CONTROLLED FORMATION OF MODEL HOMODIMER AND HETERODIMER COILED-COIL POLYPEPTIDES

Sequence-simplified coiled coil polypeptides were synthesized and their folding properties characterized in order to define the role of charged border residues at the coiled coil interface for the controlled formation of homodimer and heterodimer structures. Three peptides were designed to form parallel coiled coils with valine and leucine occupying the hydrophobic interface positions a and d, respectively, of the heptad repeat abcdefg. The polypeptide designated E/K42, with the heptad repeat sequence VSSLESK, contained glutamate and lysine in the interface border positions e and g, respectively, and was designed to form a coiled coil homodimer at neutral pH. Two other polypeptides, designated E/E35 and K/K35, have the heptad repeats VSSLESE and VSSLKSK, respectively. E/E35 contains only glutamic acid at both e and g positions; K/K35, only lysine. E/E35 and K/K35 were designed to form a stable coiled coil heterodimer when combined at neutral pH. All three polypeptides were prepared by solid-phase synthesis and purified by reverse-phase high-performance liquid chromatography followed by size-exclusion chromatography. E/K42 formed a stable dimeric coiled coil structure as determined by circular dichroism and size-exclusion chromatography. The alpha-helical content of E/K42 was highest at neutral pH and decreased at extremes of pH. The alpha-helical structure of E/K42 at micromolar concentrations had a T(m) of 62-65-degrees-C and exhibited a concentration dependence of thermal denaturation consistent with dimer formation. In contrast to results with E/K42, a mixture of E/E35 and K/K35, but neither alone, forms alpha-helix at neutral pH. At micromolar concentrations the E/E35:K/K35 mixture had a T(m) of 60-63-degrees-C and eluted as a dimer in gel filtration chromatography, suggesting that the peptides form a stable coiled coil heterodimer. Hence, for two peptides, each with a single type of charged residue at all e and g positions but oppositely charged with respect to each other, heterodimers can be stabilized and homodimers destabilized by charge attraction and repulsion, respectively. In support of this conclusion, the acidic polypeptide E/E35 forms alpha-helical structure at low pH, while the basic polypeptide K/K35 forms alpha-helical structure at high pH. The results argue that positions e and g of the heptad repeat of coiled coil peptides can be varied to control heterodimer and homodimer formation.