Single-domain VHH antibodies are promising reagents for medical therapy. A conserved disulfide bond within the VHH framework region is known to be critical for thermal stability, however, no prior studies have investigated its influence on the stability of VHH antibody–antigen complexes under mechanical load. Here, we used single-molecule force spectroscopy to test the influence of a VHH domain’s conserved disulfide bond on the mechanical strength of the interaction with its antigen mCherry. We found that although removal of the disulfide bond through cysteine-to-alanine mutagenesis significantly lowered VHH domain denaturation temperature, it had no significant impact on the mechanical strength of the VHH:mCherry interaction with complex rupture occurring at ∼60 pN at 103–104 pN/sec regardless of disulfide bond state. These results demonstrate that mechanostable binding interactions can be built on molecular scaffolds that may be thermodynamically compromised at equilibrium.

folding and unfolding rates, [9] adoption of short-lived intermediate folded states, [10] response of DNA helices to torque and tension, [11] directionally anisotropic response of proteins to forces, [12][13][14] and mechanically activated catch bonding, [15,16] to name but a few. The associated techniques used for performing SMFS all have in common the coupling of molecules to nano-tomicroscale force transducers. Magnetic small particles, [17] optically trapped beads, [18] microfabricated silicon nitride cantilevers, [19][20][21] and beads tethered to a surface under centrifugal forces [22,23] are all examples of the diversity of methods used for SMFS.When attempting to measure individual molecules using SMFS, often times, it is necessary to have a signal that discriminates specific molecular behaviors (e.g., protein unfolding events, complex unbinding) from the unavoidable signals that arise due to nonspecific interactions between the force transducer and the surface. To address this issue, SMFS with the atomic force microscope (AFM) typically relies on internal control elements that are genetically encoded into the sequence of a so-called "polyprotein." [24] Polyproteins are large multidomain proteins containing a domain of interest sandwiched between multiple tandem copies of independently foldable marker domains. The marker domains, sometimes referred to as "fingerprint" domains, aid in AFM-SMFS data analysis by providing clear features such as contour length increments, unfolding forces, and/or the presence of unfolding substeps in the trace that can be searched for in an algorithmic way. Characterization of the molecular response of polyproteins has been instrumental in the development of the field of nanomechanics and mechanobiology. [25][26][27] Production of polyproteins had in the past been achieved in a variety of ways. Due to the repetitive nature of the DNA sequences encoding polyproteins, care must be taken during polymerase chain reaction (PCR) and cloning such that multiple primer annealing sites are avoided. A restriction digest and ligation-based cloning system involving unique restriction sites flanking tandem I27 modules was among the earliest reported techniques [28] for forming polyprotein gene sequences. 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. B...

The opportunistic pathogen Staphylococcus epidermidis utilizes a multidomain surface adhesin protein to bind host components and adhere to tissues. While it is known that the interaction between the SdrG receptor and its fibrinopeptide target (FgB) is exceptionally mechanostable (∼2 nN), the influence of downstream B domains (B1 and B2) is unclear. Here, we studied the mechanical relationships between folded B domains and the SdrG receptor bound to FgB. We used protein engineering, single-molecule force spectroscopy (SMFS) with an atomic force microscope (AFM), and Monte Carlo simulations to understand how the mechanical properties of folded sacrificial domains, in general, can be optimally tuned to match the stability of a receptor–ligand complex. Analogous to macroscopic suspension systems, sacrificial shock absorber domains should neither be too weak nor too strong to optimally dissipate mechanical energy. We built artificial molecular shock absorber systems based on the nanobody (VHH) scaffold and studied the competition between domain unfolding and receptor unbinding. We quantitatively determined the optimal stability of shock absorbers that maximizes work dissipation on average for a given receptor and found that natural sacrificial domains from pathogenic S. epidermidis and Clostridium perfringens adhesins exhibit stabilities at or near this optimum within a specific range of loading rates. These findings demonstrate how tuning the stability of sacrificial domains in adhesive polyproteins can be used to maximize mechanical work dissipation and serve as an adhesion strategy by bacteria.