title: Amyloid-forming peptides selected proteolytically from phage display library.
abstract: We demonstrated that amyloid-forming peptides could be selected from phage-displayed library via proteolysis-based selection protocol. The library of 28-residue peptides based on a sequence of the second zinc finger domain of Zif268, and computationally designed betabetaalpha peptide, FSD-1, was presented monovalently on the surface of M13 phage. The library coupled the infectivity of phage particles to proteolytic stability of a peptide introduced into the coat protein III linker. It was designed to include variants with a strong potential to fold into betabetaalpha motif of zinc finger domains, as expected from secondary structure propensities, but with no structure stabilization via zinc ion coordination. As our primary goal was to find novel monomeric betabetaalpha peptides, the library was selected for stable domains with the assumption that folded proteins are resistant to proteolysis. After less than four rounds of proteolytic selection with trypsin, chymotrypsin, or proteinase K, we obtained a number of proteolysis-resistant phage clones containing several potential sites for proteolytic attack with the proteinases. Eight peptides showing the highest proteolysis resistance were expressed and purified in a phage-free form. When characterized, the peptides possessed proteolytic resistance largely exceeding that of the second zinc finger domain of Zif268 and FSD-1. Six of the characterized peptides formed fibrils when solubilized at high concentrations. Three of them assembled into amyloids as determined through CD measurements, Congo red and thioflavin T binding, and transmission electron microscopy.
authors.affiliation: Laboratory of Protein Engineering, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, Wroclaw 50-137, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Koscielska-Kasprzak
authors.forename: Jacek
authors.initials: J
authors.lastname: Otlewski
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017104:Bacteriophage M13; D002942:Circular Dichroism; D010450:Endopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D000682:Amyloid; D019151:Peptide Library; D010455:Peptides; D010450:Endopeptidases