Identifying Structural Features of Fibrillar Islet Amyloid Polypeptide Using Site-directed Spin Labeling

Abstract

Pancreatic amyloid deposits, composed primarily of the 37-residue islet amyloid polypeptide (IAPP), are a characteristic feature found in more than 90% of patients with type II diabetes. Although IAPP amyloid deposits are associated with areas of pancreatic islet ␤-cell dysfunction and depletion and are thought to play a role in disease, their structure is unknown. We used electron paramagnetic resonance spectroscopy to analyze eight spin-labeled derivatives of IAPP in an effort to determine structural features of the peptide. In solution, all eight derivatives gave rise to electron paramagnetic resonance spectra with sharp lines indicative of rapid motion on the sub-nanosecond time scale. These spectra are consistent with a rapidly tumbling and highly dynamic peptide. In contrast, spectra for the fibrillar form exhibit reduced mobility and the presence of strong intermolecular spin-spin interactions. The latter implies that the peptide subunits are ordered and that the same residues from neighboring peptides are in close proximity to one another. Our data are consistent with a parallel arrangement of IAPP peptides within the amyloid fibril. Analysis of spin label mobility indicates a high degree of order throughout the peptide, although the N-terminal region is slightly less ordered. Possible similarities with respect to the domain organization and parallelism of Alzheimer's amyloid ␤ peptide fibrils are discussed.Pancreatic amyloid deposits have been identified as a hallmark of non-insulin-dependent (type II) diabetes mellitus. More than 90% of patients with type II diabetes exhibit amyloid deposits in areas of ␤-cell dysfunction and death (1, 2). The major component of these deposits is the 37-residue islet amyloid polypeptide (IAPP) 1 (3,4), and multiple lines of investigation have provided experimental evidence implicating human IAPP amyloid deposits in type II diabetes (5-8).Although pancreatic amyloid deposits in a diabetic patient were first identified more than a century ago, structural features of these deposits were not described until recently. Electron microscopy (EM) studies of IAPP deposits revealed the presence of long fibrillar structures (9 -11). Fourier transform infrared and circular dichroism (CD) analysis of IAPP fibrils made from full-length as well as shorter fragments indicates the presence of significant amounts of ␤-sheet structure in the fibrillar form (12, 13). X-ray and electron diffraction studies using aligned fibrils have shown that the peptide chains are arranged in a cross-␤-conformation with the individual ␤-strands perpendicular to the fibril axis (14,15). Beyond these observations, little is known about the molecular details of IAPP in the fibrillar form.Amyloid deposits have been identified in a number of human diseases, such as Alzheimer's disease, Parkinson's disease, and familial amyloidotic polyneuropathy (16). Although the primary structure of amyloid forming proteins varies widely, most amyloid deposits appear to share common characteristics, such as the cross-␤-str...

Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR

“…An alternative, "β-serpentine" model for amylin fibrils has been proposed by Kajava et al (90), based on analysis of the amylin sequence, experimental data on fibril formation by amylin fragments, evidence for parallel β-sheets from EPR (36), STEM data from Goldsbury et al (41), and analogies to known structures of β-helical proteins. The model of Kajava et al contains three β-strands, formed by residues 12-17, 22-27, and 31-37 and separated by short chain-reversing bends in which His18, Asn21, and Thr30 have non-β-strand backbone conformations.…”

“…EPR data for spin-labeled amylin fibrils also indicate a parallel β-sheet structure (36). The EPR measurements were carried out on samples that were polymorphic, based on the reported TEM images (36), and were prepared from peptides that lacked the native disulfide bond.…”

“…The EPR measurements were carried out on samples that were polymorphic, based on the reported TEM images (36), and were prepared from peptides that lacked the native disulfide bond. The solid state NMR data in Figure 10 therefore strengthen the existing evidence for inregister parallel β-sheets in amylin fibrils, particularly for fibrils with the striated ribbon morphology and the native disulfide bond.…”

“…From the standpoint of molecular structure, the defining feature of an amyloid fibril is the presence of cross-β supramolecular structure, meaning that the β-sheets within the fibril are formed by β-strand segments that run approximately perpendicular to the long axis of the fibril and are linked by hydrogen bonds that run approximately parallel to this axis (11)(12)(13). Although determination of the molecular structures of amyloid fibrils is made difficult by their inherent noncrystallinity and insolubility, techniques such as solid state nuclear magnetic resonance (NMR) (12,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), electron paramagnetic resonance (EPR) (34)(35)(36), electron microscopy (37)(38)(39)(40)(41)(42)(43), hydrogen/deuterium exchange (29,(44)(45)(46)(47), scanning mutagenesis (48), chemical crosslinking (27,49,50), and x-ray diffraction of amyloid-like crystals …”

Amyloid of Rnq1p, the basis of the [ PIN + ] prion, has a parallel in-register β-sheet structure

“…Electron spin resonance studies have been used to study amylin (IAPP) and ␣-synuclein (17,18) as well as amyloid of PrP (19). Solid-state NMR has been particularly useful for the elucidation of the structure of amyloids (reviewed in ref.…”