We describe the design and exploration of new buried polar groups to control coiled-coil dimerization. Employing our recently described method for on-resin guanidinylation, we have prepared coiled-coil peptides with a single core guanidine, spaced from the backbone by 1 -3 methylene groups. Heterodimeric mixtures of these sequences with guanidine, amide, and carboxylic acid binding partners form a large number of reasonably stable coiled coils (T_m≥ 60 ℃). A detailed stability trend examination reveals that asparagine/acid pairs are sharply sensitive to acid residue chain length (Asn/Asp much worse than Asn/Glu), while guanidine/acid pairs are largely insensitive. This has been exploited to create orthogonal recognition pairs which establish the capacity to form two distinct heterodimeric coiled coils by simple mixing of four different peptides. One dimer has buried core asparagines, while the other pairs aspartic acid with any of three guanidinylated side chains. Specificity of this behavior is underscored by failure of glutamic acid substituted sequences to perform accordingly. The successful alternate pairs are further characterized by various biophysical methods (circular dichroism, ultracentrifugation, thermal and chemical denaturation, affinity tags).
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