Microelectrophoresis of a bilayer-coated silica bead in an optical trap: application to enzymology.

We describe an apparatus that combines microelectrophoresis and laser trap technologies to monitor the activity of phosphoinositide-specific phospholipase C-delta1 (PLC-delta) on a single bilayer-coated silica bead with a time resolution of approximately 1 s. A 1-microm-diameter bead was coated with a phospholipid bilayer composed of electrically neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol 4,5-bisphosphate (2% PIP2) and captured in a laser trap. When an AC field was applied (160 Hz, 20 V/cm), the electrophoretic force produced a displacement of the bead, Delta(x), from its equilibrium position in the trap; Delta(x), which was measured using a fast quadrant diode detector, is proportional to the zeta potential and thus to the number of PIP2 molecules on the outer leaflet (initially, approximately 10(5)). When a solution containing PLC-delta flows past the bead, the enzyme adsorbs to the surface and hydrolyzes PIP2 to form the neutral lipid diacylglycerol. We observed a nonexponential decay of PIP2 on the bead with time that is consistent with a model based on the known structural properties of PLC-delta.

The effector domain of myristoylated alanine-rich C kinase substrate binds strongly to phosphatidylinositol 4,5-bisphosphate.

Both the myristoylated alanine-rich protein kinase C substrate protein (MARCKS) and a peptide corresponding to its basic effector domain, MARCKS-(151-175), inhibit phosphoinositide-specific phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in vesicles (Glaser, M., Wanaski, S., Buser, C. A., Boguslavsky, V., Rashidzada, W., Morris, A., Rebecchi, M., Scarlata, S. F., Runnels, L. W., Prestwich, G. D., Chen, J., Aderem, A., Ahn, J., and McLaughlin, S. (1996) J. Biol. Chem. 271, 26187-26193). We report here that adding 10-100 nm MARCKS-(151-175) to a subphase containing either PLC-delta or -beta inhibits hydrolysis of PIP(2) in a monolayer and that this inhibition is due to the strong binding of the peptide to PIP(2). Two direct binding measurements, based on centrifugation and fluorescence, show that approximately 10 nm PIP(2), in the form of vesicles containing 0.01%, 0.1%, or 1% PIP(2), binds 50% of MARCKS-(151-175). Both electrophoretic mobility measurements and competition experiments suggest that MARCKS-(151-175) forms an electroneutral complex with approximately 4 PIP(2). MARCKS-(151-175) binds equally well to PI(4,5)P(2) and PI(3,4)P(2). Local electrostatic interactions of PIP(2) with MARCKS-(151-175) contribute to the binding energy because increasing the salt concentration from 100 to 500 mm decreases the binding 100-fold. We hypothesize that the effector domain of MARCKS can bind a significant fraction of the PIP(2) in the plasma membrane, and release the bound PIP(2) upon interaction with Ca(2+)/calmodulin or phosphorylation by protein kinase C.

Membrane binding of peptides containing both basic and aromatic residues. Experimental studies with peptides corresponding to the scaffolding region of caveolin and the effector region of MARCKS.

We have studied the binding of peptides containing both basic and aromatic residues to phospholipid vesicles. The peptides caveolin(92-101) and MARCKS(151-175) both contain five aromatic residues, but have 3 and 13 positive charges, respectively. Our results show the aromatic residues insert into the bilayer and anchor the peptides weakly to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC). Incorporation of a monovalent acidic lipid (e.g., phosphatidylserine, PS) into the vesicles enhances the binding of both peptides via nonspecific electrostatic interactions. As predicted from application of the Poisson-Boltzmann equation to atomic models of the peptide and membranes, the enhancement is larger (e.g., 10(4)- vs 10-fold for 17% PS) for the more basic MARCKS(151-175). Replacing the five Phe with five Ala residues in MARCKS(151-175) decreases the binding to 10:1 PC/PS vesicles only slightly (6-fold). This result is also consistent with the predictions of our theoretical model: the loss of the attractive hydrophobic energy is partially compensated by a decrease in the repulsive Born/desolvation energy as the peptide moves away from the membrane surface. Incorporating multivalent phosphatidylinositol 4, 5-bisphosphate (PIP(2)) into PC vesicles produces dramatically different effects on the membrane binding of the two peptides: 1% PIP(2) enhances caveolin(92-101) binding only 3-fold, but increases MARCKS(151-175) binding 10(4)-fold. The strong interaction between the effector region of MARCKS and PIP(2) has interesting implications for the cellular function of MARCKS.

Fluorescently labeled neomycin as a probe of phosphatidylinositol-4, 5-bisphosphate in membranes.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a minor component of the plasma membrane, is important in signal transduction, exocytosis, and ion channel activation. Thus fluorescent probes suitable for monitoring the PI(4,5)P(2) distribution in living cells are valuable tools for cell biologists. We report here three experiments that show neomycin labeled with either fluorescein or coumarin can be used to detect PI(4,5)P(2) in model phospholipid membranes. First, addition of physiological concentrations of PI(4,5)P(2) (2%) to lipid vesicles formed from mixtures of phosphatidylcholine (PC) and phosphatidylserine (PS) enhances the binding of labeled neomycin significantly (40-fold for 5:1 PC/PS vesicles). Second, physiological concentrations of inositol-1,4,5-trisphosphate (10 microM I(1,4,5)P(3)) cause little translocation of neomycin from PC/PS/PI(4,5)P(2) membranes to the aqueous phase, whereas the same concentrations of I(1,4,5)P(3) cause significant translocation of the green fluorescent protein/phospholipase C-delta pleckstrin homology (GFP-PH) constructs from membranes (Hirose et al., Science, 284 (1999) 1527). Third, fluorescence microscopy observations confirm that one can distinguish between PC/PS vesicles containing either 0 or 2% PI(4, 5)P(2) by exposing a mixture of the vesicles to labeled neomycin. Thus fluorescently labeled neomycin could complement GFP-PH constructs to investigate the location of PI(4,5)P(2) in cell membranes.

Electrostatic properties of membranes containing acidic lipids and adsorbed basic peptides: theory and experiment.

The interaction of heptalysine with vesicles formed from mixtures of the acidic lipid phosphatidylserine (PS) and the zwitterionic lipid phosphatidylcholine (PC) was examined experimentally and theoretically. Three types of experiments showed that smeared charge theories (e.g., Gouy-Chapman-Stern) underestimate the membrane association when the peptide concentration is high. First, the zeta potential of PC/PS vesicles in 100 mM KCl solution increased more rapidly with heptalysine concentration (14.5 mV per decade) than predicted by a smeared charge theory (6.0 mV per decade). Second, changing the net surface charge density of vesicles by the same amount in two distinct ways produced dramatically different effects: the molar partition coefficient decreased 1000-fold when the mole percentage of PS was decreased from 17% to 4%, but decreased only 10-fold when the peptide concentration was increased to 1 microM. Third, high concentrations of basic peptides reversed the charge on PS and PC/PS vesicles. Calculations based on finite difference solutions to the Poisson-Boltzmann equation applied to atomic models of heptalysine and PC/PS membranes provide a molecular explanation for the observations: a peptide adsorbing to the membrane in the presence of other surface-adsorbed peptides senses a local potential more negative than the average potential. The biological implications of these "discreteness-of-charge" effects are discussed.

Pore-forming action of mastoparan peptides on liposomes: a quantitative analysis.

We have investigated the wasp venom peptides mastoparan X and polistes mastoparan regarding their apparent potential to induce pore-like defects in phosphatidylcholine unilamellar vesicles. Based on a fundamental theoretical model, the pore activation and deactivation kinetics have been evaluated from the observed efflux of liposome entrapped carboxyfluorescein in relation to the bound peptide to lipid ratio. We can quantitatively describe our experimental data very well in terms of a specific reaction scheme resulting in only a few short-lived pores. They evidently emerge rapidly from a prepore nucleus being produced by two rate-limiting monomeric states of bound peptide. These peculiar states would be favorably populated in an early stage of bilayer perturbation, but tend to die out in the course of a peptide/lipid restabilization process.

MARCKS, membranes, and calmodulin: kinetics of their interaction.

It is well documented that membrane binding of MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) requires both hydrophobic insertion of the N-terminal myristate into the bilayer and electrostatic interaction of the basic effector region with acidic lipids. The structure of a membrane-bound peptide corresponding to the effector region, residues 151-175 of bovine MARCKS, was recently determined using spin-labeled peptides and EPR. The kinetics of the peptide-membrane interaction were determined from stopped-flow fluorescence measurements; the adsorption of the peptide onto phospholipid vesicles is a diffusion-limited process. Five microM Ca2+-calmodulin decreases the lifetime of the peptide on a 100 nm diameter 10:1 PC/PS vesicle from 0.1 s to 0.01 s by rapidly pulling the peptide off the membrane. We propose a molecular mechanism, based on previous work by M. Eigen and colleagues, by which calmodulin may remove MARCKS(151-175) from the membrane at a diffusion-limited rate. Calmodulin may also use this mechanism to remove the pseudosubstrate region from the substrate binding site of enzymes such as calmodulin kinase II and myosin light chain kinase.

Lipid and cell membranes in the presence of gadolinium and other ions with high affinity to lipids. 2. A dipole component of the boundary potential on membranes with different surface charge.

When Gd3+, a trivalent lanthanide, binds phospholipids with a high affinity, it elicits strong electrostatic effects on the surface of the lipid bilayer. Two experimental methods were applied to monitor the changes in the boundary and surface potentials induced by Gd3+ adsorption on liposomes and planar lipid bilayer membranes (BLM) made from phosphatidylserine (PS), phosphatidylcholine (PC) and their mixtures. The membrane surface charge density was changed by either varying the PS/PC ratio or by changing the degree of PS headgroup ionization in the range of pH between 2.5 and 7.5. The Gouy-Chapman-Stern (GCS) theory combined with the condition of mass balance in the experimental cell was used for quantitative treatment of ion adsorption and related changes in the diffuse part of the electrical double layer (surface potential). Data obtained using microelectrophoresis of liposome suspensions were well described within the framework of the modified GCS theory with constants of 5.10(4) and 10(3) M-1 for Gd3+ association with PS and PC, respectively (Yu. A. Ermakov, A. Z. Averbakh, and S. I. Sukharev, Biol. Membrany 14:434-445 (1997) (in Russian)). The intramembrane field compensation (IFC) technique used to study Gd3+ adsorption on planar lipid bilayers by monitoring the entire boundary potential gave completely different results. An observed drastic difference (approximately 140 mV) between the changes of boundary and surface potential was interpreted as the change in the dipole potential induced by binding of Gd3+. The magnitude of the surface dipole increased with the concentration of PS in PS/PC mixtures and became significant at most negative surface charges (more than 80% of PS in the mixture) and strongly correlated with the degree of PS ionization at different pH. The nature of structural changes at the membrane/water interface induced by Gd(3+)-PS interaction and possible lipid clusterization are discussed in the context of their biological importance.

Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin.

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, kon = 10(11) M-1 s-1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, kon is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (koff) is approximately 10 s-1 (lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca2+-calmodulin (Ca2+.CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 micrM Ca2+.CaM. Our results suggest that Ca2+.CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca2+-calmodulin could rapidly release phosphatidylinositol 4, 5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175).

Pore kinetics reflected in the dequenching of a lipid vesicle entrapped fluorescent dye.

Pore formation in lipid vesicle membranes can be monitored by the fluorescence signal F(t) arising from the induced release of a self-quenching dye in the course of the elapsed efflux time t. We present a basic theoretical analysis of pertinent experimental data allowing the quantitative evaluation of information on the pore kinetics and mechanism. This implies an investigation of the 'dynamic' quenching factor Qt exhibited by that fraction of dye which is still being retained inside the liposomes at t. It is shown how Qt depends on the mode of release which could be 'all-or-none' or more gradual as expressed by a parameter rho < or = 1 (related to the pore lifetime), i.e., the average dye retention factor in a vesicle after a single pore opening. A fit to measured values of Qt at a sufficient extent of efflux may be applied in order to determine rho. Then the pore formation rate per liposome, va(t), can be derived from the registered F(t). We give a practical demonstration of the procedures with carboxyfluorescein-loaded phosphatidylcholine liposomes of two different sizes to which the wasp venom peptide mastoparan X had been added.

On the validity of lipid dequenching assays for estimating virus fusion kinetics.

Octadecylrhodamine (R18) has often been used to measure membrane fusion of enveloped viruses by fluorescence dequenching. In order to see whether non-specific R18 exchange between non-fused membranes occurs we have measured fusion of influenza virus with erythrocyte membranes by utilizing dequenching of the non-exchangeable lipid analogue N-(lissamine-rhodamine B-sulfonyl)diacylphosphatidylethanolamine (N-Rh-PE). Rather low concentration of N-Rh-PE (< 0.1 mol%) were required to assess fusion since self-quenching in the influenza virus membrane was more efficient in comparison to R18. For both markers we observed the same kinetics as well as the same extent of fluorescence dequenching upon triggering low pH-induced fusion. Non-specific marker transfer was not observed. Haemolysis was not affected by either type of fluorophore. Our results confirm that R18 is a valuable tool to investigate membrane fusion of enveloped viruses in a quantitative manner. Differences in the efficiency of self-quenching of both markers are discussed.