A Lego Toolbox for Engineering Proteins for Single Molecule Force Spectroscopy

Abstract

The last 20 years have witnessed a revolution in our understanding of the mechanical behavior of proteins, driven largely by advances in single-molecule force spectroscopy (SMFS). However, direct single-molecule study of certain mechanical systems, such as muscle titin and the bacterial pilus, has remained out of reach due to their megadalton-scale sizes and due to difficulties of pulling at high forces. Here, we have implemented a strategy based on HaloTag and SpyCatcher-SpyTag chemistries that provides for modular hetero-polyprotein construction and fully covalent SMFS with Magnetic Tweezers (MT). We functionalized both the surface and probe with HaloTag chemistry to have end-to-end covalent attachments in MT, enabling hours-long recordings on single molecules held at high force (> 100 pN). In addition, we have incorporated the SpyCatcher-SpyTag protein conjugation system for macromolecule construction. Whereas SMFS has been limited to the study of octamers of up to ∼120 kDa, SpyCatcher-SpyTag chemistry extends the range to 2nd-order assemblies of polyproteins. We expressed polyproteins flanked with either SpyCatcher or SpyTag pairs, and assembled large heterogeneous molecules through sequential construction on MT surfaces or on magnetic beads. Applying MT-based SMFS, we observed folding/unfolding step sizes of the various constructs assembled into single hetero-polyproteins. To date, we have successfully assembled and recorded from a trimer of I27 octamers and from a hetero-trimer of LB1 and I27 polyproteins. These advances in covalent attachment and protein assembly set the stage for megadalton-scale construction of complex titin-like molecules and for their long-term study under force.