Laura Connelly

Atomic force microscopy and MD simulations reveal pore-like structures of all-D-enantiomer of Alzheimer's β-amyloid peptide: relevance to the ion channel mechanism of AD pathology.

Alzheimers disease (AD) is a protein misfolding disease characterized by a buildup of β-amyloid (Aβ) peptide as senile plaques, uncontrolled neurodegeneration, and memory loss. AD pathology is linked to the destabilization of cellular ionic homeostasis and involves Aβ peptide-plasma membrane interactions. In principle, there are two possible ways through which disturbance of the ionic homeostasis can take place: directly, where the Aβ peptide either inserts into the membrane and creates ion-conductive pores or destabilizes the membrane organization, or, indirectly, where the Aβ peptide interacts with existing cell membrane receptors. To distinguish between these two possible types of Aβ-membrane interactions, we took advantage of the biochemical tenet that ligand-receptor interactions are stereospecific; L-amino acid peptides, but not their D-counterparts, bind to cell membrane receptors. However, with respect to the ion channel-mediated mechanism, like L-amino acids, D-amino acid peptides will also form ion channel-like structures. Using atomic force microscopy (AFM), we imaged the structures of both D- and L-enantiomers of the full length Aβ(1-42) when reconstituted in lipid bilayers. AFM imaging shows that both L- and D-Aβ isomers form similar channel-like structures. Molecular dynamics (MD) simulations support the AFM imaged 3D structures. Previously, we have shown that D-Aβ(1-42) channels conduct ions similarly to their L- counterparts. Taken together, our results support the direct mechanism of Aβ ion channel-mediated destabilization of ionic homeostasis rather than the indirect mechanism through Aβ interaction with membrane receptors.

Antibiofouling, sustained antibiotic release by Si nanowire templates.

Loading or filling nanostructures with antibiotics can be one of the relevant approaches for obtaining a controlled drug release rate. Vertically aligned silicon nanowire (SiNW) arrays with 10-40 nm diameter wires having 1-3 microm in length obtained by the electroless etching (EE) technique are used in this study as novel nanostructures for mediating drug delivery. Here we report controlled antibiotic activity and sustained bioavailability from SiNW arrays and also show microstructural manipulations for a tunable release rate. As well, we have demonstrated biodegradability of SiNWs in phosphate buffer saline (PBS) solution. Strikingly suppressed cell and protein adhesion was observed on our SiNW surface, which indicates a reduced probability for biofouling and drug release impediments. Such antibiotic release from the nanowire-structured surface can provide more reliable antibiotic protection at a targeted implantation or biosensor site.

Effects of point substitutions on the structure of toxic Alzheimer's β-amyloid channels: atomic force microscopy and molecular dynamics simulations.

Alzheimers disease (AD) is a misfolded protein disease characterized by the accumulation of β-amyloid (Aβ) peptide as senile plaques, progressive neurodegeneration, and memory loss. Recent evidence suggests that AD pathology is linked to the destabilization of cellular ionic homeostasis mediated by toxic pores made of Aβ peptides. Understanding the exact nature by which these pores conduct electrical and molecular signals could aid in identifying potential therapeutic targets for the prevention and treatment of AD. Here using atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we compared the imaged pore structures with models to predict channel conformations as a function of amino acid sequence. Site-specific amino acid (AA) substitutions in the wild-type Aβ(1-42) peptide yield information regarding the location and significance of individual AA residues to its characteristic structure-activity relationship. We selected two AAs that our MD simulation predicted to inhibit or permit pore conductance. The substitution of Phe19 with Pro has previously been shown to eliminate conductance in the planar lipid bilayer system. Our MD simulations predict a channel-like shape with a collapsed pore, which is supported by the AFM channel images. We suggest that proline, a known β-sheet breaker, creates a kink in the center of the pore and prevents conductance via blockage. This residue may be a viable target for drug development studies aiming to inhibit Aβ from inducing ionic destabilization toxicity. The substitution of Phe20 with Cys exhibits pore structures indistinguishable from the wild type in AFM images. MD simulations predict site 20 to face the solvated pore. Overall, the mutations support the previously predicted β-sheet-based channel structure.

All-d-Enantiomer of β-Amyloid Peptide Forms Ion Channels in Lipid Bilayers.

Alzheimer’s disease (AD) is the most common type of senile dementia in aging populations. Amyloid β (Aβ)-mediated dysregulation of ionic homeostasis is the prevailing underlying mechanism leading to synaptic degeneration and neuronal death. Aβ-dependent ionic dysregulation most likely occurs either directly via unregulated ionic transport through the membrane or indirectly via Aβ binding to cell membrane receptors and subsequent opening of existing ion channels or transporters. Receptor binding is expected to involve a high degree of stereospecificity. Here, we investigated whether an Aβ peptide enantiomer, whose entire sequence consists of d-amino acids, can form ion-conducting channels; these channels can directly mediate Aβ effects even in the absence of receptor–peptide interactions. Using complementary approaches of planar lipid bilayer (PLB) electrophysiological recordings and molecular dynamics (MD) simulations, we show that the d-Aβ isomer exhibits ion conductance behavior in the bilayer indistinguishable from that described earlier for the l-Aβ isomer. The d isomer forms channel-like pores with heterogeneous ionic conductance similar to the l-Aβ isomer channels, and the d-isomer channel conductance is blocked by Zn2+, a known blocker of l-Aβ isomer channels. MD simulations further verify formation of β-barrel-like Aβ channels with d- and l-isomers, illustrating that both d- and l-Aβ barrels can conduct cations. The calculated values of the single-channel conductance are approximately in the range of the experimental values. These findings are in agreement with amyloids forming Ca2+ leaking, unregulated channels in AD, and suggest that Aβ toxicity is mediated through a receptor-independent, nonstereoselective mechanism.

Graphene Nanopore Support System for Simultaneous High-Resolution AFM Imaging and Conductance Measurements

Accurately defining the nanoporous structure and sensing the ionic flow across nanoscale pores in thin films and membranes has a wide range of applications, including characterization of biological ion channels and receptors, DNA sequencing, molecule separation by nanoparticle films, sensing by block co-polymers films, and catalysis through metal–organic frameworks. Ionic conductance through nanopores is often regulated by their 3D structures, a relationship that can be accurately determined only by their simultaneous measurements. However, defining their structure–function relationships directly by any existing techniques is still not possible. Atomic force microscopy (AFM) can image the structures of these pores at high resolution in an aqueous environment, and electrophysiological techniques can measure ion flow through individual nanoscale pores. Combining these techniques is limited by the lack of nanoscale interfaces. We have designed a graphene-based single-nanopore support (∼5 nm thick with ∼20 nm pore diameter) and have integrated AFM imaging and ionic conductance recording using our newly designed double-chamber recording system to study an overlaid thin film. The functionality of this integrated system is demonstrated by electrical recording (<10 pS conductance) of suspended lipid bilayers spanning a nanopore and simultaneous AFM imaging of the bilayer.

Insulated Conducting Cantilevered Nanotips and Two-Chamber Recording System for High Resolution Ion Sensing AFM

Biological membranes contain ion channels, which are nanoscale pores allowing controlled ionic transport and mediating key biological functions underlying normal/abnormal living. Synthetic membranes with defined pores are being developed to control various processes, including filtration of pollutants, charge transport for energy storage, and separation of fluids and molecules. Although ionic transport (currents) can be measured with single channel resolution, imaging their structure and ionic currents simultaneously is difficult. Atomic force microscopy enables high resolution imaging of nanoscale structures and can be modified to measure ionic currents simultaneously. Moreover, the ionic currents can also be used to image structures. A simple method for fabricating conducting AFM cantilevers to image pore structures at high resolution is reported. Tungsten microwires with nanoscale tips are insulated except at the apex. This allows simultaneous imaging via cantilever deflections in normal AFM force feedback mode as well as measuring localized ionic currents. These novel probes measure ionic currents as small as picoampere while providing nanoscale spatial resolution surface topography and is suitable for measuring ionic currents and conductance of biological ion channels.

Structure–Function Studies of Amyloid Pores in Alzheimer’s Disease as a Case Example of Neurodegenerative Diseases

Abstract Amyloidogenic proteins, characterized by their ability to form fibrilar aggregates with β-sheet configurations, play an important role in several neurodegenerative diseases, including Alzheimer’s disease. Although their exact mechanism of toxicity is not yet known, considerable experimental evidence, including planar lipid bilayer (BLM), calcium imaging, atomic force microscopy (AFM) and electron microscopy (EM), links small oligomers to membrane permeabilization, leading to cytotoxicity by loss of ionic homeostasis. A possible mechanism of this toxicity involves amyloids incorporated as pore-forming structures in the lipid environment of the membrane. Such pores produce stepwise increases in current across lipid bilayers in BLM data and can be recognized as small protrusions in AFM images. Unlike classic ion channels, these pores exhibit multiple ionic conductances and they have a variable number of subunits. Molecular dynamics (MD) simulations have provided atomistic models that capture the essential features of the pores. In these simulations, monomers adopt the U-shaped, β-strand-turn-β-strand motif as a general feature of amyloid organization.