title: Charge and charge-pair mutations alter the rate of assembly and structural properties of apolipoprotein C-II amyloid fibrils.
abstract: The misfolding, aggregation, and accumulation of proteins as amyloid fibrils is a defining characteristic of several debilitating diseases. Human apolipoprotein C-II (apoC-II) amyloid fibrils are representative of the fibrils formed by a number of plasma apolipoproteins implicated in amyloid-related disease. Previous structural analyses identified a buried charge pair between residues K30 and D69 within apoC-II amyloid fibrils. We have investigated the effects of amino acid substitutions of these residues on apoC-II fibril formation. Two point mutations of apoC-II, D69K and K30D, as well as a reversed ion-pair mutant containing both mutations (KDDK) were generated. Fibril formation by the double mutant, apoC-II KDDK, and apoC-II D69K was enhanced compared to that of wild-type (WT) apoC-II, while apoC-II K30D lacked the ability to form fibrils under standard conditions. Structural analyses showed that WT apoC-II, apoC-II D69K, and apoC-II KDDK fibrils have similar secondary structures and morphologies. Size distribution analyses revealed that apoC-II D69K fibrils have a broader range of fibril sizes while apoC-II KDDK fibrils showed an increased frequency of closed fibrillar loops. ApoC-II D69K fibrils exhibited reduced thioflavin T binding capacity compared to that of fibrils formed by WT apoC-II and apoC-II KDDK. These results indicate that specific charge and charge-pair mutations within apoC-II significantly alter the ability to form fibrils and that position 69 within apoC-II plays a key role in the rate-limiting step of apoC-II fibril formation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, Victoria 3010, Australia.
authors.forename: Yu
authors.initials: Y
authors.lastname: Mao
authors.forename: Chai Lean
authors.initials: CL
authors.lastname: Teoh
authors.forename: Shuo
authors.initials: S
authors.lastname: Yang
authors.forename: Courtney O
authors.initials: CO
authors.lastname: Zlatic
authors.forename: Zachary K
authors.initials: ZK
authors.lastname: Rosenes
authors.forename: Paul R
authors.initials: PR
authors.lastname: Gooley
authors.forename: Geoffrey J
authors.initials: GJ
authors.lastname: Howlett
authors.forename: Michael D W
authors.initials: MD
authors.lastname: Griffin
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D053304:Apolipoprotein C-II / Q000737:chemistry* / Q000235:genetics; D005453:Fluorescence; D009154:Mutation
chemical_list: D000682:Amyloid; D053304:Apolipoprotein C-II