Betabellins 15D and 16D, de novo designed beta-sandwich proteins that haveamyloidogenic properties

The betabellin structure is a de novo designed beta-sandwich protein consis ting of two 32-residue beta-sheets packed against one another by hydrophobi c interactions. D-Amino acid residues are used to energetically favor forma tion of type-I' beta turns. Air oxidation of betabellin 15S (B15S) (HSLTAKI p-kLTFSLAphTYTCAVpkYTAKVSH, where p denotes D-Pro, h denotes D-His, and k d enotes D-Lys) yields betabellin 15D (B15D), a 64-residue disulfide-bridged protein. The amino acid sequence of B15D contains a conformationally constr ained D-Pro residue at the i + 1 position of each type-I' beta turn. To tes t whether D-Pro residues are necessary for folding at these positions, the six D-Pro residues of B15D are replaced by D-Ala residues in betabellin 16D (B16D). Previously, transmission electron microscopy showed that B15D form s unbranched, 35-Angstrom wide fibrils that associate into bundles in 5.0 m M 3-(N-morpholino)propanesulfonate and 250 mM. NaCl at pH 7; under these co nditions, B16D forms ribbon-like assemblies. The B15D fibrils resemble the protofilaments that constitute amyloid fibrils, The present studies show th at both B15D and B16D have characteristics of amyloidogenic proteins: the u nbranched fibrils and ribbons stained with Congo red and displayed a green birefringence, exhibited a cross-beta structure, and bound 1-anilino-8-naph thalenesulfonate. Thus, these de novo designed beta-sandwich proteins shoul d provide useful models for studying the mechanism of amyloid protofilament formation and assembly into amyloid fibrils and for designing potential in hibitors of amyloidogenesis. (C) 2000 Academic Press.