Membrane protein biosensing with plasmonic nanopore arrays and pore-spanning lipid membranes.
ABSTRACT: Integration of solid-state biosensors and lipid bilayer membranes is important for membrane protein research and drug discovery. In these sensors, it is critical that the solid-state sensing material does not have adverse effects on the conformation or functionality of membrane-bound molecules. In this work, pore-spanning lipid membranes are formed over an array of periodic nanopores in free-standing gold films for surface plasmon resonance (SPR) kinetic binding assays. The ability to perform kinetic assays with a transmembrane protein is demonstrated with ?-hemolysin (?-HL). The incorporation of ?-HL into the membrane followed by specific antibody binding (anti-?-HL) red-shifts the plasmon resonance of the gold nanopore array, which is optically monitored in real time. Subsequent fluorescence imaging reveals that the antibodies primarily bind in nanopore regions, indicating that ?-HL incorporation preferentially occurs into areas of pore-spanning lipid membranes.
Project description:Most experiments on nanopores have concentrated on the pore-forming protein ?-haemolysin (?HL) and on artificial pores in solid-state membranes. While biological pores offer an atomically precise structure and the potential for genetic engineering, solid-state nanopores offer durability, size and shape control, and are also better suited for integration into wafer-scale devices. However, each system has significant limitations: ?HL is difficult to integrate because it relies on delicate lipid bilayers for mechanical support, and the fabrication of solid-state nanopores with precise dimensions remains challenging. Here we show that these limitations may be overcome by inserting a single ?HL pore into a solid-state nanopore. A double-stranded DNA attached to the protein pore is threaded into a solid-state nanopore by electrophoretic translocation. Protein insertion is observed in 30-40% of our attempts, and translocation of single-stranded DNA demonstrates that the hybrid nanopore remains functional. The hybrid structure offers a platform to create wafer-scale device arrays for genomic analysis, including sequencing.
Project description:Nanopore-based sensors are advancing the sensitivity and selectivity of single-molecule detection in molecular medicine and biotechnology. Current electrical sensing devices are based on either membrane protein pores supported in planar lipid bilayers or solid-state (SS) pores fabricated in thin metallic membranes. While both types of nanosensors have been used in a variety of applications, each has inherent disadvantages that limit its use. Hybrid nanopores, consisting of a protein pore supported within a SS membrane, combine the robust nature of SS membranes with the precise and simple engineering of protein nanopores. We demonstrate here a novel lipid-free hybrid nanopore comprising a natural DNA pore from a thermostable virus, electrokinetically inserted into a larger nanopore supported in a silicon nitride membrane. The hybrid pore is stable and easy to fabricate, and, most importantly, exhibits low peripheral leakage allowing sensing and discrimination among different types of biomolecules.
Project description:Design and fabrication of three-dimensionally structured, gold membranes containing hexagonally close-packed microcavities with nanopores in the base, are described. Our aim is to create a nanoporous structure with localized enhancement of the fluorescence or Raman scattering at, and in the nanopore when excited with light of approximately 600 nm, with a view to provide sensitive detection of biomolecules. A range of geometries of the nanopore integrated into hexagonally close-packed assemblies of gold micro-cavities was first evaluated theoretically. The optimal size and shape of the nanopore in a single microcavity were then considered to provide the highest localized plasmon enhancement (of fluorescence or Raman scattering) at the very center of the nanopore for a bioanalyte traversing through. The optimized design was established to be a 1200 nm diameter cavity of 600 nm depth with a 50 nm square nanopore with rounded corners in the base. A gold 3D-structured membrane containing these sized microcavities with the integrated nanopore was successfully fabricated and 'proof of concept' Raman scattering experiments are described.
Project description:Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein's insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal's DNA-ratchet function.
Project description:?-Hemolysin (?HL), a ?-barrel pore-forming toxin (?PFT), is secreted as a water-soluble monomer by Staphylococcus aureus. Upon binding to receptors on target cell membranes, ?HL assembles to form heptameric membrane-spanning pores. We have previously engineered ?HL to create a protease-activatable toxin that is activated by site-specific proteolysis including by tumor proteases. In this study, we redesigned ?HL so that it requires 2-fold activation on target cells through (i) binding to specific receptors, and (ii) extracellular proteolytic cleavage. To assess our strategy, we constructed a fusion protein of ?HL with galectin-1 (?HLG1, ?HL-Galectin-1 chimera). ?HLG1 was cytolytic toward cells that lack a receptor for wild-type ?HL. We then constructed protease-activatable mutants of ?HLG1 (PAM?HLG1s). PAM?HLG1s were activated by matrix metalloproteinase 2 (MMP-2) and had approximately 50-fold higher cytolytic activity toward MMP-2 overexpressing cells (HT-1080 cells) than toward non-overexpressing cells (HL-60 cells). Our approach provides a novel strategy for tailoring pore-forming toxins for therapeutic applications.
Project description:We have developed a novel approach for creating membrane-spanning protein-based pores. The construction principle is based on using well-defined, circular DNA nanostructures to arrange a precise number of pore-forming protein toxin monomers. We can thereby obtain, for the first time, protein pores with specifically set diameters. We demonstrate this principle by constructing artificial alpha-hemolysin (?HL) pores. The DNA/?HL hybrid nanopores composed of twelve, twenty or twenty-six monomers show stable insertions into lipid bilayers during electrical recordings, along with steady, pore size-dependent current levels. Our approach successfully advances the applicability of nanopores, in particular towards label-free studies of single molecules in large nanoscaled biological structures.
Project description:Technologies based on surface plasmon resonance (SPR) have allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions. SPR has become the gold standard in industrial and academic settings, in which the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, SPR is only beginning to be adapted to the needs of membrane-bound proteins which are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcoreTM, have been adapted for membrane protein analysis by building supported lipid layers or capturing lipid vesicles on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of G protein coupled receptor ligands and applications in basic cellular biology.
Project description:We propose and demonstrate a new method of an all-optical, contactless, one-step injection of single gold nanoparticles through phospholipid membranes. The method is based on the combination of strong optical forces acting on and simultaneous optical heating of a gold nanoparticle exposed to laser light tuned to the plasmon resonance of the nanoparticle. A focused laser beam captures single nanoparticles from the colloidal suspension, guides them toward a phospholipid vesicle and propels them through the gel-phase membrane, resulting in the nanoparticle internalization into the vesicle. Efficient resonant optical heating of the gold nanoparticle causes a pore to form in the gel-phase membrane, a few-hundred nanometers in size, which remains open for several minutes.
Project description:Pneumolysin (PLY), a major virulence factor of Streptococcus pneumoniae, perforates cholesterol-rich lipid membranes. PLY protomers oligomerize as rings on the membrane and then undergo a structural transition that triggers the formation of membrane pores. Structures of PLY rings in prepore and pore conformations define the beginning and end of this transition, but the detailed mechanism of pore formation remains unclear. With atomistic and coarse-grained molecular dynamics simulations, we resolve key steps during PLY pore formation. Our simulations confirm critical PLY membrane-binding sites identified previously by mutagenesis. The transmembrane ?-hairpins of the PLY pore conformation are stable only for oligomers, forming a curtain-like membrane-spanning ?-sheet. Its hydrophilic inner face draws water into the protein-lipid interface, forcing lipids to recede. For PLY rings, this zone of lipid clearance expands into a cylindrical membrane pore. The lipid plug caught inside the PLY ring can escape by lipid efflux via the lower leaflet. If this path is too slow or blocked, the pore opens by membrane buckling, driven by the line tension acting on the detached rim of the lipid plug. Interestingly, PLY rings are just wide enough for the plug to buckle spontaneously in mammalian membranes. In a survey of electron cryo-microscopy (cryo-EM) and atomic force microscopy images, we identify key intermediates along both the efflux and buckling pathways to pore formation, as seen in the simulations.
Project description:Protein nanopores show great potential as low-cost detectors in DNA sequencing devices. To date, research has largely focused on the staphylococcal pore ?-hemolysin (?HL). In the present study, we have developed simplified models of the wild-type ?HL pore and various mutants in order to study the translocation dynamics of single-stranded DNA under the influence of an applied electric field. The model nanopores reflect the experimentally measured conductance values in planar lipid bilayers. We show that interactions between rings of cationic amino acids and DNA backbone phosphates result in metastable tethering of nucleic acid molecules within the pore, leading us to propose a "binding and sliding" mechanism for translocation. We also observe folding of DNA into nonlinear conformational intermediates during passage through the confined nanopore environment. Despite adopting nonlinear conformations, the DNA hexamer always exits the pore in the same orientation as it enters (3' to 5') in our simulations. The observations from our simulations help to rationalize experimentally determined trends in residual current and translocation efficiency for ?HL and its mutants.