Daniel A. Bricarello

Reconstituted lipoprotein: a versatile class of biologically-inspired nanostructures.

One of biologys most pervasive nanostructures, the phospholipid membrane, represents an ideal scaffold for a host of nanotechnology applications. Whether engineering biomimetic technologies or designing therapies to interface with the cell, this adaptable membrane can provide the necessary molecular-level control of membrane-anchored proteins, glycopeptides, and glycolipids. If appropriately prepared, these components can replicate in vitro or influence in vivo essential living processes such as signal transduction, mass transport, and chemical or energy conversion. To satisfy these requirements, a lipid-based, synthetic nanoscale architecture with molecular-level tunability is needed. In this regard, discrete lipid particles, including reconstituted high density lipoprotein (HDL), have emerged as a versatile and elegant solution. Structurally diverse, native biological HDLs exist as discoidal lipid bilayers of 5-8 nm diameter and lipid monolayer-coated spheres 10-15 nm in diameter, all belted by a robust scaffolding protein. These supramolecular assemblies can be reconstituted using simple self-assembly methods to incorporate a broad range of amphipathic molecular constituents, natural or artificial, and provide a generic platform for stabilization and transport of amphipathic and hydrophobic elements capable of docking with targets at biological or inorganic surfaces. In conjunction with top-down or bottom-up engineering approaches, synthetic HDL can be designed, arrayed, and manipulated for a host of applications including biochemical analyses and fundamental studies of molecular structure. Also highly biocompatible, these assemblies are suitable for medical diagnostics and therapeutics. The collection of efforts reviewed here focuses on laboratory methods by which synthetic HDLs are produced, the advantages conferred by their nanoscopic dimension, and current and emerging applications.

A differential association of Apolipoprotein E isoforms with the amyloid‐β oligomer in solution

The molecular pathogenesis of disorders arising from protein misfolding and aggregation is difficult to elucidate, involving a complex ensemble of intermediates, whose toxicity depends upon their state of progression along distinct processing pathways. To address the complex misfolding and aggregation that initiates the toxic cascade resulting in Alzheimers disease (AD), we have developed a 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid spin‐labeled amyloid‐β (Aβ) peptide to observe its isoform‐dependent interaction with the apoE protein. Although most individuals carry the E3 isoform of apoE, ∼15% of humans carry the E4 isoform, which is recognized as the most significant genetic determinant for Alzheimers. ApoE is consistently associated with the amyloid plaque marker for AD. A vital question centers on the influence of the two predominant isoforms, E3 and E4, on Aβ peptide processing and hence Aβ toxicity. We used electron paramagnetic resonance (EPR) spectroscopy of incorporated spin labels to investigate the interaction of apoE with the toxic oligomeric species of Aβ in solution. EPR spectra of the spin‐labeled side chain report on side chain and backbone dynamics as well as the spatial proximity of spins in an assembly. Our results indicate oligomer binding involves the C‐terminal domain of apoE, with apoE3 reporting a much greater response through this conformational marker. Coupled with SPR binding measurements, apoE3 displays a higher affinity and capacity for the toxic Aβ oligomer. These findings support the hypothesis that apoE polymorphism and Alzheimers risk can largely be attributed to the reduced ability of apoE4 to function as a clearance vehicle for the toxic form of Aβ. Proteins 2011.

pH Responsive Polymer Cushions for Probing Membrane Environment Interactions

A robust and straightforward method for the preparation of lipid membranes upon dynamically responsive polymer cushions is reported. Structural characterization demonstrates that complete, well-packed membranes with tunable mobility can be constructed on the polymeric cushion. With this system, membrane conformational changes induced by cellular cytoskeleton interactions can be modeled. The membrane can be tailored to screen the cushion from changes in pH or allow rapid response to the pH environment by incorporation of protein ion channels. This elementary system offers a means to replicate the conformational changes that occur with the cellular cytoskeleton and has great potential for fundamental biophysical studies of membrane properties and membrane-protein interactions decoupled from the underlying solid support.

Ganglioside embedded in reconstituted lipoprotein binds cholera toxin with elevated affinity

The ability to exogenously present cell-surface receptors in high-affinity conformations in a synthetic system offers an opportunity to provide host cells with protection from pathogenic toxins. This strategy requires improvement of the synthetic receptor binding affinity against its native counterpart, particularly with polyvalent toxins where clustering of membrane receptors can hinder binding. Here we demonstrate that reconstituted lipoprotein, nanometer-sized discoidal lipid bilayers bounded by apolipoprotein and functionalized by incorporation of pathogen receptors, provides a means to enhance toxin-receptor binding through molecular-level control over the receptor microenvironment (specifically, its rigidity, composition, and heterogeneity). Using a Foerster Resonance Energy Transfer (FRET)-based assay, we found that reconstituted lipoprotein incorporating low concentrations of ganglioside monosialotetrahexosylganglioside (GM1) binds polymeric cholera toxin with significantly higher affinity than liposomes or supported lipid bilayers, most likely a result of the enhanced control over receptor clustering provided by the lipoprotein platform. Using wide-area epifluorescence, we found that this enhanced binding capacity can be effectively utilized to divert cholera toxin away from populations of healthy mammalian cells. In summary, we found that reconstitutions of high-density lipoprotein can be engineered to include specific pathogen receptors; that their pathogen binding affinity is altered, presumably due to attenuation of receptor aggregation; and that these assemblies are effective at protecting cells from biological toxins.

The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aβ peptide.

Background The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimers disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species. Methodology/Principal Findings To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species. Conclusions Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species.

Frustrated phase transformations in supported, interdigitating lipid bilayers

In free bilayers, the fluid to gel main phase transition of a monofluorinated phospholipid (F-DPPC) transforms a disordered fluid bilayer into a fully interdigitated monolayer consisting of ordered acyl tails. This transformation results in an increase in molecular area and decrease in bilayer thickness. We show that when confined in patches near a solid surface this reorganization proceeds under constraints of planar topography and total surface area. One consequence of these constraints is to limit the complete formation of the energetically favored, interdigitated gel phase. The noninterdigitated lipids experience enhanced lateral tension, due to the expansion of the growing interdigitated phase within the constant area. The corresponding rise in equilibrium transition temperatures produces supercooled lipids that vitrify when cooled further. Ultimately, this frustrated phase change reflects a coupling between dynamics and thermodynamics and gives rise to an unusual phase coexistence characterized by the presence of two qualitatively different gel phases.

Bridging across length scales: Multi-scale ordering of supported lipid bilayers via lipoprotein self-assembly and surface patterning

We show that a two-step process, involving spontaneous self-assembly of lipids and apolipoproteins and surface patterning, produces single, supported lipid bilayers over two discrete and independently adjustable length scales. Specifically, an aqueous phase incubation of DMPC vesicles with purified apolipoprotein A-I results in the reconstitution of high density lipoprotein (rHDL), wherein nanoscale clusters of single lipid bilayers are corralled by the protein. Adsorption of these discoidal particles to clean hydrophilic glass (or silicon) followed by direct exposure to a spatial pattern of short-wavelength UV radiation directly produces microscopic patterns of nanostructured bilayers. Alternatively, simple incubation of aqueous phase rHDL with a chemically patterned hydrophilic/hydrophobic surface produces a novel compositional pattern, caused by an increased affinity for adsorption onto hydrophilic regions relative to the surrounding hydrophobic regions. Further, by simple chemical denaturation of the boundary protein, nanoscale compartmentalization can be selectively erased, thus producing patterns of laterally fluid, lipid bilayers structured solely at the mesoscopic length scale. Since these aqueous phase microarrays of nanostructured lipid bilayers allow for membrane proteins to be embedded within single nanoscale bilayer compartments, they present a viable means of generating high-density membrane protein arrays. Such a system would permit in-depth elucidation of membrane protein structure-function relationships and the consequences of membrane compartmentalization on lipid dynamics.

Inhibiting host–pathogen interactions using membrane-based nanostructures

Virulent strains of bacteria and viruses recognize host cells by their plasma membrane receptors and often exploit the native translocation machinery to invade the cell. A promising therapeutic concept for early interruption of pathogen infection is to subvert this pathogenic trickery using exogenously introduced decoys that present high-affinity mimics of cellular receptors. This review highlights emerging applications of molecularly engineered lipid-bilayer-based nanostructures, namely (i) functionalized liposomes, (ii) supported colloidal bilayers or protocells and (iii) reconstituted lipoproteins, which display functional cellular receptors in optimized conformational and aggregative states. These decoys outcompete host cell receptors by preferentially binding to and neutralizing virulence factors of both bacteria and viruses, thereby promising a new approach to antipathogenic therapy.