Howard L. Brockman

Lipid monolayers: why use half a membrane to characterize protein-membrane interactions?

Variants of membrane-active proteins and peptides are increasingly available through synthesis and molecular engineering. When determining the effects of structural changes upon the interaction of these proteins with lipid membranes, monomolecular films of lipids at the air-water interface have significant advantages over bilayers and other lipid dispersions. In the past year, a variety of protein-lipid interactions has been characterized successfully using relatively simple surface measurements.

Dipole potential of lipid membranes

Of the individual potentials which comprise the potential profile of a membrane, the least well understood is the dipole potential. In general, the dipole potential is manifested between the hydrocarbon interior of the membrane and the first few water layers adjacent to the lipid head groups. Changes in dipole potential caused by spreading a lipid at an air- or oil-water interface can be measured directly and the dipole potential of bilayers can be estimated from the conductances of hydrophobic ions. For a typical phospholipid, like phosphatidylcholine, its measured value is approximately 400 mV in monomolecular films and approximately 280 mV in bilayer membranes, with the hydrocarbon region being positive relative to the aqueous phase. The difference between dipole potentials measured in monolayers and bilayer membranes appears to arise from the use of the lipid-free air- or oil-water interface as the reference point for monolayer measurements and can be corrected for. The species-specific correction term is a lipid concentration-independent potential, the existence of which suggests the ability of lipid headgroups to globally reorganize water structure at the interface. The dipole potential arises from the functional group dipoles of the terminal methyl groups of aliphatic chains, the glycerol-ester region of the lipids and the hydrated polar head groups. Classical methods for obtaining partial dipole moments for each of the three contributing regions are all based on questionable assumptions and give conflicting results. More sophisticated mean-field models of dipole potential origin recognize the important role of interfacial water in determining its value but still cannot adequately describe the microscopic nature of the interactions from which it arises. In part this is because the dipole potential develops in a region over which the dielectric constant of the medium is changing from 2 to 80. Despite of our limited understanding of the dipole potential, it is an important regulator of membrane structure and function. Membrane-membrane and membrane-ligand interactions are regulated by the hydration force, the value of which can be related to the dipole potential of the membrane. For thermotropically phase-separated or multicomponent membranes the size and shape of lipid domains is controlled by the balance between the line tension at the domain borders and the difference in dipole density between the domains. Line tension tends to make the domains compact and circular whereas dipole repulsion promotes transitions to complex domain shapes with larger perimeters.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol.

The effect of cholesterol on the interfacial elastic packing interactions of various molecular species of phosphatidylcholines (PCs) has been investigated by using a Langmuir-type film balance and analyzing the elastic area compressibility moduli (Cs(-1)) as a function of average cross-sectional molecular area. Emphasis was on the high surface pressure regions (pi > or = 30 mN/m) which are thought to mimic biomembrane conditions. Increasing levels of cholesterol generally caused the in-plane elasticity of the mixed monolayers to decrease. Yet, the magnitude of the cholesterol-induced changes was markedly dependent upon PC hydrocarbon structure. Among PC species with a saturated sn-1 chain but different sn-2 chain cis unsaturation levels [e.g., myristate (14:0), oleate (18:1delta9(c), linoleate (18:2delta9,12(c), arachidonate (20:4delta5,8,11,14(c), or docosahexenoate (22:6delta4,7,10,13,16,19(c)], the in-plane elasticity moduli of PC species with higher sn-2 unsaturation levels were less affected by high cholesterol mol fractions (e.g., >30 mol %) than were the more saturated PC species. The largest cholesterol-induced decreases in the in-plane elasticity were observed when both chains of PC were saturated (e.g., di-14:0 PC). When both acyl chains were identically unsaturated, the resulting PCs were 20-25% more elastic in the presence of cholesterol than when their sn-1 chains were long and saturated (e.g., palmitate). The mixing of cholesterol with PC was found to diminish the in-plane elasticity of the films beyond what was predicted from the additive behavior of the individual lipid components apportioned by mole and area fraction. Deviations from additivity were greatest for di-14:0 PC and were least for diarachidonoyl PC and didocosahexenoyl PC. In contrast to Cs(-1) analyses, sterol-induced area condensations were relatively unresponsive to subtle structural differences in the PCs at high surface pressures. Cs(-1) versus average area plots also indicated the presence of cholesterol concentration-dependent, low-pressure (<14 mN/m) phase boundaries that became more prominent as PC acyl chain unsaturation increased. Hence, area condensations measured at low surface pressures often do not accurately portray which lipid structural features are important in the lipid-sterol interactions that occur at high membrane-like surface pressures.

Cholesterol Depletion Results in Site-specific Increases in Epidermal Growth Factor Receptor Phosphorylation due to Membrane Level Effects STUDIES WITH CHOLESTEROL ENANTIOMERS

In A431 cells, depletion of cholesterol with methyl-β-cyclodextrin induced an increase in both basal and epidermal growth factor (EGF)-stimulated EGF receptor phosphorylation. This increase in phosphorylation was site-specific, with significant increases occurring at Tyr845, Tyr992, and Tyr1173, but only minor changes at Tyr1045 and Tyr1068. The elevated level of receptor phosphorylation was associated with an increase in the intrinsic kinase activity of the EGF receptor kinase, possibly as a result of the cyclodextrin-induced enhancement of the phosphorylation of Tyr845, a site in the kinase activation loop known to be phosphorylated by pp60src. Cholesterol and its enantiomer (ent-cholesterol) were used to investigate the molecular basis for the modulation of EGF receptor function by cholesterol. Natural cholesterol (nat-cholesterol) was oxidized substantially more rapidly than ent-cholesterol by cholesterol oxidase, a protein that contains a specific binding site for the sterol. By contrast, the ability of nat- and ent-cholesterol to interact with sphingomyelins and phosphatidylcholine and to induce lipid condensation in a monolayer system was the same. These data suggest that, whereas cholesterol-protein interactions may be sensitive to the absolute configuration of the sterol, sterol-lipid interactions are not. nat- and ent-cholesterol were tested for their ability to physically reconstitute lipid rafts following depletion of cholesterol. nat- and ent-cholesterol reversed to the same extent the enhanced phosphorylation of the EGF receptor that occurred following removal of cholesterol. Furthermore, the enantiomers showed similar abilities to reconstitute lipid rafts in cyclodextrin-treated cells. These data suggest that cholesterol most likely affects EGF receptor function because of its physical effects on membrane properties, not through direct enantioselective interactions with the receptor.

Sphingomyelin Interfacial Behavior: The Impact of Changing Acyl Chain Composition ☆

Sphingomyelins (SMs) containing homogeneous acyl chains with 12, 14, 16, 18, 24, or 26 carbons were synthesized and characterized using an automated Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at constant temperatures between 10 degrees C and 30 degrees C. SM containing lauroyl (12:0) acyl chains displayed only liquid-expanded behavior. Increasing the length of the saturated acyl chain (e.g., 14:0, 16:0, or 18:0) resulted in liquid-expanded to condensed two-dimensional phase transitions at many temperatures in the 10-30 degrees C range. Similar behavior was observed for SMs with lignoceroyl (24:0) or (cerotoyl) 26:0 acyl chains, but isotherms showed only condensed behavior at 10 and 15 degrees C. Insights into the physico-mechanical in-plane interactions occurring within the different SM phases and accompanying changes in SM phase state were provided by analyzing the interfacial area compressibility moduli. At similar surface pressures, SM fluid phases were less compressible than those of phosphatidylcholines with similar chain structures. The area per molecule and compressibility of SM condensed phases depended upon the length of the saturated acyl chain and upon spreading temperature. Spreading of SMs with very long saturated acyl chains at temperatures 30-35 degrees below T(m) resulted in condensed films with lower in-plane compressibilities, but consistently larger cross-sectional molecular areas than the condensed phases achieved by spreading at temperatures only 10-20 degrees below T(m). This behavior is discussed in terms of the enhancement of SM lateral aggregation by temperature reduction, a common approach used during domain isolation from biomembranes.

Molecular cloning and expression of cDNA for rat pancreatic cholesterol esterase.

A full-length cDNA complementary to the rat pancreatic cholesterol esterase mRNA was isolated by screening a rat pancreatic cDNA expression library in lambda gt11 vector with antibodies against the porcine pancreatic cholesterol esterase. The isolated cholesterol esterase cDNA is 2050 bp in length and contains an open reading frame coding for a protein of 612 amino acids. A 20-amino acid hydrophobic leader sequence is predicted, based on the position of the first ATG initiation codon upstream from the sequenced amino terminus of the isolated cholesterol esterase. The cholesterol esterase cDNA was subcloned into a mammalian expression vector, pSVL, for transfection studies. Expression of the cDNA in COS cells resulted in the production of bile salt-stimulated cholesterol esterase. Comparison of the cholesterol esterase cDNA sequence with other proteins revealed that the pancreatic cholesterol esterase is identical to rat pancreatic lysophospholipase. The primary structure of cholesterol esterase displayed no significant homology with other lipases, although the putative lipid interfacial recognition site of G-X-S-X-G is present in the cholesterol esterase sequence. However, the cholesterol esterase sequence revealed a 63-amino-acid domain which is highly homologous to the active site domain of other serine esterases. These data suggest that cholesterol esterase may be a member of the serine esterase supergene family. Analysis of the cholesterol esterase structure also revealed a repetitive sequence enriched with Pro, Asp, Glu, Ser, and Thr residues at the C-terminal end of the protein. This sequence is reminiscent of the PEST-rich sequences in short-lived proteins, suggesting that cholesterol esterase may have a short half-life in vivo. Northern blot hybridization showed that the bile salt-stimulated cholesterol esterase mRNA is present in liver suggesting that this protein may also be synthesized by liver cells.

Interfacial Interactions of Ceramide with Dimyristoylphosphatidylcholine: Impact of the N-Acyl Chain

The mixing behavior of dimyristoylphosphatidylcholine (DMPC) with either N-palmitoyl-sphingosine (C16:0-ceramide) or N-nervonoyl-sphingosine (C24:1-ceramide) was examined using monomolecular films. While DMPC forms highly elastic liquid-expanded monolayers, both neat C16:0-ceramide and C24:1-ceramide yield stable solid condensed monomolecular films with small areas and low interfacial elasticity. Compression isotherms of mixed C16:0-ceramide/DMPC films exhibit an apparent condensation upon increasing X(cer16:0) at all surface pressures. The average area isobars, coupled with the lack of a liquid-expanded to condensed phase transition as X(cer16:0) is increased, are indicative of immiscibility of the lipids at all surface pressures. In contrast, isobars for C24:1-ceramide/DMPC mixtures show surface pressure-dependent apparent condensation or expansion and surface pressure-area isotherms show a composition and surface pressure-dependent phase transition. This suggests miscibility, albeit non-ideal, of C24:1-ceramide and DMPC in both liquid and condensed surface phases. The above could be verified by fluorescence microscopy of the monolayers and measurements of surface potential, which revealed distinctly different domain morphologies and surface potential values for the DMPC/C16:0- and DMPC/C24:1-ceramide monolayers. Taken together, whereas C16:0-ceramide and DMPC form immiscible pseudo-compounds, C24:1-ceramide and DMPC are partially miscible in both the liquid-expanded and condensed phases, and a composition and lateral pressure-dependent two-phase region is evident between the liquid-expanded and condensed regimes. Our results provide novel understanding of the regulation of membrane properties by ceramides and raise the possibility that ceramides with different acyl groups could serve very different functions in cells, relating to their different physicochemical properties.

The role of cholesteryl ester hydrolysis and synthesis in cholesterol transport across rat intestinal mucosal membrane: a new concept.

Abstract The rate of accumulation of cholesteryl ester in rat intestinal sacs is enhanced up to 3-fold by the addition of porcine cholesterol esterase to the uptake medium. For this the enzyme must be catalytically active and does not exert its effect via its activity in the medium. A mechanism for this rate enhancement is proposed in which the action of cholesterol esterases on both sides of a bilayer membrane can catalyze the net transmembrane movement of cholesterol.

Surface dipole moments of lipids at the argon-water interface. Similarities among glycerol-ester-based lipids

Surface potential-surface pressure-area isotherms at the argon-buffer interface have been determined for 38 lipid species comprising 19 chemical classes. These lipids all exhibited a finite range of liquid-expanded surface pressure-area behavior. For most species, the linearity of surface potential with reciprocal area was excellent, but nonzero intercepts were obtained. This suggests a lipid-induced reorganization of interfacial water molecules which is area independent. The linearity of the data permits calculation of the surface dipole moment, mu perpendicular, for each lipid. The values of mu perpendicular for a series of oleoyl-containing acylglycerols, dioleoyl phosphatidylcholine, and dioleoyl phosphatidylethanolamine exhibit acylglycerol ester group mu perpendiculars which are generally consistent with known conformational properties of such lipids. The values are 132 mD for the perpendicular oleoyl glycerol-ester group and 252 mD for that in the kinked-chain conformation. Comparison of mu perpendiculars calculated using these values with homologues confirms the approximate independence of mu perpendicular from aliphatic chain length and permits identification of exceptions with possible conformational or orientational differences. Notably, diphytanoyl phosphatidylcholine shows a 45% larger mu perpendicular than predicted. Differences in mu perpendicular among lipid classes allow estimation of the electrical consequences of lipid metabolism and exchange. Calculations show that reactions such as the generation of 1,2-diacylglycerol from diacyl glycerophosphocholine or diacyl glycerophosphoinositol should produce surface potential changes of -127 and +42 mV, respectively. Thus, the two phospholipids are not simply alternative sources of diacylglycerol with respect to processes dependent on surface potential.

New BODIPY lipid probes for fluorescence studies of membranes

Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8) at the end of C3-, C5-, C7-, or C9-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me4-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me4-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me4-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and ∼506–515 nm) but also showed the absence of the 620–630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me4-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.