Toxic Fibrils Research Articles

Elucidation of the Effect of Phospholipid Charge on the Rate of Insulin Aggregation and Structure and Toxicity of Amyloid Fibrils.

The plasma membrane is a dynamic structure that separates the cell interior from the extracellular space. The fluidity and plasticity of the membrane determines a large number of physiologically important processes ranging from cell division to signal transduction. In turn, membrane fluidity is determined by phospholipids that possess different charges, lengths, and saturation states of fatty acids. A growing body of evidence suggests that phospholipids may play an important role in the aggregation of misfolded proteins, which causes pathological conditions that lead to severe neurodegenerative diseases. In this study, we investigate the role of the charge of the most abundant phospholipids in the plasma membrane: phosphatidylcholine and phosphatidylethanolamine, zwitterions: phosphatidylserine and phosphatidylglycerol, lipids that possess a negative charge, and cardiolipin that has double negative charge on its polar head. Our results show that both zwitterions strongly inhibit insulin aggregation, whereas negatively charged lipids accelerate fibril formation. We also found that in the equimolar presence of zwitterions insulin yields oligomers that exert significantly lower cell toxicity compared to fibrils that were grown in the lipid-free environment. Such aggregates were not formed in the presence of negatively charged lipids. Instead, long insulin fibrils that had strong cell toxicity were grown in the presence of such negatively charged lipids. However, our results showed no correlation between the charge of the lipid and secondary structure and toxicity of the aggregates formed in its presence. These findings show that the secondary structure and toxicity are determined by the chemical structure of the lipid rather than by the charge of the phospholipid polar head.

Protein-to-lipid ratio uniquely changes the rate of lysozyme aggregation but does not significantly alter toxicity of mature protein aggregates

Irreversible aggregation of misfolded proteins is the underlying molecular cause of numerous pathologies, including diabetes type 2, Alzheimer's, and Parkinson's diseases. Such an abrupt protein aggregation results in the formation of small oligomers that can propagate into amyloid fibrils. A growing body of evidence suggests that protein aggregation can be uniquely altered by lipids. However, the role of the protein-to-lipid (P:L) ratio on the rate of protein aggregation, as well as the structure and toxicity of corresponding protein aggregates remains poorly understood. In this study, we investigate the role of the P:L ratio of five different phospho- and sphingolipids on the rate of lysozyme aggregation. We observed significantly different rates of lysozyme aggregation at 1:1, 1:5, and 1:10 P:L ratios of all analyzed lipids except phosphatidylcholine (PC). However, we found that at those P:L ratios, structurally and morphologically similar fibrils were formed. As a result, for all studies of lipids except PC, mature lysozyme aggregates exerted insignificantly different cell toxicity. These results demonstrate that the P:L ratio directly determines the rate of protein aggregation, however, has very little if any effect on the secondary structure of mature lysozyme aggregates. Furthermore, our results point to the lack of a direct relationship between the rate of protein aggregation, secondary structure, and toxicity of mature fibrils.

Lipids uniquely alter the secondary structure and toxicity of amyloid beta 1-42 aggregates.

Abrupt aggregation of amyloid β1-42 (Aβ) peptide is a hallmark of Alzheimer's disease (AD), a severe pathology that affects more than 44million people worldwide. A growing body of evidence suggests that lipids can uniquely alter rates of Aβ1-42 aggregation. However, it remains unclear whether lipids only alter rates of protein aggregation or also uniquely modify the secondary structure and toxicity of Aβ1-42 oligomers and fibrils. In this study, we investigated the effect of phosphatidylcholine (PC), cardiolipin (CL), and cholesterol (Chol) on Aβ1-42 aggregation. We found that PC, CL and Chol strongly accelerated the rate of fibril formation compared to the rate of Aβ1-42 aggregation in the lipid-free environment. Furthermore, anionic CL enabled the strongest acceleration of Aβ1-42 aggregation compared to zwitterionic PC and uncharged Chol. We also found that PC, CL and Chol uniquely altered the secondary structure of early-, middle- and late-stage Aβ1-42 aggregates. Specifically, CL and Chol drastically increased the amount of parallel β-sheet in Aβ1-42 oligomers and fibrils grown in the presence of these lipids. This caused a significant increase in the toxicity of Aβ:CL and Aβ:Chol compared to the toxicity of Aβ:PC and Aβ1-42 aggregates formed in the lipid-free environment. These results demonstrate that toxicity of Aβ aggregates correlates with the amount of their β-sheet content, which, in turn, is determined by the chemical structure of lipids present at the stage of Aβ1-42 aggregation.

Probing Interfacial Charge Transfer between Amyloid-β and Graphene during Amyloid Fibrillization Using Raman Spectroscopy.

Charge transfer plays a key role in the structural transformation of amyloid-β proteins (Aβs), as it fibrillizes from small monomers to intermediate oligomers and to ordered fibrils. While the protein fibrillization states have been identified using cryo-electron microscopy, X-ray diffraction, Raman, infrared, terahertz spectroscopies, etc., there is little known about the electronic states during the fibrilization of Aβ protein. Here, we probe the charge transfer of Aβ42 proteins at different aggregation stages adsorbed on monolayer graphene (Gr) and molybdenum disulfide (MoS2) using Raman spectroscopy. Monomers, oligomers, and fibrils prepared in buffer solutions were deposited and dried separately on Gr and MoS2 where well-established characteristic Raman modes (G, 2D for Gr and E2g, A1g for MoS2) were monitored. The shifts in Raman parameters showed that the small Aβ monomers withdraw electrons, whereas fibrils donate electrons to Gr and MoS2. Oligomers undergo transient charge states near the neutrality point. This is explained in terms of modulated carrier concentration in Gr and MoS2. This finding provides insight into the electronic properties of Aβs that could be essential to identifying the onset of toxic fibril forms and developing a straightforward, label-free diagnosis using Gr and MoS2.

How oxidized EGCG remodels α-synuclein fibrils into non-toxic aggregates: insights from computational simulations.

The misfolding and aggregation of the presynaptic protein α-synuclein (α-syn) is a pathological hallmark of Parkinson's disease (PD). Targeting α-syn has emerged as a promising therapeutic strategy for PD. Emerging in vitro evidence supports a dual action of epigallocatechin-3-gallate (EGCG) against amyloid neurotoxicity. EGCG can halt the formation of toxic aggregates by redirecting the amyloid fibril aggregation pathway toward non-toxic aggregates and remodeling the existing toxic fibrils into non-toxic aggregates. Moreover, EGCG oxidation can enhance fibril's remodeling by forming Schiff bases, leading to crosslinking of the fibril. However, this covalent modification is not required for amyloid remodeling, and establishing non-specific hydrophobic interactions with sidechains seems to be the main driver of amyloid remodeling by EGCG. Thioflavin (ThT) is a gold standard probe to detect amyloid fibrils in vitro, and oxidized EGCG competes with ThT for amyloid fibrils' binding sites. In this work, we performed docking and molecular dynamics (MD) simulations to gain insights into the intermolecular interactions of oxidized EGCG and ThT with a mature α-syn fibril. We find that oxidized EGCG moves within lysine-rich sites within the hydrophobic core of the α-syn fibril, forming aromatic and hydrogen-bonding (H-bond) interactions with different residues during the whole MD simulation time. In contrast, ThT, which does not remodel amyloid fibrils, was docked to the same sites but only via aromatic interactions. Our findings suggest that non-covalent interactions play a role in oxidized EGCG binding into the hydrophobic core, including H-bond and aromatic interactions with some residues in the amyloid remodeling processes. These interactions would ultimately lead to a disturbance of structural features as determinants for stabilizing this fibril into a compact and pathogenic Greek key topology.

An unnatural amino acid modified human insulin derivative for visual monitoring of insulin aggregation.

Insulin often forms toxic fibrils during production and transportation, which are deposited as amyloids at repeated injection sites in diabetic patients. Distinguishing early fibrils from non-fibrillated insulin is difficult. Herein, we introduce a chemically modified human insulin derivative with a distinct visual colour transition upon aggregation, facilitating insulin quality assessment.

Tau liquid-liquid phase separation: At the crossroads of tau physiology and tauopathy.

Abnormal deposition of tau in neurons is a hallmark of Alzheimer's disease and several other neurodegenerative disorders. In the past decades, extensive efforts have been made to explore the mechanistic pathways underlying the development of tauopathies. Recently, the discovery of tau droplet formation by liquid-liquid phase separation (LLPS) has received a great deal of attention. It has been reported that tau condensates have a biological role in promoting and stabilizing microtubule (MT) assembly. Furthermore, it has been hypothesized that the transition of phase-separated tau droplets to a gel-like state and then to fibrils is associated with the pathology of neurodegenerative diseases. In this review, we outline LLPS, the structural disorder that facilitates tau droplet formation, the effects of posttranslational modification of tau on condensate formation, the physiological function of tau droplets, the pathways from droplet to toxic fibrils, and the therapeutic strategies for tauopathies that might evolve from toxic droplets. We expect a deeper understanding of tau LLPS will provide additional insights into tau physiology and tauopathies.

Mechanisms behind the Fibrillation and Toxicity of Insulin Fibrils on Neuron Cells by Engineered Curcumin Analogs.

Among foods, the use of plant derivatives as promising drugs and/or excipients has been considered from various perspectives. In the present study, curcumin, which is one of the most important plant derivatives for biological uses, and four curcumin-based pyrido[2,3-d]pyrimidine analogs (C2-C5) were used for investigating the mechanism of insulin fibrillation and evaluating the cytotoxicity of insulin fibrils. The synthesized analogs differed in terms of hydrophobicity and electrostatic charge. The analogs with more hydrophobicity (C1 and C4) in both acidic and neutral environments were able to reduce the rate of insulin fibrillation and the degree of cross-linking in the produced fibrils. Additionally, the toxicity of these fibrils for neural cells (N2a cell line) was very low. However, they did not show any significant effects on the toxicity of non-neural cells (HEK293 cell line), indicating the effect of the biochemical surface diversity on determining the vulnerability to fibrils and even the mechanism of action of additives on cell line survival. Although negatively charged analogs were able to reduce insulin fibrillation in the acidic environment, they indicated an opposite effect in the neutral environment. The resultant fibrils in the acidic medium appeared with a well-distinguished filament, but they were very close at neutral pH levels. Moreover, such fibrils indicated very poor toxicity against the N2a cell line and had no significant effects on HEK293 cells. Considering the docking studies, by creatively using the size exclusion chromatography, it was suggested that analogs C2 and C3 were capable of binding to the C-terminal end of the insulin B chain (low affinity) and HisB10 (high affinity). Hence, it was suggested that different compounds could play different protecting and/or destroying roles in cell toxicity by blocking some ligands at the surface of neuron cells.

De novo designed protein inhibitors of amyloid aggregation and seeding

Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-β (Aβ) in Alzheimer's disease (AD) and alpha-synuclein (αSyn) in Parkinson's disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, "capping" them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aβ, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins invitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in Caenorhabditis elegans models of PD and AD.

Lipids reverse supramolecular chirality and reduce toxicity of amyloid fibrils.

Abrupt aggregation of misfolded proteins is a hallmark of many medical pathologies including diabetes type 2, Alzheimer and Parkinson diseases. This results in the formation of amyloid fibrils, protein aggregates with distinct supramolecular chirality. A growing body of evidence suggests that lipids can alter rates of protein aggregation. In this study, we investigated whether lipids could alter the supramolecular chirality of amyloid fibrils. We found that if present at the stage of protein aggregation, phospho- and sphingolipids uniquely reversed supramolecular chirality of insulin and lysozyme fibrils. Furthermore, amyloid fibrils with opposite supramolecular chirality exerted distinctly different cell toxicity. Specifically, insulin and lysozyme fibrils with reversed supramolecular chirality were less toxic to cells than the aggregates with normal supramolecular chirality. These findings point on the important role of lipids and supramolecular chirality of amyloid fibrils in the onset and progression of amyloid diseases.

Formulation development of tocopherol polyethylene glycol nanoengineered polyamidoamine dendrimer for neuroprotection and treatment of Alzheimer disease

Amyloid-beta (Aβ) aggregates deposition at extra neuronal sites induces neurotoxicity and major hallmarks of Alzheimer’s disease (AD). To reduce the Aβ fibril toxicity, multi-functional polyamidoamine (PAMAM) dendrimer was conjugated with tocopheryl polyethylene glycol succinate-1000 (TPGS) which acts as a carrier matrix for the delivery of neuroprotective molecule piperine (PIP). This PIP-TPGS-PAMAM dendrimer was fabricated to mitigate the Aβ1–42 fibril toxicity on SHSY5Y cells. TPGS-PAMAM was fabricated through carbodiimide coupling reaction, and PIP was encapsulated in dendrimer through solvent injection method to prepare PIP-TPGS-PAMAM. Antioxidant assay of PIP-TPGS-PAMAM showed 90.18% inhibition of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radicals compared to free PIP, which was 28.27%. The SHSY5Y cells showed 37.25% for negative control group and 82.55% cell viability for PIP-TPGS-PAMAM treated group against Aβ1–42 toxicity. PIP-TPGS-PAMAM reduced the ROS activity to 15.21% and 48.5% for free PIP treated in cell group. Similarly, extent of Aβ1-42-induced apoptosis also reduced significantly from 38.2% to 12.36% in PIP-TPGS-PAMAM treated group. In addition, PIP-TPGS-PAMAM also disaggregated the Aβ1–42 fibril in SHSY5Y cells. Our findings suggested that PIP-TPGS-PAMAM showed mitigation of Aβ1-42-induced toxicity in neuronal cells, which can offer excellent prospect of neuroprotection and AD therapy.

Distinguishing islet amyloid polypeptide fibril structures with infrared isotope-label spectroscopy.

Here, we performed spectral simulations of the amide-I vibrational spectra for three proposed fibril structures of the human islet amyloid polypeptide, which is involved in type II diabetes. We modeled both the overall absorption and two-dimensional infrared spectra for these structures. We further analyzed the isotope-labeled spectra, including the variation between structures. The analysis suggests that the infrared spectra of the cryo-electron microscopy structure provide the best match with experimental data. We further simulated isotope-labeled dilution spectroscopy investigating the correlation between the predicted spectral peak shift and the coupling between the amide units. While this correlation works in most cases, failures were observed when the isotope-labeled spectra were broad compared to the coupling or exhibited structure. These findings will be useful in the quest for potential toxic fibril formation intermediates.

Rationally designed helical peptidomimetics disrupt α-synuclein fibrillation.

Misfolding of the human protein α-synuclein results in toxic fibrils and the aggregation of Lewy bodies, which are a hallmark of Parkinson's disease in brain tissue. Here we disclose a supramolecular approach where peptidomimetics are rationally designed and pre-organised to recognize the surface of native helical α-Syn by forming complementary contacts with key patches of protein surface composed of charged and hydrophobic residues. Under lipid-catalyzed conditions the mimetics slow the rate of aggregation (thioflavin-T assay) and disrupt the misfolding pathway (electron microscopy of aggregates). This hypothesis is supported by comparison with a series of negative control compounds and with circular dichroism spectroscopy. Given the approach relies on selective recognition of both amino acid sequence and conformation (helical secondary structure) there is potential to develop these compounds as tools to unravel the currently intractable structure-function relationships of (i) missense mutation, and (ii) amyloid polymorphism with disease pathogenesis.

N-Amination Converts Amyloidogenic Tau Peptides into Soluble Antagonists of Cellular Seeding.

The spread of neurofibrillary tangles composed of tau protein aggregates is a hallmark of Alzheimer's and related neurodegenerative diseases. Early oligomerization of tau involves conformational reorganization into parallel β-sheet structures and supramolecular assembly into toxic fibrils. Despite the need for selective inhibitors of tau propagation, β-rich protein assemblies are inherently difficult to target with small molecules. Here, we describe a minimalist approach to mimic the aggregation-prone modules within tau. We carried out a backbone residue scan and show that amide N-amination completely abolishes the tendency of these peptides to self-aggregate, rendering them soluble mimics of ordered β-strands from the tau R2 and R3 domains. Several N-amino peptides (NAPs) inhibit tau fibril formation in vitro. We further demonstrate that NAPs 12 and 13 are effective at blocking the cellular seeding of endogenous tau by interacting with monomeric or fibrillar forms of extracellular tau. Peptidomimetic 12 is serum stable, non-toxic to neuronal cells, and selectivity inhibits the fibrilization of tau over Aβ42. Structural analysis of our lead NAPs shows considerable conformational constraint imposed by the N-amino groups. The described backbone N-amination approach provides a rational basis for the mimicry of other aggregation-prone peptides that drive pathogenic protein assembly.

The Cellular Prion Protein Increases the Uptake and Toxicity of TDP-43 Fibrils.

Cytoplasmic aggregation of the primarily nuclear TAR DNA-binding protein 43 (TDP-43) affects neurons in most amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal lobar degeneration (FTLD) cases. The cellular prion protein, PrPC, has been recognized as a common receptor and downstream effector of circulating neurotoxic species of several proteins involved in neurodegeneration. Here, capitalizing on our recently adapted TDP-43 real time quaking induced reaction, we set reproducible protocols to obtain standardized preparations of recombinant TDP-43 fibrils. We then exploited two different cellular systems (human SH-SY5Y and mouse N2a neuroblastoma cells) engineered to express low or high PrPC levels to investigate the link between PrPC expression on the cell surface and the internalization of TDP-43 fibrils. Fibril uptake was increased in cells overexpressing either human or mouse prion protein. Increased internalization was associated with detrimental consequences in all PrP-overexpressing cell lines but was milder in cells expressing the human form of the prion protein. As described for other amyloids, treatment with TDP-43 fibrils induced a reduction in the accumulation of the misfolded form of PrPC, PrPSc, in cells chronically infected with prions. Our results expand the list of misfolded proteins whose uptake and detrimental effects are mediated by PrPC, which encompass almost all pathological amyloids involved in neurodegeneration.

Huntingtin fibrils with different toxicity, structure, and seeding potential can be interconverted

The first exon of the huntingtin protein (HTTex1) important in Huntington’s disease (HD) can form cross-β fibrils of varying toxicity. We find that the difference between these fibrils is the degree of entanglement and dynamics of the C-terminal proline-rich domain (PRD) in a mechanism analogous to polyproline film formation. In contrast to fibril strains found for other cross-β fibrils, these HTTex1 fibril types can be interconverted. This is because the structure of their polyQ fibril core remains unchanged. Further, we find that more toxic fibrils of low entanglement have higher affinities for protein interactors and are more effective seeds for recombinant HTTex1 and HTTex1 in cells. Together these data show how the structure of a framing sequence at the surface of a fibril can modulate seeding, protein-protein interactions, and thereby toxicity in neurodegenerative disease.

Identification of Transthyretin Tetramer Kinetic Stabilizers That Are Capable of Inhibiting the Retinol-Dependent Retinol Binding Protein 4-Transthyretin Interaction: Potential Novel Therapeutics for Macular Degeneration, Transthyretin Amyloidosis, and Their Common Age-Related Comorbidities.

Dissociation of transthyretin (TTR) tetramers may lead to misfolding and aggregation of proamyloidogenic monomers, which underlies TTR amyloidosis (ATTR) pathophysiology. ATTR is a progressive disease resulting from the deposition of toxic fibrils in tissues that predominantly presents clinically as amyloid cardiomyopathy and peripheral polyneuropathy. Ligands that bind to and kinetically stabilize TTR tetramers prohibit their dissociation and may prevent ATTR onset. Drawing from clinically investigated AG10, we designed a constrained congener (14) that exhibits excellent TTR tetramer binding potency, prevents TTR aggregation in a gel-based assay, and possesses desirable pharmacokinetics in mice. Additionally, 14 significantly lowers murine serum retinol binding protein 4 (RBP4) levels despite a lack of binding at that protein's all-trans-retinol site. We hypothesize that kinetic stabilization of TTR tetramers via 14 is allosterically hindering all-trans-retinol-dependent RBP4-TTR tertiary complex formation and that the compound could present ancillary therapeutic utility for indications treated with RBP4 antagonists, such as macular degeneration.

Homocysteine fibrillar assemblies display cross-talk with Alzheimer’s disease β-amyloid polypeptide

High levels of homocysteine are reported as a risk factor for Alzheimer's disease (AD). Correspondingly, inborn hyperhomocysteinemia is associated with an increased predisposition to the development of dementia in later stages of life. Yet, the mechanistic link between homocysteine accumulation and the pathological neurodegenerative processes is still elusive. Furthermore, despite the clear association between protein aggregation and AD, attempts to develop therapy that specifically targets this process have not been successful. It is envisioned that the failure in the development of efficacious therapeutic intervention may lie in the metabolomic state of affected individuals. We recently demonstrated the ability of metabolites to self-assemble and cross-seed the aggregation of pathological proteins, suggesting a role for metabolite structures in the initiation of neurodegenerative diseases. Here, we provide a report of homocysteine crystal structure and self-assembly into amyloid-like toxic fibrils, their inhibition by polyphenols, and their ability to seed the aggregation of the AD-associated β-amyloid polypeptide. A yeast model of hyperhomocysteinemia indicates a toxic effect, correlated with increased intracellular amyloid staining that could be rescued by polyphenol treatment. Analysis of AD mouse model brain sections indicates the presence of homocysteine assemblies and the interplay between β-amyloid and homocysteine. This work implies a molecular basis for the association between homocysteine accumulation and AD pathology, potentially leading to a paradigm shift in the understanding of AD initial pathological processes.

Near-Infrared-Active Copper Molybdenum Sulfide Nanocubes for Phonon-Mediated Clearance of Alzheimer's β-Amyloid Aggregates.

Ternary chalcogenide materials have attracted significant interest in recent years because of their unique physicochemical and optoelectronic properties without relying on precious metals, rare earth metals, or toxic elements. Copper molybdenum sulfide (Cu2MoS4, CMS) nanocube is a biocompatible ternary chalcogenide nanomaterial that exhibits near-infrared (NIR) photocatalytic activity based on its low band gap and electron-phonon coupling property. Here, we study the efficacy of CMS nanocubes for dissociating neurotoxic Alzheimer's β-amyloid (Aβ) aggregates under NIR light. The accumulation of Aβ aggregates in the central nervous system is known to cause and exacerbate Alzheimer's disease (AD). However, clearance of the Aβ aggregates from the central nervous system is a considerable challenge due to their robust structure formed through self-assembly via hydrogen bonding and side-chain interactions. Our spectroscopic and microscopic analysis results have demonstrated that NIR-excited CMS nanocubes effectively disassemble Aβ fibrils by changing Aβ fibril's nanoscopic morphology, secondary structure, and primary structure. We have revealed that the toxicity of Aβ fibrils is alleviated by NIR-stimulated CMS nanocubes through in vitro analysis. Moreover, our ex vivo evaluations have suggested that the amount of Aβ plaques in AD mouse's brain decreased significantly by NIR-excited CMS nanocubes without causing any macroscopic damage to the brain tissue. Collectively, this study suggests the potential use of CMS nanocubes as a therapeutic ternary chalcogenide material to alleviate AD in the future.

Understanding the Packing of Amyloidogenic Peptide Segments using Electronic Structure Calculations

It has been proposed that antiparallel amyloid fibrils are more likely to fold into potentially toxic, transient morphologies as they are typically less stable than parallel fibrils. Recent studies of some amyloidogenic protein segments revealed that crystallize as antiparallel out-of-register β-strands, unlike most amyloid fibrils that fold into an in-register β-sheet packing. Therefore, it has been suggested that such arrangement may be responsible for the cytotoxicity in amyloid diseases. Interestingly, crystal structures of the amyloidogenic peptide segments NFGAILS and FGAILSS showed antiparallel zippers assembled into out-of-register and in-register sheets, respectively, even though they differ in only one amino acid at the termini. In this work, we use density functional theory (DFT) to elucidate the molecular mechanism driving their different crystallization patterns by investigating the differences in energetics and structure between them. Our results primarily show that the electrostatic potential (ESP) at the termini in the out-of-register peptide is significantly larger than in the in-register system. These findings may help to understand the underlying molecular phenomena dictating the toxicity of amyloid fibrils.