Structural properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

Replica exchange molecular dynamics simulations (300 ns) were used to study the dimerization of amyloid β(1‐40) (Aβ(1‐40)) polypeptide. Configurational entropy calculations revealed that at physiological temperature (310 K, 37°C) dynamic dimers are formed by randomly docked monomers. Free energy of binding of the two chains to each other was ?93.56 ± 6.341 kJ mol^?1. Prevalence of random coil conformations was found for both chains with the exceptions of increased β‐sheet content from residues 16‐21 and 29‐32 of chain A and residues 15‐21 and 30‐33 of chain B with β‐turn/β‐bend conformations in both chains from residues 1‐16, 21‐29 of chain A, 1‐16, and 21‐29 of chain B. There is a mixed β‐turn/β‐sheet region from residues 33‐38 of both chains. Analysis of intra‐ and interchain residue distances shows that, although the individual chains are highly flexible, the dimer system stays in a loosely packed antiparallel β‐sheet configuration with contacts between residues 17‐21 of chain A with residues 17‐21 and 31‐36 of chain B as well as residues 31‐36 of chain A with residues 17‐21 and 31‐36 of chain B. Based on dihedral principal component analysis, the antiparallel β‐sheet‐loop‐β‐sheet conformational motif is favored for many low energy sampled conformations. Our results show that Aβ(1‐40) can form dynamic dimers in aqueous solution that have significant conformational flexibility and are stabilized by collapse of the central and C‐terminal hydrophobic cores with the expected β‐sheet‐loop‐β‐sheet conformational motif. Proteins 2017; 85:1024–1045. © 2017 Wiley Periodicals, Inc.     

Dual effects of familial Alzheimer's disease mutations (D7H,D7N,and H6R) on amyloid β peptide: Correlation dynamics and zinc binding

Although the N‐terminal region of Amyloid β (Aβ) peptides plays dual roles as metal‐coordinating sites and conformational modulator, few studies have been performed to explore the effects of mutations at this region on the overall conformational ensemble of Aβ and the binding propensity of metal ions. In this work, we focus on how three familial Alzheimer's disease mutations (D7H, D7N, and H6R) alter the structural characteristics and thermodynamic stabilities of Aβ42 using molecular dynamics simulations. We observe that each mutation displays increased β‐sheet structures in both N and C termini. In particular, both the N terminus and central hydrophobic region of D7H can form stable β‐hairpin structures with its C terminus. The conserved turn structure at Val²⁴–Lys²⁸ in all peptides and Zn²⁺‐bound Aβ42 is confirmed as the common structural motif to nucleate folding of Aβ. Each mutant can significantly increase the solvation free energy and thus enhance the aggregation of Aβ monomers. The correlation dynamics between Aβ(1–16) and Aβ(17–42) fragments are elucidated by linking the domain motions with the corresponding structured conformations. We characterize the different populations of correlated domain motions for each mutant from a more macroscopic perspective, and unexpectedly find that Zn²⁺‐bound Aβ42 ensemble shares the same populations as Aβ42, indicating that the binding of Zn²⁺ to Aβ follows the conformational selection mechanism, and thus is independent of domain motions, even though the structures of Aβ have been modified at a residue level. Proteins 2014; 82:3286–3297. © 2014 Wiley Periodicals, Inc.     

Aromatic‐interaction‐mediated inhibition of β‐amyloid assembly structures and cytotoxicity

Abnormal aggregation of β‐amyloid (Aβ) peptide plays an important role in the onset and progress of Alzheimer's disease (AD); hence, targeting Aβ aggregation is considered as an effective therapeutic strategy. Here, we studied the aromatic‐interaction‐mediated inhibitory effect of oligomeric polypeptides (K8Y8, K4Y8, K8W8) on Aβ42 fibrillization process. The polypeptides containing lysine as well as representative aromatic amino acids of tryptophan or tyrosine were found to greatly suppress the aggregation as evaluated by thioflavin T assay. Circular dichroism spectra showed that the β‐sheet formation of Aβ42 peptides decreased with the polypeptide additives. Molecular docking studies revealed that the oligomeric polypeptides could preferentially bind to Aβ42 through π–π stacking between aromatic amino acids and Phe19, together with hydrogen bonding. The cell viability assay confirmed that the toxicity of Aβ42 to SH‐SY5Y cells was markedly reduced in the presence of polypeptides. This study could be beneficial for developing peptide‐based inhibitory agents for amyloidoses. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of a β‐sheet breaker peptide with lipid membranes

Aggregation of β‐amyloid peptides into senile plaques has been identified as one of the hallmarks of Alzheimer's disease. An attractive therapeutic strategy for Alzheimer's disease is the inhibition of the soluble β‐amyloid aggregation using synthetic β‐sheet breaker peptides that are capable of binding Aβ but are unable to become part of a β‐sheet structure. As the early stages of the Aβ aggregation process are supposed to occur close to the neuronal membrane, it is strategic to define the β‐sheet breaker peptide positioning with respect to lipid bilayers. In this work, we have focused on the interaction between the β‐sheet breaker peptide acetyl‐LPFFD‐amide, iAβ5p, and lipid membranes, studied by ESR spectroscopy, using either peptides alternatively labeled at the C‐ and at the N‐terminus or phospholipids spin‐labeled in different positions of the acyl chain. Our results show that iAβ5p interacts directly with membranes formed by the zwitterionic phospholipid dioleoyl phosphatidylcholine and this interaction is modulated by inclusion of cholesterol in the lipid bilayer formulation, in terms of both peptide partition coefficient and the solubilization site. In particular, cholesterol decreases the peptide partition coefficient between the membrane and the aqueous medium. Moreover, in the absence of cholesterol, iAβ5p is located between the outer part of the hydrophobic core and the external hydrophilic layer of the membrane, while in the presence of cholesterol it penetrates more deeply into the lipid bilayer. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular mechanism of β-sheet self-organization at water-hydrophobic interfaces

The capacity to form β‐sheet structure and to self‐organize into amyloid aggregates is a property shared by many proteins. Severe neurodegenerative pathologies such as Alzheimer's disease are thought to involve the interaction of amyloidogenic protein oligomers with neuronal membranes. To understand the experimentally observed catalysis of amyloid formation by lipid membranes and other water‐hydrophobic interfaces, we examine the physico‐chemical basis of peptide adsorption and aggregation in a model membrane using atomistic molecular simulations. Blocked octapeptides with simple, repetitive sequences, (Gly‐Ala)₄, and (Gly‐Val)₄, are used as models of β‐sheet‐forming polypeptide chains found in the core of amyloid fibrils. In the presence of an n‐octane phase mimicking the core of lipid membranes, the peptides spontaneously partition at the octane‐water interface. The adsorption of nonpolar sidechains displaces the peptides' conformational equilibrium from a heterogeneous ensemble characterized by a high degree of structural disorder toward a more ordered ensemble favoring β‐hairpins and elongated β‐strands. At the interface, peptides spontaneously aggregate and rapidly evolve β‐sheet structure on a 10 to 100 ns time scale, while aqueous aggregates remain amorphous. Catalysis of β‐sheet formation results from the combination of the hydrophobic effect and of reduced conformational entropy of the polypeptide chain. While the former drives interfacial partition and displaces the conformational equilibrium of monomeric peptides, the planar interface further facilitates β‐sheet organization by increasing peptide concentration and reducing the dimensionality of self‐assembly from three to two. These findings suggest a general mechanism for the formation of β‐sheets on the surface of globular proteins and for amyloid self‐organization at hydrophobic interfaces. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Differential effects of phe19 and phe20 on fibril formation by amyloidogenic peptide Aβ16–22 (Ac‐KLVFFAE‐NH2)

The sequence KLVFFAE (Aβ16–22) in Alzheimer's β‐amyloid is thought to be a core β‐structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in β‐sheet. To elucidate the mechanism of the initial folding process, we undertook combined X‐ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized Aβ16–22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild‐type (WT) Aβ16–22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab‐like β‐crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30‐Å period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side‐chain dispositions for WT and 20CN, but not for 19CN. These X‐ray results and modeling imply that in the assembly of WT Aβ16–22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Potentially amyloidogenic conformational intermediates populate the unfolding landscape of transthyretin: Insights from molecular dynamics simulations

Protein aggregation into insoluble fibrillar structures known as amyloid characterizes several neurodegenerative diseases, including Alzheimer's, Huntington's and Creutzfeldt‐Jakob. Transthyretin (TTR), a homotetrameric plasma protein, is known to be the causative agent of amyloid pathologies such as FAP (familial amyloid polyneuropathy), FAC (familial amyloid cardiomiopathy) and SSA (senile systemic amyloidosis). It is generally accepted that TTR tetramer dissociation and monomer partial unfolding precedes amyloid fibril formation. To explore the TTR unfolding landscape and to identify potential intermediate conformations with high tendency for amyloid formation, we have performed molecular dynamics unfolding simulations of WT‐TTR and L55P‐TTR, a highly amyloidogenic TTR variant. Our simulations in explicit water allow the identification of events that clearly discriminate the unfolding behavior of WT and L55P‐TTR. Analysis of the simulation trajectories show that (i) the L55P monomers unfold earlier and to a larger extent than the WT; (ii) the single α‐helix in the TTR monomer completely unfolds in most of the L55P simulations while remain folded in WT simulations; (iii) L55P forms, early in the simulations, aggregation‐prone conformations characterized by full displacement of strands C and D from the main β‐sandwich core of the monomer; (iv) L55P shows, late in the simulations, severe loss of the H‐bond network and consequent destabilization of the CBEF β‐sheet of the β‐sandwich; (v) WT forms aggregation‐compatible conformations only late in the simulations and upon extensive unfolding of the monomer. These results clearly show that, in comparison with WT, L55P‐TTR does present a much higher probability of forming transient conformations compatible with aggregation and amyloid formation.     

Molecular insights into Aβ42 protofibril destabilization with a fluorinated compound D744: A molecular dynamics simulation study

The aggregation of amyloid β‐peptide (Aβ₄₂) into toxic oligomers, fibrils, has been identified as a key process in Alzheimer's disease (AD) progression. The role of halogen‐substituted compounds have been highlighted in the disassembly of Aβ protofibril. However, the underlying inhibitory mechanism of Aβ₄₂ protofibril destabilization remains elusive. In this regard, a combined molecular docking and molecular dynamics (MD) simulations were performed to elucidate the inhibitory mechanism of a fluorinated compound, D744 , which has been reported previously for potential in vitro and in vivo inhibitory activity against Aβ₄₂ aggregation and reduction in the Aβ‐induced cytotoxicity. The molecular docking analysis highlights that D744 binds and interacts with chain A of the protofibril structure with hydrophobic contacts and orthogonal multipolar interaction. MD simulations reveal destabilization of the protofibril structure in the presence of D744 due to the decrease in β‐sheet content and a concomitant increase of coil and bend structures, increase in the interchain D23‐K28 salt bridge distance, decrease in the number of backbone hydrogen bonds, increase in the average distance between Cα atoms, and decrease in the binding affinity between chains A and B of the protofibril structure. The binding free‐energy analysis between D744 and the protofibril structure with Molecular Mechanics Poisson‐Boltzmann Surface Area (MM‐PBSA) reveal that residues Leu17, Val18, Phe19, Phe20, Ala21, Glu22, Asp23, Leu34, Val36, Gly37, and Gly38 of chain A of the protofibril structure contribute maximum towards binding free energy (ΔG _binding  = −44.87 kcal/mol). The insights into the underlying inhibitory mechanism of small molecules that show potential in vitro anti‐aggregation activity against Aβ₄₂ will be beneficial for the current and future AD therapeutic studies.     

C‐terminal β‐strand swapping in a consensus‐derived fibronectin Type III scaffold

The crystal structures of six different fibronectin Type III consensus‐derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C‐terminal β‐strand resulting in the formation of Tencon dimers in which the target‐binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the β‐strand‐swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non‐glycine residue at position 77 and insertions of <4 residues may destabilize the β‐turn in the FG loop promoting β‐strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect β‐bridges in the FG regions that link the swapped dimers. The Tencon β‐swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain‐swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties. Proteins 2014; 82:1359–1369. © 2013 Wiley Periodicals, Inc.     

Thermodynamics and dynamics of amyloid peptide oligomerization are sequence dependent

Aggregation of the full‐length amyloid‐β (Aβ) and β2‐microglobulin (β2m) proteins is associated with Alzheimer's disease and dialysis‐related amyloidosis, respectively. This assembly process is not restricted to full‐length proteins, however, many short peptides also assemble into amyloid fibrils in vitro. Remarkably, the kinetics of amyloid‐fibril formation of all these molecules is generally described by a nucleation‐polymerization process characterized by a lag phase associated with the formation of a nucleus, after which fibril elongation occurs rapidly. In this study, we report using long molecular dynamics simulations with the OPEP coarse‐grained force field, the thermodynamics and dynamics of the octamerization for two amyloid 7‐residue peptides: the β2m83‐89 NHVTLSQ and Aβ16‐22 KLVFFAE fragments. Based on multiple trajectories run at 310 K, totaling 2.2 μs (β2m83‐89) and 4.8 μs (Aβ16‐22) and starting from random configurations and orientations of the chains, we find that the two peptides not only share common but also very different aggregation properties. Notably, an increase in the hydrophobic character of the peptide, as observed in Aβ16‐22 with respect to β2m83‐89 impacts the thermodynamics by reducing the population of bilayer β‐sheet assemblies. Higher hydrophobicity is also found to slow down the dynamics of β‐sheet formation by enhancing the averaged lifetime of all configuration types (CT) and by reducing the complexity of the CT transition probability matrix. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Discovery of amyloid‐beta aggregation inhibitors using an engineered assay for intracellular protein folding and solubility

Genetic and biochemical studies suggest that Alzheimer's disease (AD) is caused by a series of events initiated by the production and subsequent aggregation of the Alzheimer's amyloid β peptide (Aβ), the so‐called amyloid cascade hypothesis. Thus, a logical approach to treating AD is the development of small molecule inhibitors that either block the proteases that generate Aβ from its precursor (β‐ and γ‐secretases) or interrupt and/or reverse Aβ aggregation. To identify potent inhibitors of Aβ aggregation, we have developed a high‐throughput screen based on an earlier selection that effectively paired the folding quality control feature of the Escherichia coli Tat protein export system with aggregation of the 42‐residue AD pathogenesis effecter Aβ42. Specifically, a tripartite fusion between the Tat‐dependent export signal ssTorA, the Aβ42 peptide and the β‐lactamase (Bla) reporter enzyme was found to be export incompetent due to aggregation of the Aβ42 moiety. Here, we reasoned that small, cell‐permeable molecules that inhibited Aβ42 aggregation would render the ssTorA‐Aβ42‐Bla chimera competent for Tat export to the periplasm where Bla is active against β‐lactam antibiotics such as ampicillin. Using a fluorescence‐based version of our assay, we screened a library of triazine derivatives and isolated four nontoxic, cell‐permeable compounds that promoted efficient Tat‐dependent export of ssTorA‐Aβ42‐Bla. Each of these was subsequently shown to be a bona fide inhibitor of Aβ42 aggregation using a standard thioflavin T fibrillization assay, thereby highlighting the utility of our bacterial assay as a useful screen for antiaggregation factors under physiological conditions.     

Beta‐barrel models of soluble amyloid beta oligomers and annular protofibrils

Both soluble and membrane‐bound prefibrillar assemblies of Abeta (Aβ) peptides have been associated with Alzheimer's disease (AD). The size and nature of these assemblies vary greatly and are affected by many factors. Here, we present models of soluble hexameric assemblies of Aβ42 and suggest how they can lead to larger assemblies and eventually to fibrils. The common element in most of these assemblies is a six‐stranded β‐barrel formed by the last third of Aβ42, which is composed of hydrophobic residues and glycines. The hydrophobic core β‐barrels of the hexameric models are shielded from water by the N‐terminus and central segments. These more hydrophilic segments were modeled to have either predominantly β or predominantly α secondary structure. Molecular dynamics simulations were performed to analyze stabilities of the models. The hexameric models were used as starting points from which larger soluble assemblies of 12 and 36 subunits were modeled. These models were developed to be consistent with numerous experimental results. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effects of force fields on the conformational and dynamic properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

The conformational space and structural ensembles of amyloid beta (Aβ) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aβ(1‐40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER‐ff99sb‐ILDN, AMBER‐ff99sb*‐ILDN, AMBER‐ff99sb‐NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER‐ff99sb‐ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α‐helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER‐ff99sb‐NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER‐ff99sb‐NMR force field, the others tended to under estimate the expected amount of β‐sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER‐ff99sb‐NMR, reproduce a theoretically expected β‐sheet‐turn‐β‐sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C‐terminal hydrophobic cores from residues 17‐21 and 30‐36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different.     

FTIR spectroscopic studies on aggregation process of the β‐amyloid 11–28 fragment and its variants

Aggregation of Aβ peptides is a seminal event in Alzheimer's disease. Detailed understanding of the Aβ assembly process would facilitate the targeting and design of fibrillogenesis inhibitors. Here, conformational studies using FTIR spectroscopy are presented. As a model peptide, the 11–28 fragment of Aβ was used. This model peptide is known to contain the core region responsible for Aβ aggregation. The structural behavior of the peptide during aggregation provoked by the addition of water to Aβ(11–28) solution in hexafluoroisopropanol was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21–23 (A21G, E22K, E22G, E22Q and D23N). The results showed that the aggregation of the peptides proceeds via a helical intermediate, and it is possible that the formation of α‐helical structures is preceded by creation of 3₁₀‐helix/3₁₀‐turn structures. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Building a foundation for structure‐based cellulosome design for cellulosic ethanol: Insight into cohesin‐dockerin complexation from computer simulation

The organization and assembly of the cellulosome, an extracellular multienzyme complex produced by anaerobic bacteria, is mediated by the high‐affinity interaction of cohesin domains from scaffolding proteins with dockerins of cellulosomal enzymes. We have performed molecular dynamics simulations and free energy calculations on both the wild type (WT) and D39N mutant of the C. thermocellum Type I cohesin‐dockerin complex in aqueous solution. The D39N mutation has been experimentally demonstrated to disrupt cohesin‐dockerin binding. The present MD simulations indicate that the substitution triggers significant protein flexibility and causes a major change of the hydrogen‐bonding network in the recognition strips—the conserved loop regions previously proposed to be involved in binding—through electrostatic and salt‐bridge interactions between β‐strands 3 and 5 of the cohesin and α‐helix 3 of the dockerin. The mutation‐induced subtle disturbance in the local hydrogen‐bond network is accompanied by conformational rearrangements of the protein side chains and bound water molecules. Additional free energy perturbation calculations of the D39N mutation provide differences in the cohesin‐dockerin binding energy, thus offering a direct, quantitative comparison with experiments. The underlying molecular mechanism of cohesin‐dockerin complexation is further investigated through the free energy profile, that is, potential of mean force (PMF) calculations of WT cohesin‐dockerin complex. The PMF shows a high‐free energy barrier against the dissociation and reveals a stepwise pattern involving both the central β‐sheet interface and its adjacent solvent‐exposed loop/turn regions clustered at both ends of the β‐barrel structure.     

Aβ Monomers Transiently Sample Oligomer and Fibril-Like Configurations: Ensemble Characterization Using a Combined MD/NMR Approach

Amyloid β (Aβ) peptides are a primary component of fibrils and oligomers implicated in the etiology of Alzheimer's disease (AD). However, the intrinsic flexibility of these peptides has frustrated efforts to investigate the secondary and tertiary structure of Aβ monomers, whose conformational landscapes directly contribute to the kinetics and thermodynamics of Aβ aggregation. In this work, de novo replica exchange molecular dynamics (REMD) simulations on the microseconds-per-replica timescale are used to characterize the structural ensembles of Aβ42, Aβ40, and M35-oxidized Aβ42, three physiologically relevant isoforms with substantially different aggregation properties. J-coupling data calculated from the REMD trajectories were compared to corresponding NMR-derived values acquired through two different pulse sequences, revealing that all simulations converge on the order of hundreds of nanoseconds-per-replica toward ensembles that yield good agreement with experiment. Though all three Aβ species adopt highly heterogeneous ensembles, these are considerably more structured compared to simulations on shorter timescales. Prominent in the C-terminus are antiparallel β-hairpins between L17–A21, A30–L36, and V39–I41, similar to oligomer and fibril intrapeptide models that expose these hydrophobic side chains to solvent and may serve as hotspots for self-association. Compared to reduced Aβ42, the absence of a second β-hairpin in Aβ40 and the sampling of alternate β topologies by M35-oxidized Aβ42 may explain the reduced aggregation rates of these forms. A persistent V24–K28 bend motif, observed in all three species, is stabilized by buried backbone to side-chain hydrogen bonds with D23 and a cross-region salt bridge between E22 and K28, highlighting the role of the familial AD-linked E22 and D23 residues in Aβ monomer folding. These characterizations help illustrate the conformational landscapes of Aβ monomers at atomic resolution and provide insight into the early stages of Aβ aggregation pathways.     

Dissecting the behaviour of β‐amyloid peptide variants during oligomerization and fibrillation

The oligomerization and fibrillation of β‐amyloid (Aβ) peptides are important events in the pathogenesis of Alzheimer's disease. However, the motifs within the Aβ sequence that contribute to oligomerization and fibrillation and the complex interplay among these short motifs are unclear. In this study, the oligomerization and fibrillation abilities of the Aβ variants Aβ1–28, Aβ1–36, Aβ11–42, Aβ17–42, Aβ1–40 and Aβ1–42 were examined by thioflavin T fluorescence, western blotting and transmission electron microscopy. Compared with two C‐terminal‐truncated peptides (i.e. Aβ1–28 and Aβ1–36), Aβ11–42, Aβ17–42 and Aβ1–42 had stronger abilities to form oligomers. This indicated that amino acids 37–42 strengthen the β‐hairpin structure of Aβ. Both Aβ1–42 and Aβ1–40 could form fibres, but Aβ17–42 formed irregular fibres, suggesting that amino acids 1–17 were essential for Aβ fibre formation. Aβ1–28 and Aβ1–36 exhibited weak oligomerization and fibrillation, implying that they formed an unstable β‐hairpin structure owing to the incomplete C‐terminal region. Intermediate peptides were likely to form a stable structure, consistent with previous results. This work explains the roles and interplay among motifs within Aβ during oligomerization and fibrillation. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Alzheimer's Aβ42 and Aβ40 peptides form interlaced amyloid fibrils

Deposition of amyloid β (Aβ) in the brain is a pathological hallmark of Alzheimer's disease. There are two major isoforms of Aβ: the 42‐residue Aβ42 and the 40‐residue Aβ40. The only difference between Aβ42 and Aβ40 is that Aβ42 has two extra residues at the C‐terminus. The amyloid plaques in Alzheimer's brains consist of mostly Aβ42 and some plaques contain only Aβ42, even though Aβ40 concentration is several‐fold more than Aβ42. Using electron paramagnetic resonance, we studied the formation of amyloid fibrils using a mixture of Aβ42 and Aβ40 in vitro. We show that Aβ42 and Aβ40 form mixed fibrils in an interlaced manner, although Aβ40 is not as efficient as Aβ42 in terms of being incorporated into Aβ42 fibrils. Our results suggest that both Aβ42 and Aβ40 would be present in amyloid plaques if in vivo aggregation of Aβ were similar to the in vitro process. Therefore, there must be some mechanisms that lead to the preferential deposition of Aβ42 at the extracellular space. Identifying such mechanisms may open new avenues for therapeutic interventions to treat Alzheimer's disease.     

Impact of sequence on the molecular assembly of short amyloid peptides

The goal of this work is to understand how the sequence of a protein affects the likelihood that it will form an amyloid fibril and the kinetics along the fibrillization pathway. The focus is on very short fragments of amyloid proteins since these play a role in the fibrillization of the parent protein and can form fibrils themselves. Discontinuous molecular dynamics simulations using the PRIME20 force field were performed of the aggregation of 48‐peptide systems containing SNQNNF ( PrP (170–175 )), SSTSAA (RNaseA(15–20)), MVGGVV (Aβ(35–40)), GGVVIA (Aβ(37–42)), and MVGGVVIA (Aβ(35–42)). In our simulations SNQQNF, SSTTSAA, and MVGGVV form large numbers of fibrillar structures spontaneously (as in experiment). GGVVIA forms β‐sheets that do not stack into fibrils (unlike experiment). The combination sequence MVGGVVIA forms less fibrils than MVGGVV, hindered by the presence of the hydrophobic residues at the C‐terminal. Analysis of the simulation kinetics and energetics reveals why MVGGVV forms fibrils and GGVVIA does not, and why adding I and A to MVGGVVIA reduces fibrillization and enhances amorphous aggregation into oligomeric structures. The latter helps explain why Aβ(1–42) assembles into more complex oligomers than Aβ(1–40), a consequence of which is that it is more strongly associated with Alzheimer's disease. Proteins 2014; 82:1469–1483. © 2014 Wiley Periodicals, Inc.