Investigating the Effect of N-Terminal Acetylation on Coiled Coil Peptide Enzymatic Stability, Binding Strength, and Stereocomplexation-Directed Assembly

Coiled coils are well-defined helical peptides that bind strongly and specifically, allowing for their incorporation as a molecular recognition platform in applications like biosensing, drug delivery, and protein purification. Traditional coiled coils contain only L-peptides which are well understood but limited by their poor enzymatic stability. To overcome these enzymatic stability challenges, this work seeks to incorporate the principle of stereocomplexation, which has increased enzymatic stability and binding strength in other peptide materials, into coiled coil design. To align the hydrophobic residues that hold the L- and D-coils (heterochiral) together on the same face of the helix, the seven amino acid repeating pattern of traditional homochiral coiled coils must be redesigned to an eleven amino acid (hendecad) repeating pattern. Beyond this, the design rules for hendecad coiled coil systems are not well defined. Since N-terminal acetylation is known to increase peptide helicity, we anticipated that acetylation would generate more regular helical structures that better align the hydrophobic residues and lead to greater binding strengths, enzymatic stability, and assembly.

To study the effects of N-terminal acetylation in homochiral and heterochiral systems, four coiled coils, each containing a cationic peptide rich in lysine and an anionic peptide rich in glutamic acid were synthesized. Following synthesis, purification, and characterization, circular dichroism (CD) was used to measure the helical content of the individual peptides. The acetylated peptides were more helical than their unacetylated counterparts, with the lysine rich peptides seeing a larger increase in helicity relative to the glutamic acid rich peptides. With respect to binding strength, both acetylated systems saw increased heats of binding relative to their unacetylated analogs, as measured with isothermal titration calorimetry (ITC). To assess stability, the acetylated and unacetylated systems were challenged with a proteolytic enzyme for 24 hours. The homochiral acetylated pair saw a noticeable increase in stability compared to the unacetylated system: at the 6-hour time point, the acetylated system had 76% cationic and 61% anionic coil remaining, while the unacetylated system degraded completely. The heterochiral acetylated pair also saw improved enzymatic stabilities at short times. Notably, after 4 hours in solution, the heterochiral acetylated pair formed a turbid dispersion. The turbidity, which we observed only in the acetylated heterochiral system, suggests that both stereocomplexation and acetylation strengthen intermolecular interactions. Together, we found that N-terminal acetylation results in more helical peptides which bind stronger and are more resistant to proteolytic degradation. These studies expand our knowledge of the design rules for hendecad coiled coils to produce stronger binding, more enzymatically stable molecular recognition platforms.