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Engineering B family DNA polymerases to utilize artificial nucleotides with unique large group labeling is limited by a general lack of understanding about the mechanism of the structural determinants that define substrate specificity. In this study, we report the engineering process of a natural DNA polymerase (KOD pol) fromThermococcus Kodakaraenisby using semi-rational design strategies. The essential residues around the active pocket and DNA binding site of KOD pol are characterized by using an established high-throughput microwell-based screening method. We obtained a five-mutant variant by performing site-directed saturation mutagenesis and combinatorial mutations of motif A and position 485, including two previously reported mutation sites (141/143) with inactive exonuclease activity. Then we selected more mutants, which were expected to increase the catalytic activity based on computational simulations, to perform experimental verification. By stepwise combinatorial mutation, we obtained an eleven-mutation variant E10 with over 25 times improvement of kinetic efficiency which has similar and satisfactory performances on both the BGI NGS platform (SE50) and MGI CoolMPSTMplatform (PE100). The results indicated that E10 is a potential candidate for commercialization. The beneficial mutation sites identified in this study might be shared with other archaeal B-family DNA polymerases, which can provide a guide for the mutagenesis of other types of enzymes.

Nanoparticles (NPs) can modulate protein aggregation and fibril formation in the context of amyloid diseases. Understanding the mechanism of this action remains a critical next step in developing nanomedicines for the treatment or prevention of Parkinson's disease. α-Synuclein (α-Syn) can undergo interactions of different strength with nanoparticles, and these interactions can be prevented by the presence of a protein corona (PC) acquired during the exposure of NPs to serum proteins. Here, we develop a method to attach the PC irreversibly to the NPs, which enables us to study in detail the interaction of α-Syn and polyethylenimine-coated carboxyl-modified polystyrene NPs (PsNPs-PEI) and the role of the dynamics of the interactions. Analysis of the kinetics of fibril formation reveals that the NPs surface promotes the primary nucleation step of amyloid fibril formation without significantly affecting the elongation and fragmentation steps or the final equilibrium. Furthermore, the results show that even though α-Syn can access the surface of NPs that are precoated with a PC, due to the dynamic nature of the PC proteins, the PC nevertheless reduces the acceleratoring effect of the NPs. This effect is likely to be caused by reducing the overall amount of weakly interacting α-Syn molecules on the NP surface and the access of further α-Syn required for fibril elongation. Our experimental approach provides microscopic insight into how serum proteins can modulate the complex interplay between NPs and amyloid proteins.

Aggregation of the major whey protein in bovine milk, β-lactoglobulin (β-Lg) is strongly influenced by association with caseins (CNs). Here, by using combined differential scanning fluorimetry and dynamic light scattering, the conformational stability and aggregation propensity of β-Lg and three types of CNs (α, β and ĸCNs) as well as their mixture have been systematically evaluated at different temperatures and Ca2+ concentrations in a multi-parametric approach. While β-Lg was affected significantly through denaturation and resulting aggregation by heat treatment with little dependency on Ca2+, αCN and βCN were influenced considerably by Ca2+. Through modifying the aggregation of β-Lg, CNs showed a different chaperone-like activity among the three types which were markedly dependent on the temperature and Ca2+ concentration. The presence of CNs resulted in smaller mixed aggregates compared to pure β-Lg aggregates, mainly through interaction of CNs with unfolded β-Lg and also by influencing the process of β-Lg unfolding. This was further confirmed by small angle X-ray scattering and isothermal titration calorimetry indicating that Ca2+ enhanced the interaction between β-Lg and CNs. Our experimental approach sheds light on molecular understanding of CN- β-Lg interactions and provides insight into how micro-structural assembly of milk proteins can be modulated to enable different functionalities in milk-based products.

Microfluidic systems under laminar flow conditions provide in-solution information about species size and binding affinities at very modest sample costs. Flow-induced dispersion analysis directly measures the spread of the analyte profile using Taylor dispersion analysis, whereas microfluidic diffusional sizing quantifies the transfer of analyte from one phase to another. Species of sizes between 0.5 and 1000 nm can be analyzed, and different populations resolved. Both techniques also allow analysis in complex media and medium throughput analysis. These properties make them valuable complements to existing approaches to measure biomolecular interactions.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method. All authors acknowledge the COST Action project ARBRE-MOBIEU CA15126 under the auspices of whose Working Group 4 this study was carried out. ARBRE-MOBIEU COST Action CA15126 funded meetings in the Center for Protein Research, University of Copenhagen (ECOST-MEETING-CA15126-280618-098884) to organize this study and in the Vienna Biocenter Core Facilities GmbH (ECOST-MEETING-CA15126-121119-111544) to discuss the results of the benchmark. Ondřej Vaněk acknowledges support from the Ministry of Education, Youth and Sports of the Czech Republic (LTC17065 in frame of the COST Action CA15126). Jitka Holková and Josef Houser acknowledge CF Biomolecular Interactions and Crystallization of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127). Jasmina Rokov-Plavec acknowledges support from the Croatian Science Foundation (IP-2016-06-6272). Alexander K. Buell thanks the Novo Nordisk Foundation (grant number NNFSA170028392) for funding. Li Peng Lundgren acknowledges for preliminary MST/TRIC experiments (STMS 37745 in the frame of the COST Action CA15126) and the COST Action (CA15126) for a STMS (STMS 44783) for the data analysis. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001). Katja Pirc and Marjetka Podobnik acknowledge the grant by the Slovenian Research Agency P1-0391 (Title: Molecular Interactions). This research was funded by Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, “Investing in your future” PI18/00349 and Diputación General de Aragón Digestive Pathology Group B25_17R to O.A. Spanish Ministry of Science, Innovation and Universities, FPI Predoctoral Research Contract BES-2017-080739 to D.O.A. This research was funded by project no 2015/17/B/NZ2/01160 (granted to MA)

The aggregation of the amyloid-β (Aβ) peptide is a major hallmark of Alzheimer's disease. This peptide can aggregate into oligomers, proto-fibrils, and mature fibrils, which eventually assemble into amyloid plaques. The peptide monomers are the smallest assembly units and play an important role in most of the individual processes involved in amyloid fibril formation, such as primary and secondary nucleation and elongation. Several d-peptides have been confirmed as promising candidates to inhibit the aggregation of Aβ into toxic oligomers and fibrils by specifically interacting with monomeric species. In this work, we elucidate the structural interaction and thermodynamics of binding between three d-peptides (D3, ANK6, and RD2) and Aβ42 monomers by means of enhanced molecular dynamics simulations. Our study derives thermodynamic energies in good agreement with experimental values and suggests that there is an enhanced binding for D3 and ANK6, which leads to more stable complexes than for RD2. The binding of D3 to Aβ42 is shown to be weakly exothermic and mainly entropically driven, whereas the complex formation between the ANK6 and RD2 with the Aβ42 free monomer is weakly endothermic. In addition, the changes in the solvent-accessible surface area and the radius of gyration support that the binding between Aβ42 and d-peptides is mainly driven by electrostatic and hydrophobic interactions and leads to more compact conformations.

α-Synuclein (α-Syn) liquid-liquid phase separation (LLPS) leads to irreversible amyloid fibril formation associated with Parkinson’s disease pathogenesis. Critical concentrations of α-Syn LLPS are relatively high under physiological solution conditions. Moreover, α-Syn exhibits delayed LLPS kinetics under certain conditions which deviates from the behaviour predicted by classical homogeneous nucleation theory. In the current body of work, using interferometric light scattering (iSCAT), also known as mass photometry, we experimentally probe that α-Syn can form nanoscale phase separated assemblies/clusters, containing tens to hundreds of molecules— both above and below the critical LLPS concentration down to physiologically relevant scales. The formation of these clusters is instantaneous, even under conditions where the formation of microscopically visible droplets takes several days. However, they account for a very small volume fraction below saturation concentration. The slow growth of the nanoclusters can be attributed to a kinetic barrier which can be overcome by increasing the solution temperature to just below the droplet melting point. We provide reasons for caution in quantifying dilute phase concentrations for α-Syn LLPS samples containing nanoscale droplets—which can only be separated using ultracentrifugation. In addition, we also delineate that the presence of certain surfaces facilitates α-Syn droplet nucleation under conditions of delayed kinetics but is not a mandatory prerequisite for nanocluster formation. Taken together, our findings reveal that phase separation of α-Syn occurs at a wider range of solution conditions than predicted so far and provides an important step towards understanding α-Syn LLPS within physiological scales.Abstract Figure

Cell migration plays an important role in physiological and pathological processes where the fibrillar morphology of extracellular matrices (ECM) could regulate the migration dynamics. To mimic the morphological characteristics of fibrillar matrix structures, low-voltage continuous electrospinning was adapted to construct straight, wavy, looped and gridded fibre patterns made of polystyrene (of fibre diameter ca. 3 μm). Cells were free to explore their different shapes in response to the directly-adhered fibre, as well as to the neighbouring patterns. For all the patterns studied, analysing cellular migration dynamics of MDA-MB-231 (a highly migratory breast cancer cell line) demonstrated two interesting findings: first, although cells dynamically adjust their shapes and migration trajectories in response to different fibrillar environments, their average step speed is minimally affected by the fibre global pattern; secondly, a switch in behaviour was observed when the pattern features approach the upper limit of the cell body’s minor axis, reflecting that cells’ ability to divert from an existing fibre track is limited by the size along the cell body’s minor axis. It is therefore concluded that the upper limit of cell body’s minor axis might act as a guide for the design of microfibre patterns for different purposes of cell migration.

The deposition of coassemblies made of the small presynaptic protein, α-synuclein, and lipids in the brains of patients is the hallmark of Parkinson's disease. In this study, we used natural abundance 13C and 31P magic-angle spinning nuclear magnetic resonance spectroscopy together with cryo-electron microscopy and differential scanning calorimetry to characterize the fibrils formed by α-synuclein in the presence of vesicles made of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine or 1,2-dilauroyl-sn-glycero-3-phospho-L-serine. Our results show that these lipids coassemble with α-synuclein molecules to give thin and curly amyloid fibrils. The coassembly leads to slower and more isotropic reorientation of lipid molecular segments and a decrease in both the temperature and enthalpy of the lipid chain-melting compared with those in the protein-free lipid lamellar phase. These findings provide new insights into the properties of lipids within protein-lipid assemblies that can be associated with Parkinson's disease.

Amyloid fibrils are β-sheet-rich linear protein polymers that can be formed by a large variety of different proteins. These assemblies have received much interest in recent decades, due to their role in a range of human disorders. However, amyloid fibrils are also found in a functional context, whereby their structural, mechanical and thermodynamic properties are exploited by biological systems. Amyloid fibrils form through a nucleated polymerisation mechanism with secondary processes acting in many cases to amplify the number of fibrils. The filamentous nature of amyloid fibrils implies that the fibril growth rate is, by several orders of magnitude, the fastest step of the overall aggregation reaction. This article focusses specifically on in vitro experimental studies of the process of amyloid fibril growth, or elongation, and summarises the state of knowledge of its kinetics and mechanisms. This work attempts to provide the most comprehensive summary, to date, of the available experimental data on amyloid fibril elongation rate constants and the temperature and concentration dependence of amyloid fibril elongation rates. These data are compared with those from other types of protein polymers. This comparison with data from other polymerising proteins is interesting and relevant because many of the basic ideas and concepts discussed here were first introduced for non-amyloid protein polymers, most notably by the Japanese school of Oosawa and co-workers for cytoskeletal filaments.

The deposition of immunoglobulin light chains (IgLCs) in the form of amorphous aggregates or amyloid fibrils in different tissues of patients can lead to severe and potentially fatal organ damage, requiring transplantation in some cases. There has been great interest in recent years to elucidate the origin of the very different in vivo solubilities of IgLCs, as well as the molecular determinants that drive either the formation of ordered amyloid fibrils or disordered amorphous aggregates. It is commonly thought that the reason of this differential aggregation behaviour is to be found in the amino acid sequences of the respective IgLCs, i.e. that some sequences display higher intrinsic tendencies to form amyloid fibrils. Here we perform in depth Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip) of 9 multiple myeloma patient-derived IgLCs, the amino acid sequences of all of which we have solved by de novo protein sequencing with mass spectrometry. The latter technique was also used for one IgLc from a patient with AL amyloidosis. We find that all samples also contain proteases that fragment the proteins under physiologically relevant mildly acidic pH conditions, leading to amyloid fibril formation in all cases. Our results suggest that while every pathogenic IgLC has a unique ThAgg fingerprint, all sequences have comparable amyloidogenic potential. Therefore, extrinsic factors, in particular presence of, and susceptibility to, proteolytic cleavage is likely to be a strong determinant of in vivo aggregation behaviour. The important conclusion, which is corroborated by systematic analysis of our sequences, as well as many sequences of IgLCs from amyloidosis patients reported in the literature, challenges the current paradigm of the link between sequence and amyloid fibril formation of pathogenic light chains.

Liquid-liquid phase separation (LLPS) of proteins is a field of mounting importance to life sciences. We present a new method, Capflex, that can easily be automated, allowing rapid and accurate quantification of key parameters for LLPS. Dilute phase concentration, relative droplet size distributions and the kinetics of droplet formation are quantified. Uniquely, the binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We applied Capflex to characterize the LLPS of Ddx4n1 and found that PEG3000 and Ca2+ promotes LLPS while ssDNA is detrimental. Furthermore, we characterized the membraneless organelle model system RP3 and provide the first experimentally recorded affinity of RP3 for DNA. We believe the high information content and high throughput of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

Amyloid fibril formation of proteins is of great concern in neurodegenerative disease and can be detrimental to the storage and stability of biologics. Recent evidence suggests that insulin fibril formation reduces the efficacy of type II diabetes management and may lead to several complications. To develop anti-amyloidogenic compounds of endogenous origin, we have utilized the hydrogen bond anchoring, π stacking ability of porphyrin, and investigated its role on the inhibition of insulin amyloid formation. We report that hydroxylation and metal removal from the heme moiety yields an excellent inhibitor of insulin fibril formation. Thioflavin T, tyrosine fluorescence, Circular Dichorism (CD) spectroscopy, Field emission scanning electron microscopy (FESEM) and molecular dynamics (MD) simulation studies suggest that hematoporphyrin (HP) having hydrogen bonding ability on both sides is a superior inhibitor compared to hemin and protoporphyrin (PP). Experiments with hen egg white lysozyme (HEWL) amyloid fibril formation also validated the efficacy of endogenous porphyrin based small molecules. Our results will help to decipher a general therapeutic strategy to counter amyloidogenesis.

Amyloid β (Aβ) monomers are the smallest assembly units, and play an important role in most of the individual processes involved in amyloid fibril formation. An important question is whether the monomer can adopt transient fibril-like conformations in solution. Here we use enhanced sampling simulations to study the Aβ42 monomer structural flexibility. We show that the monomer frequently adopts conformations with the N-terminus region structured very similarly to the conformation it adopts inside the fibril. This intrinsic propensity of monomeric Aβ to adopt fibril-like conformations could explain the low free energy barrier for Aβ42 fibril elongation.

Amyloid fibrils are ordered, non-covalent polymers of proteins that are linked to a range of diseases, as well as biological functions. Amyloid fibrils are often considered thermodynamically so stable that they appear to be irreversible, explaining why very few quantitative thermodynamic studies have been performed on amyloid fibrils, compared to the very large body of kinetic studies. Here we explore the thermodynamics of amyloid fibril formation by the protein PI3K-SH3, which forms amyloid fibrils under acidic conditions. We use quartz crystal microbalance (QCM) and develop novel temperature perturbation experiments based on differential scanning fluorimetry (DSF) to measure the temperature dependence of the fibril growth and dissociation rates, allowing us to quantitatively describe the thermodynamic stability of PI3K-SH3 amyloid fibrils between 10 and 75°C.

The aggregation of the amyloid β (Aβ) peptide is a major hallmark of Alzheimer’s disease. This peptide can aggregate into oligomers, proto-fibrils, and mature fibrils, which eventually assemble into amyloid plaques. The peptide monomers are the smallest assembly units, and play an important role in most of the individual processes involved in amyloid fibril formation, such as primary and secondary nucleation and elongation. The structure of the Aβ monomer has been shown to be very dynamic and mostly disordered, both in experimental and in computational studies, similar to a random coil. This structural state of the monomer contrasts with the very stable and well defined structural core of the amyloid fibrils. An important question is whether the monomer can adopt transient fibril-like conformations in solution and what role such conformations might play in the aggregation process. Here we use enhanced and extensive molecular dynamics simulations to study the Aβ42 monomer structural flexibility with different force fields, water models and salt concentrations. We show that the monomer behaves as a random coil under different simulation conditions. Importantly, we find a conformation with the N-terminal region structured very similarly to that of recent experimentally determined fibril models. This is to the best of our knowledge the first monomeric structural ensemble to show such a similarity with the fibril structure.

Amyloid fibrils are homo-molecular protein polymers that play an important role in disease and biological function. While much is known about their kinetics and mechanisms of formation, the origin and magnitude of their thermodynamic stability has received significantly less attention. This is despite the fact that the thermodynamic stability of amyloid fibrils is an important determinant of their lifetimes and processing in vivo. Here we use depolymerization by chemical denaturants of amyloid fibrils of two different proteins (PI3K-SH3 and glucagon) at different concentrations and show that the previously applied isodesmic linear polymerization model is an oversimplification that does not capture the concentration dependence of chemical depolymerization of amyloid fibrils. We show that cooperative polymerization, which is compatible with the picture of amyloid formation as a nucleated polymerization process, is able to quantitatively describe the thermodynamic data. We use this combined experimental and conceptual framework in order to probe the ionic strength dependence of amyloid fibril stability. In combination with previously published data on the ionic strength dependence of amyloid fibril growth kinetics, our results provide strong evidence for the product-like nature of the transition state of amyloid fibril growth.

The amyloid fibril formation by &alpha, synuclein is a hallmark of various neurodegenerative disorders, most notably Parkinson&rsquo, s disease. Epigallocatechin gallate (EGCG) has been reported to be an efficient inhibitor of amyloid formation by numerous proteins, among them &alpha, synuclein. Here, we show that this applies only to a small region of the relevant parameter space, in particular to solution conditions where EGCG readily oxidizes, and we find that the oxidation product is a much more potent inhibitor compared to the unmodified EGCG. In addition to its inhibitory effects, EGCG and its oxidation products can under some conditions even accelerate &alpha, synuclein amyloid fibril formation through facilitating its heterogeneous primary nucleation. Furthermore, we show through quantitative seeding experiments that, contrary to previous reports, EGCG is not able to re-model &alpha, synuclein amyloid fibrils into seeding-incompetent structures. Taken together, our results paint a complex picture of EGCG as a compound that can under some conditions inhibit the amyloid fibril formation of &alpha, synuclein, but the inhibitory action is not robust against various physiologically relevant changes in experimental conditions. Our results are important for the development of strategies to identify and characterize promising amyloid inhibitors.

The amyloid fibril formation by α -synuclein is a hallmark of various neurodegenerative disorders, most notably Parkinson's disease. Epigallocatechin gallate (EGCG) has been reported to be an efficient inhibitor of amyloid formation by numerous proteins, among them α -synuclein. Here, we show that this applies only to a small region of the relevant parameter space, in particular to solution conditions where EGCG readily oxidizes, and we find that the oxidation product is a much more potent inhibitor compared to the unmodified EGCG. In addition to its inhibitory effects, EGCG and its oxidation products can under some conditions even accelerate α -synuclein amyloid fibril formation through facilitating its heterogeneous primary nucleation. Furthermore, we show through quantitative seeding experiments that, contrary to previous reports, EGCG is not able to re-model α -synuclein amyloid fibrils into seeding-incompetent structures. Taken together, our results paint a complex picture of EGCG as a compound that can under some conditions inhibit the amyloid fibril formation of α -synuclein, but the inhibitory action is not robust against various physiologically relevant changes in experimental conditions. Our results are important for the development of strategies to identify and characterize promising amyloid inhibitors.

Inhibition of the self-assembly process of amyloid-beta and even more the removal of already existing toxic amyloid-beta assemblies represent promising therapeutic strategies against Alzheimer's disease. To approach this aim, we selected a d-enantiomeric peptide by phage-display based on the interaction with amyloid-beta monomers. This lead compound was successfully optimized by peptide microarrays with respect to its affinity and specificity to the target resulting in d-peptides with both increased hydrophobicity and net charge. Here, we present a detailed biophysical characterization of the interactions between these optimized d-peptides and amyloid-beta monomers in comparison to the original lead compound in order to obtain a more thorough understanding of the physicochemical determinants of the interactions. Kinetics and apparent stoichiometry of complex formation were studied using surface plasmon resonance. Potential modes of binding to amyloid-beta were identified, and the influences of ionic strength on complex stability, as well as on the inhibitory effect on amyloid-beta aggregation were investigated. The results reveal a very different mode of interaction of the optimized d-peptides based on a combination of electrostatic and hydrophobic interactions as compared to the mostly electrostatically driven interaction of the lead compound. These conclusions were supported by the thermodynamic profiles of the interaction between optimized d-peptides and Aβ monomers, which indicate an increase in binding entropy with respect to the lead compound.

Elucidation of the fundamental interactions of proteins with biological membranes under native conditions is crucial for understanding the molecular basis of their biological function and malfunction. Notably, the large surface to volume ratio of living cells provides a molecular landscape for significant interactions of cellular components with membranes, thereby potentially modulating their function. However, such interactions can be challenging to probe using conventional biophysical methods due to the heterogeneity of the species and processes involved. Here, we use direct measurements of micron scale molecular diffusivity to detect and quantify the interactions of α-synuclein, associated with the etiology of Parkinson's disease, with negatively charged lipid vesicles. We further demonstrate that this microfluidic approach enables the characterization of size distributions of different binary mixtures of vesicles, which are not readily accessible using conventional light scattering techniques. Finally, the size distributions of the two α-synuclein conformations, free α-synuclein and membrane-bound α-synuclein, were resolved under varying lipid:protein ratios, thus, allowing the determination of the dissociation constant and the binding stoichiometry associated with this protein-lipid system. The microfluidic diffusional sizing platform allows these measurements to be performed on a time scale of minutes using microlitre volumes, thus, establishing the basis for an approach for the study of molecular interactions of heterogeneous systems under native conditions.

Parkinson's disease is one of the major neurodegenerative disorders affecting the ageing populations of the modern world. One of the hallmarks of this disease is the deposition of aggregates, mainly of the small pre-synaptic protein α-synuclein (AS), in the brains of patients. Several very significantly modified forms of AS have been found in these deposits including those resulting from truncations of the protein at its C-terminus. Here, we report how two physiologically relevant C-terminal truncations of AS, AS(1-119) and AS(1-103), where either half or virtually all of the C-terminal domain, respectively, has been truncated, affect the mechanism of AS aggregation and the properties of the fibrils formed. In particular, we have found that the deletion of these C-terminal residues induces a shift of the pH region where autocatalytic secondary processes dominate the kinetics of AS aggregation towards higher pH values, from AS wild-type (pH 3.6-5.6) to AS(1-119) (pH 4.2-7.0) and AS(1-103) (pH 5.6-8.0). In addition, we found that both truncated variants formed protofibrils in the presence of lipid vesicles, but only those formed by AS(1-103) had the capacity to convert readily into mature fibrils. These results suggest that electrostatics play an important role in secondary nucleation, a key factor in aggregate proliferation, and in the conversion of AS fibrils from protofibrils to mature fibrils. In particular, our results demonstrate that sequence truncations of AS can shift the pH range where autocatalytic proliferation of fibrils is possible into the neutral, physiological regime, thus providing an explanation of the increased propensity of the C-truncated variants to aggregate in vivo.

The amyloid fibril formation by α -synuclein is a hallmark of various neurodegenerative disorders, most notably Parkinson's disease. Epigallocatechin gallate (EGCG) has been reported to be an efficient inhibitor of amyloid formation by numerous proteins, among them α -synuclein. Here, we show that this applies only to a small region of the relevant parameter space, in particular to solution conditions where EGCG readily oxidizes, and we find that the oxidation product is a much more potent inhibitor compared to the unmodified EGCG. In addition to its inhibitory effects, EGCG and its oxidation products can under some conditions even accelerate α -synuclein amyloid fibril formation through facilitating its heterogeneous primary nucleation. Furthermore, we show through quantitative seeding experiments that, contrary to previous reports, EGCG is not able to re-model α -synuclein amyloid fibrils into seeding-incompetent structures. Taken together, our results paint a complex picture of EGCG as a compound that can under some conditions inhibit the amyloid fibril formation of α -synuclein, but the inhibitory action is not robust against various physiologically relevant changes in experimental conditions. Our results are important for the development of strategies to identify and characterize promising amyloid inhibitors.

Many proteins have the potential to aggregate into amyloid fibrils, protein polymers associated with a wide range of human disorders such as Alzheimer’s and Parkinson’s disease. The thermodynamic stability of amyloid fibrils, in contrast to that of folded proteins, is not well understood: the balance between entropic and enthalpic terms, including the chain entropy and the hydrophobic effect, are poorly characterised. Using a combination of theory, in vitro experiments, simulations of a coarse-grained protein model and meta-data analysis, we delineate the enthalpic and entropic contributions that dominate amyloid fibril elongation. Our prediction of a characteristic temperature-dependent enthalpic signature is confirmed by the performed calorimetric experiments and a meta-analysis over published data. From these results we are able to define the necessary conditions to observe cold denaturation of amyloid fibrils. Overall, we show that amyloid fibril elongation is associated with a negative heat capacity, the magnitude of which correlates closely with the hydrophobic surface area that is buried upon fibril formation, highlighting the importance of hydrophobicity for fibril stability., Author summary Most proteins fold in the cell into stable, compact structures. Nevertheless, many proteins also have the ability to stick together, forming long fibrillar structures that are associated with a wide range of human disorders including Alzheimer’s and Parkinson’s disease. The exact nature of the amyloid-causing stickiness is not well understood, nevertheless amyloid fibrils show some very specific thermodynamic characteristics. Some fibrils even destabilise at low temperatures. In this work we translate hydrophobic theory previously used to model protein folding to fibril formation. We combine this theory with experimental measurements, simulations and meta-data analysis of different types of fibrils. This allowed us to unravel the nature of the stickiness in amyloid fibrils by observing the effect of temperature changes, specifically at low temperatures, on hydrophobicity.

α-synuclein (α-syn) is a natively unfolded protein predominantly localized in the presynaptic terminals of neurons. It has been shown that α-syn fibrils are the major component of abnormal neuronal aggregates known as Lewy bodies, the characteristic hallmark of Parkinson’s disease. Amyloid fibrils arise through primary nucleation from monomers, which in the case of α-syn is accelerated by suitable surfaces with an affinity for the protein, followed by the elongation of the nuclei by monomer addition. Secondary nucleation, on the other hand, corresponds to the formation of new fibrils when it is facilitated by pre-existing fibrils. Whileα-synuclein (α-syn) is a natively unfolded protein predominantly localized in the presynaptic terminals of neurons. It has been shown that α-syn fibrils are the major component of abnormal neuronal aggregates known as Lewy bodies, the characteristic hallmark of Parkinson’s disease. Amyloid fibrils arise through primary nucleation from monomers, which in the case of α-syn is accelerated by suitable surfaces with an affinity for the protein, followed by the elongation of the nuclei by monomer addition. Secondary nucleation, on the other hand, corresponds to the formation of new fibrils when it is facilitated by pre-existing fibrils. While it is well-established that the newly added monomer in the process of fibril elongation adopts the conformation of the monomers in the seed, often called templating, it is still unclear under which conditions fibrils formed through secondary nucleation of monomers on the surface of fibrils copy the structure of the parent. Here we show by biochemical and microscopical methods that the secondary nucleation of α-syn, enabled at mildly acidic pH, leads to fibrils that structurally resemble more closely those formed de novo under the same conditions, rather than the seeds if these are formed under different solution condition. This result has important implications for the mechanistic understanding of the secondary nucleation of amyloid fibrils and its role in the propagation of aggregate pathology in protein misfolding diseases.

In light chain (LC) diseases, monoclonal immunoglobulin LCs are abundantly produced with the consequence in some cases to form deposits of a fibrillar or amorphous nature affecting various organs, such as heart and kidney. The factors that determine the solubility of any given LC in vivo are still not well understood. We hypothesize that some of the biochemical properties of the LCs that have been shown to correlate with amyloid fibril formation in patients also can be used as predictors for the degree of kidney damage in a patient group that is only biased by protein availability. We performed detailed biochemical and biophysical investigations of light chains extracted and purified from the urine of a group of 20 patients with light chain disease. For all samples that contained a sufficiently high concentration of LC, we quantified the unfolding temperature of the LCs, the monomer-dimer distribution, the digestibility by trypsin and the formation of amyloid fibrils under various conditions of pH and reducing agent. We correlated the results of our biophysical and biochemical experiments with the degree of kidney damage in the patient group and found that most of these parameters do not correlate with kidney damage as defined by clinical parameters. However, the patients with the greatest impairment of kidney function have light chains which display very poor digestibility by trypsin. Most of the LC properties reported before to be predictors of amyloid formation cannot be used to assess the degree of kidney damage. Our finding that poor trypsin digestibility correlates with kidney damage warrants further investigation in order to probe a putative mechanistic link between these factors.

Amyloid fibrils are homo-molecular protein polymers that play an important role in disease and biological function. While much is known about their kinetics and mechanisms of formation, the origin and magnitude of their thermodynamic stability has received significantly less attention. This is despite the fact that the thermodynamic stability of amyloid fibrils is an important determinant of their lifetimes and processing in vivo. Here we use depolymerization by chemical denaturants of amyloid fibrils of two different proteins (PI3K-SH3 and glucagon) at different concentrations and show that the previously applied linear polymerization model is an oversimplification that does not capture the concentration dependence of chemical depolymerization of amyloid fibrils. We show that cooperative polymerization, which is compatible with the picture of amyloid formation as a nucleated polymerization process, is able to quantitatively describe the thermodynamic data. We use this combined experimental and conceptual framework in order to probe the ionic strengthdependence of amyloid fibril stability. In combination with previously published data on the ionic strength dependence of amyloid fibril growth kinetics, our results provide strong evidence for the product-like nature of the transition state of amyloid fibril growth.

The aggregation into amyloid fibrils of amyloid-β (Aβ) peptides is a hallmark of Alzheimer's disease. A variety of Aβ peptides have been discovered in vivo, with pyroglutamate-modified Aβ (pEAβ) forming a significant proportion. pEAβ is mainly localized in the core of plaques, suggesting a possible role in inducing and facilitating Aβ oligomerization and accumulation. Despite this potential importance, the aggregation mechanism of pEAβ and its influence on the aggregation kinetics of other Aβ variants have not yet been elucidated. Here we show that pEAβ(3-42) forms fibrils much faster than Aβ(1-42) and the critical concentration above which aggregation was observed was drastically decreased by one order of magnitude compared to Aβ(1-42). We elucidated the co-aggregation mechanism of Aβ(1-42) with pEAβ(3-42). At concentrations at which both species do not aggregate as homofibrils, mixtures of pEAβ(3-42) and Aβ(1-42) aggregate, suggesting the formation of mixed nuclei. We show that the presence of pEAβ(3-42) monomers increases the rate of primary nucleation of Aβ(1-42) and that fibrils of pEAβ(3-42) serve as highly efficient templates for elongation and catalytic surfaces for secondary nucleation of Aβ(1-42). On the other hand, the addition of Aβ(1-42) monomers drastically decelerates the primary and secondary nucleation of pEAβ(3-42) while not altering the pEAβ(3-42) elongation rate. In addition, even moderate concentrations of fibrillar Aβ(1-42) prevent pEAβ(3-42) aggregation, likely due to non-reactive binding of pEAβ(3-42) monomers to the surfaces of Aβ(1-42) fibrils. Thus, pEAβ(3-42) accelerates aggregation of Aβ(1-42) by affecting all individual reaction steps of the aggregation process while Aβ(1-42) dramatically slows down the primary and secondary nucleation of pEAβ(3-42).

Parkinson's disease is a highly debilitating neurodegenerative condition whose pathological hallmark is the presence in nerve cells of proteinacious deposits, known as Lewy bodies, composed primarily of amyloid fibrils of α-synuclein. Several missense mutations in the gene encoding α-synuclein have been associated with familial variants of Parkinson's disease and have been shown to affect the kinetics of the aggregation of the protein. Using a combination of experimental and theoretical approaches, we present a systematic in vitro study of the influence of disease-associated single-point mutations on the individual processes involved in α-synuclein aggregation into amyloid fibrils. We find that lipid-induced fibril production and surface catalyzed fibril amplification are the processes most strongly affected by these mutations and show that familial mutations can induce dramatic changes in the crucial processes thought to be associated with the initiation and spreading of the aggregation of α-synuclein.

The measurement of molecular interactions with pathological protein aggregates, including amyloid fibrils, is of central importance in the context of the development of diagnostic and therapeutic strategies against protein misfolding disorders. Probing such interactions by conventional methods can, however, be challenging because of the supramolecular nature of protein aggregates, their heterogeneity, and their often dynamic nature. Here we demonstrate that direct measurement of diffusion on a microfluidic platform enables the determination of affinity and kinetics data for ligand binding to amyloid fibrils in solution. This method yields rapid binding information from only microlitres of sample, and is therefore a powerful technique for identifying and characterising molecular species with potential therapeutic or diagnostic application.