Amyloid Protofibril Research Articles

Destabilization potential of beta sheet breaker peptides on Abeta fibril structure: an insight from molecular dynamics simulation study.

Alzheimer's disease is characterized by amyloid-β aggregation. Currently, all the approved medications are to treat the symptoms but there is no clinically approved treatment for the cure or to prevent the progression of Alzheimer's disease (AD). Earlier reports suggest the use of small molecules and peptides to target and destabilize the amyloid fibril. The use of Beta Sheet Breaker (BSB) peptides seems to be a promising and attractive therapeutic approach as it can strongly bind and destabilize the preformed amyloid fibril. There are experimental studies describing the destabilization role of various BSB peptides, but the exact mechanism remains elusive. In the current work, an attempt is made to study the destabilization mechanism of different BSB peptides on preformed amyloid protofibril using molecular docking and simulations. Molecular docking of eight different BSB peptides of varying length (5-mer to 10-mer) on the Abeta protofibril was done. Docking was followed by multiple sets of molecular simulations for the Abeta protofibril–BSB peptide complex for each of the top ranked poses of the eight BSB peptides. As a control, multiple sets of simulations for the Abeta protofibril (APO) were also carried out. An increase in the RMSD, decrease in the number of interchain hydrogen bonds, destabilization of important salt bridge interactions (D23–K28), and destabilization of interchain hydrophobic interactions suggested the destabilization of Abeta protofibril by BSB peptides. The MM-GBSA free energy of binding for each of the BSB peptides was calculated to measure the binding affinity of BSB peptides to Abeta protofibril. Further residue wise contribution of free energy of binding was also calculated. The study showed that 7-mer peptides tend to bind strongly to Abeta protofibril as compared to other BSB peptides. The KKLVFFA peptide showed better destabilization potential as compared to the other BSB peptides. The details about the destabilization mechanism of BSB peptides will help in the design of other peptides for the therapeutic intervention for AD.

Molecular dynamics simulations of amyloid-β(16-22) peptide aggregation at air-water interfaces.

Oligomers of amyloid-β (Aβ) peptides are known to be related to Alzheimer's disease, and their formation is accelerated at hydrophilic-hydrophobic interfaces, such as the cell membrane surface and air-water interface. Here, we report molecular dynamics simulations of aggregation of Aβ(16-22) peptides at air-water interfaces. First, 100 randomly distributed Aβ(16-22) peptides moved to the interface. The high concentration of peptides then accelerated their aggregation and formation of antiparallel β-sheets. Two layers of oligomers were observed near the interface. In the first layer from the interface, the oligomer with less β-bridges exposed the hydrophobic residues to the air. The second layer consisted of oligomers with more β-bridges that protruded into water. They are more soluble in water because the hydrophobic residues are covered by N- and C-terminal hydrophilic residues that are aligned well along the oligomer edge. These results indicate that amyloid protofibril formation mainly occurs in the second layer.

Computational Study on Structure and Aggregation Pathway of Aβ42 Amyloid Protofibril.

Amyloid deposits of Aβ protein in neuronal cells are known to be a major symptom of Alzheimer's disease. In particular, Aβ42 shows relatively high toxicity among the different Aβ isoforms, and its toxicity is thought to be because of its structural features. Recent ssNMR and cryo-EM experiments identified that Aβ42 shows an S-shaped triple-β structure, in contrast to the previously suggested U-shaped β-arch structure. In order to associate the high toxicity of Aβ42 with its structural features, it is essential to explain the conformational stability and aggregation mechanisms of this triple-β motif. We utilized several different simulation methods, including extensive straight molecular dynamics simulation, steered molecular dynamics simulation, and replica-exchange molecular dynamics simulation. The S-shaped triple-β motif showed remarkable structural stability because of its complex residual interactions that form stable hydrophobic cores. The triple-β structure of Aβ42 is primarily made up of three β-sheet regions and two hydrophobic cores formed between β-sheet regions. Our analysis of β-sheet rupture patterns between adjacent chains showed that its two hydrophobic cores have different degrees of stability, indicating a lock phase mechanism. Our analysis of the docking pathway of monomeric Aβ42 to the fibril motif using REMD simulations showed that each of the three β-sheet sequences plays a distinct role in the docking process by changing their conformational features. Our results provide an understanding for the stability and consequent high toxicity of the triple-β structure Aβ42.

Conformation-specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen.

We engineered and employed a chaperone‐like amyloid‐binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross‐reacted with amyloid beta‐peptide (Aβ42) protofibrils, but not with Aβ40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1‐hIAPP complex cross‐react with Aβ42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation‐specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation‐sensitive and sequence‐independent and can target more than one type of protofibril species.

高圧力電気泳動法を用いたタンパク質解離会合系の定量的解析

High-pressure electrophoresis was designed to investigate the association system of proteins quantitatively. In this methodology, pressure and temperature precisely perturbs the association equilibrium of supermolecular protein assembly, whose decomposed fractions can be in-situ analyzed by native polyacrylamide gel electrophoresis (PAGE). The bands of these fractions allow us not only to unravel the elementary process of assembly formation but also to quantify various volumetric properties including thermal expansion change of each process. In this article, we report its application to the problem of amyloid protofibril formation.

Multistep Modeling Strategy To Improve the Binding Affinity Prediction of PET Tracers to Aβ42: Case Study with Styrylbenzoxazole Derivatives

Positron emission tomography (PET) tracers play an important role in the diagnosis of Alzheimer's disease, a condition that leads to progressive dementia and memory loss. A high binding affinity and specificity of the PET tracers to amyloid oligomers and fibrils are crucial for their successful application as diagnostic agents. In this sense, it is essential to design PET tracers with enhanced binding affinities, which can lead to more precise and earlier detection of Alzheimer's disease conditions. The application of in silico methodology for the design and development of efficient PET tracers may serve as an important route to improved Alzheimer's disease diagnosis. In this work, the performance of widely used computational methods is explored for predicting experimental binding affinities of styrylbenzoxazole (SB) derivatives against a common amyloid protofibril. By performing docking, molecular dynamics, and quantum chemistry calculations in sequence their combined predictive performance is explored. The present work emphasizes the merits as well as limitations of these simulation strategies in the realm of designing PET tracers for Alzheimer's disease diagnosis.

Influence of Aromatic Residues on the Material Characteristics of Aβ Amyloid Protofibrils at the Atomic Scale.

Amyloid fibrils, which cause a number of degenerative diseases, are insoluble under physiological conditions and are supported by native contacts. Recently, the effects of the aromatic residues on the Aβ amyloid protofibril were investigated in a ThT fluorescence study. However, the relationship between the material characteristics of the Aβ protofibril and its aromatic residues has not yet been investigated on the atomic scale. Here, we successfully constructed wild-type (WT) and mutated types of Aβ protofibrils by using molecular dynamics simulations. Through principle component analysis, we established the structural stability and vibrational characteristics of F20L Aβ protofibrils and compared them with WT and other mutated models such as F19L and F19LF20L. In addition, structural stability was assessed by calculating the elastic modulus, which showed that the F20L model has higher values than the other models studied. From our results, it is shown that aromatic residues influence the structural and material characteristics of Aβ protofibrils.

Negative thermal expansibility change for dissociation of lysozyme variant amyloid protofibril.

A disulfide-deficient variant of hen lysozyme, 0SS, is known to form an amyloid protofibril spontaneously, and to dissociate into monomers at high hydrostatic pressure. We carried out native PAGE at various temperatures (20-35°C) and pressures (0.1-200 MPa), to characterize the dissociation equilibrium of disulfide-deficient variant of hen lysozyme amyloid protofibril. Based on the density profiles, the partial molar volume and thermal expansibility changes for dissociation, ΔvD and ΔeD , were obtained to be -74 cm(3) /mol at 25°C and -2.3 cm(3) mol(-1) K(-1) , respectively. The dissociation of amyloid fibril destroys the cross β-structure, and such conformational destruction in native protein fold rarely accompanies negative thermal expansibility change. We discussed the negative thermal expansibility change in terms of hydration and structural packing of the amyloid protofibril.

How to Determine the Size of Folding Nuclei of Protofibrils from the Concentration Dependence of the Rate and Lag-Time of Aggregation. I. Modeling the Amyloid Protofibril Formation

The question about the size of nuclei of formation of protofibrils (which constitute mature amyloid fibrils) formed by different proteins and peptides is yet open and debatable because of the absence of solid knowledge of underlying mechanisms of amyloid formation. In this work, a kinetic model of the process of formation of amyloid protofibrils is suggested, which allows calculation of the size of the nuclei using only kinetic data. In addition to the stage of primary nucleation, which is believed to be present in many protein aggregation processes, the given model includes both linear growth of protofibrils (proceeding only at the cost of attaching of monomers to the ends) and exponential growth of protofibrils at the cost of growth from the surface, branching, and fragmentation with the secondary nuclei. Theoretically, only the exponential growth is compatible with the existence of a pronounced lag-period (which can take much more time then the growth of aggregates themselves). The obtained analytical solution allows us to determine the size of the primary and secondary nuclei from the experimentally obtained concentration dependences of the time of growth and the new parameter-the ratio Lrel of the lag-time duration to the time of growth of amyloid protofibrils.

Solvent-Induced Tuning of Internal Structure in a Protein Amyloid Protofibril

An important goal in studies of protein aggregation is to obtain an understanding of the structural diversity that is characteristic of amyloid fibril and protofibril structures at the molecular level. In this study, what to our knowledge are novel assays based on time-resolved fluorescence anisotropy decay and dynamic quenching measurements of a fluorophore placed at different specific locations in the primary structure of a small protein, barstar, have been used to determine the extent to which the protein sequence participates in the structural core of protofibrils. The fluorescence measurements reveal the structural basis of how modulating solvent polarity results in the tuning of the protofibril conformation from a pair of parallel β-sheets in heat-induced protofibrils to a single parallel β-sheet in trifluorethanol-induced protofibrils. In trifluorethanol-induced protofibrils, the single β-sheet is shown to be built up from in-register β-strands formed by nearly the entire protein sequence, while in heat-induced protofibrils, the pair of β-sheets motif is built up from β-strands formed by only the last two-third of the protein sequence.

Fluoroalcohol-Induced Modulation of the Pathway of Amyloid Protofibril Formation by Barstar

To understand how the conformational heterogeneity of protofibrils formed by any protein, as well as the mechanisms of their formation, are modulated by a change in aggregation conditions, we studied the formation of amyloid protofibrils by barstar at low pH by multiple structural probes in the presence of hexafluoroisopropanol (HFIP). In the presence of 10% HFIP, aggregation proceeds with the transient formation of spherical oligomers and leads to the formation of both protofibrils and fibrils. Curly short protofibrils and fibrils are seen to form early during the aggregation reaction, and both are seen to grow gradually in length during the course of the reaction. Atomic force microscopy images reveal that the HFIP-induced protofibrils are long (∼300 nm in length), curly, and beaded and appear to be composed primarily of β-sheet bilayers, with heights of ∼2.4 nm. The protofibrils formed in the presence of HFIP differ in both their structures and their stabilities from the protofibrils formed either in the absence of alcohol or in the presence of a related alcohol, trifluoroethanol (TFE). Aggregation appears to proceed via an isodesmic polymerization mechanism. Internal structure in the growing aggregates changes in two stages during protofibril formation. In the first stage, an α-helix-rich oligomeric intermediate is formed. In the second stage, the level of β-sheet structure increases at the expense of some α-helical structure. The second stage itself appears to occur in two distinct steps. The creation of thioflavin T binding sites occurs concomitantly with aggregate elongation and is seen to precede the change in secondary structure. The long straight fibrils with characteristic heights of 8-10 nm, which form in the course of the HFIP-induced aggregation reaction, have not been observed to form either in the absence of alcohol or in the presence of TFE.

1P052 高静水圧電気泳動法によるリゾチーム変異体アミロイド原繊維の解離平衡の研究(蛋白質-物性(安定性,折れたたみなど),第48回日本生物物理学会年会)

1P052 高静水圧電気泳動法によるリゾチーム変異体アミロイド原繊維の解離平衡の研究(蛋白質-物性(安定性,折れたたみなど),第48回日本生物物理学会年会)

Structurally Distinct Amyloid Protofibrils Form on Separate Pathways of Aggregation of a Small Protein

Understanding the structural as well as mechanistic basis of the conformational polymorphism evident during amyloid protofibril and fibril formation by proteins is an important goal in the study of protein aggregation. In this report, we compare two separate routes to amyloid protofibril formation by the small protein barstar, one induced by the addition of trifluoroethanol (TFE) and the other by heat. The study reveals that the TFE-induced aggregation of barstar leads to protofibrils that differ from heat-induced protofibrils in their external dimensions and internal structures as well as in the mechanisms of their formation. Atomic force microscopy reveals that the TFE-induced protofibrils have about half the thickness of the heat-induced protofibrils. The thickness of the TFE-induced protofibrils (1.14 +/- 0.24) suggests that they form a beta-sheet monolayer, while the thickness of the heat-induced protofibrils (2.56 +/- 0.32) suggests that they are built up from a pair (bilayer) of beta-sheets. Fourier-transform infrared (FTIR) as well as circular dichroism (CD) spectroscopy shows that the heat-induced protofibrils are not pure beta-sheet structures but that they also contain other structures (alpha-helix and/or random coil). In contrast, the TFE-induced protofibrils contain more beta-sheet structures and less of other structures, if any. The FTIR and CD spectra also reveal that the two differently created protofibrils differ in the internal structures of their beta-sheets. The TFE-induced protofibrils differ from the heat-induced protofibrils also in the kinetics of their formation. For the heat-induced reaction, the kinetics are monophasic without any lag phase, while the kinetics of the formation of TFE-induced protofibrils are sigmoidal with an initial lag phase. It appears that the TFE-induced and the heat-induced reactions involve distinct pathways for the formation of amyloid protofibrils. The existence of alternative pathways leading to amyloid protofibrils of distinct structures has important implications in understanding the kinetic origin of amyloid polymorphism.

Conformational Conversion May Precede or Follow Aggregate Elongation on Alternative Pathways of Amyloid Protofibril Formation

A major goal in the study of protein aggregation is to understand how the conformational heterogeneity characteristic of the process leads to structurally distinct amyloid fibrils. The small protein barstar is known to form amyloid protofibrils in multiple steps at low pH: a small oligomer, the A-form, first transforms into a larger spherical higher oligomeric intermediate (HOI), which then self-associates to form the elongated protofibril. To determine how the conformational conversion reaction during aggregation is coupled to the process of protofibril formation, cysteine-scanning mutagenesis was first used to identify specific residue positions in the protein sequence, which are important in defining the nature of the aggregation process. Two classes of mutant proteins, which are distinguished by their kinetics of aggregation at high protein concentration, have been identified: Class I mutant proteins undergo conformational conversion, as measured by an increase in thioflavin T binding ability and an increase in circular dichroism at 216 nm, significantly faster than Class II mutant proteins. At low protein concentration, the rates of conformational conversion are, however, identical for both classes of mutant proteins. At high protein concentration, the two classes of mutant proteins can be further distinguished on the basis of their rates of protofibril growth, as determined from dynamic light-scattering measurements. For Class I mutant proteins, protofibril elongation occurs at the same, or slightly faster, rate than conformational conversion. For Class II mutant proteins, protofibril elongation is significantly slower than conformational conversion. Dynamic light scattering measurements and atomic force microscopy imaging indicate that for the Class I mutant proteins, conformational conversion occurs concurrently with the self-association of prefibrillar HOIs into protofibrils. On the other hand, for the Class II mutant proteins, the prefibrillar HOI first undergoes conformational conversion, and the conformationally converted HOIs then self-associate to form protofibrils. The two classes of mutant proteins appear, therefore, to use structurally distinct pathways to form amyloid protofibrils. On one pathway, conformational conversion occurs along with, or after, elongation of the oligomers; on the other pathway, conformational conversion precedes elongation of the oligomers. Single mutations in the protein can cause aggregation to switch from one pathway to the other. Importantly, the protofibrils formed by the two classes of mutant proteins have significantly different diameters and different internal structures.

Amyloid Protofibril is Highly Voluminous and Compressible

We report here results of the first direct measurement of partial volume and compressibility changes of a protein as it forms an amyloid protofibril. We use a high precision density meter and an ultrasonic velocity meter on a solution of intrinsically denatured, disulfide-deficient variant of hen lysozyme, and follow the time-dependent changes in volume and compressibility, as the protein spontaneously forms a protofibril. We have found a large increase in partial specific volume with time from 0.684 to 0.724 mL x g-1 (Deltanu = 0.040 mL x g-1 corresponding to 570 mL x (mol monomer)-1) and in partial specific adiabatic compressibility coefficient from -7.48 x 10(-12) to +1.35 x 10(-12) cm2 x dyn-1 (Deltabetas = 8.83 x 10(-12) x cm2 x dyn-1) as the monomer transforms into a protofibril. The results demonstrate that the protofibril is a highly voluminous and compressible entity, disclosing a cavity-rich, fluctuating nature for the amyloid protofibril. The volume and compressibility changes occur in two phases, the faster one preceding the major development of the beta-structure in the protofibril as monitored by CD.

Dual Binding Modes of Congo Red to Amyloid Protofibril Surface Observed in Molecular Dynamics Simulations

Congo red has been used to identify amyloid fibrils in tissues for more than 80 years and is also a weak inhibitor to both amyloid-beta fibril formation and toxicity. However, the specificity of the binding and its inhibition mechanism remain unclear. Using all-atom molecular dynamics simulations with the explicit solvent model, we have identified and characterized two specific binding modes of Congo red molecules to a protofibril formed by an amyloidogenic fragment (GNNQQNY) of the yeast prion protein Sup35. The observation of dual-mode was consistent with the experimentally observed dual-mode binding to Abeta fibrils by a series of compounds similar to Congo red. In the primary mode, Congo red bound to a regular groove formed by the first three residues (GNN) of the beta-strands along the beta-sheet extension direction. Comparative simulations demonstrated that Thioflavin T also bound to the grooves on KLVFFAE protofibril surface. Because of the ubiquitous long grooves on the amyloid fibril surface, we propose that this binding interaction could be a general recognition mode of amyloid fibrils by Congo red, Thioflavin T, and other long flat molecules. In the secondary mode, Congo red bound parallel to the beta-strands on the edge or in the middle of a beta-sheet. The primary binding mode of Congo red and GNNQQNY protofibril was more stable than the secondary mode by -5.7 kcal/mol as estimated by the MM-GBSA method. Detailed analysis suggests that the hydrophobic interactions play important roles for burial of the hydrophobic part of the Congo red molecules. Two potential inhibition mechanisms of disrupting beta-sheet stacking were inferred from the primary mode, which could be exploited for the development of non-peptidic amyloid-specific inhibitors.

Kinetic Analysis of Amyloid Protofibril Dissociation and Volumetric Properties of the Transition State

We present here the first detailed kinetic analysis of the dissociation reaction of amyloid protofibrils by utilizing pressure as an accelerator of the reaction. The experiment is carried out on an excessively diluted typical protofibril solution formed from an intrinsically denatured disulfide-deficient variant of hen lysozyme with Trp fluorescence as the reporter in the pressure range 3–400 MPa. From the analysis of the time-dependent fluorescence decay and the length distribution of the protofibrils measured on atomic force microscopy, we conclude that the protofibril grows or decays by attachment or detachment of a monomer at one end of the protofibril with a monomer dissociation rate independent of the length of the fibril. Furthermore, we find that the dissociation reaction is strongly dependent on pressure, characterized with a negative activation volume Δ V o‡ = −50.5 ± 1.60 ml mol −1 at 0.1 MPa and with a negative activation compressibility Δ κ ‡ = −0.013 ± 0.001 ml mol −1 bar −1 or −0.9 × 10 −6 ml g −1 bar −1. These results indicate that the protofibril is a highly compressible high-volume state, but that it becomes less compressible and less voluminous in the transition state, most probably due to partial hydration of the existing voids. The system eventually reaches the lowest-volume state with full hydration of the monomer in the dissociated state.

Folding, Misfolding, and Amyloid Protofibril Formation of WW Domain FBP28

We study the folding mechanism of a triple β-strand WW domain from the Formin binding protein 28 (FBP28) at atomic resolution with explicit water model using replica exchange molecular dynamics computer simulations. Extended sampling over a wide range of temperatures to obtain the free energy, enthalpy, and entropy surfaces as a function of structural coordinates has been performed. Simulations were started from different configurations covering the folded and unfolded states. In the free energy landscape a transition state is identified and its structures and ϕ-values are compared with experimental data from a homologous protein, the prolyl-isomerase Pin1 WW domain. A stable intermediate state is found to accumulate during the simulation characterized by the carboxyl-terminal β-strand 3 having misregistered hydrogen bonds and where the structural heterogeneity is due to nonnative turn II formation. Furthermore, the aggregation behavior of the FBP28 WW domain may be related to one such misfolded structure, which has a much lower free energy of dimer formation than that of the native dimer. Based on the misfolded dimer, aggregation to form protofibril structure is discussed.

Apomyoglobin reveals a random-nucleation mechanism in amyloid protofibril formation

Protofibrils (PFs) represent the earliest fibrillar species that occur in the course of amyloid fibril formation. Using apomyoglobin, we report here that PFs arise from a multi-step reaction and that they are preceded by an ensemble of non-fibrillar particles (NFPs). These intermediate aggregates encompass nascent elements of amyloid structure and can act as seeds in PF formation. Taken together with the observation that PFs often protrude from NFPs, our data suggest that PFs form by a random nucleation mechanism in which the polypeptide chains sample many different aggregated conformations. Once the appropriate structural characteristics are acquired, PFs are formed by addition of further polypeptide chains.

Are amyloid diseases caused by protein aggregates that mimic bacterial pore-forming toxins?

Protein fibrillization is implicated in the pathogenesis of most, if not all, age-associated neurodegenerative diseases, but the mechanism(s) by which it triggers neuronal death is unknown. Reductionist in vitro studies suggest that the amyloid protofibril may be the toxic species and that it may amplify itself by inhibiting proteasome-dependent protein degradation. Although its pathogenic target has not been identified, the properties of the protofibril suggest that neurons could be killed by unregulated membrane permeabilization, possibly by a type of protofibril referred to here as the 'amyloid pore'. The purpose of this review is to summarize the existing supportive circumstantial evidence and to stimulate further studies designed to test the validity of this hypothesis.