ratio between the two dominating lipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol, is decreased when anteiso acyl chains replace iso acyl chains in the lipids, and when the acyl chains are elongated from 15 to 17 carbon atoms; (2) the ratio iso / anteiso acyl chains is reduced when the growth temperature is decreased; (3) the average length of the de novo synthesized branched fatty acids is decreased when the growth temperature is lowered, and when anteiso-C 5 is exchanged for iso-C 5 in the growth medium. The lipid regulation mechanisms are interpreted in terms of lipid molecular geometry and self-assembly of lipid molecules. It is concluded that iso and anteiso fatty acids have distinctively different packing properties in a biological membrane, and they mimic straight-chain saturated and cis -monounsaturated fatty acids, respectively, as seen by the way they affect the physicochemical properties of membrane lipids and by the way they are used in lipid regulation mechanisms.

Nonlamellar phases formed by membrane lipids

A brief review of membrane lipids forming cubic and reversed hexagonal phases is presented. An emphasis is made on anionic lipids and particular microbial lipids.

Regulation and Physicochemical Properties of the Polar Lipids in Acholeplasma laidlawii

Although Acholeplasma laidlawii was originally isolated from sewage, its continuous isolation from different mammalian, plant, and insect hosts clearly points to its broad parasitic and pathogenic character (Tully et al., 1990). In addition, A. laidlawii is one of the mycoplasma species commonly isolated as contaminants in eukaryotic cell cultures (McGarrity et al., 1985). Most mycoplasmas, including A. laidlawii, are usually found in close association with the membrane surface of their hosts (Neupert and Sterba, 1983; McGarrity et al., 1985). This association, in combination with the depletion of important metabolites consumed by the mycoplasmas, has deleterious effects for the host cells. Toxic effects, such as that of H2O2 from the metabolism of fermentative mycoplasmas, and less well characterized immunological signals and interactions, are also involved (Razin, 1981; Chowdhury et al., 1990). The close association of the parasites and hosts is perhaps best illustrated by the observed exchanges of membrane protein and lipid constituents between the mycoplasma and the eukaryotic host cell membranes (Powell et al., 1976; Wise et al., 1978; Tarshis et al., 1981). Several mycoplasmas were recently also shown to be able to fuse with a membrane-surrounded animal virus (Citovsky et al., 1988).

Structures of glucolipids from the membrane of Acholeplasma laidlawii strain A-EF22. III. Monoglucosyldiacylglycerol, diglucosyldiacylglycerol, and monoacyldiglucosyldiacylglycerol

The structures of three glucolipids from the membrane of Acholeplasma laidlawii, strain A-EF22, were determined by high resolution 1H-NMR and 13C-NMR spectroscopy. The two most abundant glucolipids in this organism were shown to be 1,2-diacyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerol (MGlcDAG) and 1,2-diacyl-3-O-[alpha-D-glucopyranosyl-(1 --> 2)-O-alpha-D- glucopyranosyl]-sn-glycerol (DGlcDAG). These structures agree with those determined previously by chemical analyses of the two most abundant glucolipids synthesized by the B strain of A. laidlawii. The structure of a newly discovered glucolipid in A. laidlawii strain A-EF22 was also determined. This lipid is an acylated derivative of DGlcDAG with the structure 1,2-diacyl-3-O-[alpha-D-glucopyranosyl-(1 --> 2)-O-(6-O-acyl-alpha-D- glucopyranosyl)]-sn-glycerol. The existence of this lipid was detected by 1H-NMR spectroscopy in preparations of MGlcDAG which had been judged by thin-layer chromatography to be pure. The biosynthesis of the glucolipids and their role in the metabolic lipid regulation are briefly discussed.