Polymorphism Of Amyloid Fibrils Research Articles

Exploration of Insulin Amyloid Polymorphism Using Raman Spectroscopy and Imaging

We aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na2SO4) by heating at 60, 70, 80, and 90°C. The intensity ratio of two Raman bands at 1672 and 1657 cm−1 due to antiparallel β-sheet and α-helix structures, respectively, was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the β-sheet structure was higher in the case with salts, especially at a higher temperature with Na2SO4. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond (∼514 cm−1) and the ratio of the tyrosine doublet I850/I826 were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis. Especially, amyloid fibrils with Na2SO4 media formed the gauche-gauche-gauche conformation of disulfide bond at a higher rate, but without any salts, the gauche-gauche-gauche conformation was partially transformed into the gauche-gauche-trans conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three- dimensional structural differences of amyloid polymorphisms. These results demonstrate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.

The Influence of Pathogenic Mutations in α-Synuclein on Biophysical and Structural Characteristics of Amyloid Fibrils.

Proteinaceous deposits of α-synuclein amyloid fibrils are a hallmark of human disorders including Parkinson's disease. The onset of this disease is also associated with five familial mutations of the gene encoding the protein. However, the mechanistic link between single point mutations and the kinetics of aggregation, biophysical properties of the resulting amyloid fibrils, and an increased risk of disease is still elusive. Here, we demonstrate that the disease-associated mutations of α-synuclein generate different amyloid fibril polymorphs compared to the wild type protein. Remarkably, the α-synuclein variants forming amyloid fibrils of a comparable structure, morphology, and heterogeneity show similar microscopic steps defining the aggregation kinetics. These results demonstrate that a single point mutation can significantly alter the distribution of fibrillar polymorphs in α-synuclein, suggesting that differences in the clinical phenotypes of familial Parkinson's disease could be associated with differences in the mechanism of formation and the structural characteristics of the aggregates.

Regulation of Metabolism and Structural Polymorphism of Amyloid Fibrils

Regulation of Metabolism and Structural Polymorphism of Amyloid Fibrils

Concentration-dependent polymorphism of insulin amyloid fibrils.

Protein aggregation into highly structured fibrils has long been associated with several neurodegenerative disorders, such as Alzheimer’s or Parkinson’s disease. Polymorphism of amyloid fibrils increases the complexity of disease mechanisms and may be one of the reasons for the slow progress in drug research. Here we report protein concentration as another factor leading to polymorphism of insulin amyloid fibrils. Moreover, our data suggests that insulin amyloid conformation can self-replicate only via elongation, while seed-induced nucleation will lead to environment-defined conformation of fibrils. As similar observations were already described for a couple of other amyloid proteins, we suggest it to be a generic mechanism for self-replication of different amyloid fibril conformations.

Amyloid Fibril Design: Limiting Structural Polymorphism in Alzheimer's Aβ Protofilaments.

Nanoscale fibrils formed by amyloid peptides have a polymorphic character, adopting several types of molecular structures in similar growth conditions. As shown by experimental (e.g., solid-state NMR) and computational studies, amyloid fibril polymorphism hinders both the structural characterization of Alzheimer's Aβ amyloid protofilaments and fibrils at a molecular level, as well as the possible applications (e.g., development of drugs or biomarkers) that rely on similar, controlled molecular arrangements of the Aβ peptides in amyloid fibril structures. We have explored the use of several contact potentials for the efficient identification of minimal sequence mutations that could enhance the stability of specific fibril structures while simultaneously destabilizing competing topologies, controlling thus the amount of structural polymorphism in a rational way. We found that different types of contact potentials, while having only partial accuracy on their own, lead to similar results regarding ranking the compatibility of wild-type (WT) and mutated amyloid sequences with different fibril morphologies. This approach allows exhaustive screening and assessment of possible mutations and the identification of minimal consensus mutations that could stabilize fibrils with the desired topology at the expense of other topology types, a prediction that is further validated using atomistic molecular dynamics with explicit water molecules. We apply this two-step multiscale (i.e., residue and atomistic-level) approach to predict and validate mutations that could bias either parallel or antiparallel packing in the core Alzheimer's Aβ9-40 amyloid fibril models based on solid-state NMR experiments. Besides shedding new light on the molecular origins of structural polymorphism in WT Aβ fibrils, our study could also lead to efficient tools for assisting future experimental approaches for amyloid fibril determination, and for the development of biomarkers or drugs aimed at interfering with the stability of amyloid fibrils, as well as for the future design of amyloid fibrils with a controlled (e.g., reduced) level of structural polymorphism.

Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T

In this work, α-synuclein amyloid fibrils—the formation of which is a biomarker of Parkinson’s disease—were investigated using the fluorescent probe thioflavin T (ThT). The experimental conditions of protein fibrillogenesis were chosen so that a sufficient number of continuous measurements could be performed to characterize and analyze all stages of this process. The reproducibility of fibrillogenesis and the structure of the obtained aggregates (which is a critical point for further investigation) were proven using a wide range of physical-chemical methods. For the determination of ThT-α-synuclein amyloid fibril binding parameters, the sample and reference solutions were prepared using equilibrium microdialysis. By utilizing absorption spectroscopy of these solutions, the ThT-fibrils binding mode with a binding constant of about 104 M−1 and stoichiometry of ThT per protein molecule of about 1:8 was observed. Fluorescence spectroscopy of the same solutions with the subsequent correction of the recorded fluorescence intensity on the primary inner filter effect allowed us to determine another mode of ThT binding to fibrils, with a binding constant of about 106 M−1 and stoichiometry of about 1:2500. Analysis of the photophysical characteristics of the dye molecules bound to the sites of different binding modes allowed us to assume the possible localization of these sites. The obtained differences in the ThT binding parameters to the amyloid fibrils formed from α-synuclein and other amyloidogenic proteins, as well as in the photophysical characteristics of the bound dye, confirmed the hypothesis of amyloid fibril polymorphism.

Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils

Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quantitatively in vitro, specifically probing the fibrillar cross-β-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT–amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphology on ThT fluorescence and ThT binding sites, with two morphologically distinct but chemically identical α-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their β-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound ThT reveal the presence of at least two qualitatively different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphology dependent, thus determining the observed polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphology in ThT-based amyloid studies, underline the relevance of polymorphs in ThT–amyloid interaction and can explain the variability often observed in ThT amyloid binding assays.

Amyloid fibril polymorphism: a challenge for molecular imaging and therapy.

The accumulation of misfolded proteins (MPs), both unique and common, for different diseases is central for many chronic degenerative diseases. In certain patients, MP accumulation is systemic (e.g. TTR amyloid), and in others, this is localized to a specific cell type (e.g. Alzheimer's disease). In neurodegenerative diseases, NDs, it is noticeable that the accumulation of MP progressively spreads throughout the nervous system. Our main hypothesis of this article is that MPs are not only markers but also active carriers of pathogenicity. Here, we discuss studies from comprehensive molecular approaches aimed at understanding MP conformational variations (polymorphism) and their bearing on spreading of MPs, MP toxicity, as well as MP targeting in imaging and therapy. Neurodegenerative disease (ND) represents a major and growing societal challenge, with millions of people worldwide suffering from Alzheimer's or Parkinson's diseases alone. For all NDs, current treatment is palliative without addressing the primary cause and is not curative. Over recent years, particularly the shape-shifting properties of misfolded proteins and their spreading pathways have been intensively researched. The difficulty in addressing ND has prompted most major pharma companies to severely downsize their nervous system disorder research. Increased academic research is pivotal for filling this void and to translate basic research into tools for medical professionals. Recent discoveries of targeting drug design against MPs and improved model systems to study structure, pathology spreading and toxicity strongly encourage future studies along these lines to provide an opportunity for selective imaging, prognostic diagnosis and therapy.

Physical basis of amyloid fibril polymorphism

Polymorphism is a key feature of amyloid fibril structures but it remains challenging to explain these variations for a particular sample. Here, we report electron cryomicroscopy-based reconstructions from different fibril morphologies formed by a peptide fragment from an amyloidogenic immunoglobulin light chain. The observed fibril morphologies vary in the number and cross-sectional arrangement of a structurally conserved building block. A comparison with the theoretically possible constellations reveals the experimentally observed spectrum of fibril morphologies to be governed by opposing sets of forces that primarily arise from the β-sheet twist, as well as peptide–peptide interactions within the fibril cross-section. Our results provide a framework for rationalizing and predicting the structure and polymorphism of cross-β fibrils, and suggest that a small number of physical parameters control the observed fibril architectures.

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Insulin and Lispro Insulin: What is Common and Different in their Behavior?

There are different insulin analogues with various pharmacokinetic characteristics, such as, rapid-acting, long-acting, or intermediate-acting analogues. Since insulin tends to form amyloid aggregates, it is of particular interest to measure characteristic times of formation of amyloid aggregates and compare those to action times for insulin and its analogues. For the study we have chosen one of the insulin analogues - insulin Lispro, which is a fast acting insulin analog. It is usually thought of amyloid aggregation as a nucleation-dependent process. We have estimated the size of the primary nucleus to be one monomer and the size of the secondary nucleus to be around zero in both insulin and Lispro insulin aggregation processes. The main structural element of insulin and Lispro insulin amyloid fibrils is a rounded ring oligomer of about 6-7 nm in diameter, about 2-3 nm in height and about 2 nm in diameter of the hole. Fibrils of several μm in length are produced due to interaction of such oligomers. The packing of ring oligomers in fibrils differs because of the difference in their orderliness. Though the initial stages of fibril formation (monomer, oligomer) are similar, the further process depends on the unique sequence of each peptide. Namely the sequence affects the final morphology of mature amyloids. These observations allow us to conclude that formation of fibrils by short peptides occurs via and by means of oligomer ring structures. Such an important issue as the nature of polymorphism of insulin amyloid fibrils has been settled by us. The role of early oligomeric aggregates in such processes as nucleation and aggregation of amyloid fibrils has been examined.

Structural Characterization of Fibrils from Recombinant Human Islet Amyloid Polypeptide by Solid-State NMR: The Central FGAILS Segment Is Part of the β-Sheet Core.

Amyloid deposits formed from islet amyloid polypeptide (IAPP) are a hallmark of type 2 diabetes mellitus and are known to be cytotoxic to pancreatic β-cells. The molecular structure of the fibrillar form of IAPP is subject of intense research, and to date, different models exist. We present results of solid-state NMR experiments on fibrils of recombinantly expressed and uniformly 13C, 15N-labeled human IAPP in the non-amidated, free acid form. Complete sequential resonance assignments and resulting constraints on secondary structure are shown. A single set of chemical shifts is found for most residues, which is indicative of a high degree of homogeneity. The core region comprises three to four β-sheets. We find that the central 23-FGAILS-28 segment, which is of critical importance for amyloid formation, is part of the core region and forms a β-strand in our sample preparation. The eight N-terminal amino acid residues of IAPP, forming a ring-like structure due to a disulfide bridge between residues C2 and C7, appear to be well defined but with an increased degree of flexibility. This study supports the elucidation of the structural basis of IAPP amyloid formation and highlights the extent of amyloid fibril polymorphism.

Multimodal Spectroscopic Study of Amyloid Fibril Polymorphism.

Amyloid fibrils are a large class of self-assembled protein aggregates that are formed from unstructured peptides and unfolded proteins. The fibrils are characterized by a universal β-sheet core stabilized by hydrogen bonds, but the molecular structure of the peptide subunits exposed on the fibril surface is variable. Here we show that multimodal spectroscopy using a range of bulk- and surface-sensitive techniques provides a powerful way to dissect variations in the molecular structure of polymorphic amyloid fibrils. As a model system, we use fibrils formed by the milk protein β-lactoglobulin, whose morphology can be tuned by varying the protein concentration during formation. We investigate the differences in the molecular structure and composition between long, straight fibrils versus short, wormlike fibrils. We show using mass spectrometry that the peptide composition of the two fibril types is similar. The overall molecular structure of the fibrils probed with various bulk-sensitive spectroscopic techniques shows a dominant contribution of the β-sheet core but no difference in structure between straight and wormlike fibrils. However, when probing specifically the surface of the fibrils with nanometer resolution using tip-enhanced Raman spectroscopy (TERS), we find that both fibril types exhibit a heterogeneous surface structure with mainly unordered or α-helical structures and that the surface of long, straight fibrils contains markedly more β-sheet structure than the surface of short, wormlike fibrils. This finding is consistent with previous surface-specific vibrational sum-frequency generation (VSFG) spectroscopic results ( VandenAkker et al. J. Am. Chem. Soc. , 2011 , 133 , 18030 - 18033 , DOI: 10.1021/ja206513r ). In conclusion, only advanced vibrational spectroscopic techniques sensitive to surface structure such as TERS and VSFG are able to reveal the difference in structure that underlies the distinct morphology and rigidity of different amyloid fibril polymorphs that have been observed for a large range of food and disease-related proteins.

Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies

Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid aggregates, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid aggregates, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified the morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (β-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have β-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of β-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface.

Physical basis for the ofloxacin-induced acceleration of lysozyme aggregation and polymorphism in amyloid fibrils

Aggregation of globular proteins is an intractable problem which generally originates from partially folded structures. The partially folded structures first collapse non-specifically and then reorganize into amyloid-like fibrils via one or more oligomeric intermediates. The fibrils and their on/off pathway intermediates may be toxic to cells and form toxic deposits in different human organs. To understand the basis of origins of the aggregation diseases, it is vital to study in details the conformational properties of the amyloidogenic partially folded structures of the protein. In this work, we examined the effects of ofloxacin, a synthetic fluoroquinolone compound on the fibrillar aggregation of hen egg-white lysozyme. Using two aggregation conditions (4M GuHCl at pH 7.0 and 37 °C; and pH 1.7 at 65 °C) and a number of biophysical techniques, we illustrate that ofloxacin accelerates fibril formation of lysozyme by binding to partially folded structures and modulating their secondary, tertiary structures and surface hydrophobicity. We also demonstrate that Ofloxacin-induced fibrils show polymorphism of morphology, tinctorial properties and hydrophobic surface exposure. This study will assist in understanding the determinant of fibril formation and it also indicates that caution should be exercised in the use of ofloxacin in patients susceptible to various aggregation diseases.

Rapid Filament Supramolecular Chirality Reversal of HET-s (218-289) Prion Fibrils Driven by pH Elevation.

Amyloid fibril polymorphism is not well understood despite its potential importance for biological activity and associated toxicity. Controlling the polymorphism of mature fibrils including their morphology and supramolecular chirality by postfibrillation changes in the local environment is the subject of this study. Specifically, the effect of pH on the stability and dynamics of HET-s (218-289) prion fibrils has been determined through the use of vibrational circular dichroism (VCD), deep UV resonance Raman, and fluorescence spectroscopies. It was found that a change in solution pH causes deprotonation of Asp and Glu amino acid residues on the surface of HET-s (218-289) prion fibrils and triggers rapid transformation of one supramolecular chiral polymorph into another. This process involves changes in higher order arrangements like lateral filament and fibril association and their supramolecular chirality, while the fibril cross-β core remains intact. This work suggests a hypothetical mechanism for HET-s (218-289) prion fibril refolding and proposes that the interconversion between fibril polymorphs driven by the solution environment change is a general property of amyloid fibrils.

Exploring the structure and formation mechanism of amyloid fibrils by Raman spectroscopy: a review.

Amyloid fibrils are β-sheet rich protein aggregates that are strongly associated with various neurodegenerative diseases. Raman spectroscopy has been broadly utilized to investigate protein aggregation and amyloid fibril formation and has been shown to be capable of revealing changes in secondary and tertiary structures at all stages of fibrillation. When coupled with atomic force (AFM) and scanning electron (SEM) microscopies, Raman spectroscopy becomes a powerful spectroscopic approach that can investigate the structural organization of amyloid fibril polymorphs. In this review, we discuss the applications of Raman spectroscopy, a unique, label-free and non-destructive technique for the structural characterization of amyloidogenic proteins, prefibrilar oligomers, and mature fibrils.

Physical and structural basis for polymorphism in amyloid fibrils.

As our understanding of the molecular structures of amyloid fibrils has matured over the past 15 years, it has become clear that, while amyloid fibrils do have well-defined molecular structures, their molecular structures are not uniquely determined by the amino acid sequences of their constituent peptides and proteins. Self-propagating molecular-level polymorphism is a common phenomenon. This article reviews current information about amyloid fibril structures, variations in molecular structures that underlie amyloid polymorphism, and physical considerations that explain the development and persistence of amyloid polymorphism. Much of this information has been obtained through solid state nuclear magnetic resonance measurements. The biological significance of amyloid polymorphism is also discussed briefly. Although this article focuses primarily on studies of fibrils formed by amyloid-β peptides, the same principles apply to many amyloid-forming peptides and proteins.

The mechanical response of hIAPP nanowires based on different bending direction simulations.

Amyloid proteins, implicated in numerous aging-related diseases, possess remarkable mechanical properties. Polymorphism leads to different arrangements of β sheets in amyloid fibrils, which changes the characteristics of the hydrogen bond network that determines their mechanical properties and structural characteristics. We performed bending simulations using molecular dynamics methods under constant-velocity conditions in different bending directions. Two different fibril structures, parallel/homo and parallel/hetero, of hIAPP amyloids were considered. Though the bending configuration influences the toughness of the material, our results indicate that the basic material behavior is affected by the β-sheet arrangement that is determined by the type of polymorphism in amyloid fibrils.