Structural characterization and cryo-electron tomography analysis of human islet amyloid polypeptide suggest a synchronous process of the hIAPP1-37 amyloid fibrillation.

Revealing the aggregation and fibrillation process of variant amyloid proteins is critical for understanding the molecular mechanism of related amyloidosis diseases. Here we characterized the fibrillation morphology and kinetics of type 2 diabetes (T2D) related human islet amyloid polypeptide (hIAPP1-37) fibril formation process using negative staining transmission electron microscopy (NS-TEM), cryo-electron microscopy (cryo-EM) analysis, and 3D cryo-electron tomography (cryo-ET) reconstruction, together with circular dichroism (CD) and Thioflavin-T (ThT) assays. Our results showed that various amyloid fibrils can be observed at different time points of hIAPP1-37 fibrillization process, while the winding of protofibrils presents in different growth stages, which suggests a synchronous process of hIAPP1-37 amyloid fibrillization. This work provides insights into the understanding of hIAPP1-37 amyloid aggregation process and the pathogenesis of Type 2 diabetes disease.

Fractal self-assembly and aggregation of human amylin.

Human amylin is an intrinsically disordered protein believed to have a central role in Type-II diabetes mellitus (T2DM). The formation of intermediate oligomers is a seminal event in the eventual self-assembled fibril structures of amylin. However, the recent experimental investigations have shown the presence of different self-assembled (oligomers, protofilaments, and fibrils) and aggregated structures (amorphous aggregates) of amylin formed during its aggregation. Here, we show that amylin under diffusion-limited conditions leads to fractal self-assembly. The pH and solvent sensitive fractal self-assemblies of amylin were observed using an optical microscope. Confocal microscopy and scanning electron microscopy (SEM) with energy dispersion X-ray analysis (EDAX) were used to confirm the fractal self-assembly of amylin in water and PBS buffer, respectively. The fractal characteristics of the self-assemblies and the aggregates formed during the aggregation of amylin under different pH conditions were investigated using laser light scattering. The hydropathy and the docking study indicated the interactions between the anisotropically distributed hydrophobic residues and polar/ionic residues on the solvent-accessible surface of the protein as the crucial interaction hot-spots for driving the self-assembly and aggregation of human amylin. The simultaneous presence of various self-assemblies of human amylin was observed through different microscopy techniques. The present study may help in designing different fractal-like nanomaterials with potential applications in drug delivery, sensing, and tissue engineering.

Chitosan Oligosaccharides Attenuate Amyloid Formation of hIAPP and Protect Pancreatic ß-Cells from Cytotoxicity.

The deposition of aggregated human islet amyloid polypeptide (hIAPP) in the pancreas, that has been associated with ß-cell dysfunction, is one of the common pathological features of patients with type 2 diabetes (T2D). Therefore, hIAPP aggregation inhibitors hold a promising therapeutic schedule for T2D. Chitosan oligosaccharides (COS) have been reported to exhibit a potential antidiabetic effect, but the function of COS on hIAPP amyloid formation remains elusive. Here, we show that COS inhibited the aggregation of hIAPP and disassembled preformed hIAPP fibrils in a dose-dependent manner by thioflavin T fluorescence assay, circular dichroism spectroscopy, and transmission electron microscope. Furthermore, COS protected mouse ß-cells from cytotoxicity of amyloidogenic hIAPP, as well as apoptosis and cycle arrest. There was no direct binding of COS and hIAPP, as revealed by surface plasmon resonance analysis. In addition, both chitin-oligosaccharide and the acetylated monosaccharide of COS and glucosamine had no inhibition effect on hIAPP amyloid formation. It is presumed that, mechanistically, COS regulate hIAPP amyloid formation relating to the positive charge and degree of polymerization. These findings highlight the potential role of COS as inhibitors of hIAPP amyloid formation and provide a new insight into the mechanism of COS against diabetes.

Quantification of protein aggregation rates and quenching effects of amylin-inhibitor complexes.

The formation of amyloid aggregates is the hallmark of many protein misfolding diseases, including Type-II diabetes mellitus, which is caused by the fibrillation of amylin protein. It is established that nano-sized ligands such as curcumin, resveratrol and graphene quantum dots can modify protein aggregation rates. In this article, we report a comparative study of these ligands to estimate their protein aggregation rates and fluorescence quenching using various experimental techniques. Through light scattering experiments, the RH of bare amylin was found to increase at a rate of 43% per day, whereas in the presence of the ligands in different molar ratios (A1C10, A1R10 and A1GQDs20), the sizes of the complexes were found to grow at rates of 7%, 8% and 13% per day, respectively. We observed fluorescence quenching using photoluminescence experiments for all three protein-ligand complexes. The protein aggregation rate and fluorescence quenching exhibited a concentration-dependent competitive role in the inhibition process. Interestingly, for graphene quantum dots, the protein aggregation rate is more affected at lower concentrations, while fluorescence quenching dominates at higher concentrations; this is in contrast to curcumin and resveratrol, where fluorescence quenching dominates at all concentrations of the ligands in the complex. The FTIR data showed appreciable conversion of ß-sheets into less aggregation-prone secondary structures for all three amylin-ligand ratios; however, the inhibition performance of curcumin overshadowed those of the other two inhibitors. The inhibition behavior of these three ligands was corroborated by analysis of analytical and high-resolution TEM images of the fibrils.

Kinetic and Conformational Insights into Islet Amyloid Polypeptide Self-Assembly Using a Biarsenical Fluorogenic Probe.

Amyloid fibril formation and tissue deposition are associated with many diseases. Studies have shown that prefibrillar intermediates, such as oligomers, are the most toxic proteospecies of the amyloidogenic cascade. Thus, understanding the mechanisms of formation and the conformational ensemble of prefibrillar species is critical. Due to their transient and heterogeneous nature, detection and characterization of prefibrillar species remain challenging. The fluorogenic probe fluorescein arsenical hairpin (FlAsH), which recognizes a tetracysteine motif, has been recently used to detect the oligomerization of amyloidogenic peptides encompassing a Cys-Cys tag. In this study, we extended the FlAsH detection method to gain novel kinetic and conformational insights into the self-assembly of islet amyloid polypeptide (IAPP), a 37-residue peptide hormone whose deposition is associated with type II diabetes. By positional scanning of the Cys-Cys motif, the stability of the noncontiguous tetracysteine FlAsH-binding sites formed during self-assembly was evaluated and revealed rapid monomer self-recognition through the convergence of C-terminal domains. On the other hand, the N-terminal domains come close to each other only upon the formation of the cross-ß-sheet amyloid structure. We demonstrated that this method is well-suited to detect thioflavin T-negative fibrils and to screen inhibitors of amyloid formation. This study highlights that with positional scanning of the split-tetracysteine motif (Cys-Cys), the FlAsH detection method offers unique time-dependent conformational insights on the proteospecies assembled throughout the amyloidogenic pathway.

Directly probing the dissociation effects of graphene oxide nanosheets on hIAPP fibrils.

The aggregation of human islet amyloid polypeptides (hIAPP) to mature fibrils is considered as the main cause of type II diabetes. Therefore destroying the pre-formed hIAPP fibrils is expected to be a promising strategy for therapeutic treatments. In this work, the dissociation effects of graphene oxide (GO) nanosheets on hIAPP mature fibrils are investigated. The results clearly demonstrate that hIAPP fibrils can be quickly adsorbed on the GO surface and efficiently broken into short fragments. Meanwhile, the ß-sheet structures of hIAPP fibrils are greatly destroyed. Particularly, in situ atomic force microscopy was applied to monitor the real-time interaction between hIAPP fibrils and GO nanosheets. It provides distinct evidence that the disruption of hIAPP fibrils by GO nanosheets mainly occurs at the GO edges. Size-dependent experiments further justify the interfere of edge contribution, which suggest small-sized GO nanosheets exhibit better dissociation ability than large-sized ones. Therefore, this study not only provides valuable information that GO nanosheets (especially small-sized ones) can act as efficient nanoblades to break hIAPP fibrils, but also suggests a powerful and widely available methodology for investigating real-time interaction between nanomaterials and biomolecules.

Diabetes Drug Discovery: hIAPP1-37 Polymorphic Amyloid Structures as Novel Therapeutic Targets.

Human islet amyloid peptide (hIAPP1-37) aggregation is an early step in Diabetes Mellitus. We aimed to evaluate a family of pharmaco-chaperones to act as modulators that provide dynamic interventions and the multi-target capacity (native state, cytotoxic oligomers, protofilaments and fibrils of hIAPP1-37) required to meet the treatment challenges of diabetes. We used a cross-functional approach that combines in silico and in vitro biochemical and biophysical methods to study the hIAPP1-37 aggregation-oligomerization process as to reveal novel potential anti-diabetic drugs. The family of pharmaco-chaperones are modulators of the oligomerization and fibre formation of hIAPP1-37. When they interact with the amino acid in the amyloid-like steric zipper zone, they inhibit and/or delay the aggregation-oligomerization pathway by binding and stabilizing several amyloid structures of hIAPP1-37. Moreover, they can protect cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP1-37 oligomers. The modulation of proteostasis by the family of pharmaco-chaperones A-F is a promising potential approach to limit the onset and progression of diabetes and its comorbidities.

Peptide Conjugates of Benzene Carboxylic Acids as Agonists and Antagonists of Amylin Aggregation.

Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37 residue peptide hormone that is stored and co-secreted with insulin. hIAPP plays a pivotal role in type 2 diabetes and is the major component of amyloid deposits found in the pancreas of patients afflicted with the disease. The self-assembly of hIAPP and the formation of amyloid is linked to the death of insulin producing ß-cells. Recent findings suggest that soluble hIAPP oligomers are the cytotoxic species responsible for ß-cell loss whereas amyloid fibrils themselves may indeed be innocuous. Potential avenues of therapeutic intervention include the development of compounds that prevent hIAPP self-assembly as well as those that reduce or eliminate lag time and rapidly accelerate the formation of amyloid fibrils. Both of these approaches minimize temporal exposure to soluble cytotoxic hIAPP oligomers. Toward this end our laboratory has pursued an electrostatic repulsion approach to the development of potential inhibitors and modulators of hIAPP self-assembly. Peptide conjugates were constructed in which benzene carboxylic acids of varying charge were employed as electrostatic disrupting elements and appended to the N-terminal of the hIAPP22-29 (NFGAILSS) self-recognition sequence. The self-assembly kinetics of conjugates were characterized by turbidity measurements and the structure of aggregates probed by Raman and CD spectroscopy while the morphology was assessed using transmission electron microscopy. Several benzene carboxylic acid peptide conjugates failed to self-assemble and some were found to inhibit the aggregation of full-length amylin while others served to enhance the rate of amyloid formation and/or increase the yield of amyloid produced. Studies reveal that the geometric display of free carboxylates on the benzene ring of the conjugates plays an important role in the activity of conjugates. In addition, a number of free benzene carboxylic acids were found to modulate amylin self-assembly on their own. The results of these investigations confirm the viability of the electrostatic repulsion approach to the modulation of amyloid formation and may aid the design and development of potential therapeutic agents.

Thioflavin T fluorescence to analyse amyloid formation kinetics: Measurement frequency as a factor explaining irreproducibility.

The most frequent method to monitor amyloid formation relies on the fluorescence of thioflavin T (ThT). The present study reports a novel factor of irreproducibility in ThT kinetic assays performed in microplate. Discrepancies among kinetics of amyloid assembly, performed under quiescent conditions, were associated with the frequency of fluorescence measurement. Evaluating self-assembly of the islet amyloid polypeptide at short intervals hastened its fibrillization. This observation was confirmed by transmission electron microscopy, circular dichroism spectroscopy and 8-anilino-1-naphthalenesulfonic acid fluorescence. This effect, attributed to agitation during microplate displacements between fluorescence measurements, reinforces the importance of a better standardization in amyloid formation assays.

Amylin Amyloid Inhibition by Flavonoid Baicalein: Key Roles of Its Vicinal Dihydroxyl Groups of the Catechol Moiety.

Amyloid formation of the 37-residue amylin is involved in the pathogenesis of type 2 diabetes and, potentially, diabetes-induced neurological deficits. Numerous flavonoids exhibit inhibitory effects against amylin amyloidosis, but the mechanisms of inhibition remain unclear. Screening a library of natural compounds uncovered a potent lead compound, the flavone baicalein. Baicalein inhibits amylin amyloid formation and reduces amylin-induced cytotoxicity. Analogue analyses demonstrated, for the first time, key roles of the vicinal hydroxyl groups on the A-ring. We provided mass spectrometric evidence that incubating baicalein and amylin leads to their conjugation, consistent with a Schiff base mechanism.

A Comparison of Three Fluorophores for the Detection of Amyloid Fibers and Prefibrillar Oligomeric Assemblies. ThT (Thioflavin T); ANS (1-Anilinonaphthalene-8-sulfonic Acid); and bisANS (4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic Acid).

Amyloid fiber formation is a key event in many misfolding disorders. The ability to monitor the kinetics of fiber formation and other prefibrillar assemblies is therefore crucial for understanding these diseases. Here we compare three fluorescent probes for their ability to monitor fiber formation, ANS (1-anilinonaphthalene-8-sulfonic acid) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) along with the more widely used thioflavin T (ThT). For this, we have used two highly amyloidogenic peptides: amyloid-ß (Aß) from Alzheimer's disease and islet amyloid polypeptide (IAPP) associated with type II diabetes. Using a well-plate reader, we show all three fluorophores can report the kinetics of fiber formation. Indeed, bis-ANS is markedly more sensitive to fiber detection than ThT and has a submicromolar affinity for Aß fibers. Furthermore, we show that fluorescence detection is very sensitive to the presence of excess fluorophore. In particular, beyond a 1:1 stoichiometry these probes demonstrate marked fluorescence quenching, for both Aß and IAPP. Indeed, the fiber-associated fluorescence signal is almost completely quenched in the presence of excess ThT. There is also intense interest in the detection of prefibrillar amyloid assemblies, as oligomers and protofibrils are believed to be highly cytotoxic. We generate stable, fiber-free, prefibrillar assemblies of Aß and survey their fluorescence with ANS and bis-ANS. Fluorescence from ANS has often been used as a marker for oligomers; however, we show ANS can fluoresce more strongly in the presence of fibers and should therefore be used as a probe for oligomers with caution.

Mutational analysis of the ability of resveratrol to inhibit amyloid formation by islet amyloid polypeptide: critical evaluation of the importance of aromatic-inhibitor and histidine-inhibitor interactions.

The process of amyloid formation by the normally soluble hormone islet amyloid polypeptide (IAPP) contributes to ß-cell death in type 2 diabetes and in islet transplants. There are no clinically approved inhibitors of islet amyloidosis, and the mode of action of existing inhibitors is not well-understood. Resveratrol, a natural polyphenol, has been reported to inhibit amyloid formation by IAPP and by the Alzheimer's disease Aß peptide. The mechanism of action of this compound is not known, nor is its mode of interaction with IAPP. In this study, we use a series of IAPP variants to examine possible interactions between resveratrol and IAPP. Fluorescence assays, transmission electron microscopy, and mass spectrometry demonstrate that resveratrol is much less effective as an inhibitor of IAPP amyloid formation than the polyphenol (-)-epigallocatechin 3-gallate (EGCG) and, unlike EGCG, does not significantly disaggregate preformed IAPP amyloid fibrils. Resveratrol is also shown to interfere with thioflavin-T assays. His-18 mutants, a truncation mutant, mutants of each of the aromatic residues, and mutants of Arg-11 of IAPP were examined. Mutation of His to Gln or Leu weakens the ability of resveratrol to inhibit amyloid formation by IAPP, as do mutations of Arg-11, Phe-15, or Tyr-37 to Leu, and truncation to form the variant Ac 8-37-IAPP, which removes the first seven residues to eliminate Lys-1 and the N-terminal amino group. In contrast, replacement of Phe-23 with Leu has a smaller effect. The data highlight Phe-15, His-18, and Tyr-37 as being important for IAPP-resveratrol interactions and are consistent with a potential role of the N-terminus and Arg-11 in polypeptide-resveratrol interactions.

Analysis of the ability of pramlintide to inhibit amyloid formation by human islet amyloid polypeptide reveals a balance between optimal recognition and reduced amyloidogenicity.

The hormone human islet amyloid polypeptide (hIAPP or amylin) plays a role in glucose metabolism, but forms amyloid in the pancreas in type 2 diabetes (T2D) and is associated with ß-cell death and dysfunction in the disease. Inhibitors of islet amyloid have therapeutic potential; however, there are no clinically approved inhibitors, and the mode of action of existing inhibitors is not well understood. Rat IAPP (rIAPP) differs from hIAPP at six positions, does not form amyloid, and is an inhibitor of amyloid formation by hIAPP. Five of the six differences are located within the segment of residues 20-29, and three of them are Pro residues, which are well-known disruptors of ß-sheet structure. rIAPP is thus a natural example of a "ß-breaker inhibitor", a molecule that combines a recognition element with an entity that inhibits ß-sheet formation. Pramlintide (PM) is a peptide drug approved for use as an adjunct to insulin therapy for treatment of diabetes. PM was developed by introducing the three Pro substitutions found in rIAPP into hIAPP. Thus, it more closely resembles the human peptide than does rIAPP. Here we examine and compare the ability of rIAPP, PM, and a set of designed analogues of hIAPP to inhibit amyloid formation by hIAPP, to elucidate the factors that lead to effective peptide-based inhibitors. Our results reveal, for this class of molecules, a balance between the reduced amyloidogenicity of the inhibitory sequence on one hand and its ability to recognize hIAPP on the other.

Monitoring and targeting the initial dimerization stage of amyloid self-assembly.

Amyloid deposits are pathological hallmark of a large group of human degenerative disorders of unrelated etiologies. While accumulating evidence suggests that early oligomers may account for tissue degeneration, most detection tools do not allow the monitoring of early association events. Here we exploit bimolecular fluorescence complementation analysis to detect and quantify the dimerization of three major amyloidogenic polypeptides; islet amyloid polypeptide, ß-amyloid and α-synuclein. The constructed systems provided direct visualization of protein-protein interactions in which only assembled dimers display strong fluorescent signal. Potential inhibitors that interfere with the initial intermolecular interactions of islet amyloid polypeptide were further identified using this system. Moreover, the identified compounds were able to inhibit the aggregation and cytotoxicity of islet amyloid polypeptide, demonstrating the importance of targeting amyloid dimer formation for future drug development.

Evaluation of membrane models and their composition for islet amyloid polypeptide-membrane aggregation.

Human islet amyloid polypeptide (IAPP) forms amyloid fibrils in the pancreatic islets of patients suffering from type 2 diabetes mellitus (T2DM). The formation of IAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet ß-cells during the pathogenesis of T2DM. Several studies have demonstrated a clear interaction between IAPP and lipid membranes. However the effect of different lipid compositions and of various membrane mimetics (including micelles, bicelles, SUV and LUV) on fibril formation kinetics and fibril morphology has not yet systematically been analysed. Here we report that the interaction of IAPP with various membrane models promoted different processes of fibril formation. Our data reveal that in SDS and DPC micelles, IAPP adopts a stable α-helical structure for several days, suggesting that the micelle models may stabilize monomeric or small oligomeric species of IAPP. In contrast, zwitterionic DMPC/DHPC bicelles and DOPC SUV accelerate the fibril formation compared to zwitterionic DOPC LUV, indicating that the size of the membrane model and its curvature influence the fibrillation process. Negatively charged membranes decrease the lag-time of the fibril formation kinetics while phosphatidylethanolamine and cholesterol have an opposite effect, probably due to the modulation of the physical properties of the membrane and/or due to direct interactions with IAPP within the membrane core. Finally, our results show that the modulation of lipid composition influences not only the growth of fibrils at the membrane surface but also the interactions of ß-sheet oligomers with membranes.

Defining the molecular basis of amyloid inhibitors: human islet amyloid polypeptide-insulin interactions.

Human islet amyloid polypeptide (hIAPP or Amylin) is a 37 residue hormone that is cosecreted with insulin from the pancreatic islets. The aggregation of hIAPP plays a role in the progression of type 2 diabetes and contributes to the failure of islet cell grafts. Despite considerable effort, little is known about the mode of action of IAPP amyloid inhibitors, and this has limited rational drug design. Insulin is one of the most potent inhibitors of hIAPP fibril formation, but its inhibition mechanism is not understood. In this study, the aggregation of mixtures of hIAPP with insulin, as well as with the separate A and B chains of insulin, were characterized using ion mobility spectrometry-based mass spectrometry and atomic force microscopy. Insulin and the insulin B chain target the hIAPP monomer in its compact isoform and shift the equilibrium away from its extended isoform, an aggregation-prone conformation, and thus inhibit hIAPP from forming ß-sheets and subsequently amyloid fibrils. All-atom molecular modeling supports these conclusions.

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 ß 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 ß-sheet arrangement that is determined by the type of polymorphism in amyloid fibrils.

Role of aromatic interactions in amyloid formation by islet amyloid polypeptide.

Aromatic-aromatic and aromatic-hydrophobic interactions have been proposed to play a role in amyloid formation by a range of polypeptides, including islet amyloid polypeptide (IAPP or amylin). IAPP is responsible for amyloid formation in patients with type 2 diabetes. The polypeptide is 37 residues long and contains three aromatic residues, Phe-15, Phe-23, and Tyr-37. The ability of all single aromatic to leucine mutants, all double aromatic to leucine mutants, and the triple leucine mutant to form amyloid were examined. Amyloid formation was almost twice as rapid for the F15L mutant as for the wild type but was almost 3-fold slower for the Y37L mutant and almost 2-fold slower for the F23L mutant. Amyloid fibrils formed from each of the single mutants were effective at seeding amyloid formation by wild-type IAPP, implying that the fibril structures are similar. The F15L/F23L double mutant has a larger effect than the F15L/Y37L double mutant on the rate of amyloid formation, even though a Y37L substitution has more drastic consequences in the wild-type background than does the F23L mutation, suggesting nonadditive effects between the different sites. The triple leucine mutant and the F23L/Y37L double mutant are the slowest to form amyloid. F15 has been proposed to make important contacts early in the aggregation pathway, but the data for the F15L mutant indicate that they are not optimal. A set of variants containing natural and unnatural amino acids at position 15, which were designed to conserve hydrophobicity, but alter α-helix and ß-sheet propensity, were analyzed to determine the properties of this position that control the rate of amyloid formation. There is no correlation between ß-sheet propensity at this position and the rate of amyloid formation, but there is a correlation with α-helical propensity.

Evidence of π-stacking interactions in the self-assembly of hIAPP(22-29).

The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self-assembly of human islet amyloid polypeptide (hIAPP)22-29 , peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe-23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe-23 virtually abolish the ability of hIAPP22-29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in ß-sheet secondary structure. Transmission electron microscopy images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the CC ring mode (ca. 1600 cm(-1) ) upon aggregation that has previously been shown to be indicative of π-stacking. While previous work has demonstrated that π-stacking is not an absolute requirement for fibrillization, our findings indicate that Phe-23 also contributes to fibril formation through π-stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self-assembly of hIAPP22-29 . © Proteins 2013. © 2012 Wiley Periodicals, Inc.

Ionic strength effects on amyloid formation by amylin are a complicated interplay among Debye screening, ion selectivity, and Hofmeister effects.

Amyloid formation plays a role in a wide range of human diseases. The rate and extent of amyloid formation depend on solution conditions, including pH and ionic strength. Amyloid fibrils often adopt structures with parallel, in-register ß-sheets, which generate quasi-infinite arrays of aligned side chains. These arrangements can lead to significant electrostatic interactions between adjacent polypeptide chains. The effect of ionic strength and ion composition on the kinetics of amyloid formation by islet amyloid polypeptide (IAPP) is examined. IAPP is a basic 37-residue polypeptide responsible for islet amyloid formation in type 2 diabetes. Poisson-Boltzmann calculations revealed significant electrostatic repulsion in a model of the IAPP fibrillar state. The kinetics of IAPP amyloid formation are strongly dependent on ionic strength, varying by a factor of >10 over the range of 20-600 mM NaCl at pH 8.0, but the effect is not entirely due to Debye screening. At low ionic strengths, the rate depends strongly on the identity of the anion, varying by a factor of nearly 4, and scales with the electroselectivity series, implicating anion binding. At high ionic strengths, the rate varies by only 8% and scales with the Hofmeister series. At intermediate ionic strengths, no clear trend is detected, likely because of the convolution of different effects. The effects of salts on the growth phase and lag phase of IAPP amyloid formation are strongly correlated. At pH 5.5, where the net charge on IAPP is higher, the effect of different anions scales with the electroselectivity series at all salt concentrations.