Folded-state compatibility and unfolded-state constraint govern staple-based stabilization: guidelines from a coiled-coil model.

Peptide stapling has emerged as a powerful strategy for stabilizing protein conformation, improving proteolytic resistance, and enhancing biomolecular recognition. Yet design principles for selecting staple sites remain elusive, so advances in stapling have depended largely on trial and error. Here we establish quantitative guidelines for staple placement by exploiting the well-defined geometry of an α-helical coiled coil to compare alternative staple sites in a controlled way. Using both experimental measurements and molecular simulations, we find that (1) staples that link residue pairs that normally form interhelical salt bridges yield greater stabilization than those linking non-salt-bridged pairs; (2) N-terminal staples are more stabilizing than C-terminal staples, where an existing interhelical disulfide constraint reduces their impact; and (3) mismatches between the staple length and site spacing can cause destabilization by forcing the structure into a compressed, non-native geometry. Together, these results show that staple-based stabilization depends on two underlying factors: unfolded-state constraint (the entropic advantage gained when the staple limits how far apart the linked residues can separate in the unfolded ensemble) and folded-state compatibility (how well the staple's maximum accessible span matches the native separation of those residues in the folded structure). These principles provide a predictive framework for rational stapled peptide design, offering a path beyond empirical screening toward principle-guided development of stabilized peptide therapeutics.