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Multiplexed protein force spectroscopy reveals equilibrium protein folding dynamics and the low-force response of von Willebrand factor

Achim Löf,P. Walker,Steffen M. Sedlak,Sophia Gruber,T. Obser,M. Brehm,M. Benoit,J. Lipfert

Published 2019 in Proceedings of the National Academy of Sciences of the United States of America

ABSTRACT

Significance The physiological function of proteins is often critically regulated by mechanical forces acting on them. Single-molecule manipulation techniques such as atomic force microscopy or optical tweezers have enabled unprecedented insights into the molecular mechanisms underlying such force regulation. Current limitations include the resolution at low forces and low throughput. We here introduce a versatile, modular approach for force measurements on proteins in magnetic tweezers that allows for probing dozens of single molecules in parallel, in a wide force range including very low forces <1 pN. We demonstrate the utility of this assay by elucidating regulatory low-force transitions within von Willebrand factor, a vascular protein that is activated for its critical role in hemostasis by hydrodynamic forces in the bloodstream. Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force regulation, and function. So far, the field has relied primarily on atomic force microscope and optical tweezers assays that, while powerful, are limited in force resolution, throughput, and require feedback for constant force measurements. Here, we present a modular approach based on magnetic tweezers (MT) for highly multiplexed protein force spectroscopy. Our approach uses elastin-like polypeptide linkers for the specific attachment of proteins, requiring only short peptide tags on the protein of interest. The assay extends protein force spectroscopy into the low force (<1 pN) regime and enables parallel and ultra-stable measurements at constant forces. We present unfolding and refolding data for the small, single-domain protein ddFLN4, commonly used as a molecular fingerprint in force spectroscopy, and for the large, multidomain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. For both proteins, our measurements reveal exponential force dependencies of unfolding and refolding rates. We 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’s mechano-activation. Probing the force-dependent lifetime of biotin–streptavidin bonds, we find that monovalent streptavidin constructs with specific attachment geometry are significantly more force stable than commercial, multivalent streptavidin. We expect our modular approach to enable multiplexed force-spectroscopy measurements for a wide range of proteins, in particular in the physiologically relevant low-force regime.

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