| Abstract
| - Coiled coils, which mediate the associations and regulate the functions of various proteins, have arepresentative amino acid sequence of (defgabc)n heptad repeats and usually have hydrophobic residues at thea and d positions. We have designed a triple-stranded parallel α-helical coiled coil, in which the amino acidsequence is YGG(IEKKIEA)4. To construct a peptide that undergoes metal ion induced self-assembly into atriple-stranded coiled coil, we engineered a metal binding site in the hydrophobic core of the coiled coil. Wereplaced two Ile residues of the third heptad with His residues. The peptide had a random structure in aqueoussolution. In contrast, in the presence of a transition metal ion, the peptide exhibited an α-helical conformation.The metal-complexed peptide was triple stranded and had a parallel orientation, as determined by sedimentationequilibrium and fluorescence quenching analyses. Metal ion titrations monitored by circular dichroism revealedthat the dissociation constants, Kd, were 35 ± 1 μM for Co(II), 5.0 ± 0.3 μM for Ni(II), 17 ± 1 μM for Cu(II),and 23 ± 2 μM for Zn(II). The Ni(II) binds to the His residues, as judged by both pH titration monitored bycircular dichroism and metal ion titration monitored by nuclear magnetic resonance. The highest affinity forNi(II) suggests that the metal binding site has six-coordinated octahedral geometry. Thus, the peptide is auseful tool to control the associations of functional domains attached to the peptide.
|
