Spatial Separation of β-Sheet Domains of β-Amyloid: Disruption of Each β-Sheet by N-Methyl Amino Acids†
In a recent model of ?-amyloid (A?) fibrils, based mainly on solid-state NMR data, a molecular layer consists of two ?-sheets (residues 12?23 and 31?40 of A?1?40), folded onto one another by a connecting ?bend? structure (residues 25?29) in the side-chain dimension. In this paper, we use two N-methyl amino acids to disrupt each of the two ?-sheets individually (2NMe(NTerm), residues 17 and 19; and 2NMe(CTerm), residues 37 and 39), or both of them at the same time (4NMe, with the above four N-methylated residues). Our data indicate that incorporation of two N-methyl amino acids into one ?-sheet is sufficient to disrupt that sheet while leaving the other, unmodified ?-sheet intact and able to form fibrils. We show, however, that disruption of each of the two ?-sheets has strikingly different effects on fibrillogenesis kinetics and fibril morphology. Both 2NMe(NTerm) and 2NMe(CTerm) form fibrils at similar rates, but more slowly than that of unmodified A?1?40. Electron microscopy shows that 2NMe(NTerm) forms straight fibrils with fuzzy amorphous material coating the edges, while 2NMe(CTerm) forms very regular, highly twisted fibrilsin both cases, distinct from the morphology of A?1?40 fibrils. Both 2NMe peptides show a ?CMC? approximately four times greater than that of A?1?40. CD spectra of these peptides also evolve differently in time:? whereas the CD spectra of 2NMe(NTerm) evolve little over 10 days, those of 2NMe(CTerm) show a transition to high ?-sheet content at about day 4?5. We also show that disruption of both ?-sheet domains, as in 4NMe, prevents fibril formation altogether, and renders A?1?40 highly water soluble and monomeric, and with solvent-exposed side chains. In summary, our data show (1) that the two ?-sheet domains fold in a semiautonomous manner, since disrupting each one still allows the other to fold; (2) that disruption of the N-terminal ?-sheet has a more profound effect on fibrillogenesis than disruption of the C-terminal ?-sheet, suggesting that the former is the more critical for the overall structure of the fibril; and (3) that disruption of both ?-sheet domains renders the peptide monomeric and unable to form fibrils.