Amyloid ß-Protein Assembly and Alzheimer's Disease: Dodecamers of Aß42, but Not of Aß40, Seed Fibril Formation.

Evidence suggests that oligomers of the 42-residue form of the amyloid ß-protein (Aß), Aß42, play a critical role in the etiology of Alzheimer's disease (AD). Here we use high resolution atomic force microscopy to directly image populations of small oligomers of Aß42 that occur at the earliest stages of aggregation. We observe features that can be attributed to a monomer and to relatively small oligomers, including dimers, hexamers, and dodecamers. We discovered that Aß42 hexamers and dodecamers quickly become the dominant oligomers after peptide solubilization, even at low (1 µM) concentrations and short (5 min) incubation times. Soon after (≥10 min), dodecamers are observed to seed the formation of extended, linear preprotofibrillar ß-sheet structures. The preprotofibrils are a single Aß42 layer in height and can extend several hundred nanometers in length. To our knowledge this is the first report of structures of this type. In each instance the preprotofibril is associated off center with a single layer of a dodecamer. Protofibril formation continues at longer times, but is accompanied by the formation of large, globular aggregates. Aß40, by contrast, does not significantly form the hexamer or dodecamer but instead produces a mixture of smaller oligomers. These species lead to the formation of a branched chain-like network rather than discrete structures.

Investigation of arrays of photosynthetically active heterostructures using conductive probe atomic force microscopy.

Control and optimization of optically excited charge and energy transport across solid-liquid interfaces are essential for many applications including artificial photosynthesis, photocatalysis, and photopolymerization. Nanostructures are especially suited for this purpose, because the exciton diffusion length is typically much larger than the dimension of the particle, enabling efficient charge transport from the bulk to the nanoparticle surface for use in chemical transformations. However, characterization of charge transfer processes at nanoscale interfaces involving either isolated or assembled optoelectronic components remains a major challenge. Here, we use conductive probe-atomic force microscopy (cp-AFM) to spatially characterize the photovoltaic and photoelectrochemical properties of individual nanostructured photosynthetically active heterostructure (PAH) units in large area scans and compare them to thin-film photoelectrode devices. For CuInSe2/Au Schottky barrier PAH devices electrochemically synthesized inside porous anodic aluminum oxide, we observed a significant increase in solid-state photovoltages (∼0.5 V) and applied bias photocurrents (∼5 pA at +2 V) with much less spatial variation compared to thin film devices (<0.1 V and ∼2 pA at +2 V). We identified that the key reasons for the low performance of CuInSe2/Au thin film devices were an increased number of short-circuit pathways formed as a result of the fabrication process, and a lower density of grain boundaries leading to reduced photoelectrochemically active surface area. When photoanodes were fabricated with these PAH units, the electrodes showed superior and stable photoelectrochemical performance due to their inherent fault tolerance. Our results demonstrate the potential of using cp-AFM as a tool to characterize spatially resolved photoelectrochemical performance over device structures designed for areal production of chemicals and to provide us with a means of investigating optimal structural configurations and to better understand charge transfer processes across solid-liquid interface.

Ion mobility spectrometry reveals the mechanism of amyloid formation of Aß(25-35) and its modulation by inhibitors at the molecular level: epigallocatechin gallate and scyllo-inositol.

Amyloid cascades leading to peptide ß-sheet fibrils and plaques are central to many important diseases. Recently, intermediate assemblies of these cascades were identified as the toxic agents that interact with the cellular machinery. The relationship between the transformation from natively unstructured assembly to the ß-sheet oligomers to disease is important in understanding disease onset and the development of therapeutic agents. Research on this early oligomeric region has largely been unsuccessful since traditional techniques measure only ensemble average oligomer properties. Here, ion mobility methods are utilized to deduce the modulation of peptide self-assembly pathways in the amyloid-ß protein fragment Aß(25-35) by two amyloid inhibitors (epigallocatechin gallate and scyllo-inositol) that are currently in clinical trials for Alzheimer's Disease. We provide evidence that suppression of ß-extended oligomers from the onset of the conversion into ß-oligomer conformations is essential for effective attenuation of ß-structured amyloid oligomeric species often associated with oligomer toxicity. Furthermore, we demonstrate the ease with which ion mobility spectrometry-mass spectrometry can guide the development of therapeutic agents and drug evaluation by providing molecular level insight into the amyloid formation process and its modulation by small molecule assembly modulators.

Investigation of Humidity Dependent Surface Morphology and Proton Conduction in Multi-Acid Side Chain Membranes by Conductive Probe Atomic Force Microscopy.

In this report, we employ phase-contrast tapping mode and conductive probe atomic force microscopy (cp-AFM) as tools to investigate the nanoscale morphology and proton conductance of a 3M perfluoro-imide acid (PFIA) membrane (625 EW) over a large range of relative humidity (3-95% RH). As a point of comparison, we also investigate 3M perfluorosulfonic acid (PFSA) (825 EW) and Nafion 212. With AFM, we assess the membrane's water retention and mechanical stability at low RH and high RH, respectively. Cp-AFM allows us to spatially resolve the hydrophilic and electrochemically active domains under a similar set of conditions and observe directly the ties between membrane morphology and proton conductance. From our data, we are able to correlate the improved water retention indicated by the size of the hydrophilic domains with the proton conductance in the PFIA membrane at elevated temperature and compare the result with that observed for the PFSA and Nafion. At high RH conditions, we see evidence of a nearly continuous hydrophilic phase, which indicates a high degree of swelling.

Interactions between amyloid-ß and Tau fragments promote aberrant aggregates: implications for amyloid toxicity.

We have investigated at the oligomeric level interactions between Aß(25-35) and Tau(273-284), two important fragments of the amyloid-ß and Tau proteins, implicated in Alzheimer's disease. We are able to directly observe the coaggregation of these two peptides by probing the conformations of early heteroligomers and the macroscopic morphologies of the aggregates. Ion-mobility experiment and theoretical modeling indicate that the interactions of the two fragments affect the self-assembly processes of both peptides. Tau(273-284) shows a high affinity to form heteroligomers with existing Aß(25-35) monomer and oligomers in solution. The configurations and characteristics of the heteroligomers are determined by whether the population of Aß(25-35) or Tau(273-284) is dominant. As a result, two types of aggregates are observed in the mixture with distinct morphologies and dimensions from those of pure Aß(25-35) fibrils. The incorporation of some Tau into ß-rich Aß(25-35) oligomers reduces the aggregation propensity of Aß(25-35) but does not fully abolish fibril formation. On the other hand, by forming complexes with Aß(25-35), Tau monomers and dimers can advance to larger oligomers and form granular aggregates. These heteroligomers may contribute to toxicity through loss of normal function of Tau or inherent toxicity of the aggregates themselves.

Effects of pH and charge state on peptide assembly: the YVIFL model system.

Peptide oligomerization is necessary but not sufficient for amyloid fibril formation. Here, we use a combination of experiments and simulations to understand how pH influences the aggregation properties of a small hydrophobic peptide, YVIFL, which is a mutant form of [Leu-5]-Enkephalin. Transmission electron microscopy and atomic force microscopy measurements reveal that this peptide forms small aggregates under acidic conditions (pH = 2), but that extensive fibrillization only occurs under basic conditions (pH = 9 and 11). Ion-mobility mass spectrometry identifies key oligomers in the oligomerization process, which are further characterized at an atomistic level by molecular dynamics simulations. These simulations suggest that terminal charges play a critical role in determining aggregation propensity and aggregate morphology. They also reveal the presence of steric zipper oligomers under basic conditions, a possible precursor to fibril formation. Our experiments suggest that multiple aggregation pathways can lead to YVIFL fibrils, and that cooperative and multibody interactions are key mechanistic elements in the early stages of aggregation.

Factors that drive peptide assembly and fibril formation: experimental and theoretical analysis of Sup35 NNQQNY mutants.

Residue mutations have substantial effects on aggregation kinetics and propensities of amyloid peptides and their aggregate morphologies. Such effects are attributed to conformational transitions accessed by various types of oligomers such as steric zipper or single ß-sheet. We have studied the aggregation propensities of six NNQQNY mutants: NVVVVY, NNVVNV, NNVVNY, VIQVVY, NVVQIY, and NVQVVY in water using a combination of ion-mobility mass spectrometry, transmission electron microscopy, atomic force microscopy, and all-atom molecular dynamics simulations. Our data show a strong correlation between the tendency to form early ß-sheet oligomers and the subsequent aggregation propensity. Our molecular dynamics simulations indicate that the stability of a steric zipper structure can enhance the propensity for fibril formation. Such stability can be attained by either hydrophobic interactions in the mutant peptide or polar side-chain interdigitations in the wild-type peptide. The overall results display only modest agreement with the aggregation propensity prediction methods such as PASTA, Zyggregator, and RosettaProfile, suggesting the need for better parametrization and model peptides for these algorithms.