Direct detection of transient a-helical states in isletamyloid polypeptideJESSICA A. WILLIAMSON AND ANDREW D. MIRANKERDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA(RECEIVED August 7, 2006; FINAL REVISION October 11, 2006; ACCEPTED October 16, 2006)AbstractThe protein islet amyloid polypeptide (IAPP) is a glucose metabolis m a ssociated hormone cosecretedwith insulin by the b-cells of the pancreas. In humans w ith type 2 diabetes, IAPP deposits as amyloidfibers. The assembly intermediates of thi s process are associated with b-cell death. Here, we examinethe rat IAPP sequence variant under physiological solution conditions. Rat IAPP is mechanisticallyinformative for fibrillogenesis, as it samples intermediate-like states but does not progress to formamyloid. A central challe nge was the development o f a bacterial expression system to generate iso-topically labeled IAPP without terminal tags, but which does include a eukaryotic post-translationalmodification. While optical spectroscopy shows IAPP to be natively unfolded, NMR chemical shifts ofbackbone and b-carbon resonances reveal the sampling of a-helical states across a continuous stretchcomprising ;40% of the protein. In addition, the manifestation of nonrandom coil chemical shifts isconfirmed 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, sug-gesting a functional role for t his conformational bias. The inability of rat IAPP to s elf a ssemble can beascribed, in part, to several slowly exchanging conformations evident as multiple chemical shift assign-ments in the immediate vicinity of three p roline residues residing outside of this helical region.Keywords: amylin; amyloid; IAPP; NMR; a-helix; type 2 diabetes; protein folding; intrinsic disorderSupplemental material: see www.proteinscience.orgIslet amyloid polypeptide (IAPP) is a 37-residue peptidehormone cosecreted with insulin by the endocrine b-cel lsof the pancreas (Jaikaran and Clark 2001). This protein isa member of the calcitonin gene-related peptide (CGRP)family (Muff et al. 2004). These proteins are hormones,display sequence homology, and are further character-ized by C-terminal amidation and a tight disulfide bondseparated by five residues. T he hormonal a ction s a scrib edto IAPP are diverse (Hay et al. 2004) and include, forexample, control of gastric emptying and paracrine/autocrine signaling upon insulin release by the b -cell(Cooper 1994).In solution, IAPP is widely regarded as a nativelyunstructured protein (Kayed et al. 1999; Dunker et al.2001; Jaikaran and Clark 2001; Padrick and Miranker2001). However, in humans with type 2 diabetes, IAPPundergoes conformational changes to form b-sheets or-ganized into amyloid fi bers. The process of IAPP amyloidformation is correlated with pancreatic b-cell dysfunction(Hoppener et al. 2000; Hull et al. 2004) including anincreased rate of apoptosis and a reduction in b-cell mass.At a minimum, amyloid cytotoxicity contributes to dia-betes pathology by increasing the requirement for insulinreplacement therapy. A number of animal models supportthese conclusions (Westermark et al. 2000; Wang et al. 2001;Butler et al. 2004). For example, the HIP rat is transgenicfor human IAPP and spontaneously develops pathologyps0624869 Williamson and Miranker ARTICLE RAReprint requests to: Andrew D. Miranker, Department of MolecularBiophysics and Biochemistry, Yale University, 260 Wh itney Avenue,New Haven, CT 06520-8114, USA; e-mail: [email protected];fax: (203) 432-5175.Article published online ahead of print. Arti cle and publi cation dateare at http://www.prot einscience.org/cgi/doi /10.1110/ ps.062486907.110Protein Science (2007), 16:110–117.Published by Cold Spring Harbor Laboratory Press.Copyright Ó 2007 The Protein SocietyJOBNAME: PROSCI 16#1 2006 PAGE: 1 OUTPUT: Wednesday December 13 12:52:48 2006csh/PROSCI/127806/ps0624869typical of human diabetes (Butler et al. 2004) includingelements of b-cell dysfunction and systemic insulin re-sistance. The latter result is particularly remarkable as itenables the investigators to suggest that IAPP associated b-cell impairment can be causal to systemic manifestations ofthis disease (Matveyenko and Butler 2006).Amyloid fibers themselves are highly ordered, allow-ing for recent structural studies in a number of systemsincluding Ab from Alzheimer’s disease (Petkova et al.2005) and Sup35 from yeast (Nelson et al. 2005). How-ever, the kinetics of amyloid assembly are nucleationdependent, resulting in poorly populated, transient, andpartially structured intermediates (Padrick and Miranker2002; Uversky and Fink 2004; Chiti and Dobson 2006).Structur al studies of t hese intermediate sta te s are there-fore challenging. The principle approaches to this challengehave used mutagenesis to evaluate the rate and capacity ofa protein to self assemble. For example, exhaustive muta-genesis of a model peptide (Lopez de la Paz et al. 2005) hasenabled amyloid determinants to be characterized in a man-ner suitabl e for proteome analysis. Mutagenesis can also beused in a site-directed manner to stabilize an analog of anintermediate state. For folded and globular amyloid precur-sors, such as b-2 microglobulin, this has enabled mecha-nistic insights at atomic resolution (Eakin et al. 2006).In the case of IAPP, the rat seque nce variant is arelevant and useful tool for molecular insight. Rat IAPPdiffers from human at six residues (Fig. 1) and does notform amyloid fibers (Westermark et al. 1990). Theprinciple, but not exclusive, origin of this difference isthree proline residues at positions 25, 28, and 29; forexample, human IAPP mutated to contain the three pro-line residues aggregates, but to a greatly reduced extent(Green et al. 2003). Our own investigations show that ratIAPP adopts structur es similar to prefibrillar states ofhuman IAPP (Padrick and Miranker 2001). Notably, bothrat and human IAPP in physiological buffer demonstratefluorescence resonance energy transfer between r esiduePhe15 and Tyr37, indicative of long-range structure. Inhuman IAPP, t his interaction bec omes more robust upontransition to the fiber state. Recentl y, we have shown thatboth rat and human IAPP adopt comparable structuresupon binding phopholipid bilayers (Knight et al. 2006),namely, they both cooperatively assemble