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Sedimentation Velocity Analysis of Flexible Macromolecules: Self-Association and Tangling of Amyloid Fibrils

Christopher A. MacRaild^*, Danny M. Hatters^*, Lynne J. Lawrence and Geoffrey J. Howlett^*

^* Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria 3010, Australia; and Commonwealth Scientific & Industrial Research Organisation, Parkville, Victoria 3052, Australia

Correspondence: Address reprint requests to Geoffrey J. Howlett, Tel.: +61-3-8344-7632; Fax: +61-3-9347-7730; E-mail: ghowlett{at}unimelb.edu.au.

A novel bead modeling technique has been developed for the analysis of the sedimentation velocity behavior of flexible fibrils. The method involves the generation of a family of bead models representing a sample of the conformations available to the molecule and the calculation of the sedimentation coefficients of these models by established techniques. This approach has been used to investigate the size distribution of amyloid fibrils formed by human apolipoprotein C-II (apoC-II). ApoC-II fibrils have a simple and homogeneous ribbon morphology with no evidence of amorphous aggregation. Freshly prepared apoC-II forms fibrils with systematically larger sedimentation coefficients upon increasing protein concentration (modes of 100, 300, and 800 for apoC-II concentrations of 0.3, 0.7, and 1.0 mg/mL, respectively). The sedimentation coefficient distributions are not affected by rotor speed, and are not significantly changed by dilution once the fibrils are formed. The kinetics of aggregation (1 mg/mL apoC-II) as assessed using thioflavin T and preparative pelleting assays reveal that monomeric apoC-II is depleted after 12 h incubation at room temperature. In contrast, the sedimentation coefficient distribution of fibrils continues to grow larger over a period of 48 h to an average value of 800 S. Calculations using the bead modeling procedure suggest maximum sedimentation coefficients for individual apoC-II fibrils to be around 100 S. The larger experimentally observed sedimentation coefficients for apoC-II fibrils indicate an extensive and time-dependent tangling or association of the fibrils to form specific networks.

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