Side-Chain Dynamics Reveals Transient Association of Aβ_1–40 Monomers with Amyloid Fibers

Low-lying excited states that correspond to rare conformations or transiently bound species have been hypothesized to play an important role for amyloid nucleation. Despite their hypothesized importance in amyloid formation, transiently occupied states have proved difficult to detect directly. To experimentally characterize these invisible states, we performed a series of Carr–Purcell–Meiboom–Gill (CPMG)-based relaxation dispersion NMR experiments for the amyloidogenic Aβ_1–40 peptide implicated in Alzheimer’s disease. Significant relaxation dispersion of the resonances corresponding to the side-chain amides of Q15 and N27 was detected before the onset of aggregation. The resonances corresponding to the peptide backbone did not show detectable relaxation dispersion, suggesting an exchange rate that is not within the practical limit of detection. This finding is consistent with the proposed “dock and lock” mechanism based on molecular dynamics simulations in which the Aβ_1–40 monomer transiently binds to the Aβ_1–40 oligomer by non-native contacts with the side chains before being incorporated into the fiber through native contacts with the peptide backbone

Lipid Composition-Dependent Membrane Fragmentation and Pore-Forming Mechanisms of Membrane Disruption by Pexiganan (MSI-78)

The potency and selectivity of many antimicrobial peptides (AMPs) are correlated with their ability to interact with and disrupt the bacterial cell membrane. <i>In vitro</i> experiments using model membranes have been used to determine the mechanism of membrane disruption of AMPs. Because the mechanism of action of an AMP depends on the ability of the model membrane to accurately mimic the cell membrane, it is important to understand the effect of membrane composition. Anionic lipids that are present in the outer membrane of prokaryotes but are less common in eukaryotic membranes are usually thought to be key for the bacterial selectivity of AMPs. We show by fluorescence measurements of peptide-induced membrane permeabilization that the presence of anionic lipids at high concentrations can actually inhibit membrane disruption by the AMP MSI-78 (pexiganan), a representative of a large class of highly cationic AMPs. Paramagnetic quenching studies suggest MSI-78 is in a surface-associated inactive mode in anionic sodium dodecyl sulfate micelles but is in a deeply buried and presumably more active mode in zwitterionic dodecylphosphocholine micelles. Furthermore, a switch in mechanism occurs with lipid composition. Membrane fragmentation with MSI-78 can be observed in mixed vesicles containing both anionic and zwitterionic lipids but not in vesicles composed of a single lipid of either type. These findings suggest membrane affinity and membrane permeabilization are not always correlated, and additional effects that may be more reflective of the actual cellular environment can be seen as the complexity of the model membranes is increased

Inhibition and Degradation of Amyloid Beta (Aβ40) Fibrillation by Designed Small Peptide: A Combined Spectroscopy, Microscopy, and Cell Toxicity Study

A designed nontoxic, nonhemolytic 11-residue peptide, NF11 (NAVR­WSLM­RPF), not only inhibits the aggregation of amyloid beta (Aβ40) protein but also disaggregates the preformed oligomers and mature Aβ fibrils, thereby reducing associated-toxicity. NMR experiments provide evidence of NF11’s ability to inhibit fibril formation, primarily through interaction with the N-terminus region as well as the central hydrophobic cluster of Aβ40. NF11 has micromolar binding affinity toward both monomeric and aggregated species for efficient clearance of toxic aggregates. From these in vitro results, the future development of a next generation peptidomimetic therapeutic agent for amyloid disease may be possible

Supplemental Material - Discrete wavelet transform based processing of embroidered textile-electrode EMG signal acquired with load and pressure effect

Supplemental Material for Discrete wavelet transform based processing of embroidered textile-electrode EMG signal acquired with load and pressure effect by Bulcha Belay Etana, Ahmed Ali Ali Dawud, Benny Malengier, Sitek Wojciech, Wendimu Fanta Gemechu, Janarthanan Krishnamoorthy and Lievavan Langenhove in Journal of Industrial Textiles</p

Probing the Sources of the Apparent Irreproducibility of Amyloid Formation: Drastic Changes in Kinetics and a Switch in Mechanism Due to Micellelike Oligomer Formation at Critical Concentrations of IAPP

The aggregation of amyloidogenic proteins is infamous for being highly chaotic, with small variations in conditions sometimes leading to large changes in aggregation rates. Using the amyloidogenic protein IAPP (islet amyloid polypeptide protein, also known as amylin) as an example, we show that a part of this phenomenon may be related to the formation of micellelike oligomers at specific critical concentrations and temperatures. We show that pyrene fluorescence can sensitively detect micellelike oligomer formation by IAPP and discriminate between micellelike oligomers from fibers and monomers, making pyrene one of the few chemical probes specific to a prefibrillar oligomer. We further show that oligomers of this type reversibly form at critical concentrations in the low micromolar range and at specific critical temperatures. Micellelike oligomer formation has several consequences for amyloid formation by IAPP. First, the kinetics of fiber formation increase substantially as the critical concentration is approached but are nearly independent of concentration below it, suggesting a direct role for the oligomers in fiber formation. Second, the critical concentration is strongly correlated with the propensity to form amyloid: higher critical concentrations are observed for both IAPP variants with lower amyloidogenicity and for native IAPP at acidic pH in which aggregation is greatly slowed. Furthermore, using the DEST NMR technique, we show that the pathway of amyloid formation switches as the critical point is approached, with self-interactions primarily near the N-terminus below the critical temperature and near the central region above the critical temperature, reconciling two apparently conflicting views of the initiation of IAPP aggregation

Resolution of Oligomeric Species during the Aggregation of Aβ_1–40 Using ¹⁹F NMR

In the commonly used nucleation-dependent model of protein aggregation, aggregation proceeds only after a lag phase in which the concentration of energetically unfavorable nuclei reaches a critical value. The formation of oligomeric species prior to aggregation can be difficult to detect by current spectroscopic techniques. By using real-time ¹⁹F NMR along with other techniques, we are able to show that multiple oligomeric species can be detected during the lag phase of Aβ_1–40 fiber formation, consistent with a complex mechanism of aggregation. At least six types of oligomers can be detected by ¹⁹F NMR. These include the reversible formation of large β-sheet oligomer immediately after solubilization at high peptide concentration, a small oligomer that forms transiently during the early stages of the lag phase, and four spectroscopically distinct forms of oligomers with molecular weights between ∼30 and 100 kDa that appear during the later stages of aggregation. The ability to resolve individual oligomers and track their formation in real-time should prove fruitful in understanding the aggregation of amyloidogenic proteins and in isolating potentially toxic nonamyloid oligomers

Rational Design of a Structural Framework with Potential Use to Develop Chemical Reagents That Target and Modulate Multiple Facets of Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by multiple, intertwined pathological features, including amyloid-β (Aβ) aggregation, metal ion dyshomeostasis, and oxidative stress. We report a novel compound (<b><b>ML</b></b>) prototype of a rationally designed molecule obtained by integrating structural elements for Aβ aggregation control, metal chelation, reactive oxygen species (ROS) regulation, and antioxidant activity within a single molecule. Chemical, biochemical, ion mobility mass spectrometric, and NMR studies indicate that the compound <b><b>ML</b></b> targets metal-free and metal-bound Aβ (metal–Aβ) species, suppresses Aβ aggregation in vitro, and diminishes toxicity induced by Aβ and metal-treated Aβ in living cells. Comparison of <b><b>ML</b></b> to its structural moieties (i.e., 4-(dimethylamino)­phenol (<b>DAP</b>) and (8-aminoquinolin-2-yl)­methanol (<b>1</b>)) for reactivity with Aβ and metal–Aβ suggests the synergy of incorporating structural components for both metal chelation and Aβ interaction. Moreover, <b><b>ML</b></b> is water-soluble and potentially brain permeable, as well as regulates the formation and presence of free radicals. Overall, we demonstrate that a rational structure-based design strategy can generate a small molecule that can target and modulate multiple factors, providing a new tool to uncover and address AD complexity