Using a set of natural coiled-coil proteins as models, a series of recombinant proteins were designed and expressed in E. coli. These proteins contain a consensus coiled-coil sequence as a framework into which were incorporated positively or negatively charged residues at selected positions. A mixed-site genetic strategy was used to generate DNA fragments encoding over 4,000 different combinations of charged residues within the coiled-coil motif. A subset of these genes was used to produce recombinant coiled-coil proteins with well-defined variations in charge pattern and composition. Variants of each sequence containing a unique cysteine at the C-terminus were oxidized to the disulfide-linked dimer, and characterization of their physical properties support the proposed parallel orientation of protein chains. In all cases, equilibrium populations of dimeric and tetrameric structures were observed under physiological conditions, with dimer-to-tetramer dissociation constants in the range of 50-190 riM. Significant differences in complex stability were seen with different charge patterns. Contrary to expectations, no linear relationship was observed between net ionic interaction and any measure of complex stability, arguing that a more subtle set of rules governs these interactions. This work has revealed two important aspects of coiled-coil interactions: the observed relationship between charge interactions and complex stability shows considerable nonlinearity, and the presence of higher order interactions in coiled-coil motifs may be more widespread than is currently suspected. The relationships described here have broad relevance, especially in the areas of protein folding, protein-based materials design, antibody-antigen and receptor-ligand interactions, and rational drug design.
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