Contribution of the intrinsic disulfide to the assembly mechanism of islet amyloid.
Amyloidogenesis from soluble protein requires conformational and oligomeric assembly steps. In systems where the precursor protein is natively unfolded, such as islet amyloid polypeptide (IAPP), forces and structural changes relevant to protein unfolding are not thought to participate in the assembly mechanism. Thus, fiber core structure elements should provide the dominant contributions to assembly kinetics. Here we show, however, that residues outside the amyloid core can influence the mechanism of IAPP fiber assembly. IAPP possesses an intramolecular disulfide bond between residues 2 and 7. This short-range disulfide prohibits the N-terminal region from adopting the beta-strand structure of an amyloid. We examined the role of this disulfide in fiber formation by generating a truncated construct (IAPP(8-37)) and a stable reduced form of the full-length protein (IAPP(CAM)). The fiber structures and assembly kinetics of these variants were assessed via optical and mass spectroscopy. Our data confirm that the disulfide does not contribute to the amyloid fiber core structure. Remarkably, however, it plays a central role in the assembly mechanism. First, loss of the disulfide substantially reduces fiber formation by secondary nucleation, i.e., the ability of pre-existing fibers to participate in the formation of new fibers. Second, the bypass of nucleation by seed addition is a two-step process, termed activation. Loss of the disulfide eliminates this two-step nature of seeded kinetics.
Koo
Bon W
BW
Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Ave., New Haven, CT 06520-8114, USA.
Miranker
Andrew D
AD
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D058228:Islet Amyloid Polypeptide; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D000682:Amyloid; D004220:Disulfides; D058228:Islet Amyloid Polypeptide