Protein misfolding and amyloid formation is implicated in debilitating diseases affecting many Australians. Apolipoproteins are a group of lipid-binding proteins associated with amyloid plaque formation, and in lipid-poor states have a propensity to form amyloid fibrils. Apolipoprotein C-II (apoC-II) is found co-localized in atherosclerotic plaques and has more recently been implicated in a novel type of renal amyloidosis. Under standard fibril forming conditions apoC-II forms well-defined amyloid fibrils with cross-β structure that is accompanied by the burial of two charged residues in the fibril core, K30 and D69.
Three variants that probe the importance of this charge pair interaction were generated: D69K and K30D apoC-II, and a reversed ion-pair mutant (D69K/K30D) KDDK apoC-II. Both D69K and KDDK apoC-II mutants form fibrils rapidly, but have reduced fibril stability compared to WT apoC-II. K30D apoC-II is unable to form fibrils under standard conditions but able to at low pH and at high salt and protein concentrations. We studied the possibility of overcoming the inability of K30D apoC-II to form fibrils under standard conditions by mixing K30D apoC-II with other apoC-II variants in order to introduce attractive intra- and inter-subunit interactions within the fibril core. Numerous experiments performed on WT+K30D, D69K+K30D and KDDK+K30D apoC-II mixtures demonstrated successful incorporation of K30D apoC-II into hybrid fibrils. Results show fibrils have increased stability compared to homogenous K30D apoC-II fibrils, and molecular dynamics simulations revealed that hybrid fibril species with increased inter-subunit interactions had increased stability. Given hybrid fibrils exist in other amyloid systems, our work highlights that both intra- and inter-subunit interactions within the amyloid core region of fibrils may be a key determinant of a proteins ability to form amyloid fibrils.