Chaperones mainly suppress primary nucleation during formation of functional amyloid required for bacterial biofilm formation

Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated. However, it is unclear whether functional aggregation is inhibited by chaperones targeting pathological misfolding and if so by what mechanism. Here we analyze how four entirely different human chaperones or protein modulators (transthyretin, S100A9, Bri2 BRICHOS and DNAJB6) and bacterial CsgC affect CsgA and FapC fibrillation. CsgA is more susceptible to inhibition than FapC and the chaperones vary considerably in the efficiency of their inhibition. However, mechanistic analysis reveals that all predominantly target primary nucleation rather than elongation or secondary nucleation, while stoichiometric considerations suggest that DNAJB6 and CsgC target nuclei rather than monomers. Inhibition efficiency broadly scales with the chaperones'' affinity for monomeric CsgA and FapC. The chaperones tend to target the most aggregation-prone regions of CsgA, but do not display such tendencies towards the more complex FapC sequence. Importantly, the most efficient inhibitors (Bri2 BRICHOS and DNAJB6) significantly reduce bacterial biofilm formation. This commonality of chaperone action may reflect the simplicity of functional amyloid formation, driven largely by primary nucleation, as well as the ability of non-bacterial chaperones to deploy their proteostatic capacities across biological kingdoms.

Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated.     

The extent of protein hydration dictates the preference for heterogeneous or homogeneous nucleation generating either parallel or antiparallel β-sheet α-synuclein aggregates

α-Synuclein amyloid self-assembly is the hallmark of a number of neurodegenerative disorders, including Parkinson''s disease, although there is still very limited understanding about the factors and mechanisms that trigger this process. Primary nucleation has been observed to be initiated in vitro at hydrophobic/hydrophilic interfaces by heterogeneous nucleation generating parallel β-sheet aggregates, although no such interfaces have yet been identified in vivo. In this work, we have discovered that α-synuclein can self-assemble into amyloid aggregates by homogeneous nucleation, without the need of an active surface, and with a preference for an antiparallel β-sheet arrangement. This particular structure has been previously proposed to be distinctive of stable toxic oligomers and we here demonstrate that it indeed represents the most stable structure of the preferred amyloid pathway triggered by homogeneous nucleation under limited hydration conditions, including those encountered inside α-synuclein droplets generated by liquid–liquid phase separation. In addition, our results highlight the key role that water plays not only in modulating the transition free energy of amyloid nucleation, and thus governing the initiation of the process, but also in dictating the type of preferred primary nucleation and the type of amyloid polymorph generated depending on the extent of protein hydration. These findings are particularly relevant in the context of in vivo α-synuclein aggregation where the protein can encounter a variety of hydration conditions in different cellular microenvironments, including the vicinity of lipid membranes or the interior of membraneless compartments, which could lead to the formation of remarkably different amyloid polymorphs by either heterogeneous or homogeneous nucleation.     

Molecular Dynamics Simulation Studies on the Aggregation of Amyloid-β Peptides and Their Disaggregation by Ultrasonic Wave and Infrared Laser Irradiation

Alzheimer’s disease is understood to be caused by amyloid fibrils and oligomers formed by aggregated amyloid-β (Aβ) peptides. This review article presents molecular dynamics (MD) simulation studies of Aβ peptides and Aβ fragments on their aggregation, aggregation inhibition, amyloid fibril conformations in equilibrium, and disruption of the amyloid fibril by ultrasonic wave and infrared laser irradiation. In the aggregation of Aβ, a β-hairpin structure promotes the formation of intermolecular β-sheet structures. Aβ peptides tend to exist at hydrophilic/hydrophobic interfaces and form more β-hairpin structures than in bulk water. These facts are the reasons why the aggregation is accelerated at the interface. We also explain how polyphenols, which are attracting attention as aggregation inhibitors of Aβ peptides, interact with Aβ. An MD simulation study of the Aβ amyloid fibrils in equilibrium is also presented: the Aβ amyloid fibril has a different structure at one end from that at the other end. The amyloid fibrils can be destroyed by ultrasonic wave and infrared laser irradiation. The molecular mechanisms of these amyloid fibril disruptions are also explained, particularly focusing on the function of water molecules. Finally, we discuss the prospects for developing treatments for Alzheimer’s disease using MD simulations.     

Functional amyloids from bacterial biofilms – structural properties and interaction partners

Protein aggregation and amyloid formation have historically been linked with various diseases such as Alzheimer''s and Parkinson''s disease, but recently functional amyloids have gained a great deal of interest in not causing a disease and having a distinct function in vivo. Functional bacterial amyloids form the structural scaffold in bacterial biofilms and provide a survival strategy for the bacteria along with antibiotic resistance. The formation of functional amyloids happens extracellularly which differs from most disease related amyloids. Studies of functional amyloids have revealed several distinctions compared to disease related amyloids including primary structures designed to optimize amyloid formation while still retaining a controlled assembly of the individual subunits into classical cross-β-sheet structures, along with a unique cross-α-sheet amyloid fold. Studies have revealed that functional amyloids interact with components found in the extracellular matrix space such as lipids from membranes and polymers from the biofilm. Intriguingly, a level of complexity is added as functional amyloids also interact with several disease related amyloids and a causative link has even been established between functional amyloids and neurodegenerative diseases. It is hence becoming increasingly clear that functional amyloids are not inert protein structures found in bacterial biofilms but interact with many different components including human proteins related to pathology. Gaining a clear understanding of the factors governing the interactions will lead to improved strategies to combat biofilm associated infections and the correlated antibiotic resistance. In the current review we summarize the current state of the art knowledge on this exciting and fast growing research field of biofilm forming bacterial functional amyloids, their structural features and interaction partners.

Functional bacterial amyloids forming biofilms have unique structural characteristics while still being similar to pathological ones. Through many identified interaction partners, they emerge as complex and essential components of biofilms.     

Antimicrobial α-defensins as multi-target inhibitors against amyloid formation and microbial infection

Amyloid aggregation and microbial infection are considered as pathological risk factors for developing amyloid diseases, including Alzheimer''s disease (AD), type II diabetes (T2D), Parkinson''s disease (PD), and medullary thyroid carcinoma (MTC). Due to the multifactorial nature of amyloid diseases, single-target drugs and treatments have mostly failed to inhibit amyloid aggregation and microbial infection simultaneously, thus leading to marginal benefits for amyloid inhibition and medical treatments. Herein, we proposed and demonstrated a new “anti-amyloid and antimicrobial hypothesis” to discover two host-defense antimicrobial peptides of α-defensins containing β-rich structures (human neutrophil peptide of HNP-1 and rabbit neutrophil peptide of NP-3A), which have demonstrated multi-target, sequence-independent functions to (i) prevent the aggregation and misfolding of different amyloid proteins of amyloid-β (Aβ, associated with AD), human islet amyloid polypeptide (hIAPP, associated with T2D), and human calcitonin (hCT, associated with MTC) at sub-stoichiometric concentrations, (ii) reduce amyloid-induced cell toxicity, and (iii) retain their original antimicrobial activity upon the formation of complexes with amyloid peptides. Further structural analysis showed that the sequence-independent amyloid inhibition function of α-defensins mainly stems from their cross-interactions with amyloid proteins via β-structure interactions. The discovery of antimicrobial peptides containing β-structures to inhibit both microbial infection and amyloid aggregation greatly expands the new therapeutic potential of antimicrobial peptides as multi-target amyloid inhibitors for better understanding pathological causes and treatments of amyloid diseases.

We report a new “anti-amyloid and antimicrobial hypothesis” by discovering host-defense antimicrobial peptides of α-defensins containing β-sheet structures, which possess inhibition functions against amyloid aggregation and microbial infection.     

Conformational Variability of Amyloid-β and the Morphological Diversity of Its Aggregates

Protein folding is the most fundamental and universal example of biomolecular self-organization and is characterized as an intramolecular process. In contrast, amyloidogenic proteins can interact with one another, leading to protein aggregation. The energy landscape of amyloid fibril formation is characterized by many minima for different competing low-energy structures and, therefore, is much more enigmatic than that of multiple folding pathways. Thus, to understand the entire energy landscape of protein aggregation, it is important to elucidate the full picture of conformational changes and polymorphisms of amyloidogenic proteins. This review provides an overview of the conformational diversity of amyloid-β (Aβ) characterized from experimental and theoretical approaches. Aβ exhibits a high degree of conformational variability upon transiently interacting with various binding molecules in an unstructured conformation in a solution, forming an α-helical intermediate conformation on the membrane and undergoing a structural transition to the β-conformation of amyloid fibrils. This review also outlines the structural polymorphism of Aβ amyloid fibrils depending on environmental factors. A comprehensive understanding of the energy landscape of amyloid formation considering various environmental factors will promote drug discovery and therapeutic strategies by controlling the fibril formation pathway and targeting the consequent morphology of aggregated structures.     

Implementing Complementary Approaches to Shape the Mechanism of α-Synuclein Oligomerization as a Model of Amyloid Aggregation

The aggregation of proteins into amyloid fibers is linked to more than forty still incurable cellular and neurodegenerative diseases such as Parkinson’s disease (PD), multiple system atrophy, Alzheimer’s disease and type 2 diabetes, among others. The process of amyloid formation is a main feature of cell degeneration and disease pathogenesis. Despite being methodologically challenging, a complete understanding of the molecular mechanism of aggregation, especially in the early stages, is essential to find new biological targets for innovative therapies. Here, we reviewed selected examples on α-syn showing how complementary approaches, which employ different biophysical techniques and models, can better deal with a comprehensive study of amyloid aggregation. In addition to the monomer aggregation and conformational transition hypothesis, we reported new emerging theories regarding the self-aggregation of α-syn, such as the alpha-helix rich tetramer hypothesis, whose destabilization induce monomer aggregation; and the liquid-liquid phase separation hypothesis, which considers a phase separation of α-syn into liquid droplets as a primary event towards the evolution to aggregates. The final aim of this review is to show how multimodal methodologies provide a complete portrait of α-syn oligomerization and can be successfully extended to other protein aggregation diseases.     

Near-Wall Aggregation of Amyloidogenic Aβ 1-40 Peptide: Direct Observation by the FRET

The formation of amyloid fibrils is one of the variants of the self-organization of polypeptide chains. For the amyloid aggregation, the solution must be oversaturated with proteins. The interface of the liquid (solution) and solid (vessel walls) phases can trigger the adsorption of protein molecules, and the resulting oversaturation can initiate conformational transitions in them. In any laboratory experiment, we cannot exclude the presence of surfaces such as the walls of vessels, cuvettes, etc. However, in many works devoted to the study of amyloid formation, this feature is not considered. In our work, we investigated the behavior of the Aβ 1-40 peptide at the water–glass, water–quartz, and water–plastic interface. We carried out a series of simple experiments and showed that the Aβ 1-40 peptide is actively adsorbed on these surfaces, which leads to a significant interaction and aggregation of peptides. This means that the interface can be the place where the first amyloid nucleus appears. We suggest that this effect may also be one of the reasons for the difficulty of reproducing kinetic data when studying the aggregation of the amyloid of the Aβ 1-40 peptide and other amyloidogenic proteins     

Valorization of Apple Peels through the Study of the Effects on the Amyloid Aggregation Process of κ-Casein

Waste valorization represents one of the main social challenges when promoting a circular economy and environmental sustainability. Here, we evaluated the effect of the polyphenols extracted from apple peels, normally disposed of as waste, on the amyloid aggregation process of κ-casein from bovine milk, a well-used amyloidogenic model system. The effect of the apple peel extract on protein aggregation was examined using a thioflavin T fluorescence assay, Congo red binding assay, circular dichroism, light scattering, and atomic force microscopy. We found that the phenolic extract from the peel of apples of the cultivar “Fuji”, cultivated in Sicily (Caltavuturo, Italy), inhibited κ-casein fibril formation in a dose-dependent way. In particular, we found that the extract significantly reduced the protein aggregation rate and inhibited the secondary structure reorganization that accompanies κ-casein amyloid formation. Protein-aggregated species resulting from the incubation of κ-casein in the presence of polyphenols under amyloid aggregation conditions were reduced in number and different in morphology.     

Molecular mechanisms of amyloid formation in living systems

Fibrillar protein aggregation is a hallmark of a variety of human diseases. Examples include the deposition of amyloid-β and tau in Alzheimer''s disease, and that of α-synuclein in Parkinson''s disease. The molecular mechanisms by which soluble proteins form amyloid fibrils have been extensively studied in the test tube. These investigations have revealed the microscopic steps underlying amyloid formation, and the role of factors such as chaperones that modulate these processes. This perspective explores the question to what extent the mechanisms of amyloid formation elucidated in vitro apply to human disease. The answer is not yet clear, and may differ depending on the protein and the associated disease. Nevertheless, there are striking qualitative similarities between the aggregation behaviour of proteins in vitro and the development of the related diseases. Limited quantitative data obtained in model organisms such as Caenorhabditis elegans support the notion that aggregation mechanisms in vivo can be interpreted using the same biophysical principles established in vitro. These results may however be biased by the high overexpression levels typically used in animal models of protein aggregation diseases. Molecular chaperones have been found to suppress protein aggregation in animal models, but their mechanisms of action have not yet been quantitatively analysed. Several mechanisms are proposed by which the decline of protein quality control with organismal age, but also the intrinsic nature of the aggregation process may contribute to the kinetics of protein aggregation observed in human disease.

The molecular mechanisms of amyloid formation have been studied extensively in test tube reactions. This perspective article addresses the question to what extent these mechanisms apply to the complex situation in living cells and organisms.     

Extract from the Marine Seaweed Padina pavonica Protects Mitochondrial Biomembranes from Damage by Amyloidogenic Peptides

The identification of compounds which protect the double-membrane of mitochondrial organelles from disruption by toxic confomers of amyloid proteins may offer a therapeutic strategy to combat human neurodegenerative diseases. Here, we exploited an extract from the marine brown seaweed Padina pavonica (PPE) as a vital source of natural bioactive compounds to protect mitochondrial membranes against insult by oligomeric aggregates of the amyloidogenic proteins amyloid-β (Aβ), α-synuclein (α-syn) and tau, which are currently considered to be major targets for drug discovery in Alzheimer’s disease (AD) and Parkinson’s disease (PD). We show that PPE manifested a significant inhibitory effect against swelling of isolated mitochondria exposed to the amyloid oligomers, and attenuated the release of cytochrome c from the mitochondria. Using cardiolipin-enriched synthetic lipid membranes, we also show that dye leakage from fluorophore-loaded vesicles and formation of channel-like pores in planar bilayer membranes are largely prevented by incubating the oligomeric aggregates with PPE. Lastly, we demonstrate that PPE curtails the ability of Aβ42 and α-syn monomers to self-assemble into larger β-aggregate structures, as well as potently disrupts their respective amyloid fibrils. In conclusion, the mito-protective and anti-aggregator biological activities of Padina pavonica extract may be of therapeutic value in neurodegenerative proteinopathies, such as AD and PD.     

Self-assembly and morphological characterization of two-component functional amyloid proteins

Functional amyloid has been increasingly applied as self-assembling nanostructures to construct multifunctional biomaterials. However, little has been known how different side domains, varied fusion positions and subunits affect self-assembly and morphologies of amyloid fibrils. Here, we constructed three groups of two-component amyloid proteins based on CsgA, the major protein components of Escherichia coli biofilms, to bridge these gaps. We showed that all fusion proteins have amyloid features, as indicated by Congo red assay. Atomic force microscopy (AFM) indeed reveals that these fusion proteins are able to self-assemble into fibrils, with an average diameter of 0.5-2 nm and length of hundreds of nanometers to several micrometers. The diameter of fibrils increases with the increase of the molecular weight of fusion domains, while the dynamic assembly of recombinant proteins was delayed as a result of the introduction of fusion domains. Moreover, fusion of the same functional domains but at intermediate position seems to cause the most interference on fibril assembly compared with those fused at C or Nterminus, as mainly short and irregular fibrils were detected. This phenomenon appears more pronounced for randomly coiled mussel foot proteins (Mfps) than for rigid chitin-binding domain (CBD). Finally, increase of the molecular weight of tandem repeats in protein monomer seemed to increase the fibril diameter of the resultant fibrils, but either reduction of the tandem repeats of CsgA to one single belta-sheet loop or increase in the number of tandem repeats of CsgAs from one to four produced shorter and intermittent fibrils compared with CsgA control protein. These studies therefore provide insights into self-assembly of two-component amyloid proteins and lay the foundation for rational design of multifunctional molecular biomaterials.     

Does liquid–liquid phase separation drive peptide folding?

Proline–arginine (PR) dipeptide repeats have been shown to undergo liquid–liquid phase separation and are an example of a growing number of intrinsically disordered proteins that can assemble into membraneless organelles. These structures have been posited as nucleation sites for pathogenic protein aggregation. As such, a better understanding of the effects that the increased local concentration and volumetric crowding within droplets have on peptide secondary structure is necessary. Herein we use Fourier transform infrared (FTIR) and two-dimensional infrared (2DIR) spectroscopy to show that formation of droplets by PR₂₀ accompanies changes in the amide-I spectra consistent with folding into poly-proline helical structures.

Two-dimensional infrared spectroscopy reveals folding of an intrinsically disordered peptide when sequestered into a model “membrane-less” organelle.     

Repurposing of intestinal defensins as multi-target,dual-function amyloid inhibitors via cross-seeding

Amyloid formation and microbial infection are the two common pathological causes of neurogenerative diseases, including Alzheimer''s disease (AD), type II diabetes (T2D), and medullary thyroid carcinoma (MTC). While significant efforts have been made to develop different prevention strategies and preclinical hits for these diseases, conventional design strategies of amyloid inhibitors are mostly limited to either a single prevention mechanism (amyloid cascade vs. microbial infection) or a single amyloid protein (Aβ, hIAPP, or hCT), which has prevented the launch of any successful drug on the market. Here, we propose and demonstrate a new “anti-amyloid and anti-bacteria” strategy to repurpose two intestinal defensins, human α-defensin 6 (HD-6) and human β-defensin 1 (HBD-1), as multiple-target, dual-function, amyloid inhibitors. Both HD-6 and HBD-1 can cross-seed with three amyloid peptides, Aβ (associated with AD), hIAPP (associated with T2D), and hCT (associated with MTC), to prevent their aggregation towards amyloid fibrils from monomers and oligomers, rescue SH-SY5Y and RIN-m5F cells from amyloid-induced cytotoxicity, and retain their original antimicrobial activity against four common bacterial strains at sub-stoichiometric concentrations. Such sequence-independent anti-amyloid and anti-bacterial functions of intestinal defensins mainly stem from their cross-interactions with amyloid proteins through amyloid-like mimicry of β-sheet associations. In a broader view, this work provides a new out-of-the-box thinking to search and repurpose a huge source of antimicrobial peptides as amyloid inhibitors, allowing the blocking of the two interlinked pathological pathways and bidirectional communication between the central nervous system and intestines via the gut–brain axis associated with neurodegenerative diseases.

Amyloid formation and microbial infection are the two common pathological causes of neurogenerative diseases. Here, we proposed a new “anti-amyloid and anti-bacteria” strategy to repurpose two intestinal defensins as multiple-target, dual-function amyloid inhibitors.     

Sequestration within biomolecular condensates inhibits Aβ-42 amyloid formation

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aβ-42), the peptide strongly associated with Alzheimer''s disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aβ-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.

Biomolecular condensates sequester an aggregation-prone peptide and prevent its aggregation, showing that heterotypic interactions within the condensates can prevent the formation of amyloid fibrils, despite the local increase in concentration.     

The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ₄₀) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of ¹⁵N-labeled Aβ₄₀ and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ₄₀ (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ₄₀ and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.     

Unravelling the effect of N(ε)-(carboxyethyl)lysine on the conformation,dynamics and aggregation propensity of α-synuclein

α-Synuclein (αS) aggregation is a hallmark in several neurodegenerative diseases. Among them, Parkinson''s disease is highlighted, characterized by the intraneuronal deposition of Lewy bodies (LBs) which causes the loss of dopaminergic neurons. αS is the main component of LBs and in them, it usually contains post-translational modifications. One of them is the formation of advanced glycation end-products (mainly CEL and MOLD) arising from its reaction with methylglyoxal. Despite its biological relevance, there are no data available proving the effect of glycation on the conformation of αS, nor on its aggregation mechanism. This has been hampered by the formation of a heterogeneous set of compounds that precluded conformational studies. To overcome this issue, we have here produced αS homogeneously glycated with CEL. Its use, together with different biophysical techniques and molecular dynamics simulations, allowed us to study for the first time the effect of glycation on the conformation of a protein. CEL extended the conformation of the N-terminal domain as a result of the loss of transient N-/C-terminal long-range contacts while increasing the heterogeneity of the conformational population. CEL also inhibited the αS aggregation, but it was not able to disassemble preexisting amyloid fibrils, thus proving that CEL found on LBs must be formed in a later event after aggregation.

We study the effect of an advanced glycation end product (N(ε)-(carboxyethyl)lysine), found on the Lewy bodies of people suffering from Parkinson’s disease, on the conformational and aggregation features of alpha-synuclein.     

Single Molecule Characterization of Amyloid Oligomers

The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.     

High-Efficiency Expression and Purification of DNAJB6b Based on the pH-Modulation of Solubility and Denaturant-Modulation of Size

The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein “as is” with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.