Mutational analysis of apolipoprotein C-II amyloid fibril assembly pathways — ASN Events
  1. Schedule
  2. Session Five - Poster Session A
  3. Mutational analysis of apolipoprotein C-II amyloid fibril assembly pathways

Mutational analysis of apolipoprotein C-II amyloid fibril assembly pathways (#161)

Yu Mao 1 , Chai-Lean Teoh 1 , Shuo Yang 1 , Michael D.W Griffin 1 , Paul R Gooley 1 , Geoffrey J Howlett 1
  1. Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, Melbourne, VIC, Australia

The accumulation of amyloid fibrils formed by soluble proteins that misfold and aggregate is associated with a number of devastating human diseases. Recent studies suggest that hydrophobic, aromatic and electrostatic interactions play important roles in amyloid fibril formation and stability.  We have used a mutational strategy to explore the role of an ion-pair [1] (K30 and D69) in apolipoprotein C-II fibril self-assembly.

Three mutants of apoC-II (K30D, D69K, and K30D-D69K) were generated.  Thioflavin T (ThT) fluorescence and centrifugal pelleting assays at pH 7.4 showed that fibril formation by apoC-II(D69K) and apoC-II(K30D-D69K) was 12 and 3-fold faster than wild-type apoC-II, respectively.  ApoC-II(K30D) did not form fibrils at pH 7.4 but readily formed fibrils at pH 3 or 5.  Transmission electron microscopy indicated that the mutant fibrils had a twisted ribbon morphology similar to wild-type fibrils.  The structure and assembly pathway for the mutant fibrils were analysed using fluorescence quenching, circular dichroism and sedimentation velocity analysis.  The effects of mutations on the free pool of apoC-II and the size distribution of amyloid fibrils were interpreted using a new equilibrium model [2].  These results indicate the importance of the putative ion-pair, K30 and D69 in apoC-II self-assembly and the potential of single amino acid substitutions in altering protein aggregation in disease.

  1. Teoh CL, Pham CLL, Todorova N, Hung A, Lincoln CN, et al. (2011) A Structural Model for Apolipoprotein C-II Amyloid Fibrils: Experimental Characterization and Molecular Dynamics Simulations. Journal of Molecular Biology 405: 1246-1266.
  2. Yang S, Griffin MDW, Binger KJ, Schuck P, Howlett GJ (2012) An equilibrium model for linear and closed-loop amyloid fibril formation. Journal of Molecular Biology 421: 364-377.