Experimental and Monte Carlo simulation studies of the thermodynamics of polyethyleneglycol chains grafted to lipid bilayers.

Experimental measurements of the affinity of binding of fluorescent acylated polyethyleneglycol (PEG) conjugates to bilayers containing varying levels of phosphatidylethanolamine-PEGs (PE-PEGs) have been combined with Monte Carlo simulations to investigate the properties of the polymer chains at a PEG-grafted lipid interface. The affinity of binding of such conjugates to large unilamellar phosphatidylcholine/phosphatidylethanolamine (9:1) vesicles decreases 27-fold as the size of the coupled PEG chain increases from 1 to 114 monomer units. Incorporation of increasing amounts of PE-PEG2000 or PE-PEG5000 into the vesicles progressively reduces the affinity of binding of acylpeptide-PEG2000 or -PEG5000 conjugates. Monte Carlo simulations of surfaces with grafted PEG chains revealed no significant dependence of several characteristic properties of the polymer chains, including the average internal energy per polymer and the radii of gyration, on the grafting density in the range examined experimentally. The average conformation of a surface-grafted PEG2000 or PEG5000 chain was calculated to be fairly extended even at low grafting densities, and the projected cross-sectional areas of the grafted PEG chains are considerably smaller than those predicted on the basis of the estimated Flory radius. The experimental variation of the binding affinity of acylated conjugates for bilayers containing varying mole fractions of PE-PEG2000 or -PEG5000 is well explained by expressions treating the surface-grafted PEG polymers either as a van der Waals gas or as a system of rigid discs described by scaled particle theory. From the combined results of our experimental and simulation studies we conclude that the grafted PEG chains exist in a "mushroom" regime throughout the range of polymer densities examined experimentally and that the diminished affinity of binding of acylated-PEG conjugates to bilayers containing PE-PEGs results from occlusion of the surface area accessible for conjugate binding by the mobile PE-PEG polymer chains.     

Modulation by glycosphingolipids of membrane-membrane interactions induced by myelin basic protein and melittin.

The effect of glycosphingolipids (GSLs) with oligosaccharide chains of different length and charge on membrane-membrane interactions induced by myelin basic protein (MBP) or melittin (Mel) was comparatively investigated with small unilamellar vesicles. MBP induces a fast vesicle aggregation and close membrane apposition. Merging of lipid bilayers and vesicle fusion induced by MBP are slower and less extensive processes compared to membrane apposition. The changes of membrane permeability concomitant to these phenomena are small. The Trp region of MBP remains in a rather polar environment when interacting with vesicles; its accessibility to NO3- or acrylamide quenching depends on the type of GSLs in the membrane. The Trp region of Mel is inserted more deeply into the lipid bilayer and its accessibility to the aqueous quenchers is less dependent on variations of the oligosaccharide chain of the GSLs. Mel induces a faster and more extensive membrane apposition and bilayer merging than does MBP. Extensive vesicle disruption occurs in the presence of Mel. Negatively charged GSLs facilitate membrane proximity and vesicle aggregation but an increase of the oligosaccharide chain length of either neutral or acidic GSLs decreases the interaction among vesicles that are induced by either protein. This effect is independent of the different mode of insertion of MBP and Mel into the membrane. Our results suggest that the modulation by the oligosaccharide chain on the protein-induced interactions between bilayers containing GSLs is probably exerted beyond the level of local molecular interactions between the basic proteins and the lipids.     

An analysis of the regions of the myelin basic protein that bind to phosphatidylcholine

The interactions of phosphatidylcholine (PC) to regions of the myelin basic protein (MBP) was examined. In solid phase binding assays the nature of the binding of unilamellar vesicles of¹⁴C-labeled phosphatidylcholine to bovine 18.5 kDa MBP, its N- and C-terminal peptide fragments, photooxidized 18.5 kDa MBP and the mouse 14 kDa protein, with an internal deletion of residues 117–157, was studied. The data were analyzed by computer-generated Scatchard plots in which non-specific binding was eliminated. Non-cooperative, low affinity binding of PC vesicles to MBP was observed, and this binding found to be sensitive to pH and ionic changes. At an ionic strength of 0.1 and pH 7.4, the binding of PC to the 14 kDa mouse MBP exhibited a K_d similar to that obtained with both the N-terminal and photooxidized 18.5 kDa bovine MBP. The studies indicated that the sites of PC interaction with MBP are located in the N-terminal region of the protein. The C-terminal region appeared to modulate the strength of the interaction slightly. Under similar conditions, lysozyme did not bind PC liposomes, and histone bound them nonspecifically.     

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.     

Interaction of lipid-bound myelin basic protein with actin filaments and calmodulin

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes (OLs) and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. MBP in solution has been shown by others to bind to both G- and F-actin, to bundle F-actin filaments, and to induce polymerization of G-actin. Here we show that MBP bound to acidic lipids can also bind to both G- and F-actin and cause their sedimentation together with MBP-lipid vesicles. Thus it can simultaneously utilize some of its basic residues to bind to the lipid bilayer and some to bind to actin. The amount of actin bound to the MBP-lipid vesicles decreased with increasing net negative surface charge of the lipid vesicles. It was also less for vesicles containing the lipid composition predicted for the cytosolic surface of myelin than for PC vesicles containing a similar amount of an acidic lipid. Calmodulin caused dissociation of actin from MBP and of the MBP-actin complex from the vesicles. However, it did not cause dissociation of bundles of actin filaments once these had formed as long as some MBP was still present. These results suggest that MBP could be a membrane actin-binding protein in OLs/myelin and its actin binding can be regulated by calmodulin and by the lipid composition of the membrane. Actin binding to MBP decreased the labeling of MBP by the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine (TID), indicating that it decreased the hydrophobic interactions of MBP with the bilayer. This change in interaction of MBP with the bilayer could then create a cytosol to membrane signal caused by changes in interaction of the cytoskeleton with the membrane.     

Adhesion of acidic lipid vesicles by 21.5 kDa (recombinant) and 18.5 kDa isoforms of myelin basic protein

Myelin basic protein (MBP) is thought to be responsible for adhesion of the intracellular surfaces of compact myelin to give the major dense line. The 17 and 21.5 kDa isoforms containing exon II have been reported by others to localize to the cytoplasm and nucleus of murine oligodendrocytes and HeLa cells while the 14 and 18.5 kDa isoforms lacking exon II are confined to the plasma membrane. However, we show that the exon II(-) 18.5 kDa form and a recombinant exon II(+) 21.5 kDa isoform both caused similar aggregation of acidic lipid vesicles, indicating that they should have similar abilities to bind to the intracellular lipid surface of the plasma membrane and to cause adhesion of those surfaces to each other. The circular dichroism spectra of the two isoforms indicated that both had a similar secondary structure. Thus, both isoforms should be able to bind to and cause adhesion of the cytosolic surfaces of compact myelin. The fact that they do not could be due to differences in post-translational modification in vivo, trafficking through the cell and/or subcellular location of synthesis, but it is not due to differences in their lipid binding.     

Effect of phosphorylation of phosphatidylinositol on myelin basic protein-mediated binding of actin filaments to lipid bilayers in vitro

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also assemble actin filaments and tether them to lipid bilayers through electrostatic interactions. Here we investigate the effect of increased negative charge of the lipid bilayer due to phosphorylation of phosphatidylinositol (PI) on MBP-mediated binding of actin to the lipid bilayer, by substituting phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate for PI in phosphatidylcholine/phosphatidylglycerol lipid vesicles. Phosphorylation of PI caused dissociation of the MBP/actin complex from the lipid vesicles due to repulsion of the negatively charged complex from the negatively charged membrane surface. An effect of phosphorylation could be detected even if the inositol lipid was only 2mol% of the total lipid. Calcium-calmodulin dissociated actin from the MBP-lipid vesicles and phosphorylation of PI increased the amount dissociated. These results show that changes to the lipid composition of myelin, which could occur during signaling or other physiological events, could regulate the ability of MBP to act as a scaffolding protein and bind actin filaments to the lipid bilayer.     

Effects of lipid headgroup and packing stress on poly(ethylene glycol)-induced phospholipid vesicle aggregation and fusion.

The effect of lipid headgroup and curvature-related acyl packing stress on PEG-induced phospholipid vesicle aggregation and fusion were studied by measuring vesicle and aggregate sizes using the quasi-elastic light scattering and fluorescence energy transfer techniques. The effect of the lipid headgroup was monitored by varying the relative phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents in the vesicles, and the influence of hydrocarbon chain packing stress was controlled either by the relative amount of PE and PC content in the vesicles, or by the degree of unsaturation of the acyl chains of a series of PEs, e.g., dilinoleoylphosphatidylethanolamine (dilin-PE), lysophosphatidylethanolamine (lyso-PE), and transacylated egg phosphatidylethanolamine (TPE). The PEG threshold for aggregation depends only weakly on the headgroup composition of vesicles. However, in addition to the lipid headgroup, the curvature stress of the monolayer that forms the vesicle walls plays a very important role in fusion. Highly stressed vesicles, i.e., vesicles containing PE with highly unsaturated chains, need less PEG to induce fusion. This finding applies to the fusion of both small unilamellar vesicles and large unilamellar vesicles. The effect of electrostatic charge on vesicle aggregation and fusion were studied by changing the pH of the vesicle suspension media. At pH 9, when PE headgroups are weakly charged, increasing electrostatic repulsion between headgroups on the same bilayer surface reduces curvature stress, whereas increasing electrostatic repulsion between apposing bilayer headgroups hinders intervesicle approach, both of which inhibit aggregation and fusion, as expected.     

Binding of antigen-bearing fluorescent liposomes to the murine myeloma tumor MOPC 315.

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0 degrees C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37 degrees C. The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to "target" vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.     

Effects on Interactions of Oppositely Charged Phospholipid Vesicles of Covalent Attachment of Polyethylene Glycol Oligomers to Their Surfaces: Adhesion, Hemifusion, Full Fusion and “Endocytosis”

Oppositely charged giant vesicles are known to adhere, hemifuse and fuse, all of which depend upon the nature of surface contacts. To further understand such interactions, vesicles were surface-modified with polyethylene glycol (PEG), a moiety that reduces surface-surface interactions. Positively charged vesicles were composed of O-ethyldioleoylphosphocholine (EDOPC), dioleoylphosphatidylcholine (DOPC) and a carbocyanine dye (DiO), with and without DPPE-PEG (dipalmitoylphosphatidylethanolamine-N-PEG MW of the PEG portion = 2000). Negatively charged vesicles were composed of dioleoylphosphatidylglycerol (DOPG), DOPC and a rhodamine B dye (Rh-PE), with as well as without DPPE-PEG (MW 2,000). A microscope-mounted electrophoresis chamber allowed selected pairs of vesicles to be brought into contact while color images were collected at video rates (30 frames/s). Data collection focused on effects of PEG on vesicle interactions as a function of the surface charge density. Relative to PEG-free preparations, vesicles containing DPPE-PEG (1) formed larger contact zones, (2) underwent adhesion and fusion processes more slowly (by two to four times) and (3) at high charge density were less susceptible to rupture upon contact. Unexpectedly, PEG-containing vesicles exhibited engulfment of a smaller by a larger vesicle, a process topologically similar to cellular endocytosis. These observations are interpreted to mean that, although initial surface-surface interactions are weakened by the intervening layer of PEG chains, eventual and strong bilayer-bilayer contact is still possible, evidently because the lipid anchors of these chains can diffuse away from the contact zone.     

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.     

Calmodulin binding and protein phosphorylation in adrenal medulla coated vesicles

Coated vesicles from bovine adrenal medulla contained clathrin and major detergent-insoluble polypeptides of 120-100, 51 and 49 kDa. Intact coated vesicles and vesicles lacking clathrin light chains were bound by immobilized calmodulin in the presence of Ca2+. Clathrin in the form of 700 A cages was not bound. The calmodulin binding components in intact coated vesicles are therefore contributed by the enclosed vesicle or by the 120-100, 50 or 49 kDa polypeptides. The 51 kDa component incorporated 32Pi from labelled ATP by an endogenous kinase activity; no other coat or vesicle membrane protein was phosphorylated in vitro, either by intrinsic or exogenous kinases.     

Affinity purification and characterization of an anti-PEG IgM

Anti-PEG IgM was purified by affinity chromatography using variable length PEG chains (5, 10, 20 and 30 kDa) as affinity ligands. Maximal binding of anti-PEG IgM was observed using the 30 kDa PEG-derivatized NuGel (single passage). Purified anti-PEG IgM was characterized for binding to PEG functionalized proteins/peptides by surface plasmon resonance, western blotting and ELISA. Anti-PEG IgM, in solution and adsorbed on 20 kDa PEG-derivatized NuGel, was subjected to pepsin digestion followed by affinity chromatography. SDS-PAGE analysis of eluates in both preparations yielded one fragment that was similar in size. However, an additional lower molecular weight band was observed in solution-digested affinity purified material that was not present in the eluate from the material subjected to pepsin digestion on the affinity matrix. The lower MW fragment could be eluted under milder conditions, suggesting loss of binding multiplicity. Analysis by mass spectrometry yielded molecular weights of 132 kDa (both) and 82 kDa (solution) for the respective fragments. N-terminal sequencing of both fragments resulted in primary sequences (heavy and light chains) that were not only identical to each other but also to those of native IgM. The anti-PEG IgM fragments were characterized for binding to pegylated interferon alfa-2a by ELISA. The results from these studies suggest that affinity purified anti-PEG IgM and fragments can be used as probes in detection assays for PEG functionalized biotherapeutics in pre-clinical and clinical studies.     

Investigating the interfacial binding of bacterial phosphatidylinositol-specific phospholipase C

The interactions of PI-PLC with nonsubstrate zwitterionic [phosphatidylcholine (PC)] and anionic [phosphatidylmethanol (PMe), phosphatidylserine, phosphatidylglycerol, and phosphatidic acid] interfaces that affect the catalytic activity of PI-PLC have been examined. PI-PLC binding is strongly coupled to vesicle curvature and is tighter at acidic pH for all of the phospholipids examined. PI-PLC binds to small unilamellar vesicles (SUVs) of anionic lipids with much higher affinity (K(d) is 0.01-0.07 microM for a site consisting of n = 100 +/- 25 lipids when analyzed with a Langmuir adsorption isotherm) than to zwitterionic PC SUVs (K(d) is 5-20 microM and n = 8 +/- 3). The binding to PC surfaces is dominated by hydrophobic interactions, while binding to anionic surfaces is dominated by electrostatic interactions. The contributions of specific cationic side chains and hydrophobic groups at the rim of the alpha beta-barrel to zwitterionic and anionic vesicle binding have been assessed with mutagenesis. The results are used to explain how PC activates the enzyme for both phosphotransferase and cyclic phosphodiesterase activities.     

Avidin-biotin interactions at vesicle surfaces: adsorption and binding, cross-bridge formation, and lateral interactions.

Densely packed domains of membrane proteins are important structures in cellular processes that involve ligand-receptor binding, receptor-mediated adhesion, and macromolecule aggregation. We have used the biotin-avidin interaction at lipid vesicle surfaces to mimic these processes, including the influence of a surface grafted polymer, polyethyleneglycol (PEG). Single vesicles were manipulated by micropipette in solutions of fluorescently labeled avidin to measure the rate and give an estimate of the amount of avidin binding to a biotinylated vesicle as a function of surface biotin concentration and surface-grafted PEG as PEG-lipid. The rate of avidin adsorption was found to be four times less with 2 mol% PEG750 than for the unmodified surface, and 10 mol% PEG completely inhibited binding of avidin to biotin for a 2-min incubation. Using two micropipettes, an avidin-coated vesicle was presented to a biotinylated vesicle. In this vesicle-vesicle adhesion test, the accumulation of avidin in the contact zone was observed, again by using fluorescent avidin. More importantly, by controlling the vesicle membrane tension, this adhesion test provided a direct measure of the spreading pressure of the biotin-avidin-biotin cross-bridges confined in the contact zone. Assuming ideality, this spreading pressure gives the concentration of avidin cross-bridges in the contact zone. The rate of cross-bridge accumulation was consistent with the diffusion of the lipid-linked "receptors" into the contact zone. Once adherent, the membranes failed in tension before they could be peeled apart. PEG750 did not influence the mechanical equilibrium because it was not compressed in the contact zone, but it did perform an important function by eliminating all nonspecific adhesion. This vesicle-vesicle adhesion experiment, with a lower tension limit of 0.01 dyn/cm, now provides a new and useful method with which to measure the spreading pressures and therefore colligative properties of a range of membrane-bound macromolecules.     

Effects of valency on thermodynamic parameters of specific membrane interactions

We have measured the equilibrium binding of dioleoylphosphatidylcholine vesicles (800-A diameter) containing various densities of incorporated palmitoyl-alpha-bungarotoxin (PBGT) to acetylcholine receptor (AchR) enriched microsac membranes. We have previously shown that these PBGT vesicles bind specifically to the microsacs mediated by direct interactions with the AchRs [Grant, S. W., Babbitt, B. P., West, L. K., & Huang, L. (1982) Biochemistry 21, 1274-1279]. The percent binding of liposomal lipid and associated PBGT to excess AchR sites, as well as the inhibition of binding by pretreatment of microsacs with excess alpha-bungarotoxin (alpha BGT), was strongly dependent upon the protein/lipid molar ratio of the vesicles. In addition, there existed a threshold level of approximately six PBGT molecules per vesicle at which the binding increased dramatically. The apparent association constant, KAapp, for lipid vesicle-microsac membrane binding increased approximately 4800-fold (from 3.95 X 10(4) to 1.90 X 10(8) M-1) due to an increase of 20-fold in the vesicle-associated PBGT surface density. Direct competition for binding to microsac membranes between vesicles with different PBGT/lipid molar ratios indicated that multivalent binders could easily replace binders of lower valency when receptor sites were limited. Measurement of the temperature dependence of the KAapp indicated that weak (low valency) and medium strength (intermediate valency) PBGT vesicle binders bound to microsacs in a fashion similar to the binding of alpha BGT and PBGT to detergent-solubilized AchRs. Strong PBGT vesicle binders (high valency) appear to bind by a somewhat different mechanism. All results are discussed in terms of the effects of ligand (PBGT) valency on the binding strength of vesicles to microsac membranes.     

The concanavalin a receptor from human erythrocytes in lipid bilayer membranes. Interaction with concanavalin A and succinyl-concanavalin A

The concanavalin A receptor from human erythrocyte membranes has been isolated by affinity chromatography using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide. The purified protein has been reincorporated into large unilamellar phospholipid vesicles using a detergent dialysis technique. The mean diameter of these vesicles increases as the lipid: protein ratio decreases. Binding of succinyl-concanavalin A to these vesicles was quantitated using ¹²⁵I-labelled lectin in a filtration assay. The concanavalin A receptor in lipid bilayer vesicles provides specific high affinity binding sites for succinyl-concanavalin A with an association constant of 2.13·10⁶ M^?1. Scatchard plots indicate positive cooperativity of binding at very low lectin concentrations, a characteristic also seen in concanavalin A binding to intact human erythrocytes. The presence of bovine serum albumin has little effect on lectin binding and is not required for expression of cooperativity. Concanavalin A effectively competes with succinyl-concanavalin A for binding to the vesicles with an association constant of 4.83·10⁶ M^?1. Receptor-bearing vesicles are readily agglutinated by concanavalin A but not by its succinylated derivative. The kinetics of vesicle agglutination are biphasic, with an initial rapid phase followed by a pseudo-first order process. We suggest that studies on reassembled receptor proteins in lipid bilayers can provide valuable insight into receptor involvement in transmembrane signalling events and the factors involved in cell membrane behaviour and cell agglutination.     

Reconstitution of transmembrane K+ transport with a 53 kilodalton mitochondrial protein

A 53 kDa protein has been purified from a Triton X-100 extract of liver mitochondrial membranes, by affinity chromatography on immobilized quinine, a K+ transport inhibitor. KCl-containing lipid vesicles reconstituted with this protein lose K+ to a medium low in K+ faster than vesicles lacking protein. With bacteriorhodopsin reconstituted in vesicles containing K+, light induces faster development of a pH gradient if the 53 kDa protein is included during vesicle preparation. This effect is like that of valinomycin, which catalyzes K+ efflux, dissipating the membrane potential arising from H+ entry. Evidence that vesicles containing the 53 kDa protein are permeable to K+, but exhibit low permeability to H+, indicates that this protein acts as a K+ uniporter.     

A study of the regional effects of alpha-synuclein on the organization and stability of phospholipid bilayers

Associations between the protein alpha-synuclein (alpha-syn) and presynaptic vesicles have been implicated in synaptic plasticity and neurotransmitter release and may also affect how the protein aggregates into fibrils found in Lewy bodies, the cellular inclusions associated with neurodegenerative diseases. This work investigated how alpha-syn interacts with model phospholipid membranes and examined what effect protein binding has upon the physical properties of lipid bilayers. Wide line 2H and 31P NMR spectra of phospholipid vesicles revealed that alpha-syn associates with membranes containing lipids with anionic headgroups and can disrupt the integrity of the lipid bilayer, but the protein has little effect on membranes of zwitterionic phosphatidylcholine. A peptide, alpha-syn(10-48), which corresponds to the lysine-rich N-terminal region of alpha-syn, was found to associate with lipid headgroups with a preference for a negative membrane surface charge. Another peptide, alpha-syn(120-140), which corresponds to the glutamate-rich C-terminal region, also associates weakly with lipid headgroups but with a slightly higher affinity for membranes with no net surface charge than for negatively charged membrane surfaces. Binding of alpha-syn(10-48) and alpha-syn(120-140) to the lipid vesicles did not disrupt the lamellar structure of the membranes, but both peptides appeared to induce the lateral segregation of the lipids into clusters of acidic lipid-enriched and acidic lipid-deficient domains. From these findings, it is speculated that the N-terminal and C-terminal domains of full-length alpha-syn might act in concert to organize the membrane components during normal protein function and perhaps play a role in presynaptic vesicle synthesis, maintenance, and fusion.     

Phosphorylation reverses the membrane association of peptides that correspond to the basic domains of MARCKS and neuromodulin.

Several groups have observed that phosphorylation causes the MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) protein to move off cell membranes and phospholipid vesicles. Our working hypothesis is that significant membrane binding of MARCKS requires both hydrophobic insertion of the N-terminal myristate into the bilayer and electrostatic association of the single cluster of basic residues in the protein with acidic lipids and that phosphorylation reverses this electrostatic association. Membrane binding measurements with myristoylated peptides and phospholipid vesicles show this hydrophobic moiety could, at best, barely attach proteins to plasma membranes. We report here membrane binding measurements with basic peptides that correspond to the phosphorylation domains of MARCKS and neuromodulin. Binding of these peptides increases sigmoidally with the percent acidic lipid in the phospholipid vesicle and can be described by a Gouy-Chapman/mass action theory that explains how electrostatics and reduction of dimensionality produce apparent cooperativity. The electrostatic affinity of the MARCKS peptide for membranes containing 10% acidic phospholipids (10(4) M-1 = chi/[P], where chi is the mole ratio of peptide bound to the outer monolayer of the vesicles and [P] is the concentration of peptide in the aqueous phase) is the same as the hydrophobic affinity of the myristate moiety for bilayer membranes. Phosphorylation decreases the affinity of the MARCKS peptide for membranes containing 15% acidic lipid about 1000-fold and produces a rapid (t1/2 < 30 s) dissociation of the peptide from phospholipid vesicles.