Development of High Affinity Anti-S2 Peptides for Stabilizing the Myosin Coiled Coil

Abstract

Myosin subfragment-2 (S2), like tropomyosin, is a partially unstable coiled coil. Familial hypertrophic cardiomyopathy mutations are found in clusters near the N-terminal side of S2 suggesting that they influence the coiled coils’ stability, since deletion mutations such as delE930 and delE927 are associated with very high rates of sudden cardiac arrest based on analysis of inheritance in family pedigrees. These deletion mutations will destabilize the coiled coil formation by disrupting the heptad repeat. Synthetic peptides designed to bind with high affinity and specificity to this region of S2 can stabilize the coiled coil structure thereby counteracting the mutations. To this end, synthetic peptides were designed and tested with the aid of computational simulations. Simulated force spectroscopy of myosin S2 in the presence of the candidate peptide sequences were evaluated based on the fitted contour length from a worm-like chain model when the S2 dimer was separated by an applied force. The sequences with the longest contour lengths were synthesized and used to titrate human cardiac S2 labeled with a time-resolved fluorescent probe that was quenched upon anti-S2 peptide binding. The highest affinity anti-S2 peptide had a dissociation constant of 100 ± 20 nM to wildtype and 85 ± 10 nM to the delE930 S2 and a stoichiometry of binding consistent with atomic modeling of one anti-S2 peptide per S2 dimer. S2 with a charge change, E924K, yielded a dissociation constant of 800 ± 240 nM demonstrating the specificity of the anti-S2 peptide. A competitive ELISA assay demonstrated that the anti-S2 peptide bound to unlabeled S2 with a dissociation constant of 21 ± 8 nM, while an isomer of the anti-S2 peptide had a dissociation constant of 100 ± 25 nM. These short peptides promise significant practical applications.