Session A8 - Focus Session: Neutron Scattering and Other Studies of Phospholipids.
FOCUS session, Monday morning, March 22
510A, Palais des Congres

[A8.001] Small-Angle Neutron Scattering and Spontaneous Formation of Unilamellar Vesicles: Potential Vehicles for Drug Delivery

Unilamellar vesicles (ULVs) are single-bilayer shells with radii commonly between 10 and 100 nm, and are widely used as model membranes, drug delivery systems, microreactors and substrates for a variety of enzymes and proteins. A common method of making ULVs is the extrusion of multilamellar vesicles (MLVs) through synthetic membranes of known pore size. These extruded ULVs are invariably unstable and in due time, revert back to MLVs.

Over the years there have been reports of the spontaneous formation of stable ULVs in surfactant, lipid, and lipid/detergent mixtures. These ULVs have sometimes been shown to be monodisperse and their radii were found, almost without exception, to vary with concentration. We have carried-out small-angle neutron scattering (SANS) experiments on a biomimetic system composed of the phospholipids dimyristoyl and dihexanoyl phosphorylcholine (DMPC and DHPC, respectively). Doping DMPC/DHPC multilamellar vesicles with either the negatively charged lipid dimyristoyl phosphorylglycerol (DMPG, net charge -1) or the divalent cation, calcium (Ca2+) leads to the spontaneous formation of monodisperse unilamellar vesicles whose radii are concentration independent, in contrast to previous experimental observations.

[A8.002] Transition Temperatures of Phospholipid Bilayers Under Pressure

Near the main liquid-crystalline to gel transition of smectic stacks of phospholipids in excess water, the inter-layer distance increases in a critical-like manner. This so-called anomalous swelling phenomenon has been attributed to a hidden unbinding transition of the stack due to density fluctuations of the chains. We have studied the influence of hydrostatic pressure on anomalous swelling in phosphocholine bilayers using neutron diffraction and a specially constructed pressure cell, as a function of temperature, pressure and acyl-chain length. The power law behavior of anomalous swelling is preserved up to 240 MPa, and for PC lipids with hydrocarbon chains more than 12 carbons, the theoretical unbinding transition temperature is coupled to the main transition temperature. Lipids with shorter chains exhibit a qualitatively different trend, with the difference between these two transition temperatures decreasing with increasing pressure. We predict that at high hydrostatic pressure, complete unbinding may occur for the smaller chain lipids.

[A8.003] Structural Phase Behavior of Model Biomimetic Membranes -- ``Bicelles''

It has been shown that concentrated (> 5 wt.%) aqueous solutions of phospholipid mixtures composed of long- (14-C phosphatidylcholine) and short-chain (6-C phosphatidylcholine) phosphorylcholines, align in the presence of an applied magnetic field. These ``alignable substrates'', also known as ``bicelle'' mixtures, have been used to study the structure of membrane associated proteins and biomolecules. We observe an isotropic-to-nematic (I \rightarrow N) and a nematic-to-smectic (N \rightarrow S) transition with increasing temperature. The I \rightarrow N transition generally occurs around 20 ^oC, whereas the N \rightarrow S transition is strongly dependant on the charge density of the system, introduced by doping with the negatively charged lipid, dimyristoyl phosphatidylglycerol (DMPG). Moreover, neutron diffraction results show that a weak shear can be used to align the system, instead of a magnetic field.

[A8.004] Monte Carlo simulation of lipid bicelle phase behavior

We use a novel mesoscale Monte Carlo method to simulate a simple model of self-assembled mixed-lipid disks or "bicelles." We will discuss results from these simulations, including volume fractions, order parameters, and disk size distributions of coexisting isotropic and nematic phases. We will also discuss simulations in which the disks are allowed to include pores, and the insights these results offer into the morphological transition from disks to perforated sheets or other extended structures that have been proposed to explain experimental results in nematic bicelle phases.

[A8.005] Neutron Scattering: A Powerful and Versatile Methodology for Research on Bio, Soft and Nanophase Materials

\huge This abstract was not submitted electronically.

[A8.006] Phase-sensitive Neutron Reflectometry Studies of a Biomineralization Peptide

A phase-sensitive neutron reflectometry (NR) method was used to study the surface orientation of the 59-amino acid residue biomineralization peptide, LRAP, which is a variant of amelogenin, a protein that controls tooth enamel development. Phase-sensitive NR methods provide an essentially exact determination of the neutron scattering length density (SLD) profile perpendicular to the plane of the surface, limited only by sample integrity and the quality of the data. Peptides for the NR measurements were prepared with seven amino acids near the C-terminus deuterated in order to increase their neutron SLD with respect to the unlabeled residues. The peptide was bound to an 11Åthick C11-ethylene oxide layer with a COO- group presented at the surface. The entire system was supported on planar, gold-coated substrates and measured in a humidity chamber filled with Argon gas saturated with D_2O at 92% humidity. The phase-sensitive NR method used to determine the orientation of the LRAP peptide on this surface will be described and the resultant compositional depth profiles, derived from the SLD profiles, will be presented.

[A8.007] Single-Residue Sensitivity in Neutron Reflectivity and Resonant X-ray Reflectivity from Langmuir Monolayers of Synthetic Peptides

Labeling groups with ^2H to distinguish them in the scattering length density (SLD)

profile constitutes the chief advantage of neutron reflectivity (NR) in studying Langmuir

monolayers (LM) of lipids and proteins. Solid phase synthesis (SPPS) permits the labeling of a single residue in a peptide. Recent work demonstrates the sensitivity of NR to single ^2H-labeled residues in LM of vectorially oriented \alpha -helical bundle peptides. NR requires comparison of isomorphic samples of all-^1H and ^2H-labeled peptides. Alternately, resonant x-ray reflectivity (RXR) uses only one sample. RXR exploits energy-dependent changes in the scattering factor from heavy atoms to distinguish them within the SLD profile. Peptides may be labeled by SPPS (e.g. Br-Phe), or may have \textitinherent labels (e.g. Fe in heme proteins). As test cases, we studied LM of Br-labeled lipids and peptides with RXR. Both approaches require a model-independent means of obtaining SLD profiles from the reflectivity data. We have applied box-refinement to obtain the gradient SLD profile. This is fit uniquely with a sum of Gaussians and integrated analytically [Blasie \textitet al., PRB \textbf67 224201 (2003)] to provide the SLD profile. Label positions can then be determined to sub-Ångstrom accuracy.

This work supported by the NIH (GM55876).

[A8.008] X-ray reflectivity study of in situ polymerized phospholipid Monolayers

Supported phospholipid membranes (lipid bilayers and monolayers) on solids have proven to be useful model surfaces for the characterization of many important biological processes. Recently, we have reported a method of preparing a surface-grafted phospholipid monolayer using in situ polymerization at the interface between a mono-acrylated lipid monolayer and a methacryloyl-terminated surface. As confirmed by studies on water contact angles, atomic force microscopy, and X-ray photoelectron spectroscopy, the polymerized phospholipid monolayer (poly-PC) was chemically bonded to a solid in a substantial manner and it was very stable to organic solvents. The reduced adsorption of proteins and macrophase on the poly-PC were clearly observed by in vivo and in vitro experiment, respectively. For the potential application of a surface-grafted phospholipid monolayer, it is crucial to determine the detailed physical structure of the lipid monolayer grafted on solids. The studies are therefore concerned with a depth profiling measurement that characterizes the surface properties of bare and PEG covered phospholipid surfaces before and after in situ polymerization, where demonstrates that in situ polymerization techniques can produce a stabilized phospholipid monolayer surface. Part of this work was supported by the Korea Research Foundation.

[A8.009] Collapse of Langmuir Monolayers Spread on Calcium Solutions: A hydrophobic dimer at the air-water interface

Surface X-ray scattering experiments of collapsed-Langmuir monolayers of arachidic acid (AA) that are spread on CaCl2 solutions reveal that during the collapse process, calcium is ejected from the subphase together with the amphiphile, forming a object where two head-groups of two amphiphiles are bridged by a calcium ion, i.e., linear dimer is formed at the interface. The X-ray reflectivity measurements indicate that the hydrophobic tail of the Ca-induced dimerized AA is in contact with water. Theoretical arguments as well as the status of some follow up experiments will be presented.

[A8.010] Dynamics of Pinned Biological Membranes

We present a theoretical treatment and simulation algorithm for the dynamics of biological membranes. For our purposes, the lipid bilayer is treated as a continuous Helfrich elastic surface in the presence of hydrodynamic coupling to the surrounding solvent. We can additionally add general harmonic perturbations, which, in the limit of localized and strong interactions, can be used to pin the membrane to intracellular/intercellular structures. We consider the case of pinning to the cytoskeleton and use such a model to estimate the macroscopic diffusion constant for band 3 protein on the surface of human erythrocytes. Comparison to experimental results suggests that thermal undulations of the membrane surface should play a significant role in protein mobility on the red blood cell.

Part A of program listing