Electrostatic interactions control the parallel and antiparallel orientation of alpha-helical chains in two-stranded alpha-helical coiled-coils.

The role of interchain electrostatic interactions in orientating alpha-helical chains to form two-stranded parallel and antiparallel coiled-coils has been investigated. Four disulfide-bridged coiled-coils were designed: parallel coiled-coils with interchain electrostatic attractions (P/A) and repulsions (P/R) and antiparallel coiled-coils with interchain electrostatic attractions (AP/A) and repulsions (AP/R). These coiled-coils were made by air oxidation of two 35-residue peptides with the appropriate heptad repeat (LaEbAcLdEeGfKg or LaAbEcLdKeGfEg) to give the desired interchain electrostatic interactions, and the appropriate position of the cysteine residue (C2 or C33) to give the desired chain orientation. The coiled-coils were characterized by circular dichroism spectroscopy, and their stabilities were assessed by guanidine hydrochloride and urea denaturations. The results indicated that the favored chain orientation, that is, the major disulfide-bridged product formed under benign conditions, was the one that provides interchain electrostatic attractions between oppositely-charged amino acid residues in the e-g' and g-e' positions of the parallel coiled-coil and the g-g' and e-e' positions in the antiparallel coiled-coil. When the electrostatic interactions were similar, the antiparallel coiled-coils were more stable than the parallel coiled-coils. However, the overall stability of the coiled-coils was either increased by interchain electrostatic attractions or decreased by interchain electrostatic repulsions, as determined by urea denaturation. Thus, the order of overall stability of these coiled-coils was AP/A > P/A > AP/R > P/R. This study demonstrates the importance of interchain electrostatic interactions in determining the parallel or antiparallel orientation of alpha-helical chains in two-stranded coiled-coils.