Mark W. Vaughn

Morphology and Amine Accessibility of (3-Aminopropyl) Triethoxysilane Films on Glass Surfaces

3-Aminopropyl) triethoxysilane (APTES) is commonly used to functionalize glass substrates because it can form an amine-reactive film that is tightly attached to the surface. In this study, we investigated the morphology and chemical reactivity of APTES films prepared on glass substrates using common deposition techniques. Films were prepared using concentrated vapor-phase deposition, dilute vapor-phase deposition, anhydrous organic-phase deposition and aqueous-phase deposition. All films were annealed, or cured, at 150 degrees C. The morphology of the films was quantified by fluorescence and by atomic force microscopy (AFM). The optical equivalent of the AFM images was computed and then used to directly compare optical and AFM images. Reactive amine density was determined by a picric acid assay and by a method that employed N-succinimidyl 3-[2-pyridyldithio]-propionamido (SPDP) cross-linked rhodamine. Fluorescence and AFM images showed that silane films prepared from dilute vapor-phase and aqueous-phase deposition were more uniform and had fewer domains than those deposited by the other methods. The ratio of picric acid-accessible amino groups to SPDP cross-linked rhodamine-accessible groups varied with the preparation method, suggesting reactant size-dependent difference in amine accessibility. We found a larger number of accessible amino groups on films prepared by vapor-phase deposition than on those prepared from solution deposition. The dilute vapor-phase deposition technique produced relatively few domains, and it should be a good choice for bioconjugation applications. There were appreciable differences in the films produced by each method. We suggest that these differences originate from differences in film rearrangement during annealing.

Cholesterol Modulates the Interaction of β-Amyloid Peptide with Lipid Bilayers

The interaction of an amphiphilic, 40-amino acid beta-amyloid (Abeta) peptide with liposomal membranes as a function of sterol mole fraction (X(sterol)) was studied based on the fluorescence anisotropy of a site-specific membrane sterol probe, dehydroergosterol (DHE), and fluorescence resonance energy transfer (FRET) from the native Tyr-10 residue of Abeta to DHE. Without Abeta, peaks or kinks in the DHE anisotropy versus X(sterol) plot were detected at X(sterol) approximately 0.25, 0.33, and 0.53. Monomeric Abeta preserved these peaks/kinks, but oligomeric Abeta suppressed them and created a new DHE anisotropy peak at X(sterol) approximately 0.38. The above critical X(sterol) values coincide favorably with the superlattice compositions predicted by the cholesterol superlattice model, suggesting that membrane cholesterol tends to adopt a regular lateral arrangement, or domain formation, in the lipid bilayers. For FRET, a peak was also detected at X(sterol) approximately 0.38 for both monomeric and oligomeric Abeta, implying increased penetration of Abeta into the lipid bilayer at this sterol mole fraction. We conclude that the interaction of Abeta with membranes is affected by the lateral organization of cholesterol, and hypothesize that the formation of an oligomeric Abeta/cholesterol domain complex may be linked to the toxicity of Abeta in neuronal membranes.

Molecular dynamics simulations reveal the protective role of cholesterol in β-amyloid protein-induced membrane disruptions in neuronal membrane mimics.

Interactions of β-amyloid (Aβ) peptides with neuronal membranes have been associated with the pathogenesis of Alzheimers disease (AD); however, the molecular details remain unclear. We used atomistic molecular dynamics (MD) simulations to study the interactions of Aβ(40) and Aβ(42) with model neuronal membranes. The differences between cholesterol-enriched and depleted lipid domains were investigated by the use of model phosphatidylcholine (PC) lipid bilayers with and without 40 mol % cholesterol. A total of 16 independent 200 ns simulation replicates were investigated. The surface area per lipid, bilayer thickness, water permeability barrier, and lipid order parameter, which are sensitive indicators of membrane disruption, were significantly altered by the inserted state of the protein. We conclude that cholesterol protects Aβ-induced membrane disruption and inhibits β-sheet formation of Aβ on the lipid bilayer. The latter could represent a two-dimensional (2D) seeding template for the formation of toxic oligomeric Aβ in the pathogenesis of AD.

Evidence of meniscus interface transport in dip-pen nanolithography: An annular diffusion model

Ring shaped dots were patterned with mercaptohexadecanoic acid ink by dip-pen nanolithography. These dots have an ink-free inner core surrounded by an inked annular region, making them different from the filled dots usually obtained. This suggests a different transport mechanism than the current hypothesis of bulk water meniscus transport. A meniscus interface ink transport model is proposed, and its general applicability is demonstrated by predicting the patterned dot radii of chemically diverse inks.

Cell Detachment Model for an Antibody-Based Microfluidic Cancer Screening System

We consider cells bound to the floor of a microfluidic channel and present a model of their flow‐induced detachment. We approximate hydrodynamic force and cell elastic response using static finite‐element simulation of a single cell. Detachment is assumed to occur when hydrodynamic and adhesive forces are roughly equal. The result is extended to multiple cells at the device level using a sigmoidal curve fit. The model is applied to a microfluidic cancer‐screening device that discriminates between normal epithelial cells and cells infected with human papillomavirus (HPV), on the basis of increased expression of the transmembrane protein α6 integrin in the latter. Here, the cells to be tested are bound to a microchannel floor coated with anti α6 integrin antibodies. In an appropriate flow rate range, normal cells are washed away while HPV‐infected cells remain bound. The model allows interpolation between data points to choose the optimal flow rate and provides insight into interaction of cell mechanical properties and the flow‐induced detachment mechanism. Notably, the results suggest a significant influence of cell elastic response on detachment.

Small angle X-ray scattering analysis of the effect of cold compaction of Al/MoO3 thermite composites

Nanothermites composed of aluminum and molybdenum trioxide (MoO(3)) have a high energy density and are attractive energetic materials. To enhance the surface contact between the spherical Al nanoparticles and the sheet-like MoO(3) particles, the mixture can be cold-pressed into a pelleted composite. However, it was found that the burn rate of the pellets decreased as the density of the pellets increased, contrary to expectation. Ultra-small angle X-ray scattering (USAXS) data and scanning electron microscopy (SEM) were used to elucidate the internal structure of the Al nanoparticles, and nanoparticle aggregate in the composite. Results from both SEM imaging and USAXS analysis indicate that as the density of the pellet increased, a fraction of the Al nanoparticles are compressed into sintered aggregates. The sintered Al nanoparticles lost contrast after forming the larger aggregates and no longer scattered X-rays as individual particles. The sintered aggregates hinder the burn rate, since the Al nanoparticles that make them up can no longer diffuse freely as individual particles during combustion. Results suggest a qualitative relationship for the probability that nanoparticles will sinter, based on the particle sizes and the initial structure of their respective agglomerates, as characterized by the mass fractal dimension.

Scaling and Alpha-Helix Regulation of Protein Relaxation in a Lipid Bilayer

Protein conformation and orientation in the lipid membrane plays a key role in many cellular processes. Here we use molecular dynamics simulation to investigate the relaxation and C-terminus diffusion of a model helical peptide: beta-amyloid (Aβ) in a lipid membrane. We observed that after the helical peptide was initially half-embedded in the extracelluar leaflet of phosphatidylcholine (PC) or PC/cholesterol (PC/CHOL) membrane, the C-terminus diffused across the membrane and anchored to PC headgroups of the cytofacial lipid leaflet. In some cases, the membrane insertion domain of the Aβ was observed to partially unfold. Applying a sigmoidal fit to the process, we found that the characteristic velocity of the C-terminus, as it moved to its anchor site, scaled with θu (-4/3), where θu is the fraction of the original helix that was lost during a helix to coil transition. Comparing this scaling with that of bead-spring models of polymer relaxation suggests that the C-terminus velocity is highly regulated by the peptide helical content, but that it is independent of the amino acid type. The Aβ was stabilized by the attachment of the positive Lys28 side chain to the negative phosphate of PC or 3β oxygen of CHOL in the extracellular lipid leaflet and of the C-terminus to its anchor site in the cytofacial lipid leaflet.

Data supporting beta-amyloid dimer structural transitions and protein-lipid interactions on asymmetric lipid bilayer surfaces using MD simulations on experimentally derived NMR protein structures.

This data article supports the research article entitled “Maximally Asymmetric Transbilayer Distribution of Anionic Lipids Alters the Structure and interaction with Lipids of an Amyloidogenic Protein Dimer Bound to the Membrane Surface” [1]. We describe supporting data on the binding kinetics, time evolution of secondary structure, and residue-contact maps of a surface-absorbed beta-amyloid dimer protein on different membrane surfaces. We further demonstrate the sorting of annular and non-annular regions of the protein/lipid bilayer simulation systems, and the correlation of lipid-number mismatch and surface area per lipid mismatch of asymmetric lipid membranes.

Maximally asymmetric transbilayer distribution of anionic lipids alters the structure and interaction with lipids of an amyloidogenic protein dimer bound to the membrane surface.

We used molecular dynamics simulations to explore the effects of asymmetric transbilayer distribution of anionic phosphatidylserine (PS) lipids on the structure of a protein on the membrane surface and subsequent protein-lipid interactions. Our simulation systems consisted of an amyloidogenic, beta-sheet rich dimeric protein (D42) absorbed to the phosphatidylcholine (PC) leaflet, or protein-contact PC leaflet, of two membrane systems: a single-component PC bilayer and double PC/PS bilayers. The latter comprised of a stable but asymmetric transbilayer distribution of PS in the presence of counterions, with a 1-component PC leaflet coupled to a 1-component PS leaflet in each bilayer. The maximally asymmetric PC/PS bilayer had a non-zero transmembrane potential (TMP) difference and higher lipid order packing, whereas the symmetric PC bilayer had a zero TMP difference and lower lipid order packing under physiologically relevant conditions. Analysis of the adsorbed protein structures revealed weaker protein binding, more folding in the N-terminal domain, more aggregation of the N- and C-terminal domains and larger tilt angle of D42 on the PC leaflet surface of the PC/PS bilayer versus the PC bilayer. Also, analysis of protein-induced membrane structural disruption revealed more localized bilayer thinning in the PC/PS versus PC bilayer. Although the electric field profile in the non-protein-contact PS leaflet of the PC/PS bilayer differed significantly from that in the non-protein-contact PC leaflet of the PC bilayer, no significant difference in the electric field profile in the protein-contact PC leaflet of either bilayer was evident. We speculate that lipid packing has a larger effect on the surface adsorbed protein structure than the electric field for a maximally asymmetric PC/PS bilayer. Our results support the mechanism that the higher lipid packing in a lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions for a dimeric protein on membrane surfaces.

Characterization of 3D Voronoi tessellation nearest neighbor lipid shells provides atomistic lipid disruption profile of protein containing lipid membranes

Quantifying protein-induced lipid disruptions at the atomistic level is a challenging problem in membrane biophysics. Here we propose a novel 3D Voronoi tessellation nearest-atom-neighbor shell method to classify and characterize lipid domains into discrete concentric lipid shells surrounding membrane proteins in structurally heterogeneous lipid membranes. This method needs only the coordinates of the system and is independent of force fields and simulation conditions. As a proof-of-principle, we use this multiple lipid shell method to analyze the lipid disruption profiles of three simulated membrane systems: phosphatidylcholine, phosphatidylcholine/cholesterol, and beta-amyloid/phosphatidylcholine/cholesterol. We observed different atomic volume disruption mechanisms due to cholesterol and beta-amyloid. Additionally, several lipid fractional groups and lipid-interfacial water did not converge to their control values with increasing distance or shell order from the protein. This volume divergent behavior was confirmed by bilayer thickness and chain orientational order calculations. Our method can also be used to analyze high-resolution structural experimental data.