Fortified Coiled Coils: Enhancing Mechanical Stability with Lactam or Metal Staples

Abstract

Coiled coils (CCs) are powerful supramolecular building blocks for biomimetic materials, increasingly used for their mechanical properties. Here, we introduce helix-inducing macrocyclic constraints, so-called staples, to tune thermodynamic and mechanical stability of CCs. We show that thermodynamic stabilization of CCs against helix uncoiling primarily depends on the number of staples, whereas staple positioning controls CC mechanical stability. Inserting a covalent lactam staple at one key force application point significantly increases the barrier to force-induced CC dissociation and reduces structural deformity. A reversible His-Ni²⁺ -His metal staple also increases CC stability, but ruptures upon mechanical loading to allow helix uncoiling. Staple type, position and number are key design parameters in using helical macrocyclic templates for fine-tuning CC properties in emerging biomaterials.

Keywords: coiled coil; lactam; metal coordination; peptide stapling; single-molecule force spectroscopy.

Figure 1

Coiled coil design. A) Peptide sequences. For immobilization, Cys was inserted at the C‐terminus of the A₄ peptides and the N‐terminus of the B₄ peptides. B) Staple design. A helix‐inducing macrocycle was formed via amide bond formation between Lys and Asp side chains (X) or via Ni²⁺ coordination to two His residues (H). C) AFM‐SMFS experiments. Each peptide was coupled to the surface via Cys and the dimeric coiled coil was subjected to shear force (F).

Figure 2

Circular dichroism spectra of stapled coiled coils. A) Spectra of the lactam‐stapled CCs A_4XB₄, A₄B_4X and A_4XB_4X as well as the parent CC A₄B₄. All spectra were measured in PBS (pH 7.4, 23 °C). B) Spectrum of the His‐containing CC A_4HB₄ in the absence and presence of Ni²⁺ (150 μM NiCl₂) in PIPPS‐BS (pH 7.4, 23 °C). The reducing agent tris(2‐carboxyethyl)phosphine (TCEP) was added in all measurements to prevent disulfide bond formation.

Figure 3

AFM‐based SMFS of lactam‐stapled coiled coils. A) Rupture force histograms, obtained at a retract speed of 400 nm s⁻¹. B) Dynamic SMFS data, from three independent experiments, were fitted to the Bell‐Evans model (solid and dashed lines). All measurements were performed in PBS (pH 7.4, 23 °C).

Figure 4

AFM‐based SMFS of the His‐Ni²⁺‐His stapled coiled coil in PIPPS‐BS. A) Rupture force histogram (retract speed 400 nm s⁻¹). B) Dynamic SMFS data (n=3) fitted to the Bell‐Evans model (dashed lines). Ni²⁺: ≥500 μM NiCl₂.