Higher-order Molecular Packing in Amyloid-like Fibrils Constructed with Linear Arrangements of Hydrophobic and Hydrogen-bonding Side-chains

Various mutants of the protein fragment, barnase module-1 (1-24) were investigated in order to reveal the structural principle of amyloid-like fibrils. By means of circular dichroism spectroscopy, X-ray diffraction, electron microscopy, and thioflavin T binding assay, we found that the molecules containing two β-strands and an intervening turn structure are assembled to form a cross-β structure. Stabilization by both the hydrophobic interactions and hydrogen bonding between the respective paired side-chains on the coupled β-strands was essential for fibril formation. These two types of interaction can also arrange the corresponding residues in lines on both sheet surfaces of protofilaments with a cross-β structure. This leads to the most probable fibril structure constructed with the line-matching interactions between protofilaments. Consideration of the geometrical symmetry resulted in our finding that a limited number of essential models for molecular packing in fibril structure are stable, which would rationally explain the occurrence of two or three morphologies from an identical molecular species. The ribbon-like fibrils exhibited striped texture along the axis, which was assigned to a stacked two-sheet repeat as a structural unit. The comprehensively proposed structural model, that is, the sheet–sheet interaction between left-handed cross-β structures, results in a slightly right-handed twist of β-sheet stacking, which reasonably elucidates the intrinsic sizes of the fibril width and its helical period along the fibril axis, as the bias in the orientation of the hydrogen-bonded β-strand pair at the lateral edge is larger than that at the central protofilament.