Amyloid fibril formation by circularly permuted and C-terminally deleted mutants

Abstract

The natural N- and C-termini, i.e., the given order of secondary structure segments, are critical for protein folding and stability, as shown by several studies using circularly permuted proteins, mutants that have their N- and C-termini linked and are then digested at another site to create new termini. A previous work showed that circularly permuted mutants of sperm whale myoglobin (Mb) are functional, have native-like folding and bind heme, but are less stable than the wild-type protein and aggregate. The ability of wild-type myoglobin to form amyloid fibrils has been established recently, and because circularly permuted mutations are destabilizing, we asked whether these permutations would also affect the rate of amyloid fibril formation. Our investigations revealed that, indeed, the circularly permuted mutants formed cytotoxic fibrils at a rate higher than that of the wild-type. To further investigate the role of the C-terminus in the overall stability of the protein, we investigated two C-terminally deleted mutant, Mb1–123 and Mb1–99, and found that Mb1–123 formed cytotoxic fibrils at a higher rate than that of the wild-type while Mb1–99 formed cytotoxic fibrils at a similar rate than that of the wild-type. Collectively, our findings show that the native position of both the N-and C-termini is important for the precise structural architecture of myoglobin.