AbstractMost avidin-based handles in force spectroscopy are tetravalent biotin binders. Tetravalency presents two issues: multiple pulling geometries as well as multiple targets bound simultaneously. Additionally, such tetravalent handles require elaborate purification protocols in order to reassemble. A stoichiometric, monomeric variant of streptavidin (mcSA2) had been engineered previously. It is readily expressed and purified, and it binds biotin with a nanomolar KD. For atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), we fused the monomeric streptavidin with a small protein domain as an experimental fingerprint and to improve solubility. A ybbR-tag was additionally included for covalent site-specific tethering. Rupture forces of the mcSA2:biotin complex were found to be in a comparable range above 150 pN at force loading rates of 1E4 pN/s as for previously published, tetravalent streptavidin:biotin systems. Additionally, when tethering mcSA2 from its C-terminus, rupture forces were found to be slightly higher than when tethered N-terminally. Due to its monomeric nature, mcSA2 could also be chemically denatured and subsequently refolded - and thus regenerated during an experiment, in case the handle gets misfolded or clogged. We show that mcSA2 features a straightforward expression and purification with flexible tags, high stability, regeneration possibilities and an unambiguous pulling geometry. Combined, these properties establish mcSA2 as a reliable handle for single-molecule force spectroscopy.
Research progress of single molecule force spectroscopy technology based on atomic force microscopy in polymer materials: Structure, design strategy and probe modification
