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Amyloid fibrils result from the self-assembly of proteins into large aggregates with fibrillar morphology and common structural features. These fibrils form the major component of amyloid plaques that are associated with a number of common and debilitating diseases, including Alzheimer's disease. While a range of unrelated proteins and peptides are known to form amyloid fibrils, a common feature is the formation of aggregates of various sizes, including mature fibrils of differing length and/or structural morphology, small oligomeric precursors, and other less well-understood forms such as amorphous aggregates. These various species can possess distinct biochemical, biophysical, and pathological properties. Sedimentation velocity analysis can characterize amyloid fibril formation in exceptional detail, providing a particularly useful method for resolving the complex heterogeneity present in amyloid systems. In this chapter, we describe analytical methods for accurate quantification of both total amyloid fibril formation and the formation of distinct amyloid structures based on differential sedimentation properties. We also detail modern analytical ultracentrifugation methods to determine the size distribution of amyloid aggregates. We illustrate examples of the use of these techniques to provide biophysical and structural information on amyloid systems that would otherwise be difficult to obtain., (© 2015 Elsevier Inc. All rights reserved.)

Plasma apolipoproteins show alpha-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19-37 and 57-74 with reduced amide proton exchange rates compared to monomeric apoC-II. The C-terminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II(56-76). Synthetic apoC-II(56-76) readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to full-length apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60-70. We postulate that the ability of apoC-II(60-70) to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo.