Computationally Designed Molecules Modulate ALS-Related Amyloidogenic TDP-43307-319 Aggregation.

Abnormal cytosolic aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is observed in multiple diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration, and Alzheimer's disease. Previous studies have shown that TDP-43307-319 located at the C-terminal of TDP-43 can form higher-order oligomers and fibrils. Of particular interest are the hexamers that adopt a cylindrin structure that has been strongly correlated to neurotoxicity. In this study, we use the joint pharmacophore space (JPS) model to identify and generate potential TDP-43 inhibitors. Five JPS-designed molecules are evaluated using both experimental and computational methods: ion mobility mass spectrometry, thioflavin T fluorescence assay, circular dichroism spectroscopy, atomic force microscopy, and molecular dynamics simulations. We found that all five molecules can prevent the amyloid fibril formation of TDP-43307-319, but their efficacy varies significantly. Furthermore, among the five molecules, [AC0101] is the most efficient in preventing the formation of higher-order oligomers and dissociating preformed higher-order oligomers. Molecular dynamics simulations show that [AC0101] both is the most flexible and forms the most hydrogen bonds with the TDP-43307-319 monomer. The JPS-designed molecules can insert themselves between the ß-strands in the hexameric cylindrin structure of TDP-43307-319 and can open its structure. Possible mechanisms for JPS-designed molecules to inhibit and dissociate TDP-43307-319 oligomers on an atomistic scale are proposed.

Tau P301L mutation promotes core 4R tauopathy fibril fold through near-surface water structuring and conformational rearrangement.

Tau forms toxic fibrillar aggregates in a family of neurodegenerative diseases known as tauopathies. The faithful replication of tauopathy-specific fibril structures is a critical gap for developing diagnostic and therapeutic tools. This study debuts a strategy of identifying a critical segment of tau that forms a folding motif that is characteristic of a family of tauopathies and isolating it as a standalone peptide that form seeding-competent fibrils. The 19-residue jR2R3 peptide (295-313) spanning the R2/R3 splice junction of tau, in the presence of P301L, forms seeding-competent amyloid fibrils. This tau fragment contains the hydrophobic VQIVYK hexapeptide that is part of the core of every pathological tau fibril structure solved to-date and an intramolecular counter-strand that stabilizes the strand-loop-strand (SLS) motif observed in 4R tauopathy fibrils. This study shows that P301L exhibits a duality of effects: it lowers the barrier for the peptide to adopt aggregation-prone conformations and enhances the local structuring of water around the mutation site that facilitates site-specific dewetting and in-register stacking of tau to form cross ß-sheets. We solve a 3 Å cryo-EM structure of jR2R3-P301L fibrils with a pseudo 2 1 screw symmetry in which each half of the fibril's cross-section contains two jR2R3-P301L peptides. One chain adopts a SLS fold found in 4R tauopathies that is stabilized by a second chain wrapping around the SLS fold, reminiscent of the 3-fold and 4-fold structures observed in 4R tauopathies. These jR2R3-P301L fibrils are able to template full length tau in a prion-like fashion. Significance Statement: This study presents a first step towards designing a tauopathy specific aggregation pathway by engineering a minimal tau prion building block, jR2R3, that can template and propagate distinct disease folds. We present the discovery that P301L-among the widest used mutations in cell and animal models of Alzheimer's Disease-destabilizes an aggregation-prohibiting internal hairpin and enhances the local surface water structure that serves as an entropic hotspot to exert a hyper-localized effect in jR2R3. Our study suggests that P301L may be a more suitable mutation to include in modeling 4R tauopathies than for modelling Alzheimer's Disease, and that mutations are powerful tools for the purpose of designing of tau prion models as therapeutic tools.

Computationally Designed Small Molecules Disassemble Both Soluble Oligomers and Protofibrils of Amyloid ß-Protein Responsible for Alzheimer's Disease.

Alzheimer's disease (AD) is one of the world's most pressing health crises. AD is an incurable disease affecting more than 6.5 million Americans, predominantly the elderly, and in its later stages, leads to memory loss, dementia, and death. Amyloid ß (Aß) protein aggregates have been one of the pathological hallmarks of AD since its initial characterization. The early stages of Aß accumulation and aggregation involve the formation of oligomers, which are considered neurotoxic and play a key role in further aggregation into fibrils that eventually appear in the brain as amyloid plaques. We have recently shown by combining ion mobility mass spectrometry (IM-MS) and atomic force microscopy (AFM) that Aß42 rapidly forms dodecamers (12-mers) as the terminal oligomeric state, and these dodecamers seed the early formation of Aß42 protofibrils. The link between soluble oligomers and fibril formation is one of the essential aspects for understanding the root cause of the disease state and is critical to developing therapeutic interventions. Utilizing a joint pharmacophore space (JPS) method, potential drugs have been designed specifically for amyloid-related diseases. These small molecules were generated based on crucial chemical features necessary for target selectivity. In this paper, we utilize our combined IM-MS and AFM methods to investigate the impact of three second-generation JPS small-molecule inhibitors, AC0201, AC0202, and AC0203, on dodecamer as well as fibril formation in Aß42. Our results indicate that AC0201 works well as an inhibitor and remodeler of both dodecamers and fibril formation, AC0203 behaves less efficiently, and AC0202 is ineffective.

Effect of Cosolutes on the Aggregation of a Tau Fragment: A Combined Experimental and Simulation Approach.

The intrinsically disordered protein Tau represents the main component of neurofibrillary tangles that are a hallmark of Alzheimer's disease. A small fragment of Tau, known as paired helical filament 6 (PHF6), is considered to be important for the formation of the ß-structure core of the fibrils. Here we study the aggregation of this fragment in the presence of different cosolutes, including urea, TMAO, sucrose and 2-hydroxypropyl-ß-cyclodextrin (2-HPßCD), using both experiments and molecular dynamics simulations. A novel implicit solvation approach (MIST - Model with Implicit Solvation Thermodynamics) is used, where an energetic contribution based on the concept of transfer free energies describes the effect of the cosolutes. The simulation predictions are compared to thioflavin-T and atomic force microscopy results, and the good agreement observed confirms the predictive ability of the computational approach herein proposed. Both simulations and experiments indicate that PHF6 aggregation is inhibited in the presence of urea and 2-HPßCD, while TMAO and sucrose stabilize associated conformations. The remarkable ability of HPßCD to inhibit aggregation represents an extremely promising result for future applications, especially considering the widespread use of this molecule as a drug carrier to the brain and as a solubilizer/excipient in pharmaceutical formulations.

VO Cluster-Stabilized H2O Adsorption on a TiO2 (110) Surface at Room Temperature.

We probe the adsorption of molecular H2O on a TiO2 (110)-(1 × 1) surface decorated with isolated VO clusters using ultrahigh-vacuum scanning tunneling microscopy (UHV-STM) and temperature-programmed desorption (TPD). Our STM images show that preadsorbed VO clusters on the TiO2 (110)-(1 × 1) surface induce the adsorption of H2O molecules at room temperature (RT). The adsorbed H2O molecules form strings of beads of H2O dimers bound to the 5-fold coordinated Ti atom (5c-Ti) rows and are anchored by VO. This RT adsorption is completely reversible and is unique to the VO-decorated TiO2 surface. TPD spectra reveal two new desorption states for VO stabilized H2O at 395 and 445 K, which is in sharp contrast to the desorption of water due to recombination of hydroxyl groups at 490 K from clean TiO2(110)-(1 × 1) surfaces. Density functional theory (DFT) calculations show that the binding energy of molecular H2O to the VO clusters on the TiO2 (110)-(1 × 1) surface is higher than binding to the bare surface by 0.42 eV, and the resulting H2O-VO-TiO2 (110) complex provides the anchor point for adsorption of the string of beads of H2O dimers.

Tachykinin Neuropeptides and Amyloid ß (25-35) Assembly: Friend or Foe?

Amyloid ß (Aß) protein is responsible for Alzheimer's disease, and one of its important fragments, Aß(25-35), is found in the brain and has been shown to be neurotoxic. Tachykinin neuropeptides, including Neuromedin K (NK), Kassinin, and Substance P, have been reported to reduce Aß(25-35)'s toxicity in cells even though they share similar primary structures with Aß(25-35). Here, we seek to understand the molecular mechanisms of how these peptides interact with Aß(25-35) and to shed light on why some peptides with similar primary structures are toxic and others nontoxic. We use both experimental and computational methods, including ion mobility mass spectrometry and enhanced-sampling replica-exchange molecular dynamics simulations, to study the aggregation pathways of Aß(25-35), NK, Kassinin, Substance P, and mixtures of the latter three with Aß(25-35). NK and Substance P were observed to remove the higher-order oligomers (i.e., hexamers and dodecamers) of Aß(25-35), which are related to its toxicity, although Substance P did so more slowly. In contrast, Kassinin was found to promote the formation of these higher-order oligomers. This result conflicts with what is expected and is elaborated on in the text. We also observe that even though they have significant structural homology with Aß(25-35), NK, Kassinin, and Substance P do not form hexamers with a ß-sheet structure like Aß(25-35). The hexamer structure of Aß(25-35) has been identified as a cylindrin, and this structure has been strongly correlated to toxic species. The reasons why the three tachykinin peptides behave so differently when mixed with Aß(25-35) are discussed.

Catalytic Cross Talk between Key Peptide Fragments That Couple Alzheimer's Disease with Amyotrophic Lateral Sclerosis.

Protein aggregation is a common feature in prominent neurodegenerative diseases, usually thought to be due to the assembly of a single peptide or protein. Recent studies have challenged this notion and suggested several proteins may be involved in promoting and amplifying disease. For example, the TDP-43 protein associated with Amyotrophic Lateral Sclerosis has been found in the brain along with Aß assemblies associated with Alzheimer's disease, and those patients that show the presence of TDP-43 are 10 times more likely to demonstrate cognitive impairment compared to TDP-43-negative Alzheimer's patients. Here we examine the interactions between the amyloidogenic core of TDP-43, TDP-43307-319, and a neurotoxic physiologically observed fragment of Aß, Aß25-35. Utilizing ion mobility mass spectrometry in concert with atomic force microscopy and molecular dynamics simulations, we investigate which oligomers are involved in seeding aggregation across these two different protein systems and gain insight into which structures initiate and result from these interactions. Studies were conducted by mixing Aß25-35 with the toxic wild type TDP-43307-319 peptide and with the nontoxic synthetic TDP-43307-319 mutant, G314V. Our findings identify a strong catalytic effect of TDP-43307-319 WT monomer in the acceleration of Aß25-35 aggregation to its toxic cylindrin and ß barrel forms. This observation is unprecedented in both its speed and specificity. Interestingly, the nontoxic G314V mutant of TDP-43307-319 and dimers or higher order oligomers of WT TDP-43307-319 do not promote aggregation of Aß25-35 but rather dissociate preformed toxic higher order oligomers of Aß25-35. Reasons for these very different behaviors are reported.

Modulating ALS-Related Amyloidogenic TDP-43307-319 Oligomeric Aggregates with Computationally Derived Therapeutic Molecules.

TDP-43 aggregates are a salient feature of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and a variety of other neurodegenerative diseases, including Alzheimer's disease (AD). With an anticipated growth in the most susceptible demographic, projections predict neurodegenerative diseases will potentially affect 15 million people in the United States by 2050. Currently, there are no cures for ALS, FTD, or AD. Previous studies of the amyloidogenic core of TDP-43 have demonstrated that oligomers greater than a trimer are associated with toxicity. Utilizing a joint pharmacophore space (JPS) method, potential drugs have been designed specifically for amyloid-related diseases. These molecules were generated on the basis of key chemical features necessary for blood-brain barrier permeability, low adverse side effects, and target selectivity. Combining ion-mobility mass spectrometry and atomic force microscopy with the JPS computational method allows us to more efficiently evaluate a potential drug's efficacy in disrupting the development of putative toxic species. Our results demonstrate the dissociation of higher-order oligomers in the presence of these novel JPS-generated inhibitors into smaller oligomer species. Additionally, drugs approved by the Food and Drug Administration for the treatment of ALS were also evaluated and demonstrated to maintain higher-order oligomeric assemblies. Possible mechanisms for the observed action of the JPS molecules are discussed.

Humidity-Dependent Surface Structure and Hydroxide Conductance of a Model Quaternary Ammonium Anion Exchange Membrane.

Anion exchange membrane (AEM) fuel cells (AEMFCs) are a promising cost-effective alternative energy conversion technology because of the potential implementation of earth-abundant catalysts, obviating the need for precious metals. AEMs, however, have low conductivity and suffer from poor stability. The conductivity of the AEM is inherently tied to the complex phase-separated morphology, as its dependence on the hydration level is not well understood. In this report, we employ phase-contrast tapping mode and conductive-probe atomic force microscopy (cp-AFM) to study the nanoscale surface morphology and hydroxide conductance of a commercially available quaternary ammonium (QA) AEM by FuMA-Tech GmbH (Fumapem FAA-3). The chemical structure of FAA-3 consists of a poly(phenylene oxide) backbone with QA functionality. The morphology of FAA-3 was observed in the bromide (FAA-3-Br-) and hydroxide form (FAA-3-OH-) in dehydrated and hydrated conditions. Under dehydrated conditions, both membranes showed no phase contrast, indicating the absence of phase-separated hydrophilic domains at the surface. At hydrated conditions, FAA-3-Br- shows randomly dispersed isolated clusters, while FAA-3-OH- shows elongated fibrillar structures extending microns in length. cp-AFM of hydrated FAA-3-OH- showed that these elongated regions were insulating. These results provide morphological evidence for the conduction of hydroxide at the surface and its dependence on the hydration level.

Characterizing TDP-43307-319 Oligomeric Assembly: Mechanistic and Structural Implications Involved in the Etiology of Amyotrophic Lateral Sclerosis.

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a salient feature of amyotrophic lateral sclerosis (ALS), a debilitating neurodegenerative disorder affecting over 200 000 people worldwide. The protein undergoes both functional and pathogenic aggregation; the latter is irreversible and hypothesized to produce soluble oligomers that are toxic to neurons in addition to inclusions made of stable fibrous deposits. Despite progress made toward identifying disease-related proteins, the underlying pathogenic mechanism associated with these toxic oligomers remains elusive. Utilizing a multimodal approach that combines several measurement techniques (circular dichroism (CD), thioflavin T spectroscopy (ThT), Fourier transform infrared spectroscopy (FTIR)) and high spatial resolution imaging tools (electron microscopy (EM) and atomic force microscopy (AFM)), with soft ion mobility mass spectrometry (IM-MS) and atomistic molecular dynamics (MD) simulations, we explore the oligomerization mechanisms, structures, and assembly pathways of TDP-43307-319. This fragment is both amyloidogenic and toxic and is within the glycine-rich C-terminal domain essential for both toxicity and aggregation of the full-length protein. In addition to the wild-type peptide, two ALS-related mutants (A315T and A315E) and a non-axon-toxic mutant (G314V) were investigated to determine how mutations affect the oligomerization of TDP-43307-319 and structures of toxic oligomers. The results of our study provide new insights into how ALS-related mutants, A315T and A315E, accelerate or alter the pathogenic mechanism and highlight the role of an internal glycine, G314, in maintaining efficient packing known to be critical for functional oligomer assembly. More importantly, our data demonstrate that G314 plays a vital role in TDP-43 assembly and prevents cytotoxicity via its unique aversion to oligomers larger than trimer. Our observation is consistent with previous studies showing that G314V mutation of the full-length TDP-43 induced remediation of both axonotoxicity and neuronal apoptosis. Our findings reveal a distinct aggregation mechanism for each peptide and elucidate oligomeric species and possible structures that may be involved in the pathology of ALS.

Inhibiting and Remodeling Toxic Amyloid-Beta Oligomer Formation Using a Computationally Designed Drug Molecule That Targets Alzheimer's Disease.

Alzheimer's disease (AD) is rapidly reaching epidemic status among a burgeoning aging population. Much evidence suggests the toxicity of this amyloid disease is most influenced by the formation of soluble oligomeric forms of amyloid ß-protein, particularly the 42-residue alloform (Aß42). Developing potential therapeutics in a directed, streamlined approach to treating this disease is necessary. Here we utilize the joint pharmacophore space (JPS) model to design a new molecule [AC0107] incorporating structural characteristics of known Aß inhibitors, blood-brain barrier permeability, and limited toxicity. To test the molecule's efficacy experimentally, we employed ion mobility mass spectrometry (IM-MS) to discover [AC0107] inhibits the formation of the toxic Aß42 dodecamer at both high (1:10) and equimolar concentrations of inhibitor. Atomic force microscopy (AFM) experiments reveal that [AC0107] prevents further aggregation of Aß42, destabilizes preformed fibrils, and reverses Aß42 aggregation. This trend continues for long-term interaction times of 2 days until only small aggregates remain with virtually no fibrils or higher order oligomers surviving. Pairing JPS with IM-MS and AFM presents a powerful and effective first step for AD drug development. Graphical Abstract.

Zinc-Induced Conformational Transitions in Human Islet Amyloid Polypeptide and Their Role in the Inhibition of Amyloidosis.

Type-2 diabetes mellitus (T2DM) is a disease hallmarked by improper homeostasis within the islets of Langerhans of the pancreas. The most critical species affected is insulin, which is produced by the ß-cells of the islets, but there are a number of other species copackaged and cosecreted within the insulin granules. This includes zinc, which exists in high (millimolar) concentrations within the ß-cells, and islet amyloid polypeptide (IAPP), which is an amyloid peptide thought to induce ß-cell apoptosis through self-association into toxic amyloid oligomers. Zinc is essential in the packaging of crystalline insulin within the vesicles but it can also bind and interact with IAPP. This implies a complex relationship between all three species and diabetes, particularly in the structure and function of toxic IAPP aggregates. Atypical (low or high) concentrations of zinc generally appear to correlate with increased hIAPP aggregation, whereas physiological zinc concentrations have an inhibitory effect. To better understand how zinc ions alter the monomer and oligomer structure of hIAPP in vitro, we employ a combination of ion mobility mass spectrometry and atomic force microscopy. We observe an increase in the extended ß-hairpin conformation of hIAPP when it is bound to zinc. With sufficiently low concentrations of zinc this could result in an association site for zinc-free hIAPP, promoting amyloid aggregation. At high zinc concentrations, we see the appearance of a secondary zinc association site whose coordination could account for the loss of inhibition at high zinc concentrations. Generally, it appears that zinc preferentially stabilizes the ß-hairpin conformation of hIAPP and the population of zinc-bound hIAPP in solution determines what effect this has on amyloid aggregation.

Hetero-oligomeric Amyloid Assembly and Mechanism: Prion Fragment PrP(106-126) Catalyzes the Islet Amyloid Polypeptide ß-Hairpin.

Protein aggregation is typically attributed to the association of homologous amino acid sequences between monomers of the same protein. Coaggregation of heterogeneous peptide species can occur, however, and is implicated in the proliferation of seemingly unrelated protein diseases in the body. The prion protein fragment (PrP106-126) and human islet amyloid polypeptide (hIAPP) serve as an interesting model of nonhomologous protein assembly as they coaggregate, despite a lack of sequence homology. We have applied ion-mobility mass spectrometry, atomic force microscopy, circular dichroism, and high-level molecular modeling to elucidate this important assembly process. We found that the prion fragment not only forms pervasive hetero-oligomeric aggregates with hIAPP but also promotes the transition of hIAPP into its amyloidogenic ß-hairpin conformation. Further, when PrP106-126 was combined with non-amyloidogenic rIAPP, the two formed nearly identical hetero-oligomers to those seen with hIAPP, despite rIAPP containing ß-sheet breaking proline substitutions. Additionally, while rIAPP does not natively form the amyloidogenic ß-hairpin structure, it did so in the presence of PrP106-126 and underwent a conformational transition to ß-sheet in solution. We also find that PrP106-126 forms hetero-oligomers with the IAPP8-20 fragment but not with the "aggregation hot spot" IAPP20-29 fragment. PrP106-126 apparently induces IAPP into a ß-hairpin structure within the PrP:IAPP heterodimer complex and then, through ligand exchange, catalytically creates the amyloidogenic ß-hairpin dimer of IAPP in significantly greater abundance than IAPP does on its own. This is a new mechanistic model that provides a critical foundation for the detailed study of hetero-oligomerization and prion-like proliferation in amyloid systems.

Distal amyloid ß-protein fragments template amyloid assembly.

Amyloid formation is associated with devastating diseases such as Alzheimer's, Parkinson's and Type-2 diabetes. The large amyloid deposits found in patients suffering from these diseases have remained difficult to probe by structural means. Recent NMR models also predict heterotypic interactions from distinct peptide fragments but limited evidence of heterotypic packed sheets is observed in solution. Here we characterize two segments of the protein amyloid ß (Aß) known to form fibrils in Alzheimer's disease patients. We designed two variants of Aß(19-24) and Aß(27-32), IFAEDV (I6V) and NKGAIF (N6F) to lower the aggregation propensity of individual peptides while maintaining the similar interactions between the two segments in their native forms. We found that the variants do not form significant amyloid fibrils individually but a 1:1 mixture forms abundant fibrils. Using ion mobility-mass spectrometry (IM-MS), hetero-oligomers up to decamers were found in the mixture while the individual peptides formed primarily dimers and some tetramers consistent with a strong heterotypic interaction between the two segments. We showed by X-ray crystallography that I6V formed a Class 7 zipper with a weakly packed pair of ß-sheets and no segregated dry interface, while N6F formed a more stable Class 1 zipper. In a mixture of equimolar N6F:I6V, I6V forms a more stable zipper than in I6V alone while no N6F or hetero-typic zippers are observed. These data are consistent with a mechanism where N6F catalyzes assembly of I6V into a stable zipper and perhaps into stable, pure I6V fibrils that are observed in AFM measurements.

Imaging Channel Connectivity in Nafion Using Electrostatic Force Microscopy.

Channel connectivity is an important material property that is considered in making higher-performance proton-exchange membranes. Our group has previously demonstrated that nearly 50% of the aqueous surface domains in Nafion films do not have a connected path to the opposite side of the membrane. These so-called "dead-end" channels lead to a loss in the conductance efficiency of the membrane. Understanding the structure of these dead-end channels is an important step in improving the conductance of the membrane. Although conductive atomic force microscopy is able to provide insight into the connected channels, it does directly report on the dead-end channels. To address this, we use electrostatic force microscopy (EFM) to probe channel connectivity in a Nafion thin film (100-300 nm) under ambient conditions. EFM provided an image of the capacitive phase shift, which is influenced by surface charge, dielectric permittivity, and tip-sample geometry. We studied several individual channels and measured the quadratic dependence of the EFM signal with the bias voltage. Applying a simple parallel plate model allowed us to assign differences in the EFM signal to particular channel shapes: connected cylindrical channels, dead-end cylinder channels, and bottleneck channels.

Human Islet Amyloid Polypeptide Assembly: The Key Role of the 8-20 Fragment.

The aggregation of human islet amyloid polypeptide (hIAPP) has been closely associated with the pathogeny of type 2 diabetes mellitus (T2DM) and destruction of pancreatic islet ß-cells. Several amyloidogenic domains within the hIAPP sequence capable of self-association have been identified. Among them is the 8-20 region of hIAPP, which has formed ß-sheet fibrils despite being contained within an α-helical region of full-length hIAPP. To further understand the propensity of this region for self-assembly, two peptide fragments were compared, one consisting of the residues 8-20 (WT8-20) and a mutant fragment with a His18Pro substitution (H18P8-20). The conformational distribution and aggregation propensity of these peptides was determined using a combination of ion mobility mass spectrometry and atomic force microscopy. Our results reveal that the two peptide fragments have vastly differing assembly pathways. WT8-20 produces a wide range of oligomers up to decamer whereas the H18P8-20 mutant produces only low order oligomers. This study confirms the propensity of the 8-20 region to aggregate from its native α-helical structure into amyloid ß-sheet oligomers and highlights the significance of the charged His18 in the aggregation process.

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.

Human Islet Amyloid Polypeptide N-Terminus Fragment Self-Assembly: Effect of Conserved Disulfide Bond on Aggregation Propensity.

Amyloid formation by human islet amyloid polypeptide (hIAPP) has long been implicated in the pathogeny of type 2 diabetes mellitus (T2DM) and failure of islet transplants, but the mechanism of IAPP self-assembly is still unclear. Numerous fragments of hIAPP are capable of self-association into oligomeric aggregates, both amyloid and non-amyloid in structure. The N-terminal region of IAPP contains a conserved disulfide bond between cysteines at position 2 and 7, which is important to hIAPP's in vivo function and may play a role in in vitro aggregation. The importance of the disulfide bond in this region was probed using a combination of ion mobility-based mass spectrometry experiments, molecular dynamics simulations, and high-resolution atomic force microscopy imaging on the wildtype 1-8 hIAPP fragment, a reduced fragment with no disulfide bond, and a fragment with both cysteines at positions 2 and 7 mutated to serine. The results indicate the wildtype fragment aggregates by a different pathway than either comparison peptide and that the intact disulfide bond may be protective against aggregation due to a reduction of inter-peptide hydrogen bonding. Graphical Abstract ᅟ.

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.