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Biomimetic supported lipid bilayers with high cholesterol content formed by ?-helical peptide-induced vesicle fusion.

ABSTRACT: In this study, we present a technique to create a complex, high cholesterol-containing supported lipid bilayers (SLBs) using ?-helical (AH) peptide-induced vesicle fusion. Vesicles consisting of POPC : POPE : POPS : SM : Chol (9.35 : 19.25 : 8.25 : 18.15 : 45.00) were used to form a SLB that models the native composition of the human immunodeficiency virus-1 (HIV-1) lipid envelope. In the absence of AH peptides, these biomimetic vesicles fail to form a complete SLB. We verified and characterized AH peptide-induced vesicle fusion by quartz crystal microbalance with dissipation monitoring, neutron reflectivity, and atomic force microscopy. Successful SLB formation entailed a characteristic frequency shift of -35.4 ± 2.0 Hz and a change in dissipation energy of 1.91 ± 0.52 × 10-6. Neutron reflectivity measurements determined the SLB thickness to be 49.9 +1.9-1.5 Å, and showed the SLB to be 100 +0.0-0.1% complete and void of residual AH peptide after washing. Atomic force microscopy imaging confirmed complete SLB formation and revealed three distinct domains with no visible defects. This vesicle fusion technique gives researchers access to a complex SLB composition with high cholesterol content and thus the ability to better recapitulate the native HIV-1 lipid membrane.

SUBMITTER: Hardy GJ

PROVIDER: S-EPMC3728912 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Biomimetic supported lipid bilayers with high cholesterol content formed by α-helical peptide-induced vesicle fusion.

Hardy Gregory J GJ Nayak Rahul R Alam S Munir SM Shapter Joseph G JG Heinrich Frank F Zauscher Stefan S

Journal of materials chemistry 20120801 37

In this study, we present a technique to create a complex, high cholesterol-containing supported lipid bilayers (SLBs) using α-helical (AH) peptide-induced vesicle fusion. Vesicles consisting of POPC : POPE : POPS : SM : Chol (9.35 : 19.25 : 8.25 : 18.15 : 45.00) were used to form a SLB that models the native composition of the human immunodeficiency virus-1 (HIV-1) lipid envelope. In the absence of AH peptides, these biomimetic vesicles fail to form a complete SLB. We verified and characterized ...[more]

PMID: 23914075

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Project description:The strict requirement of constructing a native lipid environment to preserve the structure and functionality of membrane proteins is the starting constraint when building biomaterials and sensor systems from these biomolecules. To enhance the viability of supported biomembranes systems and build new ligand display interfaces, we apply rationally designed peptides partitioned into the lipid bilayer interface. Peptides designed to form membrane-spanning ?-helical anchoring domains are synthesized using solid-phase peptide synthesis. K(3)A(4)L(2)A(7)L(2)A(3)K(2)-FITC is synthesized on the 100 mg scale for use as a biomembrane anchoring molecule, where orthogonal side-chain modifications allow us to introduce probes enabling peptide localization within supported bilayers. The peptides are found to form ?-helical domains within liposomes as assessed with circular dichroism spectroscopy. These peptides are designed to be incorporated into lipid bilayers supported by microspheres and serve as biomembrane anchoring moieties to amino-terminated surfaces. Here, the silica bead surface (4.7 ?m diameter) is activated with homobifunctional NHS-PEG(3000)-NHS as "polymer cushion" spacers. This tethering to a subset of the K(3)A(4)L(2)A(7)L(2)A(3)K(2)-FITC molecules present in the bilayer is achieved by the fusion of liposomes followed by coupling of the peptide amino groups to the NHS presented from the silica microsphere surfaces. The biomembrane distributions of tethered and untethered K(3)A(4)L(2)A(7)L(2)A(3)K(2)-FITC are probed with confocal microscopy and are found to give 3D reconstructions consistent with largely homogeneous supported biomembranes. The fluidity of the untethered fraction of peptides within supported membranes is quantified using the fluorescence recovery after photobleaching (FRAP) technique. The presence of the PEG(3000) polymer cushion facilitated a 28.9% increase in peptide diffusivity over untethered bilayers at the lowest peptide to lipid ratio we examined. We show that rationally designed peptide-based anchors can be used to tether lipid bilayers, creating a polymer-cushioned lipid microenvironment on surfaces with high lateral mobility and facilitating the development of a new platform for ligand displays.

Project description:The molecular interactions between antimicrobial peptides (AMPs) and lipid A-containing supported lipid bilayers were probed using single-molecule total internal reflection fluorescence microscopy. Hybrid supported lipid bilayers with lipid A outer leaflets and phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) inner leaflets were prepared and characterized, and the spatiotemporal trajectories of individual fluorescently labeled LL37 and Melittin AMPs were determined as they interacted with the bilayer surfaces comprising either monophosphoryl or diphosphoryl lipid A (from Escherichia coli) to determine the impact of electrostatic interactions. Large numbers of trajectories were obtained and analyzed to obtain the distributions of surface residence times and the statistics of the spatial trajectories. Interestingly, the AMP species were sensitive to subtle differences in the charge of the lipid, with both peptides diffusing more slowly and residing longer on the diphosphoryl lipid A. Furthermore, the single-molecule dynamics indicated a qualitative difference between the behavior of AMPs on hybrid Lipid A bilayers and on those composed entirely of DOPE. Whereas AMPs interacting with a DOPE bilayer exhibited two-dimensional Brownian diffusion with a diffusion coefficient of ∼1.7 μm2/s, AMPs adsorbed to the lipid A surface exhibited much slower apparent diffusion (on the order of ∼0.1 μm2/s) and executed intermittent trajectories that alternated between two-dimensional Brownian diffusion and desorption-mediated three-dimensional flights. Overall, these findings suggested that bilayers with lipid A in the outer leaflet, as it is in bacterial outer membranes, are valuable model systems for the study of the initial stage of AMP-bacterium interactions. Furthermore, single-molecule dynamics was sensitive to subtle differences in electrostatic interactions between cationic AMPs and monovalent or divalent anionic lipid A moieties.

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Biomimetic supported lipid bilayers with high cholesterol content formed by ?-helical peptide-induced vesicle fusion.

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Biomimetic supported lipid bilayers with high cholesterol content formed by α-helical peptide-induced vesicle fusion.

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