Towards a new modular polyprotein system compatible with single-molecule force spectroscopy by atomic force microscopy and magnetic tweezers.

Abstract

Single-molecule force spectroscopy (SMFS) has provided multiple advancements in the understanding of the free energy landscape of proteins under force. Specific covalent tethering of proteins to surfaces has improved both the yield and the quality of data acquisition in Atomic Force Spectroscopy (AFS) and Magnetic Tweezers (MT). Given the complementary information obtained by AFS and MT, we propose a modular polyprotein system with HaloTag specific covalent tethering to surfaces, in order to enable testing of the same protein preparation by both techniques. The modular design allows to easily include the proteins of interest in the system via SnoopTag/SnoopCatcher and SpyTag/SpyCatcher orthogonal spontaneous isopeptide bonding and can exploit cohesin-dockerin reversible interaction. The system also includes fingerprinting protein markers for improved accuracy by concurrent experiments and elastin-like polypeptides to reduce non-specific interactions with the surface. In summary, the proposed modular polyprotein strategy is intended to provide an accessible and versatile setup, easily adaptable to different proteins of interest, and includes recent improvements in sample preparation to increase the yield, quality and accuracy of SMFS experiments.