Thermophoretic trap for single amyloid fibril and protein aggregation studies.

The study of the aggregation of soluble proteins into highly ordered, insoluble amyloid fibrils is fundamental for the understanding of neurodegenerative disorders. Here, we present a method for the observation of single amyloid fibrils that allows the investigation of fibril growth, secondary nucleation or fibril breakup that is typically hidden in the average ensemble. Our approach of thermophoretic trapping and rotational diffusion measurements is demonstrated for single Aß40, Aß42 and pyroglutamyl-modified amyloid-ß variant (pGlu3-Aß3-40) amyloid fibrils.

Probing the role of ceramide hydroxylation in skin barrier lipid models by 2H solid-state NMR spectroscopy and X-ray powder diffraction.

In this work, we studied model stratum corneum lipid mixtures composed of the hydroxylated skin ceramides N-lignoceroyl 6-hydroxysphingosine (Cer[NH]) and α-hydroxylignoceroyl phytosphingosine (Cer[AP]). Two model skin lipid mixtures of the composition Cer[NH] or Cer[AP], N-lignoceroyl sphingosine (Cer[NS]), lignoceric acid (C24:0) and cholesterol in a 0.5:0.5:1:1 molar ratio were compared. Model membranes were investigated by differential scanning calorimetry and 2H solid-state NMR spectroscopy at temperatures from 25 °C to 80 °C. Each component of the model mixture was specifically deuterated for selective detection by 2H NMR. Thus, the exact phase composition of the mixture at varying temperatures could be quantified. Moreover, using X-ray powder diffraction we investigated the lamellar phase formation. From the solid-state NMR and DSC studies, we found that both hydroxylated Cer[NH] and Cer[AP] exhibit a similar phase behavior. At physiological skin temperature of 32 °C, the lipids form a crystalline (orthorhombic) phase. With increasing temperature, most of the lipids become fluid and form a liquid-crystalline phase, which converts to the isotropic phase at higher temperatures (65-80 °C). Interestingly, lignoceric acid in the Cer[NH]-containing mixture has a tendency to form two types of fluid phases at 65 °C. This tendency was also observed in Cer[AP]-containing membranes at 80 °C. While Cer[AP]-containing lipid models formed a short periodicity phase featuring a repeat spacing of d = 5.4 nm, in the Cer[NH]-based model skin lipid membranes, the formation of unusual long periodicity phase with a repeat spacing of d = 10.7 nm was observed.

Pyroglutamate-Modified Amyloid ß (11- 40) Fibrils Are More Toxic than Wildtype Fibrils but Structurally Very Similar.

The morphology, structure, and dynamics of mature amyloid ß (Aß) fibrils formed by the Aß variant, which is truncated at residue 11 and chemically modified by enzymatic pyroglutamate formation (pGlu11 -Aß(11-40)), was studied along with the investigation of the toxicity of these Aß variants to neurons and astrocytes. The fibrils of pGlu11 -Aß (11-40) were more toxic than wildtype Aß (1-40) and the longer pGlu3-Aß (3-40) especially at higher concentration, whereas the overall morphology was quite similar. The secondary structure of pGlu11 -Aß (11-40) fibrils shows the typical two ß-strands connected by a short turn as known for mature fibrils of Aß (1-40) and also pGlu3 -Aß (3-40). Further insights into tertiary contacts exhibit some similarities of pGlu11 -Aß (11-40) fibrils with wildtype Aß (1-40), but also a so far not described contact between Gly25 and Ile31 . This highlights the biological importance of chemical modifications on the molecular structure of Aß.

N-terminal lipid conjugation of amyloid ß(1-40) leads to the formation of highly ordered N-terminally extended fibrils.

Fibril formation of amyloid ß(1-40) (Aß(1-40)) peptides N-terminally lipid modified with saturated octanoyl or palmitoyl lipid chains was investigated. Lipid modification of Aß(1-40) significantly accelerates the fibrillation kinetics of the Aß peptides as revealed by ThT fluorescence. Electron microscopy and X-ray diffraction results indicate a heterogeneous cross-ß structure of the fibrils formed by the lipid-conjugated peptides. Solid-state NMR was used to investigate structural features of these fibrils. The lipid moieties form dynamic and loosely structured heterogeneous lipid assemblies as inferred from 2H NMR of the deuterated lipid chains. 13C NMR studies of selected isotopic labels reveals that in addition to Phe19 and Val39, which are part of the canonical cross-ß structure, also N-terminal residues (Ala2, Phe4, Val12) are found in ß-strand conformation. This suggests that the increased hydrophobicity induced by the lipid modification, alters the energy landscape rendering an N-terminal extension of the ß-sheet structure favorable. Furthermore, the fibrils formed by the Aß-lipid hybrids are much more rigid than wildtype Aß fibrils as inferred from NMR order parameter measurements. Taken together, increasing the local hydrophobicity of the Aß N-terminus results in highly ordered but heterogeneous amyloid fibrils with extended N-terminal ß-sheet structure.

Structural characterization of amyloid fibrils from the human parathyroid hormone.

Amyloid deposits are common in various tissues as a consequence of misfolded proteins. However, secretory protein and peptides are often stored in membrane coated granules as functional amyloids. In this article, we present a detailed characterization of in vitro generated amyloid fibrils from human parathyroid hormone (hPTH(1-84)). Fully mature fibrils could be obtained after a short lag phase within less than one hour at 65°C. These fibrils showed all characteristic of a cross-ß structure. Protease cleavage combined with mass spectrometry identified the central region of the peptide hormone involved in the fibril core formation. EGCG, an inhibitor of amyloid fibril formation, showed binding to residues in the peptide monomers corresponding to the later fibril core and thus explaining the inhibition of the fibril growth. Conformational and dynamic studies by solid-state NMR further corroborated the cross-ß core of the fibrils, but also identified highly mobile segments with a random coil structure not belonging to the rigid fibril core.

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid ß (1-40).

A small library of rationally designed amyloid ß [Aß(1-40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aß(1-40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid-state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aß(1-40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aß fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross-ß structure.

Local interactions influence the fibrillation kinetics, structure and dynamics of Aß(1-40) but leave the general fibril structure unchanged.

A series of peptide mutants was studied to understand the influence of local physical interactions on the fibril formation mechanism of amyloid ß (Aß)(1-40). In the peptide variants, the well-known hydrophobic contact between residues phenylalanine 19 and leucine 34 was rationally modified. In single site mutations, residue phenylalanine 19 was replaced by amino acids that introduce higher structural flexibility by a glycine mutation or restrict the backbone flexibility by introduction of proline. Next, the aromatic phenylalanine was replaced by tyrosine or tryptophan, respectively, to probe the influence of additional hydrogen bond forming capacity in the fibril interior. Furthermore, negatively charged glutamate or positively charged lysine was introduced to probe the influence of electrostatics. In double mutants, the hydrophobic contact was replaced by a putative salt bridge (glutamate and lysine) or two electrostatically repelling lysine residues. The influence of these mutations on the fibrillation kinetics and morphology, cross-ß structure as well as the local structure and dynamics was probed using fluorescence, transmission electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. While the fibrillation kinetics and the local structure and dynamics of the peptide variants were influenced by the introduction of these local fields, the overall morphology and cross-ß structure of the fibrils remained very robust against all the probed interactions. Overall, 7 out of the 8 mutated peptides formed fibrils of very similar morphology compared to the wildtype. However, characteristic local structural and dynamical changes indicate that amyloid fibrils show an astonishing ability to respond to local perturbations but overall show a very homogenous mesoscopic organization.

The influence of the ΔK280 mutation and N- or C-terminal extensions on the structure, dynamics, and fibril morphology of the tau R2 repeat.

Tau is a microtubule-associated protein and is involved in microtubule assembly and stabilization. It consists of four repeats that bind to the microtubule. The ΔK280 deletion mutation in the tau R2 repeat region is directly associated with the development of the frontotemporal dementia parkinsonism linked to chromosome 17 (FTDP-17). This deletion mutation is known to accelerate tau R2 repeat aggregation. However, the secondary and the tertiary structures of the self-assembled ΔK280 tau R2 repeat mutant aggregates are still controversial. Moreover, it is unclear whether extensions by one residue in the N- or the C-terminus of this mutant can influence the secondary or the tertiary structure. Herein, we combine solid-state NMR, atomic force microscopy, electron microscopy and all-atom explicit molecular dynamics simulations to investigate the effects of the deletion mutation and the N- and the C-terminal extension of this mutant on the structure. Our main findings show that the deletion mutation induces the formation of small aggregates, such as oligomers, and reduces the formation of fibrils. However, the extensions in the N- or the C-terminus revealed more fibril formation than small aggregates. Further, in the deletion mutation only one structure is preferred, while the N- and the C-terminal extensions strongly lead to polymorphic states. Finally, our broad and combined experimental and computational techniques provide direct structural information regarding ΔK280 tau R2 repeat mutant aggregates and their extensions in the N- and C-terminii by one residue.

Membrane-Mediated Allosteric Action of Serotonin on a Noncognate G-Protein-Coupled Receptor.

The structure and dynamics of the lipid membrane can affect the activity of membrane proteins. Therefore, small lipophilic molecules that alter membrane properties (such as the neurotransmitter serotonin) can potentially modulate receptor activity without binding to the receptor. Here, we investigated how the activity of neuropeptide Y type 4 receptor (Y4R, reconstituted in lipid bicelles) is modulated by serotonin, which has no known interaction with Y4R. We found a serotonin-concentration-dependent decrease (down to 0.1 mM of serotonin) in the ligand affinity of Y4R. This effect correlates with a serotonin-induced reduction of the resistance of the bilayer to indentation (measured by atomic force microscopy) and bilayer thickness (measured by solid state NMR) in two different types of zwitterionic lipid bicelles. Our findings indicate a "membrane-mediated allosteric effect" of serotonin on the activation of Y4R and suggest the potential for developing pharmacophores, which can modulate cellular signaling without directly interacting with any receptor.

Mechanistic insights into the size-dependent effects of nanoparticles on inhibiting and accelerating amyloid fibril formation.

The aggregation of peptides into amyloid fibrils has been linked to ageing-related diseases, such as Alzheimer's and type 2 diabetes. Interfaces, particularly those with large nanostructured surfaces, can affect the kinetics of peptide aggregation, which ranges from complete inhibition to strong acceleration. While a number of physiochemical parameters determine interfacial effects, we focus here on the role of nanoparticle (NP) size and curvature. We used thioflavin T (ThT) fluorescence assays to demonstrate the size-dependent effects of NPs on amyloid fibril formation for the peptides Aß40, NNFGAIL, GNNQQNY and VQIYVK. While 5 nm gold NPs (AuNP-5) retarded or inhibited the aggregation of all peptides except NNFGAIL, larger 20 nm gold NPs (AuNP-20) tended to accelerate or not influence peptide aggregation. Differences in the NP effects for the peptides resulted from the different peptide properties (size, tendency to aggregate) and associated surface binding affinities. Additional dynamic light scattering (DLS), electron microscopy, and atomic force microscopy (AFM) experiments with the Aß40 peptide confirmed size-dependent NP effects on peptide aggregation, and also suggested a structural influence on the formed fibrils. NPs can serve as a surface for the adsorption of peptide monomers and enable nucleation to oligomers and fibril formation. However, molecular dynamics (MD) simulations showed that peptide oligomers were less stable at smaller NPs. High surface curvatures destabilized prefibrillar structures, which provides a possible explanation for inhibitory effects on fibril growth, provided that peptide-NP surface binding was relevant for fibril formation. These mechanistic insights can support the design of future nanostructured materials.

Free Heme and Amyloid-ß: A Fatal Liaison in Alzheimer's Disease.

While the etiology of Alzheimer's disease (AD) is still unknown, an increased formation of amyloid-ß (Aß) peptide and oxidative processes are major pathological mechanism of the disease. The interaction of Aß with free heme leads to the formation of peroxidase-active Aß-heme complexes. However, enzyme-kinetic data and systematic mutational studies are still missing. These aspects were addressed in this study to evaluate the role of Aß-heme complexes in AD. The enzyme-kinetic measurements showed peroxidase-specific pH- and H2O2-dependencies. In addition, the enzymatic activity of Aß-heme complexes constantly increased at higher peptide excess. Moreover, the role of the Aß sequence for the named enzymatic activity was tested, depicting human-specific R5, Y10, and H13 as essential amino acids. Also by studying Y10 as an endogenous peroxidase substrate for Aß-heme complexes, ratio-specific effects were observed, showing an optimal dityrosine formation at an about 40-fold peptide excess. As dityrosine formation promotes Aß fibrillation while free heme disturbs protein aggregation, we also investigated the effect of Aß-heme complex-derived peroxidase activity on the formation of Aß fibrils. The fluorescence measurements showed a different fibrillation behavior at strong peroxidase activity, leading also to altered fibril morphologies. The latter was detected by electron microscopy. As illustrated by selected in vivo measurements on a mouse model of AD, the disease is also characterized by Aß-derived microvessel destructions and hemolytic processes. Thus, thrombo-hemorrhagic events are discussed as a source for free heme in brain tissue. In summary, we suggest the formation and enzymatic activity of Aß-heme complexes as pathological key features of AD.

Amyloid ß (1-40) Toxicity Depends on the Molecular Contact between Phenylalanine 19 and Leucine 34.

The formation of the hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) of amyloid ß (1-40) (Aß(1-40)) is known to be an important step in the fibrillation of Aß(1-40) peptides. Mutations of this putatively early molecular contact were shown to strongly influence the toxicity of Aß(1-40) ( Das et al. ( 2015 ) ACS Chem. Neurosci. 6 , 1290 - 1295 ). Any mutation of residue F19 completely abolished the toxicity of Aß(1-40), suggesting that a proper F19-L34 contact is crucial also for the formation of transient oligomers. In this work, we investigate a series of isomeric substitutions of L34, namely, d-leucine, isoleucine, and valine, to study further details of this molecular contact. These replacements represent very minor alterations in the Aß(1-40) structure posing the question how these alterations challenge the fibrillation kinetics, structure, dynamics, and toxicity of the Aß(1-40) aggregates. Our work involves kinetic studies using thioflavin T, transmission electron microscopy, X-ray diffraction for the analysis of the fibril morphology, and nuclear magnetic resonance experiments for local structure and molecular dynamics investigations. Combined with cell toxicity assays of the mutated Aß(1-40) peptides, the physicochemical and biological importance of the early folding contact between F19 and L34 in Aß(1-40) is underlined. This implies that the F19-L34 contact influences a broad range of different processes including the initiation of fibrillation, oligomer stability, fibril elongation, local fibril structure, and dynamics and cellular toxicity. These processes do not only cover a broad range of diverse mechanisms, but also proved to be highly sensitive to minor modulations of this crucial contact. Furthermore, our work shows that the contact is not simply mediated by general hydrophobic interactions, but also depends on stereospecific mechanisms.

Perturbation of the F19-L34 Contact in Amyloid ß (1-40) Fibrils Induces Only Local Structural Changes but Abolishes Cytotoxicity.

We explored structural details of fibrils formed by a mutated amyloid ß (Aß(1-40)) peptide carrying a Phe19 to Lys19 mutation, which was shown to completely abolish the toxicity of the molecule. Computer models suggest that the positively charged Lys19 side chain is expelled from the hydrophobic fibril interior upon fibrillation. This can be accommodated by either a 180° flip of the entire lower ß-strand (model M1) or local perturbations of the secondary structure in the direct vicinity of the mutated site (model M2). This is accompanied by the formation of a new salt bridge between Glu22 and Lys28 in model M1. Experimentally, a novel contact between Phe20 and Leu34 as well as the significant structural perturbation of residues 20-23 could be confirmed. However, the mutated fibrils do not show the formation of any salt bridges. This demonstrates that although morphologically very robust, local perturbations of the Aß(1-40) sequence lead to moderate structural alterations with tremendous impact on the physiological importance of these aggregates, which may suggest alternative strategies for the development of a remedy against Alzheimer's disease.

Stereoisomers Probe Steric Zippers in Amyloid-ß.

Shape complementarity between close-packed residues plays a critical role in the amyloid aggregation process. Here, we probe such "steric zipper" interactions in amyloid-ß (Aß40), whose aggregation is linked to Alzheimer's disease, by replacing natural residues by their stereoisomers. Such mutations are expected to specifically destabilize the shape sensitive "packing" interactions, which may potentially increase their solubility and change other properties. We study the stereomutants DF19 and DL34 and also the DA2/DF4/DH6/DS8 mutant of Aß40. F19-L34 is a critical contact in a tightly packed region of Aß, while residues 1-9 are known to be disordered. While both DF19 and DL34 slow down the kinetics of aggregation and form amyloid fibrils efficiently, only DL34 increases the final solubility. DF19 gives rise to additional off-pathway aggregation which results in large, kinetically stable aggregates, and has lower net solubility. DA2/DF4/DH6/DS8 does not have an effect on the kinetics or the solubility. Notably, both DF19 and DL34 oligomers have a significantly lower level of interactions with lipid vesicles and live cells. We conclude that stereoisomers can cause complex site dependent changes in amyloid properties, and provide an effective tool to determine the role of individual residues in shaping the packed interiors of amyloid aggregates.

Fibrils of Truncated Pyroglutamyl-Modified Aß Peptide Exhibit a Similar Structure as Wildtype Mature Aß Fibrils.

Fibrillation of differently modified amyloid ß peptides and deposition as senile plaques are hallmarks of Alzheimer's disease. N-terminally truncated variants, where the glutamate residue 3 is converted into cyclic pyroglutamate (pGlu), form particularly toxic aggregates. We compare the molecular structure and dynamics of fibrils grown from wildtype Aß(1-40) and pGlu3-Aß(3-40) on the single amino acid level. Thioflavin T fluorescence, electron microscopy, and X-ray diffraction reveal the general morphology of the amyloid fibrils. We found good agreement between the (13)C and (15)N NMR chemical shifts indicative for a similar secondary structure of both fibrils. A well-known interresidual contact between the two ß-strands of the Aß fibrils could be confirmed by the detection of interresidual cross peaks in a (13)C-(13)C NMR correlation spectrum between the side chains of Phe 19 and Leu 34. Small differences in the molecular dynamics of residues in the proximity to the pyroglutamyl-modified N-terminus were observed as measured by DIPSHIFT order parameter experiments.

Amyloid Beta Aggregation in the Presence of Temperature-Sensitive Polymers.

The formation of amyloid fibrils is considered to be one of the main causes for many neurodegenerative diseases, such as Alzheimer's, Parkinson's or Huntington's disease. Current knowledge suggests that amyloid-aggregation represents a nucleation-dependent aggregation process in vitro, where a sigmoidal growth phase follows an induction period. Here, we studied the fibrillation of amyloid ß 1-40 (Aß40) in the presence of thermoresponsive polymers, expected to alter the Aß40 fibrillation kinetics due to their lower critical solution behavior. To probe the influence of molecular weight and the end groups of the polymer on its lower critical solution temperature (LCST), also considering its concentration dependence in the presence of buffer-salts needed for the aggregation studies of the amyloids, poly(oxazolines) (POx) with LCSTs ranging from 14.2⁻49.8 °C and poly(methoxy di(ethylene glycol)acrylates) with LCSTs ranging from 34.4⁻52.7 °C were synthesized. The two different polymers allowed the comparison of the influence of different molecular structures onto the fibrillation process. Mixtures of Aß40 with these polymers in varying concentrations were studied via time-dependent measurements of the thioflavin T (ThT) fluorescence. The studies revealed that amyloid fibrillation was accelerated in, accompanied by an extension of the lag phase of Aß40 fibrillation from 18.3 h in the absence to 19.3 h in the presence of the poly(methoxy di(ethylene glycol)acrylate) (3600 g/mol).