Molecular Tools for Advanced Single-Molecule Studies

Abstract

Specific attachment and tethering strategies are of utmost importance to study single biomolecules, e.g. in force and fluorescence spectroscopy. To this end we implement and improve molecular tools for use in e.g. AFM-based Single-Molecule Force Spectroscopy (SMFS).The biotin:(Strept)avidin interaction inevitably comes to mind, whenever detection, trapping or purification of biomolecules is desired. Derived thereof, the Strep-Tag II peptide/Strep-Tactin pair eliminates the necessity of biotin-labelling when working with protein specimens. As for (Strept)avidin, the tetravalency in Strep-Tactin is unfavourable for single-molecule applications. We therefore developed a monovalent Strep-Tactin featuring well-defined binding geometry and stoichiometry, yet unaltered affinity towards Strep-Tagged proteins. A unique Cysteine allows for selective immobilization or fluorescence labelling of this construct. We exploited the mechanical properties of the Strep-Tag II:monovalent Strep-Tactin interaction utilizing AFM-based single-molecule force spectroscopy. Rupture forces up to 200 pN are observed, which is comparable to biotin:(Strept)avidin unbinding. The applicability of the system in various force spectroscopy settings to study proteins of interest is evident. In addition, monovalent Strep-Tactin can be considered a precision tool that can replace (Strept)avidin in various applications, especially in mechanically demanding environments and when utilization of the genetically encoded Strep-Tag II is preferential to biotin-labelling.We utilize this novel tethering strategy to for example characterize force-sensing kinases, like Myosin Light Chain Kinase and Focal Adhesion Kinase in AFM SMFS experiments.