Modular, ultra-stable, and highly parallel protein force spectroscopy in magnetic tweezers using peptide linkers

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force-regulation, and function. Here, we present a modular magnetic tweezers force spectroscopy approach that uses elastin-like polypeptide linkers to provide a high yield of protein tethers. Our approach extends protein force spectroscopy into the low force (<1 pN) regime and enables ultra-stable measurements on many molecules in parallel. We present (un-)folding data for the single protein domain ddFLN4 and for the large multi-domain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. The measurements reveal exponential force-dependencies of unfolding and refolding rates, directly resolve the stabilization of the VWF A2 domain by Ca2+, and discover transitions in the VWF C-domain stem at low forces that likely constitute the first steps of VWF activation in vivo. Our modular attachment approach will enable precise and multiplexed force spectroscopy measurements for a wide range of proteins in the physiologically relevant force regime.