Off-pathway Aggregation Research Articles

Noncovalent Catalyst-cum-Inhibitor Directed Supramolecular Pathway Selection and Asymmetry Amplification by Aggregate Cross-Nucleation.

The key to any controlled supramolecular polymerization (CSP) process lies in controlling the nucleation step, which is typically achieved by sequestering monomers in a kinetically trapped state. However, kinetic traps that are shallow cannot prevent spontaneous nucleation, thus limiting the applicability of the CSP in such systems. We use a molecular additive to overcome this limitation by modifying the energy landscape of a competitive self-assembly process and increasing the kinetic stability of an otherwise short-lived trap state. The additive achieves this by simultaneously catalyzing OFF-pathway nucleation and inhibiting ON-pathway aggregation. In the process, it guides the molecular assembly exclusively toward the OFF-pathway aggregate analogue. The mechanisms of OFF-pathway catalysis and ON-pathway inhibition are elucidated. By specifically targeting the nucleation step, it was possible to achieve pathway selection at an extremely low additive-to-monomer ratio of 1:100. The generality of our approach is also demonstrated for other related molecular systems. Finally, removing the additive triggers the cross-nucleation of the ON-pathway aggregate on the surface of a less stable, OFF-pathway aggregate analogue. The resultant supramolecular polymer not only exhibits a more uniform morphology but more importantly, a marked improvement in the structural order that leads to an amplification of chiral asymmetry and a high absorption dissymmetry factor (gAbs) of ∼0.05.

An Innate Host Defense Protein β2-Microglobulin Keeps a Check on α-Synuclein amyloid Assembly: Implications in Parkinson's Disease

Amyloid formation due to protein misfolding has gained significant attention due to its association with neurodegenerative diseases. α-Synuclein (α-syn) is one such protein that undergoes a profound conformational switch to form higher order cross-β-sheet structures, resulting in amyloid formation, which is linked to the pathophysiology of Parkinson's disease (PD). The present status of research on α-syn aggregation and PD reveals that the disease progression may be linked with many other diseases, such as kidney-related disorders. Unraveling the link between PD and non-neurological diseases may help in early detection and a better understanding of PD progression. Herein, we investigated the modulation of α-syn in the presence of β2-microglobulin (β2m), a structural protein associated with dialysis-related amyloidosis. We took a multi-disciplinary approach to establish that β2m mitigates amyloid formation by α-syn. Our fluorescence, microscopy and toxicity data demonstrated that sub-stoichiometric ratio of β2m drives α-syn into off-pathway non-toxic aggregates incompetent of transforming into amyloids. Using AlphaFold2 and all-atom MD simulation, we showed that the β-strand segments (β1 and β2) of α-synuclein, which frequently engage in interactions within amyloid fibrils, interact with the last β-strand at the C-terminal of β2m. The outcome of this study will unravel the yet unknown potential linkage of PD with kidney-related disorders. Insights from the cross-talk between two amyloidogenic proteins will lead to early diagnosis and new therapeutic approaches for treating Parkinson's disease. Finally, disruption of the nucleation process of α-syn amyloids by targeting the β1-β2 region will constitute a potential therapeutic approach for inhibiting amyloid formation.

Amyloid Disassembly: What Can We Learn from Chaperones?

Protein aggregation and subsequent accumulation of insoluble amyloid fibrils with cross-β structure is an intrinsic characteristic of amyloid diseases, i.e., amyloidoses. Amyloid formation involves a series of on-pathway and off-pathway protein aggregation events, leading to mature insoluble fibrils that eventually accumulate in multiple tissues. In this cascade of events, soluble oligomeric species are formed, which are among the most cytotoxic molecular entities along the amyloid cascade. The direct or indirect action of these amyloid soluble oligomers and amyloid protofibrils and fibrils in several tissues and organs lead to cell death in some cases and organ disfunction in general. There are dozens of different proteins and peptides causing multiple amyloid pathologies, chief among them Alzheimer's, Parkinson's, Huntington's, and several other neurodegenerative diseases. Amyloid fibril disassembly is among the disease-modifying therapeutic strategies being pursued to overcome amyloid pathologies. The clearance of preformed amyloids and consequently the arresting of the progression of organ deterioration may increase patient survival and quality of life. In this review, we compiled from the literature many examples of chemical and biochemical agents able to disaggregate preformed amyloids, which have been classified as molecular chaperones, chemical chaperones, and pharmacological chaperones. We focused on their mode of action, chemical structure, interactions with the fibrillar structures, morphology and toxicity of the disaggregation products, and the potential use of disaggregation agents as a treatment option in amyloidosis.

Using T4 genetics and Laemmli’s development of high-resolution SDS gel electrophoresis to reveal structural protein interactions controlling protein folding and phage self-assembly

One of the most transformative experimental techniques in the rise of modern molecular biology and biochemistry was the development of high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis, which allowed separation of proteins—including structural proteins—in complex mixtures according to their molecular weights. Its development was intimately tied to investigations of the control of virus assembly within phage-infected cells. The method was developed by Ulrich K. Laemmli working in the virus structural group led by Aaron Klug at the famed Medical Research Council Laboratory for Molecular Biology at Cambridge, UK. While Laemmli was tackling T4 head assembly, I sat at the next bench working on T4 tail assembly. To date, Laemmli’s original paper has been cited almost 300,000 times. His gel procedure and our cooperation allowed us to sort out the sequential protein–protein interactions controlling the viral self-assembly pathways. It is still not fully appreciated that this control involved protein conformational change induced by interaction with an edge of the growing structure. Subsequent efforts of my students and I to understand how temperature-sensitive mutations interfered with assembly were important in revealing the intracellular off-pathway aggregation processes competing with productive protein folding. These misfolding processes slowed the initial productivity of the biotechnology industry. The article below describes the scientific origin, context, and sociology that supported these advances in protein biochemistry, protein expression, and virus assembly. The cooperation and collaboration that was integral to both the Laboratory for Molecular Biology culture and phage genetics fields were key to these endeavors.

Mechanistic insights into global suppressors of protein folding defects.

Most amino acid substitutions in a protein either lead to partial loss-of-function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss-of-function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.

Oligomeric procyanidins inhibit insulin fibrillation by forming unstructured and off-pathway aggregates.

β-sheet-rich amyloid fibril or aggregate accumulation has been implicated in a number of human diseases. Numerous studies demonstrate that natural polyphenols decrease the risk of degenerative diseases and inhibit in vitro amyloid formation. However, the molecular mechanism for the anti-amyloidogenesis of polyphenols is still unclear. Thus, this study investigates the effects of oligomeric procyanidins (OPCs), resveratrol, and trehalose on the amyloidogenicity of insulin via thioflavin-T (ThT) fluorescence, dynamic light scattering (DLS), circular dichroism (CD), and transmission electronic microscopy (TEM). The results demonstrate that the order of inhibitory effects on insulin amyloid fibrillation is OPCs > resveratrol > trehalose, suggesting that the polyphenolic structure is important for fibril deposition. OPCs show potent inhibitory effects at all stages of insulin fibrillation and redirect the insulin aggregation pathway via the formation of unstructured, off-pathway aggregates. These findings contribute to the development of novel anti-amyloidogenic products from naturally occurring materials.

A network thermodynamic analysis of amyloid aggregation along competing pathways

Self-assembly of proteins towards amyloid aggregates is a significant event in many neurodegenerative diseases. Aggregates of low-molecular weight called oligomers are largely the primary toxic agents in many of these maladies. Therefore, there is an increasing interest in understanding their formation and behavior. In this paper, we build on our previously established theoretical investigations on the interactions between Aβ and lipids (L) that induces off-pathway aggregates under the control of L concentrations. Here, our previously developed competing game theoretic framework between the on- and off-pathway dynamics has been expanded to understand the underlying network topological structures in the reaction kinetics of amyloid formation. The mass-action based dynamical systems are solved to identify dominant pathways in the system with fixed initial conditions, and variations in the occurrence of these dominant pathways are identified as a function of various seeding conditions. The mechanistic approach is supported by thermodynamic free energy computations which helps identify stable reactions. The resulting analysis provides possible intervention strategies that can draw the dynamics away from the off-pathways and potential toxic intermediates. We also draw upon the classic literature on network thermodynamics to suggest new approaches to better understand such complex systems.

A game-theoretic approach to deciphering the dynamics of amyloid-β aggregation along competing pathways.

Aggregation of amyloid-β (Aβ) peptides is a significant event that underpins Alzheimer's disease (AD). Aβ aggregates, especially the low-molecular weight oligomers, are the primary toxic agents in AD pathogenesis. Therefore, there is increasing interest in understanding their formation and behaviour. In this paper, we use our previously established results on heterotypic interactions between Aβ and fatty acids (FAs) to investigate off-pathway aggregation under the control of FA concentrations to develop a mathematical framework that captures the mechanism. Our framework to define and simulate the competing on- and off-pathways of Aβ aggregation is based on the principles of game theory. Together with detailed simulations and biophysical experiments, our models describe the dynamics involved in the mechanisms of Aβ aggregation in the presence of FAs to adopt multiple pathways. Specifically, our reduced-order computations indicate that the emergence of off- or on-pathway aggregates are tightly controlled by a narrow set of rate constants, and one could alter such parameters to populate a particular oligomeric species. These models agree with the detailed simulations and experimental data on using FA as a heterotypic partner to modulate the temporal parameters. Predicting spatio-temporal landscape along competing pathways for a given heterotypic partner such as lipids is a first step towards simulating scenarios in which the generation of specific ‘conformer strains’ of Aβ could be predicted. This approach could be significant in deciphering the mechanisms of amyloid aggregation and strain generation, which are ubiquitously observed in many neurodegenerative diseases.

Peptidomimetics prepared by tail-to-side chain one component peptide stapling inhibit Alzheimer's amyloid-β fibrillogenesis.

Alzheimer's disease (AD) is the most common form of dementia affecting the elderly population worldwide. Despite enormous efforts and considerable advancement in research, no therapeutic agents have come to light to date. However, many peptide-based and small molecule inhibitors interact efficiently with the amyloid-β (Aβ) peptide and alter its aggregation pathway. On the other hand, stapled peptides have been developed mainly to stabilize α-helix conformations and study protein–protein interactions. β-Sheet stabilization or destabilization by stapled peptides has not been explored enough. Herein, we describe the generation of a library of “tail-to-side chain” stapled peptides via lactamization and their application for the first time as modulators of Aβ1-40 self-association and fibrillogenesis. They also disrupt the preformed fibrillar aggregates into nontoxic species. Their stability in the presence of proteolytic enzymes is increased due to stapling. Therefore, the stapled peptides thus formed can be useful as potent amyloid aggregation inhibitors and pave a therapeutic pathway for combating amyloid-related diseases. Also, they may help in gaining insight into the process of aggregation.

Single-residue physicochemical characteristics kinetically partition membrane protein self-assembly and aggregation.

Ninety-five percent of all transmembrane proteins exist in kinetically trapped aggregation-prone states that have been directly linked to neurodegenerative diseases. Interestingly, the primary sequence almost invariably avoids off-pathway aggregate formation, by folding reliably into its native, thermodynamically stabilized structure. However, with the rising incidence of protein aggregation diseases, it is now important to understand the underlying mechanism(s) of membrane protein aggregation. Micromolecular physicochemical and biochemical alterations in the primary sequence that trigger the formation of macromolecular cross-β aggregates can be measured only through combinatorial spectroscopic experiments. Here, we developed spectroscopic thermal perturbation with 117 experimental variables to assess how subtle protein sequence variations drive the molecular transition of the folded protein to oligomeric aggregates. Using the Yersinia pestis outer transmembrane β-barrel Ail as a model, we delineated how a single-residue substitution that alters the membrane-anchoring ability of Ail significantly contributes to the kinetic component of Ail stability. We additionally observed a stabilizing role for interface aliphatics, and that interface aromatics physicochemically contribute to Ail self-assembly and aggregation. Moreover, our method identified the formation of structured oligomeric intermediates during Ail aggregation. We show that the self-aggregation tendency of Ail is offset by the evolution of a thermodynamically compromised primary sequence that balances folding, stability, and oligomerization. Our approach provides critical information on how subtle changes in protein primary sequence trigger cross-β fibril formation, with insights that have direct implications for deducing the molecular progression of neurodegeneration and amyloidogenesis in humans.

The initial stage of structural transformation of Aβ42 peptides from the human and mole rat in the presence of Fe2+ and Fe3+: Related to Alzheimer's disease

The early stage of secondary structural conversion of amyloid beta (Aβ) to misfolded aggregations is a key feature of Alzheimer's disease (AD). Under normal physiological conditions, Aβ peptides can protect neurons from the toxicity of highly concentrated metals. However, they become toxic under certain conditions. Under conditions of excess iron, amyloid precursor proteins (APP) become overexpressed. This subsequently increases Aβ production. Experimental studies suggest that Aβ fibrillation (main-pathway) and amorphous (off-pathway) aggregate formations are two competitive pathways driven by factors such as metal binding, pH and temperature. In this study, we performed molecular dynamic (MD) simulations to examine the initial stage of conformational transformations of human Aβ (hAβ) and rat Aβ (rAβ) peptides in the presence of Fe2+ and Fe3+ ions. Our results demonstrated that Fe2+ and Fe3+ play key roles in Aβs folding and aggregation. Fe3+ had a greater effect than Fe2+on Aβs’ folding during intermolecular interactions and subsequently, had a greater effect in decreasing structural diversity. Fe2+ was observed to be more likely than Fe3+ to interact with nitrogen atoms from the residues of imidazole rings of His. rAβ peptides are more energetically favorable than hAβ for intermolecular interactions and amorphous aggregations. We concluded that most hAβ structures were energetically unfavorable. However, hAβs with intermolecular β-sheet formations in the C-terminal were energetically favorable. It is notable that Fe2+ can change the surface charge of hAβ. Furthermore, Fe3+ can promote C-terminal folding by binding to Glu22 and Ala42, and by forming stable β-sheet formations on the C-terminal. Fe3+ can also pause the main-pathway by inducing random aggregations.

Mechanism of Fibril and Soluble Oligomer Formation in Amyloid Beta and Hen Egg White Lysozyme Proteins.

Assembly and deposition of insoluble amyloid fibrils with a distinctive cross-β-sheet structure is the molecular hallmark of amyloidogenic diseases affecting the central nervous system as well as non-neuropathic amyloidosis. Amyloidogenic proteins form aggregates via kinetic pathways dictated by initial solution conditions. Often, early stage, cytotoxic, small globular amyloid oligomers (gOs) and curvilinear fibrils (CFs) precede the formation of late-stage rigid fibrils (RFs). Growing experimental evidence suggests that soluble gOs are off-pathway aggregates that do not directly convert into the final stage RFs. Yet, the kinetics of RFs aggregation under conditions that either promote or suppress the growth of gOs remain incompletely understood. Here we present a self-assembly model for amyloid fibril formation in the presence and absence of early stage off-pathway aggregates, driven by our experimental results on hen egg white lysozyme (HewL) and beta amyloid (Aβ) aggregation. The model reproduces a range of experimental observations including the sharp boundary in the protein concentration above which the self-assembly of gOs occurs. This is possible when both primary and secondary RFs nucleation rates are allowed to have a nonlinear dependence on initial protein concentration, hinting toward more complex prenucleation and RFs assembly scenarios. Moreover, analysis of RFs lag period in the presence and absence of gOs indicates that these off-pathway aggregates have an inhibitory effect on RFs nucleation. Finally, we incorporate the effect of an Aβ binding protein on the aggregation process in the model that allows us to identify the most suitable solution conditions for suppressing gOs and RFs formation.

Conversion of a soluble protein into a potent chaperone in vivo

Molecular chaperones play an important role in cellular protein-folding assistance and aggregation inhibition. As a different but complementary model, we previously proposed that, in general, soluble cellular macromolecules with large excluded volume and surface charges exhibit intrinsic chaperone activity to prevent aggregation of their connected polypeptides irrespective of the connection type, thereby contributing to efficient protein folding. As a proof of concept, we here demonstrated that a model recombinant protein with a specific sequence-binding domain robustly exerted chaperone activity toward various proteins harbouring a short recognition tag of 7 residues in Escherichia coli. The chaperone activity of this protein was comparable to that of representative E. coli chaperones in vivo. Furthermore, in vitro refolding experiments confirmed the in vivo results. Our findings reveal that a soluble protein exhibits the intrinsic chaperone activity to prevent off-pathway aggregation of its interacting proteins, leading to more productive folding while allowing them to fold according to their intrinsic folding pathways. This study gives new insights into the plausible chaperoning role of soluble cellular macromolecules in terms of aggregation inhibition and indirect folding assistance.

Mechanisms of Protein Fibril Formation in Amyloid Beta and Lysozyme Proteins

Rigid fibril (RFs) formation of amyloid proteins is a precursor to many degenerative disorders, thus understanding the kinetics behind their generation could yield important information with respect to disease pathology. These amyloid proteins exhibit what is known as the “critical oligomer concentration” (COC), exhibiting similar behavior as that of micelle formation by surfactants. At low concentrations these proteins generate only smaller oligomeric species and RFs, upon crossing the COC the growth of globular oligomers (gOs) and curvilinear fibrils (CFs) begin, aggregate species that have been shown to be strongly correlated with disease pathology. Below the COC, RFs result in a sigmoidal rise with an initial lag period generated by delayed polymer nucleation. Above the COC, the rapid generation of gOs/CFs result in the elimination of the initial lag period, followed by a secondary rise due to RF growth, producing a biphasic onset. Here, we simulate the growth kinetics of amyloid beta and lysozyme proteins utilizing the theory of nucleated polymerization with off-pathway aggregation, with the assumption that on-pathway nucleation and off-pathway aggregation occur so rapidly that they can be represented as a single step. We show that in order to simulate experimental fibril formation, the primary nucleation rate must be significantly decreased at initial monomer concentration where there is a significant amount of off-pathway aggregates, resulting in a corresponding increase in initial lag periods. In addition, attempting similar fitting with models of the same type produce comparable trends, further supporting our initial findings, and hinting at a possibly inhibitory effect on RF growth by gOs/CFs.

A Computational Framework for Preferential Switching of Competing Aβ Aggregation Pathways Based on Game Theory Approach

Polymorphism among amyloid-β (Aβ) aggregates are emerging as the main contributors to the observed phenotypic diversity in sporadic Alzheimer's disease (sAD). Aggregation of Aβ is susceptible to several heterotypic factors, and lipids (LDs) are the most important physiological agents to interact with, and modulate Aβ aggregation. Therefore, understanding the mechanism of how LDs modulate Aβ aggregation to generate polymorphic strains along competing pathways is crucial for understanding AD pathogenesis. Here, we demonstrate that steady-state switching dynamics between the competing aggregation pathways thar arise due to heterotypic Aβ-LD interactions can be modeled by a novel game theory approach. The game theory model abstracts the mass-action based non-linear, coupled dynamical system of competing pathways of aggregation and the corresponding Nash equilibrium points can predict the preferred pathways as a function of LD parameters and the level of dilution of the medium. While higher LD concentration leads to the generation of more micelle-bound oligomers (promoting the off-pathway of aggregation), higher levels of dilution of the medium result in dissociating micelle bound oligomers enabling higher conversion to fibrils (promoting the traditional on-pathway of aggregation). These models were subsequently validated by our previously developed ensemble kinetics simulations (EKS) that use ordinary differential equations based models of the competing pathways to estimate the rate constants of converting oligomers between on- and off-pathway aggregates; the combined computational framework accurately predicts the temporal rates and stability of oligomers formed under conditions predicted by controlled in vitro biophysical experiments. Hence, our results demonstrate the possibility of switching oligomeric strains between different competing pathways of Aβ aggregation besides providing a novel computational framework to model its dynamics.

Carbamylation promotes amyloidogenesis and induces structural changes in Tau-core hexapeptide fibrils

BackgroundCarbamylation is a non-enzymatic post-translational modification (PTM), which involves the covalent modification of N-terminus of protein or ε-amino group of Lys. The role of carbamylation in several age-related disorders is well documented, however, the relationship between carbamylation and neurodegenerative disorders including Alzheimer's disease remains uncharted. MethodsIn the present study, using aggregation-prone tau-core hexapeptide fragments 306VQIVYK311 (PHF6) and 275VQIINK280 (PHF6*) as models, we have elucidated the effect of carbamylation on aggregation kinetics and the changes occurring in the 3-dimensional architecture of fibrils using biophysical assays and molecular dynamics simulations. ResultsWe found that carbamylation aids in amyloid formation and can convert the unstructured off-pathway aggregates into robust amyloids, which were toxic to cells. Electron microscopy images and molecular dynamics simulations of PHF6 fibrils showed that carbamylated peptides can form excess hydrogen bonds and modulate the pitch length and twist of peptides fibrils. We have also compared N-terminal carbamylation to acetylation and further extended our finding to full length tau that exhibits aggregation upon carbamylation even in the absence of any external inducer. ConclusionOur in vitro and in silico results together suggest that carbamylation can modulate the aggregation pathway of the amyloidegenic sequences and cause structural changes in fibril assemblies. General significanceCarbamylation acts as a switch, which triggers the aggregation in short amyloidogenic peptide fragments and modulate the structural changes in resulting amyloid fibrils.

Pathway Complexity Versus Hierarchical Self‐Assembly in N‐Annulated Perylenes: Structural Effects in Seeded Supramolecular Polymerization

Studies were carried out on the hierarchical self-assembly versus pathway complexity of N-annulated perylenes 1-3, which differ only in the nature of the linking groups connecting the perylene core and the side alkoxy chains. Despite the structural similarity, compounds 1 and 2 exhibit noticeable differences in their self-assembly. Whereas 1 forms an off-pathway aggregate I that converts over time (or by addition of seeds) into the thermodynamic, on-pathway product, 2 undergoes a hierarchical process in which the kinetically trapped monomer species does not lead to a kinetically controlled supramolecular growth. Finally, compound 3, which lacks the amide groups, is unable to self-assemble under identical experimental conditions and highlights the key relevance of the amide groups and their position to govern the self-assembly pathways.

Energy Landscapes for the Aggregation of Aβ17-42.

The aggregation of the Aβ peptide (Aβ1-42) to form fibrils is a key feature of Alzheimer's disease. The mechanism is thought to be a nucleation stage followed by an elongation process. The elongation stage involves the consecutive addition of monomers to one end of the growing fibril. The aggregation process proceeds in a stop-and-go fashion and may involve off-pathway aggregates, complicating experimental and computational studies. Here we present exploration of a well-defined region in the free and potential energy landscapes for the Aβ17-42 pentamer. We find that the ideal aggregation process agrees with the previously reported dock-lock mechanism. We also analyze a large number of additional stable structures located on the multifunnel energy landscape, which constitute kinetic traps. The key contributors to the formation of such traps are misaligned strong interactions, for example the stacking of F19 and F20, as well as entropic contributions. Our results suggest that folding templates for aggregation are a necessity and that aggregation studies could employ such species to obtain a more detailed description of the process.

Solvent dependent pathway complexity and seeded supramolecular polymerization.

cNDI-1 exhibits an off-pathway aggregate in cyclic hydrocarbon (MCH) but produces a helical supramolecular polymer in linear alkane (decane) by well-defined J-aggregation. Effective solvent-assisted nucleation in linear alkanes due to shape matching with the peripheral alkyl chains of a monomer is responsible for the pathway complexity. Seed, produced by sonication induced fragmentation of the fibers in decane, could initiate supramolecular polymerization of the off-pathway aggregate in MCH generating controllable helical nanostructures. The chiral seed of cNDI-1 was also successfully employed for the synthesis of a helical supramolecular polymer from the off-pathway aggregate of achiral cNDI-2.

Real-time monitoring of the aggregation of Alzheimer's amyloid-β via1H magic angle spinning NMR spectroscopy.

Proton magic-angle-spinning NMR used for real-time analysis of amyloid aggregation reveals that mechanical rotation of Aβ1-40 monomers increases the rate of formation of aggregates, and that the increasing lag-time with peptide concentration suggests the formation of growth-incompetent species. EGCG's ability to shift off-pathway aggregation is also demonstrated.