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Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N -terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant -like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this "spectator protein", exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 A & ring; . The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques.

Inhibitors targeting amyloids formed by the human Islet Amyloid Polypeptide (hIAPP) are promising therapeutic candidates for type 2 diabetes. Peptide formulations derived from the nonamyloidogenic rat IAPP (rIAPP) sequence are currently used as hIAPP mimetics to support insulin therapy. rIAPP itself acts as a peptide inhibitor; yet, the structural-level consequences of such inhibition, particularly its impact on amyloid polymorphism, have not been studied in detail. Here, we conduct coaggregation experiments with varying rIAPP-to-hIAPP concentration ratios and employ high-resolution cryo-electron microscopy (Cryo-EM) to elucidate the polymorphism of the resulting fibril structures. Our results demonstrate that the polymorphism of hIAPP amyloids is highly sensitive to the electrostatic environment, which can be modulated by buffer composition, the concentration of the inhibitor, and cosolvents such as hexafluoroisopropanol (HFIP). Under native conditions, rIAPP associates with hIAPP but does not cross-aggregate, resulting in fibrils primarily composed of hIAPP. Significant inhibition is observed at relatively high concentrations of rIAPP. However, trace amounts of HFIP disrupt this inhibition, leading to increased fibril concentrations due to the formation of cross-seeded products composed of both hIAPP and rIAPP, as evidenced by mass spectrometry and two-dimensional infrared (2D IR) spectroscopy. These findings highlight the critical role of experimental conditions, particularly the electrostatic environment, in modulating amyloid polymorphism, cross-seeding, and inhibition. By providing structural insights into these processes, this study advances our understanding of peptide aggregation and offers valuable guidance for the rational design of more effective therapeutic inhibitors targeting hIAPP-related amyloidosis.

In the pursuit of potential therapeutic agents for type 2 diabetes, non-amyloidogenic forms of the human Islet Amyloid Polypeptide (hIAPP) containing site-specific mutations are of significant interest. In the present study, we dissect the three proline mutations present in the core region of the non-amyloidogenic rat IAPP into single-point mutations at A25P, S28P, and S29P sites. We apply high-resolution cryo-electron microscopy and solve the structures of 6 polymorphs formed by these mutants, revealing the peptide's self-assembly patterns and identifying critical interactions that reinforce these structures in the presence of the b-sheet breaker. A unique trimeric aggregate with C3 symmetry was identified in the A25P mutant, which we resolved with a 3.05 A resolution, while asymmetric trimeric assemblies were observed in the other mutants. Guided by the high-resolution structural models of A25P and S28P fibrils obtained in our study, we successfully designed novel non-amyloidogenic mutants of IAPP with potential therapeutic value. Our findings demonstrate the immense potential of structure-based approaches in developing effective therapeutics against amyloid diseases. 

The human islet amyloid polypeptide (hIAPP) aggregates into amyloid fibrils that contribute to β-cell failure in type 2 diabetes. hIAPP is produced from a 67-residue precursor, proIAPP, but incomplete cleavage by prohormone convertase 2 (PC2) produces the 48-residue intermediate proIAPP(1–48), which accelerates amyloid formation in vivo. Here we show that proIAPP(1–48) assembles almost exclusively into a single fibril polymorph. Using cryo-electron microscopy we solved its structure at 3.5 Å resolution and uncovered a P-shaped, C2-symmetric dimer whose backbone and side-chain packing are nearly identical to the disease-associated TW2 polymorph propagated from pancreatic tissue, although with different helical symmetry. All eleven extra N-terminal residues remain disordered but create a weak density around His29. Based on time-averaged density derived from molecular dynamics (MD) simulations, we identified multiple hydrogen(H)-bonding interactions, which may contribute to stabilising the TW2-like fold and explain the peripheral cryo-EM density. These data establish a structural link between defective proIAPP processing and the polymorphic spectrum of islet amyloid and suggest a seeding pathway by which proIAPP(1–48) templates pathogenic architectures that fully processed hIAPP rarely adopts in vitro.