A calorimetric examination of the effect of myotoxin a on the thermotropic phase behavior of model lipid membranes

The effect of myotoxin a on the thermotropic phase behavior of aqueous dispersions of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) was examined using differential scanning calorimetry (DSC). Myotoxin a significantly altered the normal phase behavior of DMPC in a concentration dependent fashion. This effect is perturbed by Ca2+ and is sensitive to ionic strength and pH. High concentrations of toxin eliminate the characteristic pretransition associated with the polar head group of DMPC. They also increase the temperature of the main gel-to-liquid crystal transition from 23 degrees C to 32-35 degrees C. At low concentrations of toxin, the first visible effect is upon the pretransition which is split into two components that diminish with time. The main transition is less affected at low toxin concentrations, although the magnitude of the transition is reduced while it is simultaneously shifted to higher temperatures. The main transition is also split into multiple components. The toxin also had pH specific effects on the phase behavior of DMPS. Above physiological pH (8.5) the normal transition of DMPS at 36-38 degrees C was split in the presence of myotoxin a and new components appeared centered at 31 degrees C and 35 degrees C. These observations are consistent with reports that the skeletal muscle membrane system is the major site of the myonecrotic effect of myotoxin a.     

Influence of hydrophobic mismatch on the catalytic activity of Escherichia coli GlpG rhomboid protease

Rhomboids comprise a broad family of intramembrane serine proteases that are found in a wide range of organisms and participate in a diverse array of biological processes. High-resolution structures of the catalytic transmembrane domain of the Escherichia coli GlpG rhomboid have provided numerous insights that help explain how hydrolytic cleavage can be achieved below the membrane surface. Key to this are observations that GlpG hydrophobic domain dimensions may not be sufficient to completely span the native lipid bilayer. This formed the basis for a model where hydrophobic mismatch Induces thinning of the local membrane environment to promote access to transmembrane substrates. However, hydrophobic mismatch also has the potential to alter the functional properties of the rhomboid, a possibility we explore in the current work. For this purpose, we purified the catalytic transmembrane domain of GlpG into phosphocholine or maltoside detergent micelles of varying alkyl chain lengths, and assessed proteolytic function with a model water-soluble substrate. Catalytic turnover numbers were found to depend on detergent alkyl chain length, with saturated chains containing 10–12 carbon atoms supporting maximal activity. Similar results were obtained in phospholipid bicelles, with no proteolytic activity being detected in longer-chain lipids. Although differences in thermal stability and GlpG oligomerization could not explain these activity differences, circular dichroism spectra suggest that mismatch gives rise to a small change in structure. Overall, these results demonstrate that hydrophobic mismatch can exert an inhibitory effect on rhomboid activity, with the potential for changes in local membrane environment to regulate activity in vivo.     

Effects of alpha-tocopherol (vitamin E) on the stability and lipid dynamics of model membranes mimicking the lipid composition of plant chloroplast membranes

Tocopherol (vitamin E) is widely recognized as a cellular antioxidant. It is essential for human and animal health, but only synthesized in photosynthetic organisms, where it is localized in chloroplast membranes. While many studies have investigated non-antioxidative effects of tocopherol on phospholipid membranes, nothing is known about its effects on membranes containing chloroplast glycolipids. Here, liposomes resembling plant chloroplast membranes were used to investigate the effects of alpha-tocopherol on vesicle stability during freezing and on lipid dynamics. alpha-Tocopherol had a pronounced influence on membrane dynamics and showed strong interactions in its effects on membrane stability during freezing with the cryoprotectant sucrose. alpha-Tocopherol showed maximal effects at low concentrations (around 2mol%), close to its contents in chloroplast membranes.     

The effect of growth rate and other growth conditions on the lipid composition of Escherichia coli

Escherichia coli was grown as a continuous culture at various defined conditions of temperature, pH, aeration rate and dilution rate. The lipids were extracted from disrupted cells and the relative fatty acid content of the individual and total phospholipids was determined. The lipid composition of E. coli was shown to change with the fermentation conditions. Interestingly, E. coli adapted to high growth rates and to low oxygen tension by changing the lipid composition of the membrane in exactly the same way, thus indicating a common effect.     

The hydrophobic mismatch determines the miscibility of ceramides in lipid monolayers

The organization of lipids within membranes strongly depends on the interaction with other lipid and protein molecules. Sphingolipids comprise a structurally diverse family, the ceramides being some of the simplest members. Although small chemical modifications of ceramide structure, such as varying the N-acyl chain length, lead to a complex polymorphism of this lipid, only long acyl chain ceramides have usually been studied and their properties became a putative hallmark for all ceramides. In this work, we studied the mixing behavior of C10:0 Cer, which has the N-acyl chain shorter than that of the sphingosine acyl chain and displays an expanded to condensed phase transition at 25mNm(-1) at 24°C, with ceramides N-acylated with longer fatty acyl chains C12:0, C14:0 and C18:0. The N-acyl chain length determined the miscibility of ceramides in Langmuir monolayers, as it was ascertained by the dependence of the mean molecular area, perpendicular dipole moment, surface topography and film thickness with the mixture composition. We found that, as the hydrophobic mismatch in ceramides increased complete miscibility, partial or complete immiscibility can occur.     

The differential effect on two hybrid proteins of deletion mutations within the hydrophobic region of the Escherichia coli OmpA signal peptide

Oligonucleotide-directed site-specific mutagenesis was used to systematically shorten the hydrophobic region within the signal peptide of the Escherichia coli outer membrane protein OmpA. DNA encoding the wild type and mutant OmpA signal peptides were then fused in frame to DNA encoding the mature regions of Staphylococcus aureus nuclease A and TEM beta-lactamase. The ability of these signal peptides to direct processing of the resulting hybrid proteins was dependent on both their length and the protein to which they were fused. Deletion of two or more residues progressively slowed processing of pro-OmpA-nuclease. By contrast, pro-OmpA-beta-lactamase was less sensitive to the length of the hydrophobic region than to the nature of the deleted residue(s). Deletion of an Ala residue tended to reduce processing efficiency of pro-OmpA-beta-lactamase, while deletion of an Ile residue, together with the Ala residue, resulted in improvement. The loss of either 3 or 4 residues abolished processing of both hybrids. These data indicate that both the length as well as the identity of residues in the hydrophobic region are important. The relative importance of these two factors depends on the mature region of the protein being secreted.     

A molecular model for lipid-protein interaction in membranes: the role of hydrophobic mismatch.

The interaction free energy between a hydrophobic, transmembrane, protein and the surrounding lipid environment is calculated based on a microscopic model for lipid organization. The protein is treated as a rigid hydrophobic solute of thickness dP, embedded in a lipid bilayer of unperturbed thickness doL. The lipid chains in the immediate vicinity of the protein are assumed to adjust their length to that of the protein (e.g., they are stretched when dP > doL) in order to bridge over the lipid-protein hydrophobic mismatch (dP-doL). The bilayer's hydrophobic thickness is assumed to decay exponentially to its asymptotic, unperturbed, value. The lipid deformation free energy is represented as a sum of chain (hydrophobic core) and interfacial (head-group region) contributions. The chain contribution is calculated using a detailed molecular theory of chain packing statistics, which allows the calculation of conformational properties and thermodynamic functions (in a mean-field approximation) of the lipid tails. The tails are treated as single chain amphiphiles, modeled using the rotational isometric state scheme. The interfacial free energy is represented by a phenomenological expression, accounting for the opposing effects of head-group repulsions and hydrocarbon-water surface tension. The lipid deformation free energy delta F is calculated as a function of dP-doL. Most calculations are for C14 amphiphiles which, in the absence of a protein, pack at an average area per head-group ao approximately equal to 32 A2 (doL approximately 24.5 A), corresponding to the fluid state of the membrane. When dP = doL, delta F > 0 and is due entirely to the loss of conformational entropy experienced by the chains around the protein. When dP > doL, the interaction free energy is further increased due to the enhanced stretching of the tails. When dP < doL, chain flexibility (entropy) increases, but this contribution to delta F is overcounted by the increase in the interfacial free energy. Thus, delta F obtains a minimum at dP-doL approximately 0. These qualitative interpretations are supported by detailed numerical calculations of the various contributions to the interaction free energy, and of chain conformational properties. The range of the perturbation of lipid order extends typically over few molecular diameters. A rather detailed comparison of our approach to other models is provided in the discussion.     

Ca2+-induced phase separation in black lipid membranes and its effect on the transport of a hydrophobic ion

Voltage jump-current relaxation studies have been performed with dipicrylamine-doped black membranes of binary lipid mixtures. As in the case of the carrier-mediated ion transport (Schmidt, G., Eibl, H. and Knoll, W. (1982) J. Membrane Biol. 70, 147-155) no evidence was found that the neutral lipid phosphatidylcholine (DPMPC) and the charged phosphatidic acid (DPMPA) are heterogeneously distributed in the membrane over the whole range of composition. However, besides a continuous dilution of the surface charges of DPMPA by the addition of DPMPC molecules, different structural properties of mixed membranes influence the kinetics of the dipicrylamine transport. The addition of Ca2+ to the electrolyte induces a lipid phase separation within the membrane into two fluid phases of distinctly different characteristics of the translocation of hydrophobic ions. Thus, it is possible to determine a preliminary composition phase diagram for the DPMPA/DPMPC mixtures as a function of the Ca2+ concentration.     

Effect of hydrophobic mismatch and interdigitation on sterol/sphingomyelin interaction in ternary bilayer membranes

Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1(Δ4t)), and N-linked acyl chains (often 16:0, 18:0 or 24:1(Δ15c)). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane.     

The effect of changes in the lipid composition of membranes on the functional activity of platelets

The phospholipid and fatty acid composition as well as the effect of platelet lipid composition modifications on the functional parameters of platelets were studied in blood sera from healthy donors and from patients with ischemic heart disease (IHD). It was found that the content of cholesterol and phospholipid hydrolysis products in IHD patients was increased. Reconstitution of the lipid composition of donor platelets by lysophosphatidylcholines, phosphatidic acid, fatty acids and cholesterol led to the increase of the platelet functional activity. It is suggested that the increased adsorption of Ca2+ on platelet surface is due to alterations in the platelet lipid composition in IHD and after modifications.     

Channel-closing activity of porins from Escherichia coli in bilayer lipid membranes

The opening and closing of the ompF porin from Escherichia coli JF 701 was investigated by reconstituting the purified protein into planar bilayer membranes. The electrical conductance changes across the membranes at constant potential were used to analyze the size and aggregate nature of the porin channel complexes and the relative number of opening and closing events. We found that, when measured at pH 5.5, the channel conductance diminished and the number of closing events increased when the voltage was greater than 100 mV. The results suggest that the number of smaller sized conductance channels increases above this potential. There was also an increase in the smaller subunits and in the closing events when the pH was lowered to 3.5, and these changes were further enhanced by increasing the voltage. We propose that both lowering the pH and elevating the potential across the membrane stabilize the porin in a conformation in which the subunits are less tightly associated and the subunits open in a non-cooperative manner. These same conditions also appear to stabilize the closed state of the pore.     

Effect of cytochrome c on the phase behavior of charged multicomponent lipid membranes

We studied the effect of submicromolar concentrations of cytochrome c (cyt c) on the phase behavior of ternary lipid membranes composed of charged dioleoylphosphatidylglycerol, egg sphingomyelin and cholesterol. The protein was found to induce micron-sized domains in membranes belonging to the single-fluid-phase region of the protein-free ternary mixture and, as a result, to expand the region of coexistence of liquid ordered (L_o) and liquid disordered (L_d) phases. Direct observations on individual vesicles revealed that protein adsorption increases the area of L_d domains. Measurements using a fluorescent analog of cyt c showed that the protein preferentially adsorbs onto domains belonging to the L_d phase. The adsorption was quantitatively characterized in terms of partitioning ratios between the L_d and the L_o phases. The protein was also found to induce vesicle leakage even at relatively low concentrations. In eukaryotic cells under normal physiological conditions, cyt c is localized within the intermembrane space of mitochondria. During cell apoptotis, cyt c is released into the cytosol and its adsorption to intracellular membranes may strongly perturb the lipid distribution within these membranes as suggested by our results.     

Effect of the lipid phase transition on the lactose permease from Escherichia coli

The temperature dependence of lactose active transport, efflux down a concentration gradient, and equilibrium exchange were analyzed in right-side-out membrane vesicles from Escherichia coli containing wild-type lactose permease and mutant Glu325 --> Ala. With respect to uphill transport and efflux down a concentration gradient, both of which involve H(+) symport, Arrhenius plots with wild-type permease exhibit a discontinuity at 18-19 degrees C with a 7-8-fold decrease in activation energy above the phase transition. For equilibrium exchange, which does not involve H(+) symport, the change in activation energy is much less pronounced (2-3-fold) than that observed for active transport or efflux. Strikingly, mutant Glu325 --> Ala, which catalyzes equilibrium exchange as well as wild-type permease but is defective in all translocation reactions that involve net H(+) translocation, exhibits no change whatsoever in activation energy. The findings are consistent with the conclusion that the primary effect of the lipid phase transition is to alter coupling between substrate and H(+) translocation rather than the conformational change(s) responsible for translocation across the membrane.     

The interaction of hydrophobic ions with lipid bilayer membranes

Summary Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and Läuger (J. Membrane Biol. 5:225, 1971). This model visualizes as three distinct steps the interfacial adsorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions adsorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon adsorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon adsorption, of a highly ordered water structure which surrounds the hydrophobic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead be attributed to a more complex effect equivalent to a reduction of membrane fluidity.Research performed while on sabbatical leave April-September, 1974.     

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.     

Regulation of lipid composition in Acholeplasma laidlawii and Escherichia coli membranes: NMR studies of lipid lateral diffusion at different growth temperatures

Lipid lateral diffusion coefficients have been directly determined by pulsed field gradient NMR spectroscopy on macroscopically aligned, fully hydrated lamellar phases containing dimyristoylphosphatidylcholine and total lipid extracts from Acholeplasma laidlawii and Escherichia coli. The temperature dependence of the diffusion coefficient was of the Arrhenius type in the temperature interval studied. The sharp increase in the diffusion coefficient at the growth temperature of E. coli obtained by FRAP measurements, using a fluorescent probe molecule (Jin, A. J., Edidin, M., Nossal, R., and Gershfeld, N. L. (1999) Biochemistry 38, 13275-13278), was not observed. Thus, we conclude that the lipid structural properties (i.e., those affecting the lipid phase behavior), rather than the lipid dynamics, are involved in the adjustment of the membrane lipid composition. Further support for this conclusion is given by the finding that lipid extracts from A. laidlawii grown at different temperatures have about the same diffusion coefficients. Finally, the lipid lateral diffusion in bilayers of phospholipids was found to be much faster than that in bilayers of mainly glucolipids, which can be understood in terms of a free volume theory for the diffusion process.