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Direct detection of transient a-helical states in islet amyloid polypeptide



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JOBNAME PROSCI 16 1 2006 PAGE 1 OUTPUT Wednesday December 13 12 52 48 2006 csh PROSCI 127806 ps0624869 Direct detection of transient a helical states in islet amyloid polypeptide JESSICA A WILLIAMSON AND ANDREW D MIRANKER Department of Molecular Biophysics and Biochemistry Yale University New Haven Connecticut 06520 8114 USA R ECEIVED August 7 2006 F INAL R EVISION October 11 2006 ACCEPTED October 16 2006 Abstract The protein islet amyloid polypeptide IAPP is a glucose metabolism associated hormone cosecreted with insulin by the b cells of the pancreas In humans with type 2 diabetes IAPP deposits as amyloid fibers The assembly intermediates of this process are associated with b cell death Here we examine the rat IAPP sequence variant under physiological solution conditions Rat IAPP is mechanistically informative for fibrillogenesis as it samples intermediate like states but does not progress to form amyloid A central challenge was the development of a bacterial expression system to generate isotopically labeled IAPP without terminal tags but which does include a eukaryotic post translational modification While optical spectroscopy shows IAPP to be natively unfolded NMR chemical shifts of backbone and b carbon resonances reveal the sampling of a helical states across a continuous stretch comprising 40 of the protein In addition the manifestation of nonrandom coil chemical shifts is confirmed by the relative insensitivity of the amide proton chemical shifts to alterations in temperature Intriguingly the residues displaying helical propensity are conserved with the human sequence suggesting a functional role for this conformational bias The inability of rat IAPP to self assemble can be ascribed in part to several slowly exchanging conformations evident as multiple chemical shift assignments in the immediate vicinity of three proline residues residing outside of this helical region Keywords amylin amyloid IAPP NMR a helix type 2 diabetes protein folding intrinsic disorder Supplemental material see www proteinscience org Islet amyloid polypeptide IAPP is a 37 residue peptide hormone cosecreted with insulin by the endocrine b cells of the pancreas Jaikaran and Clark 2001 This protein is a member of the calcitonin gene related peptide CGRP family Muff et al 2004 These proteins are hormones display sequence homology and are further characterized by C terminal amidation and a tight disulfide bond separated by five residues The hormonal actions ascribed to IAPP are diverse Hay et al 2004 and include for example control of gastric emptying and paracrine Reprint requests to Andrew D Miranker Department of Molecular Biophysics and Biochemistry Yale University 260 Whitney Avenue New Haven CT 06520 8114 USA e mail Andrew miranker yale edu fax 203 432 5175 Article published online ahead of print Article and publication date are at http www proteinscience org cgi doi 10 1110 ps 062486907 110 autocrine signaling upon insulin release by the b cell Cooper 1994 In solution IAPP is widely regarded as a natively unstructured protein Kayed et al 1999 Dunker et al 2001 Jaikaran and Clark 2001 Padrick and Miranker 2001 However in humans with type 2 diabetes IAPP undergoes conformational changes to form b sheets organized into amyloid fibers The process of IAPP amyloid formation is correlated with pancreatic b cell dysfunction Hoppener et al 2000 Hull et al 2004 including an increased rate of apoptosis and a reduction in b cell mass At a minimum amyloid cytotoxicity contributes to diabetes pathology by increasing the requirement for insulin replacement therapy A number of animal models support these conclusions Westermark et al 2000 Wang et al 2001 Butler et al 2004 For example the HIP rat is transgenic for human IAPP and spontaneously develops pathology Protein Science 2007 16 110 117 Published by Cold Spring Harbor Laboratory Press Copyright 2007 The Protein Society ps0624869 Williamson and Miranker ARTICLE RA JOBNAME PROSCI 16 1 2006 PAGE 2 OUTPUT Wednesday December 13 12 52 48 2006 csh PROSCI 127806 ps0624869 Intrinsic structure of IAPP typical of human diabetes Butler et al 2004 including elements of b cell dysfunction and systemic insulin resistance The latter result is particularly remarkable as it enables the investigators to suggest that IAPP associated bcell impairment can be causal to systemic manifestations of this disease Matveyenko and Butler 2006 Amyloid fibers themselves are highly ordered allowing for recent structural studies in a number of systems including Ab from Alzheimer s disease Petkova et al 2005 and Sup35 from yeast Nelson et al 2005 However the kinetics of amyloid assembly are nucleation dependent resulting in poorly populated transient and partially structured intermediates Padrick and Miranker 2002 Uversky and Fink 2004 Chiti and Dobson 2006 Structural studies of these intermediate states are therefore challenging The principle approaches to this challenge have used mutagenesis to evaluate the rate and capacity of a protein to self assemble For example exhaustive mutagenesis of a model peptide Lopez de la Paz et al 2005 has enabled amyloid determinants to be characterized in a manner suitable for proteome analysis Mutagenesis can also be used in a site directed manner to stabilize an analog of an intermediate state For folded and globular amyloid precursors such as b 2 microglobulin this has enabled mechanistic insights at atomic resolution Eakin et al 2006 In the case of IAPP the rat sequence variant is a relevant and useful tool for molecular insight Rat IAPP differs from human at six residues Fig 1 and does not form amyloid fibers Westermark et al 1990 The principle but not exclusive origin of this difference is three proline residues at positions 25 28 and 29 for example human IAPP mutated to contain the three proline residues aggregates but to a greatly reduced extent Green et al 2003 Our own investigations show that rat IAPP adopts structures similar to prefibrillar states of human IAPP Padrick and Miranker 2001 Notably both rat and human IAPP in physiological buffer demonstrate fluorescence resonance energy transfer between residue Phe15 and Tyr37 indicative of long range structure In human IAPP this interaction becomes more robust upon transition to the fiber state Recently we have shown that both rat and human IAPP adopt comparable structures upon binding phopholipid bilayers Knight et al 2006 namely they both cooperatively assemble into oligomeric a helical states


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