The electrostatic contribution to spontaneous membrane curvature is calculated within Poisson-Boltzmann theory under a variety of assumptions and emphasizing parameters in the physiological range. Asymmetric surface charges, either fixed with respect to bilayer midplane area, or with respect to the lipid-water area both induce curvature but of opposite sign. Unequal screening layers on the two sides of a vesicle (e.g. multivalent cationic proteins on one side and monovalent salt on the other) also induce bending. For reasonable parameters, tubules formed by electrostatically induced bending can have radii in the 50-100nm range, often seen in many intracellular organelles. Thus membrane associated proteins may induce curvature and subsequent budding, without themselves being intrinsically curved. Furthermore, we derive the previously unexplored effects of respecting the strict conservation of charge within the interior of a vesicle. The electrostatic component to the bending modulus is small under most of our conditions, and is left as an experimental parameter. The large parameter space of conditions is surveyed in an array of graphs.
[C21.02] Kinematics of Lipid Bilayer Adsorption Measured With a QCM
C. A. Keller, F. Höök, B. Kasemo (Chalmers University of Technology and Göteborg University)
We have performed measurements of the kinetics of formation of supported lipid bilayers from small unilamellar vesicles using a quartz crystal microbalance (QCM). Both the shift in resonant frequency and the change in energy dissipation have been monitored during bilayer formation. These parameters correspond, respectively, to adsorbed mass and to the structure and mechanical properties of the adsorbed layer during and after formation. The adsorption measured in this manner appears to be a two phase process.
Simultaneous frequency and dissipation measurements provide a two parameter signature that can be used to identify different adsorption processes. Based on our results we will discuss the ability of the QCM measurements to provide quantitative information about the kinetics of formation and the properties of the adsorbed layer.
[C21.03] X-Ray Synchrotron and Neutron Reflectivity Studies of = Polymer-Modified Lipid Monolayers on Water
G.S. Smith, J. Majewski (MLNSC, Los Alamos National Laboratory), T. Kuhl, J. Israelachvili (Department of Chemical and Nuclear Engineering, UCSB), K. Kjaer, M.C. Gerstenberg (Department of Solid State Physics, Risoe National = Laboratory, Denmark), J. Als-Nielsen (Niels Bohr Institute, Copenhagen University, Denmark)
We studied monolayers (at air-water interface) composed of mixtures of distearoyl phosphatidyl ethanolamine (DSPE) mixed with 1.3, 4.5 and 9% of the same lipid but modified by polyethylene glycol chains (PEG) covalently linked to its head group. The GID data yielded three reflections leading to a hexagonal unit cell a_H=4.7Åarea per lipid molecule = 38.3Åindependent of PEG concentration. The in-plane coherence lengths decreased from 360Åfor the pure lipid to 230Åfor 9.0% DSPE-PEG. The FWHM(q_z) of each of the Bragg rods increased with PEG-lipid concentration suggesting decreasing of the lengths of the coherently diffracting part of the hydrocarbon chains. Reflectivities show that both the density and the extension of the polymer segments increase with DSPE-PEG concentration and can be well modeled with a parabolic density profile. Our data indicates that the bulky hydrophilic polymer disrupts the lipid monolayer. This is attributed to an increase in lipid protrusions and a relaxation of the lateral force between PEG portions by staggering of the lipid headgroups.
[C21.04] Membrane Active Peptides: Modes-of-Action, Phase Transitions, and Supramolecular Assemblies
Huey W. Huang (Physics Department, Rice University, Houston, TX 77005)
Recent discoveries showed that 20-40 amino-acid peptides are used as very effective antimicrobials in the host-defense systems throughout the animal kingdom, including human. What distinguishes these peptide antimicrobials from the conventional antibiotics is that they attack the lipid matrix of the cytoplasmic membranes rather than protein targets. So the central questions are what are their modes-of-action and how do their distinguish the bacterial cell membranes from the host-cell membranes? Many of these peptides have very simple structures--they are helices with amphiphilic side chains. Despite such simplicities, their interactions with membranes are complex and interesting. The talk will discuss the optical, x-ray and neutron techniques for studying such systems and the experimental as well as theoretical results. The mode-of-action is a phase transition controlled by the concentration of the peptide bound to the membrane. The specificities with respect to membranes are achieved by having different critical concentrations for different lipid composistions of the membrane.
[C21.05] Complexes of Negatively Charged Polypeptides with Cationic Lipids
G Subramanian, Youli Li, Cyrus R Safinya (Materials and Physics Departments, Materials Research Laboratory, University of California Santa Barbara)
Complexes of cationic lipids with oppositely charged proteins are promising candidates for new biomolecular materials. In addition to being used as a direct vehicle for protein transfection, they also find applications as templates for synthesis of molecular sieves. In spite of these wide ranging applications, the structure and interactions in these complexes have largely remained unclear. Here we report on the study of complexes formed between the cationic lipid didodecyldimethylammonium bromide (DDAB) with negatively charged polypeptide poly glutamic acid (PGA) both in the presence and absence of the neutral lipid dilauroylglycerophosphocholine (DLPC). X-ray diffraction of the complexes indicates a condensed lamellar lipid structure with the polypeptide intercalated between the layers. We present a comprehensive phase diagram on this system based on X-ray diffraction data.
This work is supported in part by grants NSF DMR-9624091, PRF-31352 AC7, and CU LAR STP/UC 96-118.
[C21.06] The Interactions between SP-B Protein and Anionic Lipids Found in Human Lung Surfactant
Ka Yee C. Lee, Michael M. Lipp (), Joseph A. Zasadzinski (Dept. of Chemical Engineering, UC Santa Barbara), Alan J. Waring (Harbor-UCLA, Research & Educational Institute)
Several lung pathologies, including neonatal respiratory distress syndrome, are characterized by a failure of the lung surfactant (LS) system to function properly. Utilizing fluorescence and Brewster angle microscopy, we have investigated the phase behavior of monolayers of binary mixtures of anionic lipids found in LS (palmitic acid, and both saturated and unsaturated phosphatidylglycerol) with both the full length SP-B protein and a shorter, 25-amino acid sequence based on its amino terminus. We found that both protein candidates interact specifically yet differently with each of the lipid components, altering their phase behavior to resemble more closely to that of an ideal LS monolayer. The SP-B protein incorporates itself in the lipid monolayer in all cases, and partitions preferentially into the fluid-type phases during phase transitions; its presence drastically changes the collapse mechanism of the monolayer.
[C21.07] The In-Situ Structure of Cationic Lipid/DNA Complexes in Animal Cells: Applications to Gene Therapy
Alison J. Lin, Nelle L. Slack, S.H.J. Idziak, C. X. George, C. E. Samuel, C. R. Safinya (University of California, Santa Barbara)
Gene therapy has been the focus of many recent investigations. One promising technique is to use cationic lipids as vectors for DNA transfection. However, the exact mechanism of DNA uptake is unknown, due to a lack of knowledge regarding interactions and structures of DNA and cationic lipids. We are developing x-ray and optical microscopy techniques to directly image the temporal and spatial distribution of cationic lipid/DNA complexes (CL-DNA) during the various stages of transfection in mouse L-cells. The structure of these complexes in water have been shown by x-ray studies to consist of alternating lipid bilayers and DNA monolayers.(J. Radler, I. Koltover, T. Salditt, C. R. Safinya, Science (January 1997)) We demonstrate the feasibility of in-situ x-ray diffraction studies of CL-DNA complexes in L-cells. The x-ray data implies that complexes are taken up by endocytosis and DOPE destabilizes the endosomal membrane. Results from optical microscopy studies and X-Gal staining of transfected cells support the x-ray data. Funded in part by NSF grant DMR-9624091, PRF (No. 31352-AC7), Los Alamos CULAR grant No. STB/UC: 96-118.
[C21.08] Direct Measurements of Long-Range Repulsive Interactions in the L_\alpha phase of Polymer-Coated Highly Flexible Membranes
Heidi E. Warriner (Physics Department, UCSB), Cyrus R. Safinya (Materials and Physics Departments, UCSB)
Using two complimentary techniques, we have measured repulsive interactions in the L_\alpha phase of very flexible membranes composed of the surfactant C12E5 and small amounts of polymer-lipids derived from polyethylene glycol (PEG-DMPE 5000, PEG-DMPE 2000 and PEG-DMPE 550). In the first method, the lamellar repeat distance of samples in equilibrium with a dextran solution of known osmotic pressure is determined, yielding a direct measurement of pressure versus distance. These data immediately differentiate the repulsive interaction between flexible polymer-decorated membranes from polymer-brush forces found in rigid lamellar systems. In the second method, fits to high-resolution x-ray data yield the \eta parameter, proportional to (\kappaB)^-1øver2, where B is the layer compressional modulus and \kappa is the bending rigidity of a single membrane. Combining the two types of data to eliminate B, one can quantitatively determine the \kappa of a decorated membrane as a function of
polymer-lipid concentration.
For the bare C12E5 membrane, where \kappa is known , a direct comparison of the compressibility modulus values derived via the two methods is also possible. This work supported by NSF-DMR-9624091; PRF-31352-AC7 CULAR-STB/UC:96-118.
[C21.09] Spontaneously Broken Symmetry in Membrane/Rod Mixtures
Carlos Marques (R.P.-C.N.R.S., Complex Fluid Laboratory, Cranbury, NJ 08512-7500), K. Yaman, P. Pincus (Department of Physics, University of California, Santa Barbara, California 93106--9530)
We study the contribution of the the depletion layer of rod or disk solutions to the bending elastic constants of membranes. A spontaneous curvature can be obtained when a membrane is immersed in a rod or disk solution. We also study the confinement of rods in spherical and cylindrical repulsive shells, and the depletion force between two colloidal spheres in a rod solution.
[C21.10] Reorientational Motions in the Photocycles of Wild-Type and Mutant Bacteriorhodopsin
Carey K. Johnson, Greg S. Harms (University of Kansas)
The reorientational motions of bacteriorhodopsin in the purple membrane have been investigated by time resolved linear dichroism. The anisotropies of the intermediates decrease to a minimum in the M-state and recover in the O-state, demonstrating reversible and non-diffusive reorientation. The anisotropies are 0.40\pm0.01 for the initial ground state, 0.38\pm0.01 for the K-state, 0.35\pm0.01 for the L-state, 0.32\pm0.01 for the M-state, and 0.39\pm0.01 for the O-state. These values indicate reorientation by 15^\circ to 20^\circ in the M-state relative to the ground state. Measurements in oriented samples show that this motion is predominately toward the membrane normal. The reorientations were found to depend on pH and ionic strength, suggesting correlation of the M-state reorientation with proton release. The reorientations also depend on viscosity, indicating restricted reorientation with increased viscosity. The decay of the anisotropy of ground-state bacteriorhodopsin can be explained by reorientations of unexcited spectator proteins by approximately 7^\circ. Similar reorientational motions occur for the D96N and T46V/D96N mutants of bacteriorhodopsin in purple membrane. Anisotropy measurements of T46V/D96N have allowed the N-state anisotropy to be estimated at 0.30\pm0.05.
[C21.11] Calculation of Lyotropic Phases of Biological Lipids
Michael Schick, Marcus Müller (University of Washington)
We have calculated the phase diagrams of monoacylglycerol/water systems employing a simple model in which the hydrocarbon chain is treated within the rotational isomeric state framework, and the head group as rigid. Interactions between tail segments and head, and between tail segments and water, are identical and characterized by a single parameter. The volumes of the head group and of the chain segments relative to that of water comprise the other two parameters. The system is incompressible. We employ mean-field theory, and use a partial enumeration scheme to evaluate the partition function. The calculated phase diagram displays lamellar, inverted hexagonal, and Ia3d cubic phases. Variation of the transition between lamellar and inverted hexagonal phases with chain length and head group architecture is in qualitative agreement with experiment. Detailed composition profiles are also obtained.
[C21.12] Melting of Two-Dimensional Crystals of Membrane-Protein Bacteriorhodopsin.
I. Koltover, T. Salditt, J.O. Rädler, C.R. Safinya (UCSB), K.J. Rothschild (Boston University)
Bacteriorhodopsin (bR), a light-driven proton pump, is an integral membrane protein of bacterium Halobacterium Salinarium. In the native bacterial membrane bR self-assembles into regular hexagonal crystalline arrays in the plane of membrane. This two-dimensional (2D) protein crystal undergoes a fully reversible melting transition as a function of temperature. We have conducted a synchrotron x-ray diffraction study of oriented multilayers of bR-containing native bacterial membrane patches and of of novel giant (50\mum) single-crystal fused bR membranes. The precise in-situ control of humidity and sample temperature combined with the line-shape analyses allowed us to elucidate and control the protein-protein interactions. The important findings are as follows. First, the ordered 2D self-assembled lattice of proteins exhibits diffraction patterns characteristic of a 2D solid with power-law decay of in-plane positional correlations, which allows to measure the elastic constants of protein crystal. Second, the melting temperature is a function of the multilayer hydration, with two distinct regimes of melting. We discuss the models of interactions in bR multilayers consistent with our data. Supported by NSF grant DMR-962091, and the Petroleum Research Fund (No.31352-AC7).
[C21.13] X-ray scattering from a highly aligned Bacteriorhodopsin multilayer
T. Salditt, I. Koltover, C.R. Safinya (Materials and Physics Depts., UCSB)
The purple membrane of the achebacteria Halobacterium salinarium with its native photoactive protein bacteriorhodopsin (bR) was investigated by x-ray reflectivity, nonspecular scattering, and truncation rod scattering. The application of these scattering techniques has become possible after the preparation of giant fused-patch single crystal domains (\simeq 50 \mum). These patches orient on Si-substrates with a mosaicity of less than 4/100 ^o. Experiments have been carried out on multilayer samples of about 10 periods at different hydration levels. The nonspecular (diffuse scattering) indicates a membrane corrugation on lateral length scale of 400Å\ that is conformaly replicated through the stack. The results of this first investigation and future prospects of this novel technique for the study of membrane proteins are discussed. Work supported in part by NSF grant DMR-9624091, the Petroleum research Fund (No.31352-AC7), a Los Alamos CULAR grant No.STB/UC:95-146, and a DAAD fellowship to T.S.