10.1021/acs.nanolett.9b02062.s001 Haipei Liu Haipei Liu Valentin Schittny Valentin Schittny Michael A. Nash Michael A. Nash Removal of a Conserved Disulfide Bond Does Not Compromise Mechanical Stability of a VHH Antibody Complex American Chemical Society 2019 single-molecule force spectroscopy Conserved Disulfide Bond disulfide bond Compromise Mechanical Stability VHH framework region VHH domain denaturation temperature mechanostable binding interactions VHH Antibody Complex Single-domain VHH antibodies disulfide bond state 2019-07-01 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Removal_of_a_Conserved_Disulfide_Bond_Does_Not_Compromise_Mechanical_Stability_of_a_VHH_Antibody_Complex/8792537 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 10<sup>3</sup>–10<sup>4</sup> 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.