Location and orientation of Triclosan in phospholipid model membranes

Triclosan is a hydrophobic antibacterial agent used in dermatological preparations and oral hygiene products. Although the molecular mechanism of action of this molecule has been attributed to inhibition of fatty acid biosynthesis, earlier work in our laboratories strongly suggested that the antibacterial action of Triclosan is mediated at least partly through its membranotropic effects. In order to assess its location in phospholipid membranes, high-resolution magic-angle spinning natural abundance ¹³C NMR of Triclosan embedded within egg yolk lecithin model membranes has been used to obtain ¹³C spin–lattice relaxation times for both Triclosan and lecithin carbon atoms in the presence of Gd³⁺ ions. The results indicate that Triclosan is localized in the upper region of the phospholipid membrane, its hydroxyl group residing in the vicinity of the C=O/C2 carbon atoms of the acyl chain of the phospholipid, and the rest of the Triclosan molecule is probably aligned in a nearly perpendicular orientation with respect to the phospholipid molecule. Intercalation of Triclosan into bacterial cell membranes likely compromises the functional integrity of those membranes, thereby accounting for at least some of this compounds antibacterial effects.Abbreviations COLOC correlation by long-range coupling - EYL egg yolk lecithin - HETCOR heteronuclear chemical-shift correlation - MAS magic-angle spinning - MLV multilamellar vesicles     

The cryoprotectant trehalose destabilises the bilayer organisation of Escherichia coli-derived membrane systems at elevated temperatures as determined by H and P-NMR

In this study, ²H and ³¹P-NMR techniques were used to study the effects of trehalose and glycerol on phase transitions and lipid acyl chain order of membrane systems derived from cells of E. coli unsaturated fatty acid auxotroph strain K1059, which was grown in the presence of [11,11-²H₂]-oleic acid or [11,11-²H₂]-elaidic acid. From an analysis of the temperature dependence of the quadrupolar splitting it could be concluded that neither 1 M trehalose or glycerol generally had any significant effect on the temperature of the lamellar gel to liquid-crystalline phase transition. In the case of the oleate-containing hydrated total lipid extract, glycerol but not trehalose caused a 5°C increase of this transition temperature. In general, both cryoprotectants induced an ordering of the acyl chains in the liquid-crystalline state. Trehalose and glycerol both decrease the bilayer to non-bilayer transition temperature of the hydrated lipid extract of oleate-grown cells by about 5°C, but only trehalose in addition induces an isotropic to hexagonal (H_II) phase transition. In the biological membranes, trehalose and not glycerol destabilised the lipid bilayer, and in the case of the E. coli spheroplasts, part of the induced non-bilayer structures is ascribed to a hexagonal (H_II) phase in analogy with the total lipids. Interestingly, 1 mM Mg²⁺ was a prerequisite for the destabilisation of the lipid bilayer. In the hydrated total lipid extract of E. coli grown on the more ordered elaidic acid, both transition temperatures were shifted about 20°C upwards compared with the oleate-containing lipid, but the effect of trehalose on the lipid phase behaviour was similar. The bilayer destabilising ability of trehalose might have implications for the possible protection of biological systems by (cryo-)protectants during dehydration, in that protection is unlikely to be caused by preventing the occurrence of polymorphic phase transitions.     

Phospholipid matrix as a target for sulfur mustard (HD): NMR study in model membrane systems

Although the interactions of sulfur mustard (HD) with nucleic acids and proteins have been well studied, the toxic interactions with the membrane matrix and specially the phospholipid bilayer have so far been poorly investigated. We have used several NMR techniques to study these interactions: ¹H NMR to observe the localization of HD in membranes of small unilamellar vesicles (SUV) of lecithin; ³¹P NMR to verify the hypothesis of pore formation in membranes of large unilamellar vesicles (LUV); and pseudo solid state ³¹P and ²H NMR to analyze the dynamic consequences of the presence of HD in multilayer dispersions of dimyristoylphosphatidylcholine (DMPC). Immediate and late modifications of the DMPC–HD complexes have been observed at the macroscopic and microscopic levels. After intoxication, HD is spontaneously incorporated into the membrane and locates at the level of the chain methylene groups. This incorporation occurs without formation of pores in the membrane. The presence of HD in the phospholipid dispersion differentially increases the membrane fluidity depending upon the level involved. Weak at the superficial level (phosphate group), this increase is dose-dependent on progression into the membrane. This increase is related to a lowering of transition temperature when measured at the chain level. Macroscopically, HD induces dose- and time-dependent modifications of the DMPC–HD complexes, leading to the formation of an optically transparent gel. This gel formation is confirmed at a microscopic level, where all structures disappear after intoxication. This revised version was published online in July 2006 with corrections to the Cover Date.     

Thermodynamic and structural study of the main phospholipid components comprising the mitochondrial inner membrane

Cardiolipin (CL) is a phospholipid found in the energy-transducing membranes of bacteria and mitochondria and it is thought to be involved in relevant biological processes as apoptosis. In this work, the mixing properties of CL and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) at the air-water interface, have been examined using the thermodynamic framework analysis of compression isotherms. Accordingly, the values of the Gibbs energy of mixing, the more stable monolayers assayed were: POPC:CL (0.6:0.4, mol:mol) and POPE:CL (0.8:0.2, mol:mol). The results reflect that attractive forces are the greatest contributors to the total interaction in these compositions. Supported planar bilayers (SPBs) with such compositions were examined using atomic force microscopy (AFM) at different temperatures. With the POPC:CL mixture, rounded and featureless SPBs were obtained at 4 °C and 24 °C. In contrast, the extension of the POPE:CL mixture revealed the existence of different lipid domains at 24 °C and 37 °C. Three lipid domains coexisted which can be distinguished by measuring the step height difference between the uncovered mica and the bilayer. While the low and intermediate domains were temperature dependent, the high domain was composition dependent. When cytochrome c (cyt c) was injected into the fluid cell, the protein showed a preferential adsorption onto the high domain of the POPC:CL. These results suggest that the high domain is mainly formed by CL.     

Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs

Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest.     

Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins

Phospholipid bilayer Nanodiscs are novel model membranes derived from high-density lipoprotein particles and have proven to be useful in studies of membrane proteins. Membrane protein enzymology has been hampered by the inherent insolubility of membrane proteins in aqueous environments and has necessitated the use of model membranes such as liposomes and detergent-stabilized micelles. Current model membranes display a polydisperse particle size distribution and can suffer from problems of inconsistency and instability. It is also unclear how well they mimic biological lipid bilayers. In contrast, Nanodiscs, the particle size of which is constrained by a coat of scaffold proteins, are relatively monodisperse, stable model membranes with a "nativelike" lipid bilayer. Nanodiscs have already been used to study a variety of membrane proteins, including cytochrome P450s, seven-transmembrane proteins, and bacterial chemoreceptors. These proteins are simultaneously monomerized, solubilized, and incorporated into the well-defined membrane environment provided by Nanodiscs. Nanodiscs may also provide useful insights into the thermodynamics and biophysics of biological membranes and binding of small molecules to membranes.     

Anchoring of a monotopic membrane protein: the binding of prostaglandin H2 synthase-1 to the surface of a phospholipid bilayer

Prostaglandin H₂ synthases (PGHS-1 and -2) are monotopic peripheral membrane proteins that catalyse the synthesis of prostaglandins in the arachidonate cascade. Picot et al. (1994) proposed that the enzyme is anchored to one leaflet of the bilayer by a membrane anchoring domain consisting of a right-handed spiral of amphipathic helices (residues 73–116) forming a planar motif. Two different computational approaches are used to examine the association of the PGHS-1 membrane anchoring domain with a membrane via the proposed mechanism. The electrostatic contribution to the free energy of solvation is obtained by solving numerically the finite-difference Poisson equation for the protein attached to a membrane represented as a planar slab of low dielectric. The nonpolar cavity formation and van der Waals contributions to the solvation free energy are assumed to be proportional to the water accessible surface area. Based on the optimum position determined from the continuum solvent model, two atomic models of the PGHS-1 anchoring domain associated with an explicit dimyristoylphosphatidylcholine (DMPC) bilayer differing by the thickness of the membrane bilayer were constructed. A total of 2 ns molecular dynamics simulation were performed to study the details of lipid- protein interactions at the microscopic level. In the simulations the lipid hydrocarbon chains interacting with the anchoring domain assume various shapes, suggesting that the plasticity of the membrane is significant. The hydrophobic residues in the membrane side of the helices interact with the hydrophobic membrane core, while the positively charged residues interact with the lipid polar headgroups to stabilize the anchoring of the membrane domain to the upper half of the bilayer. The phosphate headgroup of one DMPC molecule disposed at the center of the spiral formed by helices A, B, C and D interacts strongly with Arg120, a residue on helix D that has previously been identified as being important in the activity of PGHS-1. In the full enzyme structure, this position corresponds to the entrance of a long hydrophobic channel leading to the cyclooxygenase active site. These observations provide insights into the association of the arachidonic acid substrate to the cyclooxygenase active site of PGHS-1. Received: 20 December 1999 / Revised version: 26 March 2000 / Accepted: 26 March 2000     

Effects of lipophilic dications on planar bilayer phospholipid membrane and mitochondria

In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199-208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (ΔΨ), the compartment with a lower dication concentration positive. However, the ΔΨ values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a non-Ohmicity was especially strong at ΔΨ > 100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian ΔΨ, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered ΔΨ. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a time-dependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria.     

Catalytic and structural modifications of sarcoplasmic reticulum and plasma membrane (Ca2+ + Mg2+)ATPases induced by organic solutes that accumulate in living systems

Organic solutes such as urea, methylamines, polyols and amino acid can accumulate in the cytoplasm of cells to compensate for hyperosmotic conditions in the external medium. Whereas urea is considered to be typical of solutes that destabilize structure and function of proteins, methylamines, polyols and some amino acids appear to have the opposite effect, and can also compensate for the perturbing effects of urea. These effects have been extensively analyzed for a variety of proteins in terms of global changes in enzyme structure and acceleration or inhibition of overall reaction rates. Here the influence of these solutes on sarcoplasmic reticulum and plasma membrane (Ca²⁺ + Mg²⁺)ATPases is reviewed. The focus is on the changes induced by perturbing and stabilizing solutes at specific steps of the catalytic cycles of these enzymes, which can run forward (leading to ATP hydrolysis) and backward (leading to ATP synthesis). Structural changes promoted by osmolytes are correlated with functional changes, especially those that are related to energy coupling.This review is dedicated to Prof. Carlos Chagas Filho, founder of the Institute of Biophysics, on the occasion of its 50th anniversary.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions.This lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (?120°C to +120°C).Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids.Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H²O and ²H₂O media yielded a value of 1.013 ± 0.026 ml/g for the partial specific volume of this lipid.We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude.Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

Effect of micellar lipids on rabbit intestinal brush-border membrane phospholipid bilayer integrity studied by31P NMR

Summary The effect of biliary salts and fatty acids on the bilayer structure of rabbit intestinal brush-border membranes was studied using the nonperturbing probe³¹P NMR. The broad. asymmetric lineshape of the³¹P NMR spectrum of isolated brush-border vesicles demostrates that their component phospholipids are organized in extended bilayers. These membranes are not significantly perturbed by incubation with physiological concentrations of biliary salts (3, 9, 18mm), demonstrating that the vesicles are highly stable, corresponding to their biological function. However, the emergence of a narrow peak superimposed on the broad lineshape indicates that a small proportion of the membrane phospholipids has reached isotropic motion, which may correspond to external or internal micellar structures. Incubation with mixed micelles of fatty acids and taurochlorate show that long-chain fatty acids enhance the membrane-perturbing effect of taurocholate while short-chain, watersoluble fatty acids do not, suggesting a difference in the absorption mechanisms.     

Poly-l-lysines and poly-l-arginines induce leakage of negatively charged phospholipid vesicles and translocate through the lipid bilayer upon electrostatic binding to the membrane

Poly-l-lysines (PLL) and poly-l-arginines (PLA) of different polymer chain lengths interact strongly with negatively charged phospholipid vesicles mainly due to their different electrical charges. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and their mixtures (1/1 mol/mol) with the respective phosphatidylcholines of equivalent chain length were chosen as model membrane systems that form at room temperature either the fluid L_α or the gel phase L_β lipid bilayer membranes, respectively. Leakage experiments revealed that the fluid POPG membranes are more perturbed compared to the gel phase DPPG membranes upon peptide binding. Furthermore, it was found that pure PG membranes are more prone to release the vesicle contents as a result of pore formation than the lipid mixtures POPG/POPC and DPPG/DPPC. For the longer polymers (≥ 44 amino acids) maximal dye-release was observed when the molar ratio of the concentrations of amino acid residues to charged lipid molecules reached a value of R_P = 0.5, i.e. when the outer membrane layer was theoretically entirely covered by the polymer. At ratios lower or higher than 0.5 leakage dropped significantly. Furthermore, PLL and PLA insertions and/or translocations through lipid membranes were analyzed by using FITC-labeled polymers by monitoring their fluorescence intensity upon membrane binding. Short PLL molecules and PLA molecules of all lengths seemed to translocate through both fluid and gel phase lipid bilayers. Comparison of the PLL and PLA fluorescence assay results showed that PLA interacts stronger with phospholipid membranes compared to PLL. Isothermal titration calorimetry (ITC) measurements were performed to give further insight into these mechanisms and to support the findings obtained by fluorescence assays. Cryo-transmission electron microscopy (cryo-TEM) was used to visualize changes in the vesicles' morphology after addition of the polypeptides.     

Deducing the lateral distribution of proteins in lipid bilayer membranes

We consider four models of the lateral distribution of proteins in lipid bilayer membranes and study the fraction of lipids which are adjacent to at least one protein (adjacent lipids) and how this quantity depends upon protein concentration. The models are (i) hard hexagons free to move from one lattice site to another; (ii) hard disks moving on a continuum; (iii) a mixture of two sizes of nearly-hard disks moving on a continuum; (iv) a modification of (ii). The hexagons or disks represent proteins, while unocupied lattice sites or the remainder of the continuum represents lipids. In (iii) large disks represent proteins and small disks represent lipids. In (iv) some of the continuum between pairs of disks, where packing defects might occur, is not occupied by lipids. We find that an analytical expression for the adjacent lipids (Hoffmann et al. 1981), which is in excellent agreement with the results of the Hexagon model (i), breaks down at a packing density of f _A0.805, and we show by considering the hexagon pair correlation function, that this indicates the onset of random close packing, and that a transition to ordered close packing occurs at f _A=0.866. We thus obtain an operational definition for a random distribution of hexagons: distributions of packing densities0.805. We show that the Disk model (ii) gives results for adjacent lipids that are greater than the Hexagon model and compare these results to the Two Disk model (iii) which gives a result substantially less than the Hexagon model (Mountain et al. 1986). We show that the Modified Disk model (iv) gives results in essential agreement with the Hexagon model except for f _A0.77. Finally we discuss the general appearance of the motion restricted ESR spectrum and conclude that, of these four models, the Modified Disk or the Hexagon models best account for the data. We discuss why this is so with reference to the representation of a 3-dimensional membrane by a 2-dimensional plane.Abbreviations ESR Electron Spin Resonance     

The importance of the phospholipid bilayer and the length of the cholesterol molecule in membrane structure

The properties of mixtures of phosphatidylcholine and analogues of cholesterol bearing side chains of varying lengths were examined by a variety of methods. The incorporation of the analogues into sonicated liposomes and their effect on the rate of osmotic shrinking of multilamellar liposomes were determined. The ordering of a steroid spin label was studied in an oriented multibilayer system and the effect of the analogues on the phase transition of dipalmitoyl phosphatidylcholine monitored using the spin label TEMPO ($\text{2,2,6,6-}\text{tetramethylpiperidine}\text{-N-}\text{oxyl}$). Mixtures of analogues and phospholipid were also studied in monolayers.In all the bilayer systems studied cholesterol caused the greatest ‘rigidifying’ effect, the analogues with shorter or longer side chains being less effective. However, in the monolayer experiments the length of the sterol molecule was found to be much less critical. It is suggested that cholesterol is anchored in position in a phospholipid bilayer by virtue of the molecule being the precise length required to maximise interactions between neighbouring molecules without disturbing the bilayer structure.     

Folding kinetics of an alpha helical membrane protein in phospholipid bilayer vesicles

We report a detailed kinetic study of the folding of an alpha-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane alpha-helical proteins.     

Compatible solutes and their effects on phosphoenolpyruvate carboxylase of C4-halophytes

Abstract Phosphoenolpyruvate carboxylase (PEPCase), extracted from two Poaceae (Cynodon dactylon and Sporobolus pungens) grown on saline soil, was affected physiologically by betaine and proline. Its affinity for phosphoenolpyruvate (PEP) was increased and full protection against NaCl inhibition was observed; enzymic activity was also stabilized when assayed at low PEP levels. Betaine has the same effects on PEPCase extracted from a Chenopodiaceae (Salsola soda), whereas proline behaves as a competitive inhibitor, i.e. it does not protect the enzyme against NaCl and it accelerates inactivation at low PEP levels. Betaine was only compatible with PEPCase extracted from Saisola kali, without any effect on activity, protection or stabilization, but proline was again inhibitory. The levels of free proline in the two salt-stressed Poaceae were high, whereas in the Chenopodiaceae the free proline was low, as in non-stressed plants. The above data indicate that osmoregulators could not only be compatible with cytoplasmic enzymes, but they could either promote or inhibit enzyme activity, depending on the source of enzyme. Coevolution of PEPCase with the osmolyte selected for, could also be inferred.     

Osmotic stress alters the balance between organic and inorganic solutes in flax (Linum usitatissimum)

Flax (Linum usitatissimum) is grown for its oil and its fiber. This crop, cultivated in temperate regions, has seen a renewed interest due to the presence of abundant molecules of interest for many applications. Little information is available about the behavior of flax during osmotic stress; yet this is considered a major stress that causes significant yield losses in most crops. To control the presence of this stress better, flax behavior was investigated following the application of osmotic stress and the response was examined by applying increasing concentrations of PEG 8000. This resulted in the reorganization of 32 metabolites and 6 mineral ions in the leaves. The analysis of these two types of solute highlighted the contrasting behavior between a higher metabolite content (particularly fructose, glucose and proline) and a decrease in mineral ions (especially nitrate and potassium) following PEG treatment. However, this reorganization did not lead to a greater accumulation of solutes, with the total amount remaining unchanged in leaves during osmotic stress.     

The importance of the phospholipid bilayer and the length of the cholesterol molecule in membrane structure.

The properties of mixtures of phosphatidylcholine and analogues of cholesterol bearing side chains of varying lengths were examined by a variety of methods. The incorporation of the analogues into sonicated liposomes and their effect on the rate of osmotic shrinking of multilamellar liposomes were determined. The ordering of a steroid spin label was studied in an oriented multibilayer system and the effect of the analogues on the phase transition of dipalmitoyl phosphatidylcholine monitored using the spin label TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl). Mixtures of analogues and phospholipid were also studied in monolayers. In all the bilayer systems studied cholesterol caused the greatest 'rigidifying' effect, the analogues with shorter or longer side chains being less effective. However, in the monolayer experiments the length of the sterol molecule was found to be much less critical. It is suggested that cholesterol is anchored in position in a phospholipid bilayer by virtue of the molecule being the precise length required to maximise interactions between neighbouring molecules without disturbing the bilayer structure.