Designing heterodimeric two-stranded α-helical coiled-coils -: Effects of hydrophobicity and α-helical propensity on protein folding, stability, and specificity

The E/K coli, a heterodimeric colied-coli, has been designed as a universal peptide capture and delivery system for use in applications such as biosensors and as an expression and affinity purification tag. In this design, heterodimer formation is specified through the placement of charged residues at the e and g positions of the heptad repeat such that the E coli contains all glutamic acid residues at these positions, and the K coli contains all lysine residues at these positions. The affinity and stability of the E/K coli have been modified to allow a greater range of conditions for association and dissociation. Increasing the hydrophobicity of the colied-coli core, by substituting isoleucine for valine, gave increases in stability of 2.81 and 3.73 kcal/mol (0.47 kcal/ mol/substitution). Increasing the alpha-helical propensity of residues outside the core, by substituting alanine for serine, yielded increases in stability of 2.68 and 3.28 kcal/mol (0.41 and 0.45 kcal/mol/substitution). These sequence changes yielded a series of heterodimeric colied-colis whose stabilities varied from 6.8 to 11.2 kcal/mol, greatly expanding their scope for use in protein engineering and biomedical applications.