Single Bilayer Vesicles Research Articles

On the Mechanism of Bilayer Separation by Extrusion; or, Why Your Large Unilamellar Vesicles are Not Really Unilamellar

ABSTRACT Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Sub-microscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would dramatically impact interpretations from many model membrane experiments. Using three probe-free methods—small-angle X-ray and neutron scattering (SAXS and SANS) and cryogenic electron microscopy (cryoEM)—we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100 nm diameter pores. In such preparations, only ~35% of lipids are externally accessible, and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity, as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity. STATEMENT OF SIGNIFICANCE Extruded vesicles are a ubiquitous tool in membrane research. It is widely presumed that extrusion produces unilamellar (i.e., single bilayer) vesicles, an assumption that is often crucial for data analysis and interpretation. Using X-ray and neutron scattering and cryogenic electron microscopy, we show that a substantial amount of lipid remains inaccessible after extrusion due to an abundance of multilamellar vesicles (MLVs). While this is a general phenomenon for neutral lipids, MLV contamination can be reduced by several complementary approaches such as including charged lipids in the mixture, reducing the ionic strength of the aqueous medium, and reducing the extrusion pore size. These observations together suggest a mechanism by which extrusion strips MLVs of their layers.

On the Mechanism of Bilayer Separation by Extrusion, or Why Your LUVs Are Not Really Unilamellar

Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Submicroscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would impact interpretations from many model membrane experiments. Using three probe-free methods—small angle X-ray and neutron scattering and cryogenic electron microscopy—we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100-nm diameter pores. In such preparations, only ∼35% of lipids are externally accessible and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein the formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity.

Using Tension to Map Free-Energy Landscapes that Govern Pore Nucleation in Membranes

Because of the large energy, thermal activation is needed to open nanopores in membranes yet activation energy can change significantly with tension. Although models exist describing the impact of tension, experiments have failed to cover the ranges of tension and pore nucleation rate required to examine theories. Hence, a micromechanical method was established to assay rates at which unstable nanopores form in giant single-bilayer vesicles in relation to tension, covering rates from 100/s. Plotted as logarithms, rates of nanopore nucleation yield changes in activation energy (scaled by thermal energy kBT). Derivatives of activation energy with respect to tension establish the critical-pore areas that couple with tension to lower activation energy. When correlated to pore area, changes in activation energy can be transformed to free energies along a reaction coordinate defined by pore size. In this way, we have mapped free-energy landscapes for pore nucleation in several types of fluid-lipid bilayer systems (cf. figure), beginning near the symmetry break and rising by 10-20 kBT when pore sizes reach a few nm. [E. Evans and B.A. Smith, New Journal of Physics13 (2011)].View Large Image | View Hi-Res Image | Download PowerPoint Slide

Encapsulation and NMR on an Aggregating Peptide before Fibrillogenesis

The early stages of peptide and protein aggregation include the formation of soluble oligomers, some of which may be cytotoxic. There is a paucity of structural information on these oligomers, however, because they are temporally unstable and tend to aggregate further into insoluble protofibrils and fibrils. To obtain structural information on soluble oligomers, we have developed a procedure for encapsulating a fibril-forming peptide, Peptide 1 (NH2-SDDYYYGFGSNKFGRPRDD-COOH), in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine single bilayer vesicles (POPC SBVs). We also encapsulated a non-fibril forming peptide, Peptide 2 (NH2-EEWEE-COOH), in POPC SBVs. The nominal concentration of Peptide 1 in the resulting 40 nm diameter SBVs was 2.4 +/- 0.1 mM, well above the concentration at which Peptide 1 forms fibrils. We demonstrated that these peptides had indeed been encapsulated by measuring longitudinal relaxation times (T1) in the presence and absence of a paramagnetic substance, 1 mM Gd-EDTA, by NMR spectroscopy. When the peptides were free in solution, they showed the expected shortening of T1 times and broadening of NMR peaks. In contrast, peptide encapsulated in POPC SBVs were shielded from the effects of Gd-EDTA and showed preservation of T1 values and NMR line widths. To demonstrate that encapsulation inhibits fibril formation, we measured one-dimensional proton (1D-1H) NMR spectra of the peptides in solution, and of the encapsulated peptides immediately after encapsulation, and 4 days after encapsulation, because Peptide 1 forms fibrils within 1 day. A 2.8 mM solution of Peptide 1 shows the loss of NMR signal expected for a fibrillizing peptide. In contrast, the 1D-1H spectra of encapsulated Peptide 1 measured immediately after encapsulation and 4 days after encapsulation were essentially identical, with preservation of line width at 4 days, i.e., well within the time frame of most high-resolution NMR experiments. Encapsulation may provide a means to obtain high-resolution NMR data on unstable soluble oligomers of peptides implicated in amyloidoses such as Alzheimer's Disease and provide the first detailed structural information about these possibly cytotoxic species that have hitherto been inaccessible to analysis.

N-Glycosylation is Required for Secretion-Competent Human Plasma Phospholipid Transfer Protein

Human plasma phospholipid transfer protein (PLTP) contains six potential N-glycosylation sites (Asn-X-Ser). To study the role of these sites on PLTP structure and function, seven variants in which asparagine (N) residues were converted to glycine (G) were prepared by site-directed mutagenesis. These were N(47)G, N(77)G, N(100)G, N(126)G, N(228)G, N(381)G and N(47, 77, 100, 126, 228, 381)G (N(null)G). These variants and wild-type (WT) PLTP were expressed in COS-7 cells. Intracellular and secreted PLTP mass was analyzed by Western blots and quantitative enzyme-linked immunosorbent assay; PLTP activities in cellular lysates and media were based on the transfer of [(3)H]dipalmitoylphosphatidylcholine from phospholipid single bilayer vesicles to HDL. N(null)G was not detected intracellularly. N(381)G was similar to WT PLTP with respect to specific activity and secretion efficiency. The specific activities of N(47)G, N(77)G, N(100)G, N(126)G, N(228)G and N(381)G were similar in cell lysate (range = 67-90% WT) and medium (range = 65-77% WT). Intracellular masses of these PLTP variants were similar to that of WT (Mean = 103% WT); mean secreted mass was 88% WT. These results suggest that secretion-competent PLTP requires glycosylation but that no single glycosylation site is required.

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures ( d = 6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 °C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 °C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.

Interaction of ultrasound driven microbubbles and lipid membranes

Micron-sized bubbles show pronounced oscillations when submitted to ultrasound, leading to increased scattering and improved echographic contrast. It has been reported that this excitation can also alter nearby cell membranes [M. Ward, J. Wu, and J. F. Chiu, Ultrasound Med. Biol. 26, 1169–1175 (2000)], and increase the permeability for drug delivery. To elucidate the mechanisms at work in these sonoporation experiments, we developed a setup that allows for a controlled study of the interaction of single microbubbles with single lipid bilayer vesicles. Substituting vesicles for cell membranes is advantageous because the mechanical properties of vesicles are well-known. Microscopic observations reveal that vesicles near a bubble undergo vivid motion, being periodically accelerated toward and away from the bubble. This ‘‘bouncing’’ of vesicles is a vivid motion that we attribute to a streaming flow field set up by the bubble oscillation. Some vesicles undergo dramatic deformations as they follow the flow, reflecting the high shear rates attained. Break-up of vesicles could also be observed.

Modulation of the phospholipid transfer protein-mediated transfer of phospholipids by diacylglycerols

Previous studies have shown that diacylglycerols (DAG) are formed during triglyceride hydrolysis in very low density lipoproteins (VLDL), a process that is accompanied by an elevated phospholipid transfer protein (PLTP)-mediated transfer of phopholipids (PL) from VLDL to high density lipoprotein. Because PLTP has been also shown to transfer DAG, we hypothetized that DAG might modulate PL transfer through a mechanism of competition with respect to PLTP. To address this question we performed in vitro PL transfer assays using specifically designed PL donor particles. These were single bilayer vesicles (SBV) and large (EM-L) or small (EM-S) lipid emulsions, containing various proportions of DAG. The PLTP-mediated transfers of PL decreased as the volumes of the particle cores increased (SBV > EM-S > EM-L). In all cases, these transfers were inhibited by DAG in a concentration-dependent manner. We determined the core-to-surface distribution of DAG and we measured their relative affinity for PLTP by comparison with that of PL. From these parameters, we calculated the theoretical effects of DAG on PL transfers that would result from a competition mechanism. The experimental data showed that the inhibiting effects of DAG on PL transfers were much more important than those predicted from our calculations. Additional data showed that a large part of DAG effects was in fact due to their ability to increase the viscosity of the particle PL surfaces, as calculated from electron spin resonance experiments. These results show that DAG can modulate the PLTP-dependent PL transfers, both by competition with PL and by increasing the viscosity of the particle surfaces. These findings might be physiopathologically relevant in situations where elevated plasma concentrations of DAG might result from hypertriglyceridemia.—Lalanne, F., C. Motta, Y. Pafumi, D. Lairon, and G. Ponsin. Modulation of the phospholipid transfer protein-mediated transfer of phospholipids by diacylglycerols. J. Lipid Res. 2001. 42: 142–149.

Depth-dependent analysis of membranes using benzophenone-based phospholipids

Any attempt to probe the membrane hydrophobic core with chemical reagents necessitates the use of reactive intermediates like carbenes and nitrenes, which can insert into CH bonds. Several photoactivable reagents based on carbenes and nitrenes have been reported. However, the high reactivity of these reagents, often leads to very low insertion yields. We report here a high degree of cross-linking (35–40%) achieved with three benzophenone-based phospholipids and analyze the carbon functionalization data using a multiple Gaussian function. These phospholipids are so designed so as to permit depth-dependent labeling in membranes. Single bilayer vesicles were prepared from these phospholipids and dimyristoylphosphatidylcholine. The cross-linked product was isolated and characterized by mass spectroscopy. The results obtained indicated that the cross-linked product was dominated by dimeric product formed by intermolecular cross-linking. The Gaussian analysis used here provides insight into the relative depths of the probes inside the membrane.

Mechanism of the phospholipid transfer protein-mediated transfer of phospholipids from model lipid vesicles to high density lipoproteins

To study the effects of the phospholipid transfer protein (PLTP) on the thermodynamic parameters governing the transfer of phospholipids (PL) from single bilayer vesicles (SBV) to high density lipoprotein (HDL), we performed transfer measurements at various temperatures between 4 and 65°C, using a pyrenylphosphatidylcholine (Pyr-PC) as probe. The proportion of excimer ( E) to monomer ( M) fluorescence of a pyrenyl moiety constitutes a direct measure of its local concentration. The transfers of Pyr-PC were monitored by following the decrease of E/ M. The data were used to calculate the rate constants K +1 for the transfer from SBV to HDL and to generate the corresponding Arrhenius plots. The equilibrium constants, K eq, for the same reactions were also determined and used to generate Van’t Hoff plots. From these data, we calculated the thermodynamic parameters for both the whole transfer reaction and the transition state. Both K +1 and K eq values clearly varied with temperature. PLTP induced very similar decreases in the free energy for the whole reaction (Δ G) and in that for the transition state (Δ G #). At 37°C, the decreases were of 0.37 and 0.29 kcal/mol, respectively. We studied the thermal denaturation of PLTP between 37 and 65°C, and the effects of denatured PLTP samples on the PL transfer reaction were then determined. In all cases, the changes of Δ G remained comparable to those of Δ G #. Thus the essential action of PLTP is to facilitate the first step of the reaction, which can be considered as the desorption of PL molecules from the surface of donor particles.

Vesicle-Forming Single-Tail Hydrocarbon Surfactants with Sulfonium Headgroup

It is shown by small angle neutron scattering and freeze-fracture electron microscopy that sulfonium surfactants with a single surfactant tail form bilayer vesicles of narrow size distribution. This exceptional behavior is attributed to an unusually small area requirement of the polar headgroup due to the high polarizability of the sulfonium cation.

Cytoplasm to vacuole trafficking of aminopeptidase I requires a t-SNARE-Sec1p complex composed of Tlg2p and Vps45p.

Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non-classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway. Newly synthesized precursor API is sequestered in double-membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with the vacuole, releasing single-membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API. Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non-selective mechanism that shares many similarities with the Cvt pathway. Here we show that Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy. Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle. Thus, the early Vps45-Tlg2p-dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy. Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API maturation is not blocked in a pep12(ts) mutant, indicating that Vps45p independently regulates the function of multiple t-SNARES at distinct trafficking steps.

Surface Properties of Native Human Plasma Lipoproteins and Lipoprotein Models

Plasma lipoprotein surface properties are important but poorly understood determinants of lipoprotein catabolism. To elucidate the relation between surface properties and surface reactivity, the physical properties of surface monolayers of native lipoproteins and lipoprotein models were investigated by fluorescent probes of surface lipid fluidity, surface lateral diffusion, and interfacial polarity, and by their reactivity to Naja melanoleuca phospholipase A 2 (PLA 2). Native lipoproteins were human very low, low-, and subclass 3 high-density lipoproteins (VLDL, LDL, and HDL 3); models were 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) or its ether analog in single-bilayer vesicles, large and small microemulsions of POPC and triolein, and reassembled HDL (apolipoprotein A-I plus phospholipid). Among lipoproteins, surface lipid fluidity increased in the order HDL 3 < LDL < VLDL, varying inversely with their (protein + cholesterol)/phospholipid ratios. Models resembled VLDL in fluidity. Both lateral mobility in the surface monolayer and polarity of the interfacial region were lower in native lipoproteins than in models. Among native lipoproteins and models, increased fluidity in the surface monolayer was associated with increased reactivity to PLA 2. Addition of cholesterol (up to 20 mol%) to models had little effect on PLA 2 activity, whereas the addition of apolipoprotein C-III stimulated it. Single-bilayer vesicles, phospholipid-triolein microemulsions, and VLDL have surface monolayers that are quantitatively similar, and distinct from those of LDL and HDL 3. Surface property and enzymatic reactivity differences between lipoproteins and models were associated with differences in surface monolayer protein and cholesterol contents. Thus differences in the surface properties that regulate lipolytic reactivity are a predictable function of surface composition.

Structural studies of detergent-solubilized and vesicle-reconstituted low-density lipoprotein (LDL) receptor.

The low-density lipoprotein (LDL) receptor plays a key role in maintaining circulating and cellular cholesterol homeostasis. The LDL receptor is a transmembrane glycoprotein whose biochemical and genetic properties have been extensively studied notably by Brown, Goldstein and colleagues [Brown, M. S., & Goldstein, J. L., (1986) Science 232, 34-47]. However, few if any structural studies of the LDL receptor have been reported, and details of its secondary and tertiary structure are lacking. In an attempt to determine the low-resolution structure of the LDL receptor, we have purified the receptor from bovine adrenal cortices using modifications of the method of Schneider et al. [Schneider, W. J., Goldstein, J. L., & Brown, M. S. (1985) Methods in Enzymol.109, 405-417]. Using circular dichroism, the secondary structure of the detergent-solubilized bovine LDL receptor at 25 degrees C was shown to be 19% alpha-helix, 42% beta-sheet, and 39% random coil. Interestingly, the detergent-solubilized receptor appeared to be quite resistant to changes in secondary structure over the temperature range 10-90 degrees C, with only minor but reversible changes being observed. In contrast, a more pronounced unfolding of the detergent-solubilized receptor was observed in the presence of guanidinium hydrochloride. Using the complete sequence of the human LDL receptor, sequence analysis by the Chou-Fasman prediction algorithm showed quite good agreement with the experimentally determined secondary structure of the bovine LDL receptor at 25 degrees C. Finally, the purified, bovine LDL receptor was reconstituted into large unilamellar vesicles of egg yolk phosphatidylcholine using a procedure exploiting preformed vesicles and detergent dialysis. We showed previously using negative stain electron microscopy that reconstituted vesicles bind LDL. Now, using cryoelectron microscopy of frozen hydrated reconstituted vesicles evidence of an extended, stick-like morphology (length approximately 120 A) for the extracellular domain of the LDL receptor has been obtained. Successful purification of the receptor, its incorporation into single bilayer vesicles, and its direct visualization by cryoelectron microscopy pave the way for more detailed structural studies of the LDL receptor and the receptor-LDL complex.

Sans Investigation of the Pressure- and Temperature-Dependent Structure of the Bile Salt/Lecithin System

We used small angle neutron scattering to investigate the pressure and temperature dependent particle structure in mixed colloids of egg yolk phosphatidylcholine with the bile salt, glycocholate in D2O. The concentrations and compositions of the mixtures, at ambient temperature and pressure, corresponded to the mixed vesicle region of the isotropic (I) phase. Upon increasing the hydrostatic pressure at ambient temperature, interesting morphological changes were observed. At a pressure of 6.9 MPa, the system is best described by a coexistence of the mixed, spherical single bilayer vesicle, stacked lamellar and stacked ribbon phases. At higher pressures, the system transformed to a stacked ribbon phase. Analysis of scattering data as a function of temperature, at ambient pressure, revealed that a vesicle phase remained stable with the average spherical radius decreasing as the temperature was increased from 15 to 40°C.

Phospholipid Transfer Protein Mediates Transfer of not Only Phosphatidylcholine but Also Cholesterol from Phosphatidylcholine-Cholesterol Vesicles to High Density Lipoproteins

Phospholipid transfer protein (PLTP) purified from human plasma was found to enhance the transfer of cholesterol from single bilayer vesicles containing phosphatidylcholine and cholesterol to high density lipoprotein-3. The rate of cholesterol transfer was greatly influenced by the cholesterol content of the donor vesicles. The maximal rate was observed with the vesicles containing 20-25 mol % cholesterol. This was in contrast to a progressive decline in the rate of phosphatidylcholine transfer with an increase in the cholesterol content. To determine the binding of cholesterol and phosphatidylcholine to PLTP, the mixtures of PLTP and the vesicles containing 3H-labeled phosphatidylcholine and 14C-labeled cholesterol were incubated and subjected to sucrose density gradient centrifugation. Determination of the label profiles showed that cholesterol as well as phosphatidylcholine were transferred from the vesicles to PLTP. The reversible nature of the binding was shown by the transfer of labeled cholesterol and phosphatidylcholine bound to PLTP to the acceptor vesicles or low density lipoprotein. Isothermal equilibrium binding of PLTP for cholesterol and phosphatidylcholine showed that PLTP possessed a considerably higher affinity and binding capacity for phosphatidylcholine than for cholesterol. The phosphatidylcholine binding affinity and capacity were greater when PLTP was incubated with phosphatidylcholine vesicles without cholesterol. A possible importance of PLTP-mediated cholesterol transfer in the circulation was described.

Phosphatidylinositol 4,5-Bisphosphate Binding to the Pleckstrin Homology Domain of Phospholipase C-δ1 Enhances Enzyme Activity

The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some co-complexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-delta1 (PLCdelta1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in a dose-dependent manner with an EC50 = 1 microM (0.3 mol%), up to 9-fold higher when 5 microM (1.5 mol%) of PIP2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 microM of PI with a molar ratio of PI:PS:PC = 1:5:5). Stimulation was specific for PIP2, since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP2-mediated stimulation was, however, inhibited by inositol 1,4, 5-triphosphate (IP3) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLCdelta1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP2-mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP2. Analysis of PIP2-stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (Ks) of PLCdelta1 for the PI/PS/PC vesicles was reduced from 558 microM to 53 microM, and the interfacial Michaelis constant (Km) was reduced from 0.21 to 0.06 by PIP2. The maximum rate of PI hydrolysis (Vmax) was not affected by PIP2. These results demonstrate that a major function of the PH domain of PLCdelta1 is to modulate enzyme activity. Further, our results identify PIP2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP2.

Probing self assembly in biological mixed colloids by SANS, deuteration, and molecular manipulation.

Small-angle neutron scattering was used to obtain information on the form and molecular arrangement of particles in mixed colloids of bile salts with phosphatidylcholine, and bile salts with monoolein. Both types of systems showed the same general characteristics. The particle form was highly dependent on total lipid concentration. At the highest concentrations the particles were globular mixed micelles with an overall size of 50A. As the concentration was reduced the mixed micelles elongated, becoming rodlike with diameter about 50A. The rods had a radial core-shell structure in which the phosphatidylcholine or monoolein fatty tails were arranged radially to form the core with the headgroups pointing outward to form the shell. The bile salts were at the interface between the shell and core with the hydrophilic parts facing outward as part of the shell. The lengths of the rods increased and became more polydispersed with dilution. At sufficiently low concentrations the mixed micelles transformed into single bilayer vesicles. These results give insight on the physiological function of bile and on the rules governing the self assembly of bile particles in the hepatic duct and the small intestine.

Membrane-protein interaction and the molten globule state: interaction of alpha-lactalbumin with membranes.

The insertion of soluble proteins into membranes has been a topic of considerable interest. We have studied the insertion of bovine alpha-lactalbumin into single-bilayer vesicles prepared from egg phosphatidylcholine (PC). Fluorescence studies indicated rapid and tight binding of apo-alpha-lactalbumin (apo-alpha-LA) to PC vesicles as a function of pH. The binding was maximal at pH values which favor the formation of the molten globule state. As an increase of hydrophobic surface is observed in the molten globule state, this conformational state can provide a molecular basis for insertion of soluble proteins into membranes. The membrane-bound complex formed at low pH (3.0) could be isolated and was found to be stable at neutral pH. The structural characterization of the apo-alpha-LA-PC complex was studied by fluorescence quenching using iodide, acrylamide, and 9,10-dibromostearic acid. The results obtained indicated that some of the tryptophans of apo-alpha-LA were buried in the membrane interior and some were exposed on the outer side. Fluorescence quenching and CD studies indicated the membrane-bound conformation of apo-alpha-LA was some conformational state that is between the soluble, fully folded conformation and the molten globule state.

Photolabeling of a Pore-forming Toxin with the Hydrophobic Probe 2-[3H]Diazofluorene: IDENTIFICATION OF MEMBRANE-INSERTED SEGMENTS OF STAPHYLOCOCCUS AUREUS α-TOXIN

The identification of membrane-inserted segments of pore-forming soluble proteins is crucial to understanding the action of these proteins at the molecular level. A distinct member of this class of proteins is alpha-toxin, a 293-amino acid-long 33-kDa hemolytic toxin secreted by Staphylococcus aureus that can form pores in both artificial and natural membranes. We have studied the interaction of alpha-toxin with single bilayer vesicles prepared from asolectin using a hydrophobic photoactivable reagent, 2-[3H]diazofluorene ([3H]DAF) (Pradhan, D., and Lala, A. K. (1987) J. Biol. Chem. 262, 8242-8251). This reagent readily partitions into the membrane hydrophobic core and on photolysis labels the lipid and protein segments that penetrate the membrane. Current models on the mode of action of alpha-toxin indicate that, on interaction with membranes, alpha-toxin forms an oligomer, which represents the active pore. In keeping with these models, we observe that [3H]DAF photolabels the membrane-bound alpha-toxin oligomer. Cyanogen bromide fragmentation of [3H]DAF-labeled alpha-toxin gave several fragments, which were subjected to Edman degradation. We could thus sequence residues 1-19, 35-60, 114-139, 198-231, and 235-258. Radioactive analysis and phenylthiohydantoin-derivative analysis during sequencing permitted analysis of DAF insertion sites. The results obtained indicated that the N and C termini (residues 235-258) have been extensively labeled. The putative pore-forming glycine-rich central hinge region was poorly labeled, indicating that the apposing side of the lumen of the pore does not form the lipid-protein interface. The DAF labeling pattern indicated that the major structural motif in membrane-bound alpha-toxin was largely beta-sheet.