Tsunetomo Takei

Effects of chain length of an amphipathic polypeptide carrying the repeated amino acid sequence (LETLAKA)(n) on α-helix and fibrous assembly formation.

Polypeptide α3 (21 residues), with three repeats of a seven-amino-acid sequence (LETLAKA)(3), forms an amphipathic α-helix and a long fibrous assembly. Here, we investigated the ability of α3-series polypeptides (with 14-42 residues) of various chain lengths to form α-helices and fibrous assemblies. Polypeptide α2 (14 residues), with two same-sequence repeats, did not form an α-helix, but polypeptide α2L (15 residues; α2 with one additional leucine residue on its carboxyl terminal) did form an α-helix and fibrous assembly. Fibrous assembly formation was associated with polypeptides at least as long as polypeptide α2L and with five leucine residues, indicating that the C-terminal leucine has a critical element for stabilization of α-helix and fibril formation. In contrast, polypeptides α5 (35 residues) and α6 (42 residues) aggregated easily, although they formed α-helices. A 15-35-residue chain was required for fibrous assembly formation. Electron microscopy and X-ray fiber diffraction showed that the thinnest fibrous assemblies of polypeptides were about 20 Å and had periodicities coincident with the length of the α-helix in a longitudinal direction. These results indicated that the α-helix structures were orientated along the fibrous axis and assembled into a bundle. Furthermore, the width and length of fibrous assemblies changed with changes in the pH value, resulting in variations in the charged states of the residues. Our results suggest that the formation of fibrous assemblies of amphipathic α-helices is due to the assembly of bundles via the hydrophobic faces of the helices and extension with hydrophobic noncovalent bonds containing a leucine.

Role of positions e and g in the fibrous assembly formation of an amphipathic α‐helix‐forming polypeptide

We previously characterized α3, a polypeptide that has a three times repeated sequence of seven amino acids (abcdefg: LETLAKA) and forms fibrous assemblies composed of amphipathic α‐helices. Upon comparison of the amino acid sequences of α3 with other α‐helix forming polypeptides, we proposed that the fibrous assemblies were formed due to the alanine (Ala) residues at positions e and g. Here, we characterized seven α3 analog polypeptides with serine (Ser), glycine (Gly), or charged residues substituted for Ala at positions e and g. The α‐helix forming abilities of the substituted polypeptides were less than that of α3. The polypeptides with amino acid substitutions at position g and the polypeptide KEα3, in which Ala was substituted with charged amino acids, formed few fibrous assemblies. In contrast, polypeptides with Ala replaced by Ser at position e formed β‐sheets under several conditions. These results show that Ala residues at position e and particularly at position g are involved in the formation of fibrous assemblies.

pH responsiveness of fibrous assemblies of repeat‐sequence amphipathic α‐helix polypeptides

We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven‐amino‐acid sequence (LETLAKA)3, forms not only an amphipathic α‐helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α‐helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)3, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)3, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)3; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α‐helix under basic conditions and 6Sα3 formed an α‐helix under acid conditions. In 1M KCl, they both formed α‐helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α‐helices. α‐Helix and fibrous assembly formation by these polypeptides was reversible in a pH‐dependent manner. In contrast, 2S6Sα3 formed an α‐helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α‐helix formation.