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.inhibitors | aggregation pathway | vibrational coupling T he misfolding of proteins into β-sheet-rich amyloid fibrils is associated with the pathology of more than 20 human diseases, including Alzheimer's, Parkinson, and Huntington diseases (1). Amyloid plaques are formed by masses of fibrils, but growing evidence suggests that the toxic species may be prefibrillar intermediates (2, 3). As a result, there is much interest in understanding the mechanism by which these proteins form fibrils and identifying intermediates in the aggregation pathway. However, obtaining structural information about intermediate species is difficult due to their transient nature. Solid-state NMR (ssNMR) and X-ray crystallography provide high-resolution structures of fibrils (4, 5) and optical techniques can track structural changes in real time (6, 7), but few techniques have both the structural and the temporal resolution to extract specific structural details about intermediates. Fragments have been trapped in intriguing oligomeric structures that may represent intermediate states (5,8) and transient secondary structures are known to exist from circular dichroism measurements and other experiments (9-11), but for full-length proteins it has been difficult to identify the specific residues that contribute to the secondary structure and thus understand their role in the aggregation mechanism. In this paper, we use 2D infrared (2D IR) spectroscopy and isotope labeling to monitor the structural evolution of the full-length human islet amyloid polypeptide (hIAPP or amylin), a 37-residue peptide implicated in type 2 diabetes. We observe the formation of a structured prefibrillar intermediate in a region that has long been known to influence aggregation, but that does not form well-ordered cross-β structure in the amyloid fibril. Its presence provides unique structural insights into the mechanism of amyloid aggregation and helps unify many seemingly inconsistent prior studies.Many studies on hIAPP have focused ...

The amyloid protein aggregation associated with diseases such as Alzheimer’s, Parkinson’s, and type II diabetes (among many others), features a bewildering variety of β-sheet-rich structures in transition from native proteins to ordered oligomers and fibers. The variation in the amino acid sequences of the β-structures presents a challenge to developing a model system of β-sheets for the study of various amyloid aggregates. Here we introduce a family of robust β-sheet macrocycles that can serve as a platform to display a variety of heptapeptide sequences from different amyloid proteins. We have tailored these amyloid β-sheet mimics (ABSMs) to antagonize aggregation of various amyloid proteins, thereby reducing the toxicity of amyloid aggregates. We describe the structures and inhibitory properties of ABSMs containing amyloidogenic peptides from Aβ associated with Alzheimer’s disease, β2-microglobulin associated with dialysis-related amyloidosis, α-synuclein associated with Parkinson’s disease, islet amyloid polypeptide associated with type II diabetes, human and yeast prion proteins, and Tau, which forms neurofibrillary tangles.

Although aberrant protein aggregation has been conclusively linked to dozens of devastating amyloid diseases, scientists remain puzzled about the molecular features that render amyloid fibrils or small oligomers toxic. Here, we report a previously unobserved type of amyloid fibril that tests as cytotoxic: one in which the strands of the contributing β-sheets are out of register. In all amyloid fibrils previously characterized at the molecular level, only inregister β-sheets have been observed, in which each strand makes its full complement of hydrogen bonds with the strands above and below it in the fibril. In out-of-register sheets, strands are sheared relative to one another, leaving dangling hydrogen bonds. Based on this finding, we designed out-of-register β-sheet amyloid mimics, which form both cylindrin-like oligomers and fibrils, and these mimics are cytotoxic. Structural and energetic considerations suggest that out-of-register fibrils can readily convert to toxic cylindrins. We propose that out-of-register β-sheets and their related cylindrins are part of a toxic amyloid pathway, which is distinct from the more energetically favored in-register amyloid pathway.X-ray crystallography | BAMs I n contrast to infectious and metabolic disorders, for which researchers can usually uncover the causative entity and the pathway to disease, amyloid diseases have challenged scientists to identify the etiologic agents and the initial pathological events (1-3). Part of the difficulty is that pathways of protein aggregation are diverse, leading to multiple species differing in size, structure, lifetime, and cytotoxicity (4-8). As the most-studied aggregation species, amyloid fibrils have long been associated with devastating human pathologies, including Alzheimer's disease, type II diabetes, and prion disease (2). However, other proteins form amyloid-like aggregates with normal biological functions (9). Studies by NMR, EPR, X-ray diffraction, and scanning mutagenesis have shown that both deleterious and functional amyloid fibrils are made up of extended β-strands running perpendicular to the fibril axis and hydrogen bonded into β-sheets (10-14). The sheets are normally paired into steric zippers, and most often, the strands run parallel to each other and are strictly in-register (10,11,(15)(16)(17). However, in some cases, the strands are antiparallel (18), and some antiparallel fibrils have been found to be more cytotoxic than parallel counterparts (19). Studies of prion (20), HypF-N (21), and Aβ 1-40 (22) indicate that different aggregate morphologies have different levels of cytotoxicity. Therefore, it is important to investigate the various amyloid fibrils from different aggregation pathways, especially those fibrils related to toxic amyloid pathogenesis.Complicating the picture is the variety of oligomers found apparently on the pathways to fibrils, which are more toxic than the fibrils (2, 23, 24). Amyloid oligomers with distinct structural features exhibit different cytotoxicity. A diversity of structural models...

This paper elucidates the teixobactin pharmacophore by comparing the arginine analogue of teixobactin Arg10-teixobactin to seven homologues with varying structure and stereochemistry. The roles of the guanidinium group at position 10, the stereochemistry of the macrolactone ring, and the “tail” comprising residues 1–5 are investigated. The guanidinium group is not necessary for activity; Lys10-teixobactin is more active than Arg10-teixobactin against gram-positive bacteria in minimum inhibitory concentration (MIC) assays. The relative stereochemistry of the macrolactone ring is important; diastereomer l-Thr8,Arg10-teixobactin is inactive, and diastereomer d-allo-Ile11,Arg10-teixobactin is less active. The macrolactone ring is critical; seco-Arg10-teixobactin is inactive. The absolute stereochemistry is not important; the enantiomer ent-Arg10-teixobactin is comparable in activity. The hydrophobic N-terminal tail is important; truncation of residues 1–5 results in loss of activity, and replacement of residues 1–5 with a dodecanoyl group partially restores activity. These findings pave the way for developing simpler homologues of teixobactin with enhanced pharmacological properties.

Interactions among β-sheets occur widely in protein quaternary structure, protein-protein interaction, and protein aggregation and are central in Alzheimer’s and other amyloid-related diseases. This Perspective looks at the structural biology of these important yet under-appreciated interactions from a supramolecular chemist’s point of view. Common themes in the supramolecular interactions of β-sheets are identified and richly illustrated though examples from proteins, amyloids, and chemical model systems. β-Sheets interact through edge-to-edge hydrogen bonding to form extended layers and through face-to-face hydrophobic or van der Waals interactions to form layered sandwich-like structures. Side chains from adjacent layers can fit together through simple hydrophobic contacts or can participate in complementary interdigitation or knob-hole interactions. The layers can be aligned, offset, or rotated. The right-handed twist of β-sheets provides additional opportunities for stabilization of edge-to-edge contacts and rotated layered structures.