Direct detection of transient alpha-helical states in islet amyloid polypeptide.
The protein islet amyloid polypeptide (IAPP) is a glucose metabolism associated hormone cosecreted with insulin by the beta-cells of the pancreas. In humans with type 2 diabetes, IAPP deposits as amyloid fibers. The assembly intermediates of this process are associated with beta-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 beta-carbon resonances reveal the sampling of alpha-helical states across a continuous stretch comprising approximately 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.
Williamson
Jessica A
JA
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Miranker
Andrew D
AD
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D006801:Humans; D058228:Islet Amyloid Polypeptide; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D000682:Amyloid; D058228:Islet Amyloid Polypeptide; D011994:Recombinant Proteins