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

Abstract

The 37-residue amylin peptide, also known as islet amyloid polypeptide, forms fibrils that are the main peptide or protein component of amyloid that develops in the pancreas of type 2 diabetes patients. Amylin also readily forms amyloid fibrils in vitro that are highly polymorphic under typical experimental conditions. We describe a protocol for the preparation of synthetic amylin fibrils that exhibit a single predominant morphology, which we call a striated ribbon, in electron microscope and atomic force microscope images. Solid state nuclear magnetic resonance (NMR) measurements on a series of isotopically labeled samples indicate a single molecular structure within the striated ribbons. We use scanning transmission electron microscopy and several types of one-dimensional and two-dimensional solid state NMR techniques to obtain constraints on the peptide conformation and supramolecular structure in these amylin fibrils, and derive molecular structural models that are consistent with the experimental data. The basic structural unit in amylin striated ribbons, which we call the protofilament, contains four-layers of parallel β-sheets, formed by two symmetric layers of amylin molecules. The molecular structure of amylin protofilaments in striated ribbons closely resembles the protofilament in amyloid fibrils with similar morphology formed by the 40-residue β-amyloid peptide that is associated with Alzheimer's disease. Keywordsislet amyloid polypeptide; type 2 diabetes; amyloid fibril structure; electron microscopy; atomic force microscopy Amylin, also called islet amyloid polypeptide or IAPP, is a 37-residue peptide that is cosecreted with insulin by the β-cells of pancreas. The normal biological effects of amylin secretion include inhibition of glucagon secretion and reduction of the rate of gastric emptying, effects that contribute to the maintenance of postprandial glucose homeostasis (1). Amylin is the major peptide or protein component of the islet amyloid found in the pancreas of approximately 90% of type 2 (or non-insulin-dependent) diabetes patients (2,3). Fibrillar amylin has been shown * Corresponding author: Dr. Robert Tycko, National Institutes of Health, Building 5, Room 112, Bethesda, MD 20892-0520. phone 301-402-8272. fax 301-496-0825. e-mail robertty@mail.nih.gov.Supporting Information Available HPLC chromatograms from the purification of both reduced and oxidized amylin by reverse-phase chromatography ( Figure S1); FPLC chromatograms from the purification of both human and rodent amylin by size-exclusion chromatography ( Figure S2); 1D 13 C spectra of amylin fibrils with various isotopic labeling patterns, in lyophilized and rehydrated conditions ( Figures S3-S5); Table of 13 C NMR chemical shifts in amylin fibrils, TALOS predictions of backbone torsion angles based on these shifts, and torsion angles determined from 15 N fpRFDR-CT measurements (Table S1). This material is freely available on the World Wide Web at http://www.acs.org. to be toxic to cultured β-cells (4), and has been propo...

Solid‐State NMR Studies on Supramolecular Chemistry

“…Again, several types of oneand two-dimensional SSNMR techniques have been used to obtain constraints on the peptide conformation and supramolecular structure in amylin fibrils, a 37-residue peptide also called islet amyloid polypeptide or IAPP, and to derive molecular structural models that are consistent with the experimental data. 52 SSNMR measurements on a series of isotopically labeled samples indicate a single molecular structure within the striated ribbons which contains four layers of parallel β-sheets, formed by two symmetric layers of amylin molecules. …”

α-helix to β-hairpin transition of human amylin monomer

“…[8][9][10][11] For fully formed aggregates, Kajava et al 12 proposed a structure consisting of a three-strand model, with β-strands assigned to residues 12-17, 22-27, and 31-37. Luca et al 11 used solid-state NMR to propose a strand-loop-strand morphology for the hIAPP monomers in the fibrils, with residues 8-17 and 28-37 forming the β-sheets. That morphology is also supported by timeresolved two-dimensional infrared spectroscopy (2DIR) 10 and electron paramagnetic resonance (EPR) studies.…”

Tracking Fiber Formation in Human Islet Amyloid Polypeptide with Automated 2D-IR Spectroscopy

“…The hIAPP fibril structure has recently been reported and consists of four stacked β-sheets of about equal size. 7 The inner two sheets are protected from solvent and thus will have amide I frequencies 5-10 cm −1 higher in frequency. Because the shift is insufficient to resolve the individual β-sheets, stacking will appear as an increase in the line width.…”