Simultaneously Measured Kinetics of Two Amyloid Polymorphs Using Cross Peak Specific 2D IR Spectroscopy.

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.

Interspecies Variation Affects Islet Amyloid Polypeptide Membrane Binding.

The aggregation of islet amyloid polypeptide (IAPP) is associated with ß-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 ß-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.

Metastable intermediate during hIAPP aggregation catalyzed by membranes as detected with 2D IR spectroscopy.

The aggregation of human islet amyloid polypeptide (hIAPP) into amyloid fibrils involves formation of oligomeric intermediates that are thought to be the cytotoxic species responsible for ß-cell dysfunction in type 2 diabetes. hIAPP oligomers permeating or disrupting the cellular membrane may be one mechanism of toxicity and so measuring the structural kinetics of aggregation in the presence of membranes is of much interest. In this study, we use 2D IR spectroscopy and 13C18O isotope labeling to study the secondary structure of the oligomeric intermediates formed in solution and in the presence of phospholipid vesicles at sites L12A13, L16V17, G24A25 and V32G33. Pairs of labels monitor the couplings between associated polypeptides and the dihedral angles between adjacent residues. In solution, the L12A13 residues form an oligomeric ß-sheet in addition to an α-helix whereas with the phospholipid vesicles they are α-helical throughout the aggregation process. In both solution and with DOPC vesicles, L16V17 and V32G33 have disordered structures until fibrils are formed. Similarly, under both conditions, G24A25 exhibits 3-state kinetics, created by an oligomeric intermediate with a well-defined ß-sheet structure. Amyloid fibril formation is often thought to involve intermediates with exceedingly low populations that are difficult to detect experimentally. These experiments establish that amyloid fibril formation of hIAPP when catalyzed by membranes includes a metastable intermediate and that this intermediate has a similar structure at G24A25 in the FGAIL region as the corresponding intermediate in solution, thought to be the toxic species.

Application of 2D IR Bioimaging: Hyperspectral Images of Formalin-Fixed Pancreatic Tissues and Observation of Slow Protein Degradation.

We used two-dimensional IR bioimaging to study the structural heterogeneity of formalin-fixed mouse pancreas. Images were generated from the hyperspectral data sets by plotting quantities associated with the amide I vibrational mode, which is created by the backbone carbonyl stretch. Images that measure the fundamental vibrational frequencies, cross peaks, and anharmonic shifts are presented. Histograms are generated for each quantity, providing averaged values and distributions around the mean that serve as metrics for protein structures. Images were generated from tissue that had been stored in a formalin fixation for 3, 8, and 48 weeks. Over this period, all three metrics show that that the ß-sheet content of the samples increased, consistent with protein aggregation. Our results indicate that formalin fixation does not entirely arrest the degradation of a protein structure in pancreas tissue.

A Different hIAPP Polymorph Is Observed in Human Serum Than in Aqueous Buffer: Demonstration of a New Method for Studying Amyloid Fibril Structure Using Infrared Spectroscopy.

There is enormous interest in measuring amyloid fibril structures, but most structural studies measure fibril formation in vitro using aqueous buffer. Ideally, one would like to measure fibril structure and mechanism under more physiological conditions. Toward this end, we have developed a method for studying amyloid fibril structure in human serum. Our approach uses isotope labeling, antibody depletion of the most abundant proteins (albumin and IgG), and infrared spectroscopy to measure aggregation in human serum with reduced protein content. Reducing the nonamyloid protein content enables the measurements by decreasing background signals but retains the full composition of salts, sugars, metal ions, etc. that are naturally present but usually missing from in vitro studies. We demonstrate the method by measuring the two-dimensional infrared (2D IR) spectra of isotopically labeled human islet amyloid polypeptide (hIAPP or amylin). We find that the fibril structure of hIAPP formed in serum differs from that formed via aggregation in aqueous buffer at residues Gly24 and Ala25, which reside in the putative "amyloidogenic core" or FGAIL region of the sequence. The spectra are consistent with extended parallel stacks of strands consistent with ß-sheet-like structure, rather than a partially disordered loop that forms in aqueous buffer. These experiments provide a new method for using infrared spectroscopy to monitor the structure of proteins under physiological conditions and reveal the formation of a significantly different polymorph structure in the most important region of hIAPP.