Linker peptide acts as an external mechanical clamp in a designed polyprotein

Rational protein design has successfully enhanced the mechanical stability of biomaterials by optimizing the internal topology of force-bearing domains. However, current design strategies predominantly focus on isolated monomers, paying less attention to the immediate oligomeric environment, such as the connecting linkers, modulates mechanical resistance. Here, we report a “Neighbor Peptide Effect” in SuperMyo A339, a recently de novo designed mechanostable protein with an engineered shear topology. Using single-molecule force spectroscopy, we demonstrate that oligomerization via a specific flexible linker (RSGGS) amplifies mechanical stability (∼15%), elevating the unfolding force from ∼350 pN in the monomer to ∼400 pN in the tetramers. Steered molecular dynamics simulations, together with contact-occupancy analysis, elucidate the molecular mechanism underlying this enhancement, revealing a discrete, sequence-specific interaction between the linker and the force-bearing loop of the adjacent domain. Specifically, an interfacial hydrogen bond between linker residue Ser117 and domain residue Lys218, which exhibits a 0.21 occupancy increase at the transition state, acts as an external mechanical clamp. This interaction may restrict loop stochasticity, optimize the pulling geometry and stabilize the core hydrogen-bond network, raising the energy barrier for unfolding without altering the protein’s core fold. These findings indicate that interfacial peptide design may serve as an external means to adjust the mechanical properties of protein.