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The origin of in vitro amyloid fibril polymorphs is debated, in part, because few techniques can simultaneously monitor the formation kinetics of multiple amyloid polymorphs. Using a cross-peak specific polarization scheme, ⟨0°,0°,60°,-60°⟩, we resolve 22 previously unseen cross peaks in the 2D IR spectra of amyloid fibrils formed by the human islet amyloid polypeptide (hIAPP). Those cross peaks include a subset assigned to a second fibril polymorph, which forms on a slower time scale. We simulated the data with three different kinetic models for polymorph formation. Only a model based on secondary nucleation reproduces the cross peak kinetics. These experiments are evidence that fibrils formed by secondary nucleation have a different polymorphic structure than the parent fibrils and illustrate the enhanced structural resolution of this new cross peak specific polarization scheme.

The aggregation of islet amyloid polypeptide (IAPP) is associated with β-cell dysfunction in type 2 diabetes (T2D) in humans. One possible mechanism of toxicity is the interaction of IAPP oligomers with lipid membranes to disrupt the bilayer integrity and/or homeostasis of the cell. Amino acid sequence variations of IAPPs between species can greatly decrease their propensity for aggregation. For example, human IAPP is toxic to β-cells, but rat and pig IAPP are not. However, it is not clear how these differences affect membrane association. Using native mass spectrometry with lipid nanodiscs, we explored the differences in the association of human, rat, and pig IAPP with lipid bilayers. We discovered that human and rat IAPP bound nanodiscs with anionic dipalmitoyl-phosphatidylglycerol (DPPG) lipids, but pig IAPP did not. Furthermore, human and rat IAPP interacted differently with the membrane. Human IAPP show potential tetramer complexes, but rat IAPP associated with the membrane sequentially. Thus, overall IAPP-bilayer interactions are not necessarily related to disease, but small differences in oligomeric behavior at the membrane may instead play a role.

Immunosensors use antibodies to detect and quantify biomarkers of disease, though the sensors often lack structural information. We create a surface-sensitive two-dimensional infrared (2D IR) spectroscopic immunosensor for studying protein structures. We tether antibodies to a plasmonic surface, flow over a solution of amyloid proteins, and measure the 2D IR spectra. The 2D IR spectra provide a global assessment of antigen structure, and isotopically labeled proteins give residue-specific structural information. We report the 2D IR spectra of fibrils and monomers using a polyclonal antibody that targets human islet amyloid polypeptide (hIAPP). We observe two fibrillar polymorphs differing in their structure at the G24 residue, which supports the hypothesis that hIAPP polymorphs form from a common oligomeric intermediate. This work provides insight into the structure of hIAPP, establishes a new method for studying protein structures using 2D IR spectroscopy, and creates a spectroscopic immunoassay applicable for studying a wide range of biomarkers.

It is now recognized that many amyloid-forming proteins can associate into multiple fibril structures. Here, we use two-dimensional infrared spectroscopy to study two fibril polymorphs formed by human islet amyloid polypeptide (hIAPP or amylin), which is associated with type 2 diabetes. The polymorphs exhibit different degrees of structural organization near the loop region of hIAPP fibrils. The relative populations of these polymorphs are systematically altered by the presence of macrocyclic peptides which template β-sheet formation at specific sections of the hIAPP sequence. These experiments are consistent with polymorphs that result from competing pathways for fibril formation and that the macrocycles bias hIAPP aggregation toward one pathway or the other. Another macrocyclic peptide that matches the loop region but extends the lag time leaves the relative populations of the polymorphs unaltered, suggesting that the branching point for structural divergence occurs after the lag phase, when the oligomers convert into seeds that template fibril formation. Thus, we conclude that the structures of the polymorphs stem from restricting oligomers along diverging folding pathways, which has implications for drug inhibition, cytotoxicity, and the free energy landscape of hIAPP aggregation.

Transiently populated oligomers formed en route to amyloid fibrils may constitute the most toxic aggregates associated with many amyloid-associated diseases. Most nucleation theories used to describe amyloid aggregation predict low oligomer concentrations and do not take into account free energy costs that may be associated with structural rearrangements between the oligomer and fiber states. We have used isotope labeling and two-dimensional infrared spectroscopy to spectrally resolve an oligomeric intermediate during the aggregation of the human islet amyloid protein (hIAPP or amylin), the protein associated with type II diabetes. A structural rearrangement includes the F 23 G 24 A 25 I 26 L 27 region of hIAPP, which starts from a random coil structure, evolves into ordered β-sheet oligomers containing at least 5 strands, and then partially disorders in the fibril structure. The supercritical concentration is measured to be between 150 and 250 μM, which is the thermodynamic parameter that sets the free energy of the oligomers. A 3-state kinetic model fits the experimental data, but only if it includes a concentration independent free energy barrier >3 kcal/mol that represents the free energy cost of refolding the oligomeric intermediate into the structure of the amyloid fibril; i.e., "oligomer activation" is required. The barrier creates a transition state in the free energy landscape that slows fibril formation and creates a stable population of oligomers during the lag phase, even at concentrations below the supercritical concentration. Largely missing in current kinetic models is a link between structure and kinetics. Our experiments and modeling provide evidence that protein structural rearrangements during aggregation impact the populations and kinetics of toxic oligomeric species.

Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.

Transition dipoles are an underutilized quantity for probing molecular structures. The transition dipole strengths in an extended system like a protein are modulated by the couplings and thus probe the structures. Here we measure the absolute transition dipole strengths of human and rat amylin in their solution, aggregated, membrane, and micelleular bound forms, using a combination of 1D and 2D infrared spectroscopy. We find that the vibrational modes of amyloid fibers made of human amylin can extend across as many as 12 amino acids, reflecting very ordered β-sheets in the most carefully prepared samples. Rat amylin has FTIR spectra that are nearly identical in solution, micelles, and membranes. We show that the transition dipoles of rat amylin are much larger when bound to micelles and membranes than when in solution, consistent with rat amylin adopting an α-helical structure. We interpret the transition dipole strengths as experimental measurements of the inverse participation ratio often calculated in theoretical studies. The structure of aggregating and membrane-bound proteins can be difficult to identify with existing techniques, especially during kinetics. These results demonstrate how absolute transition dipoles measured via our 1D/2D spectroscopy method can provide important structural information.

By adding a mid-infrared pulse shaper to a sum-frequency generation (SFG) spectrometer, we have built a 2D SFG spectrometer capable of measuring spectra analogous to 2D IR spectra but with monolayer sensitivity and SFG selection rules. In this paper, we describe the experimental apparatus and provide an introduction to 2D SFG spectroscopy to help the reader interpret 2D SFG spectra. The main aim of this manuscript is to report 2D SFG spectra of the amyloid forming peptide FGAIL. FGAIL is a critical segment of the human islet amyloid polypeptide (hIAPP or amylin) that aggregates in people with type 2 diabetes. FGAIL is catalyzed into amyloid fibers by many types of surfaces. Here, we study the structure of FGAIL upon deposition onto a gold surface covered with a self-assembled monolayer of methyl-4-mercaptobenzoate (MMB) that produces an ester coating. FGAIL deposited on bare gold does not form ordered layers. The measured 2D SFG spectrum is consistent with amyloid fiber formation, exhibiting both the parallel (a+) and perpendicular (a-) symmetry modes associated with amyloid β-sheets. Cross peaks are observed between the ester stretches of the coating and the FGAIL peptides. Simulations are presented for two possible structures of FGAIL amyloid β-sheets that illustrate the sensitivity of the 2D SFG spectra to structure and orientation. These results provide some of the first molecular insights into surface catalyzed amyloid fiber structure.

Islet amyloid polypeptide (IAPP or Amylin) is a 37-residue, C-terminally amidated pancreatic hormone, cosecreted with insulin that forms islet amyloid in type 2 diabetes. Islet amyloid formation is complex and characterizing preamyloid oligomers is an important topic because oligomeric intermediates are postulated to be the most toxic species produced during fibril formation. A range of competing models for early oligomers have been proposed. The role of the amidated C-terminus in amyloid formation by IAPP and in stabilizing oligomers is not known. Studies with unamidated IAPP have provided evidence for formation of an antiparallel dimer at pH 5.5, stabilized by stacking of His-18 and Tyr-37, but it is not known if this interaction is formed in the physiological form of the peptide. Analysis of a set of variants with a free and with an amidated C-terminus shows that disrupting the putative His-Tyr interaction accelerates amyloid formation, indicating that it is not essential. Amidation to generate the physiologically relevant form of IAPP accelerates amyloid formation, demonstrating that the advantages conferred by C-terminal amidation outweigh increased amyloidogenicity. The analysis of this variant argues that IAPP is not under strong evolutionary pressure to reduce amyloidogenicity. Analysis of an H18Q mutant of IAPP shows that the charge state of the N-terminus is an important factor controlling the rate of amyloid formation, even though the N-terminal region of IAPP is believed to be flexible in the amyloid fibers., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Amyloid formation is implicated in more than 20 human diseases, yet the mechanism by which fibrils form is not well understood. We use 2D infrared spectroscopy and isotope labeling to monitor the kinetics of fibril formation by human islet amyloid polypeptide (hIAPP or amylin) that is associated with type 2 diabetes. We find that an oligomeric intermediate forms during the lag phase with parallel β-sheet structure in a region that is ultimately a partially disordered loop in the fibril. We confirm the presence of this intermediate, using a set of homologous macrocyclic peptides designed to recognize β-sheets. Mutations and molecular dynamics simulations indicate that the intermediate is on pathway. Disrupting the oligomeric β-sheet to form the partially disordered loop of the fibrils creates a free energy barrier that is the origin of the lag phase during aggregation. These results help rationalize a wide range of previous fragment and mutation studies including mutations in other species that prevent the formation of amyloid plaques.

Deamidation of asparagine and glutamine is the most common nonenzymatic, post-translational modification. Deamidation can influence the structure, stability, folding, and aggregation of proteins and has been proposed to play a role in amyloid formation. However there are no structural studies of the consequences of deamidation on amyloid fibers, in large part because of the difficulty of studying these materials using conventional methods. Here we examine the effects of deamidation on the kinetics of amyloid formation by amylin, the causative agent of type 2 diabetes. We find that deamidation accelerates amyloid formation and the deamidated material is able to seed amyloid formation by unmodified amylin. Using site-specific isotope labeling and two-dimensional infrared (2D IR) spectroscopy, we show that fibers formed by samples that contain deamidated polypeptide contain reduced amounts of β-sheet. Deamidation leads to disruption of the N-terminal β-sheet between Ala-8 and Ala-13, but β-sheet is still retained near Leu-16. The C-terminal sheet is disrupted near Leu-27. Analysis of potential sites of deamidation together with structural models of amylin fibers reveals that deamidation in the N-terminal β-sheet region may be the cause for the disruption of the fiber structure at both the N- and C-terminal β-sheet. Thus, deamidation is a post-translational modification that creates fibers that have an altered structure but can still act as a template for amylin aggregation. Deamidation is very difficult to detect with standard methods used to follow amyloid formation, but isotope-labeled IR spectroscopy provides a means for monitoring sample degradation and investigating the structural consequences of deamidation.

Amyloid formation has been implicated in the pathology of over 20 human diseases, but the rational design of amyloid inhibitors is hampered by a lack of structural information about amyloid-inhibitor complexes. We use isotope labelling and two-dimensional infrared spectroscopy to obtain a residue-specific structure for the complex of human amylin (the peptide responsible for islet amyloid formation in type 2 diabetes) with a known inhibitor (rat amylin). Based on its sequence, rat amylin should block formation of the C-terminal β-sheet, but at 8 h after mixing, rat amylin blocks the N-terminal β-sheet instead. At 24 h after mixing, rat amylin blocks neither β-sheet and forms its own β-sheet, most probably on the outside of the human fibrils. This is striking, because rat amylin is natively disordered and not previously known to form amyloid β-sheets. The results show that even seemingly intuitive inhibitors may function by unforeseen and complex structural processes.

The aggregation of human amylin to form amyloid contributes to islet β-cell dysfunction in type 2 diabetes. Studies of amyloid formation have been hindered by the low structural resolution or relatively modest time resolution of standard methods. Two-dimensional infrared (2DIR) spectroscopy, with its sensitivity to protein secondary structures and its intrinsic fast time resolution, is capable of capturing structural changes during the aggregation process. Moreover, isotope labeling enables the measurement of residue-specific information. The diagonal line widths of 2DIR spectra contain information about dynamics and structural heterogeneity of the system. We illustrate the power of a combined atomistic molecular dynamics simulation and theoretical and experimental 2DIR approach by analyzing the variation in diagonal line widths of individual amide I modes in a series of labeled samples of amylin amyloid fibrils. The theoretical and experimental 2DIR line widths suggest a "W" pattern, as a function of residue number. We show that large line widths result from substantial structural disorder and that this pattern is indicative of the stable secondary structure of the two β-sheet regions. This work provides a protocol for bridging MD simulation and 2DIR experiments for future aggregation studies.

A cost-efficient, time-reducing solid-phase synthesis of the amyloidogenic, 37 residue islet amyloid polypeptide (IAPP) is developed using two pseudoprolines (highlighted blue in sequence) in combination with microwave technology. A yield twice that obtained with conventional syntheses is realized. The utility of this protocol is demonstrated by the synthesis of a (13)C(18)O-labeled Ser-20 IAPP variant, a prohibitively expensive and chemically challenging site to label via other protocols. TEM analysis shows the peptide forms normal amyloid (abstract image).

Patients with type II diabetes exhibit fibrillar deposits of human amylin protein in the pancreas. It has been proposed that amylin oligomers arising along the aggregation or fibril-formation pathways are important in the genesis of the disease. In a step toward understanding these aggregation pathways, in this work we report the conformational preferences of human amylin monomer in solution using molecular simulations and infrared experiments. In particular, we identify a stable conformer that could play a key role in aggregation. We find that amylin adopts three stable conformations: one with an α-helical segment comprising residues 9-17 and a short antiparallel β-sheet comprising residues 24-28 and 31-35; one with an extended antiparallel β-hairpin with the turn region comprising residues 20-23; and one with no particular structure. Using detailed calculations, we determine the relative stability of these various conformations, finding that the β-hairpin conformation is the most stable, followed by the α-helical conformation, and then the unstructured coil. To test our predicted structure, we calculate its infrared spectrum in the amide I stretch regime, which is sensitive to secondary structure through vibrational couplings and linewidths, and compare it to experiment. We find that theoretically predicted spectra are in good agreement with the experimental line shapes presented herein. The implications of the monomer secondary structures on its aggregation pathway and on its interaction with cell membranes are discussed., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)