Ester to amide switch peptides provide a simple method for preparing monomeric islet amyloid polypeptide under physiologically relevant conditions and facilitate investigations of amyloid formation.
A major issue in studies of amyloid formation is the difficulty of preparing the polypeptide of interest in an initially monomeric state under physiologically relevant conditions. This is particularly problematic for polypeptides which are natively unfolded in their unaggregated state, and perhaps the most challenging such system is islet amyloid polypeptide (Amylin), the causative agent of amyloid formation in type-2 diabetes. Preparation of islet amyloid polypeptide with the Ser-19 Ser-20 amide bond replaced by an ester circumvents these problems. The modified peptide is unstructured and monomeric at slightly acidic pH's as judged by analytical ultracentrifugation, gel filtration, dynamic light scattering, and CD. A rapid pH jump leads to deprotonation of the Ser-20 amide group, and a subsequent rapid O to N acyl shift regenerates normal human islet amyloid polypeptide. The half time, t(1/2), for the conversion to normal islet amyloid polypeptide is 70 s at pH 7.4. The amyloid fibrils which are formed by the regenerated islet amyloid polypeptide are indistinguishable from those formed by the wild type polypeptide. The approach allows studies of amyloid formation by islet amyloid polypeptide to be carried out from a well-defined, physiologically relevant starting state in the absence of denaturants or organic cosolvents.
Cao
Ping
P
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, USA.
Raleigh
Daniel P
DP
D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D000682:Amyloid / Q000032:analysis* / Q000737:chemistry*; D004952:Esters / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D058228:Islet Amyloid Polypeptide; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding
D000577:Amides; D000682:Amyloid; D004952:Esters; D058228:Islet Amyloid Polypeptide; D010455:Peptides