Mechanical Polyprotein Assembly Using Sfp and Sortase‐Mediated Domain Oligomerization for Single‐Molecule Studies

Single‐molecule force spectroscopy (SMFS) with the atomic force microscope (AFM) provides remarkable details on the energy landscapes governing protein (un)folding and intermolecular complex dissociation. In such experiments, multidomain polyproteins consisting of multiple copies of independently foldable domains provide internal controls identifiable by characteristic contour length increments, unfolding forces, and/or unfolding substeps. Here, a new approach to polyprotein synthesis is presented relying on posttranslational enzyme‐mediated oligomerization of domains. Mutant variants of immunoglobulin 27 (I27) and a bacterial cellulose binding module (CBM) fused to an Ig‐like X‐module (XMod), and a mechanostable receptor called Dockerin (Doc) are produced with complementary peptide tags. By utilizing 4′‐phosphopantetheinyl transferase and Sortase A, the system enables I27‐domain oligomerization into polyproteins of varying lengths followed by C‐terminal capping with mechanostable Doc. The number of oligomerized domains per molecule, the unfolding forces, and the complex rupture forces of posttranslationally assembled polyproteins are characterized using >40 h automated AFM-SMFS with a Cohesin (Coh)‐modified cantilever. Use of the Coh-Doc interaction to unfold polyproteins provides a high yield of ≈ 3800 specific single‐molecule interaction curves. This approach is advantageous for assembly of polyproteins from domains that lack proper folding or are insoluble in a polyprotein format.