Fig. 1. Experimental setup. (A) Structure of a heterodimeric coiled coil in the helical wheel representation. (B) Sequences of the coiled coil heterodimers used. The sequences used in the SMFS experiments contained two glycine residues at each terminus as well as a cysteine residue for site-specific immobilization. Only one glycine and no cysteine was present in the sequences used for the SMD simulations. (C) Geometry of force application for studying the mechanical stability of coiled coils under shear loading. The force was applied at the N-terminus of peptide A₄ and at the C-termini of peptides B₄, B_3.5 and B₃, utilizing cysteine residues introduced at the respective termini. In the SMFS experiments, the spring represents a poly(ethylene glycol) linker, which was used to couple the individual peptides to the surface and the AFM cantilever. In the SMD simulations, two virtual harmonic springs were introduced. The distal end of the spring present at the N-terminus of A₄ was fixed, whereas the distal end of the spring attached at the C-terminus of the B peptides was displaced parallel to the helical axis at a constant speed. The initial structure of each coiled coil was produced using Avogadro48 and equilibrated before the SMD simulations.