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 (apoC-II) fibril assembly.
Three mutants of apoC-II, K30D, D69K, and K30D-D69K (KDDK) were generated. Thioflavin T (ThT) fluorescence assays showed that the rates of fibril formation by D69K and KDDK were faster than that of wild-type (WT), while K30D did not form fibrils at pH 7.4. Transmission electron microscopy indicated that the fibrils formed by D69K and KDDK had a twisted ribbon morphology similar to WT fibrils.
To further explore the structural similarities and differences underlying amyloid fibrils formed by apoC-II WT and D69K, we performed circular dichroism, X-ray diffraction, hydrogen/deuterium exchange (H/D), and molecular dynamic simulation (MD) analyses. These studies indicate that fibrils formed by D69K are enriched in β sheet structure, and the protected regions (β strands) are very similar to those formed by WT fibrils. H/D and MD studies also suggest that there is perturbation between β strands within D69K fibrils, which is likely to be caused by the repulsion between lysines at positions 30 and 69. Intra and inter ion-pair effects on apoC-II fibril formation were investigated by mixing K30D monomer with WT, D69K and KDDK monomer at pH 7.4. The results show that K30D, which does not form fibrils at pH 7.4, can be incorporated into these other fibril systems as demonstrated by ThT fluorescence, acetic-urea gel and mass spectrometry.
These studies emphasise 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.