Effects of sequential proline substitutions on amyloid formation by human amylin(20-29)

Amylin, also known as islet amyloid polypeptide (IAPP), is the major protei n component of the fibril deposits found in the pancreas of individuals wit h type II diabetes. The central region of amylin, residues 20-29, has been implicated as a key determinate of amyloid formation. To establish which po sitions are most important for amyloid formation, the wild-type sequence of the 20-29 fragment and a set of 10 variants have been synthesized in which a proline was placed at each position. Proline is energetically unfavorabl e in the extended cross-p structure found in amyloid. If a particular posit ion is critical for amyloid formation, then substitution with a proline sho uld inhibit amyloid formation. A proline substitution at any position inhib ited aggregation and amyloid formation. Substitution of Asn22, Gly24, and r esidues 26-28 had the largest effect. Fourier transform infrared (FTIR) spe ctroscopy showed little secondary structure in these peptides, and transmis sion electron microscopy (TEM) showed mostly amorphous material. The peptid es were much more soluble than the wild-type sequence, and no birefringence was observed with Congo Red staining. Proline substitutions at the N (resi dues 20 and 21) and C termini showed the least effect. These peptides showe d the classic fibril morphology, a significant amount of P-sheet structure, and exhibited green birefringence when stained with Congo Red. The results indicate that residues 22, 24, and 26-28 play a key role in formation of a myloid by amylin. Positions 23 and 25 also appear to be important, but may be less critical than positions 22, 24, and 26-28.