Single Molecule Probes of Lipid Membrane Structure

Biological membranes are highly heterogeneous structures that are thought to use this heterogeneity to organize and modify the function of membrane constituents. Probing membrane organization, structure, and changes therein are crucial for linking structural metrics with function in biological membranes. Single-molecule fluorescence studies were used to measure membrane structure at the molecular level. Several groups have shown that polarized total internal reflection fluorescence microscopy (PTIRF-M) using p-polarized excitation can reveal single-molecule orientations when spherical aberrations are introduced into the optics train. This approach was used here to measure the orientation of fluorescent lipid analogs doped into Langmuir-Blodgett and bilayer films of DPPC and DPPC/sterol mixed monolayers. Two commonly used fluorescent lipid analogs, BODIPY-PC and DiIC18 which have their fluorophores located in the tailgroup and headgroup, respectively were used and a variety of other probes are currently being studied. It was found that the tilt orientation of BODIPY-PC is very sensitive to the surface pressure at which the DPPC films are transferred onto the substrate. At low surface pressures, the tailgroups are largely lying in the plane of the film and evolve to an orientation normal to the surface as pressure is increased. For DiIC18, however, no evolution in orientation with surface pressure is observed which is consistent with the headgroup located fluorophore being less sensitive to changes in membrane packing. The monolayer / bilayer "equivalent surface pressure" was also found to be ~23 mN/m by directly comparing the molecular structure in the two films. Using this information, the condensing affect of cholesterol and other biologically relevant sterols on monolayers and bilayers at the equivalent surface pressure was studied. Molecular dynamics simulations were also compared with the experimental results to probe the insertion of BODIPY-PC into membrane lipids

Single molecule probes of membrane structure: Orientation of BODIPY probes in DPPC as a function of probe structure

Single molecule fluorescence measurements have recently been used to probe the orientation of fluorescent lipid analogs doped into lipid films at trace levels. Using defocused polarized total internal reflection fluorescence microscopy (PTIRF-M), these studies have shown that fluorophore orientation responds to changes in membrane surface pressure and composition, providing a molecular level marker of membrane structure. Here we extend those studies by characterizing the single molecule orientations of six related BODIPY probes doped into monolayers of DPPC. Langmuir–Blodgett monolayers transferred at various surface pressures are used to compare the response from fluorescent lipid analogs in which the location of the BODIPY probe is varied along the length of the acyl chain. For each BODIPY probe location along the chain, comparisons are made between analogs containing phosphocholine and smaller fatty acid headgroups. Together these studies show a general propensity of the BODIPY analogs to insert into membranes with the BODIPY probe aligned along the acyl chains or looped back to interact with the headgroups. For all BODIPY probes studied, a bimodal orientation distribution is observed which is sensitive to surface pressure, with the population of BODIPY probes aligned along the acyl chains increasing with elevated surface pressure. Trends in the single molecule orientations for the six analogs reveal a configuration where optimal placement of the BODIPY probe within the acyl chain maximizes its sensitivity to the surrounding membrane structure. These results are discussed in terms of balancing the effects of headgroup association with acyl chain length in designing the optimal placement of the BODIPY probe

Fuming Method for Micropatterning Structures on Langmuir- Blodgett Films

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Langmuir, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/la804104k.Lipid monolayers of L-α-dipalmitoylphosphatidylcholine (DPPC) are used to pattern substrates using the Langmuir-Blodgett (LB) technique. Lipid monolayers are deposited onto a freshly cleaved mica surface or glass capillary under conditions that lead to distinct patterns in the film. Exposure of the supported monolayer to ethyl 2-cyanoacrylate fumes leads to preferential polymerization in the more hydrated regions of the patterned monolayer. This method enables surfaces to be micropatterned where the lateral features are controlled by the structure present in the underlying LB film and the vertical feature size is controlled by the length of the fuming process. Atomic force microscopy (AFM) measurements confirm that the original structure in the LB film is preserved following fuming and that the lateral and vertical feature sizes can be controlled from nanometers to microns. This method, therefore, provides a rapid and versatile approach for micropatterning both flat and curved surfaces on a variety of substrates

Orientation of Fluorescent Lipid Analog BODIPY-PC to Probe Lipid Membrane Properties: Insights from Molecular Dynamics Simulations

Single-molecule fluorescence measurements have been used to characterize membrane properties, and recently showed a linear evolution of the fluorescent lipid analog BODIPY-PC towards small tilt angles in Langmuir-Blodgett monolayers as the lateral surface pressure is increased. In this work, we have performed comparative molecular dynamics (MD) simulations of BODIPY-PC in DPPC (dipalmitoylphosphatidylcholine) monolayers and bilayers at three surface pressures (3, 10, and 40 mN/m) to explore 1) the microscopic correspondence between monolayer and bilayer structures, 2) the fluorophore’s position within the membrane, and 3) the microscopic driving forces governing the fluorophore’s tilting. The MD simulations reveal very close agreement between the monolayer and bilayer systems in terms of the fluorophore’s orientation and lipid chain order, suggesting that monolayer experiments can be used to approximate bilayer systems. The simulations capture the trend of reduced tilt angle of the fluorophore with increasing surface pressure as seen in the experimental results, and provide detailed insights into fluorophore location and orientation, not obtainable in the experiments. The simulations also reveal that the enthalpic contribution is dominant at 40 mN/m resulting in smaller tilt angles of the fluorophore, and the entropy contribution is dominant at lower pressures resulting in larger tilt angles

The effects of starvation and alloxan diabetes on the intestinal transport of amino acids in adult male rats

The effects of total starvation for 1, 3, and 5 days on the absorption of L-alanlne, L-lyslne, L-methlonine and L-prollne were studied in adult male rats using an in vitro everted intestinal method. Active transport of these amino acids was increased significantly by fasting for 3 and 5 days. The effects of alloxan diabetes on the active transport of the same test amino acids were studied at 3, 8, 14, 30, 90 and 180 days post-alloxan using the same in vitro method. Absorption of all amino acids was enhanced in the short-term (3 day) and in the long-term (90 and 180 day) alloxan diabetics. Transport of these amino acids were below control values at 8, 14 and 30 days post-alloxan injection. This study confirmed previous published work that net transport of amino acids decreases with age in the normal rat. This study also confirms earlier published works on the effects of short-term alloxan diabetes and distinguishes a diabetic effect from a possible toxic effect of alloxan. This study confirms and extends previous work on morphological changes which occur and which may effect the transport of amino acids in the rat.Biology and Biochemistry, Department o

Peptide-Induced Domain Formation in Supported Lipid Bilayers: Direct Evidence by Combined Atomic Force and Polarized Total Internal Reflection Fluorescence Microscopy

Direct visualization of the mechanism(s) by which peptides induce localized changes to the structure of membranes has high potential for enabling understanding of the structure-function relationship in antimicrobial and cell-penetrating peptides. We have applied a combined imaging strategy to track the interaction of a model antimicrobial peptide, PFWRIRIRR-amide, with bacterial membrane-mimetic supported phospholipid bilayers comprised of POPE/TOCL. Our in situ studies revealed rapid reorganization of the POPE/TOCL membrane into localized TOCL-rich domains with a concomitant change in the organization of the membranes themselves, as reflected by changes in fluorescent-membrane-probe order parameter, upon introduction of the peptide