Is mechanical receptor ligand dissociation driven by unfolding or unbinding?

Abstract

Mechanical force can play a pivotal role in biological systems. Single Molecule Force Spectroscopy, is a powerful tool to probe the mechanics of proteins and their binding partners. Yet, it remains unclear how complex dissociation of a protein-protein interaction under mechanical forces occurs. Are receptor and ligand unbinding, or are they unfolding? We utilize an approach wherein receptor and ligand are expressed as a single molecule fused by a long flexible linker. Force is applied to the complex via an ultrastable handle. Consequently, the events during and following complex dissociation can be monitored. We investigate two high-affinity systems: The cohesin-dockerin type I interaction in which we find that a binding partner unfolds upon complex dissociation, and a colicin-immunity protein complex in which both proteins unfold completely upon unbinding. Mechanical receptor ligand dissociation thus can encompass unfolding of one or both binding partners.

Removal of a Conserved Disulfide Bond Does Not Compromise Mechanical Stability of a VHH Antibody Complex

“…When protein−protein interactions are mechanically dissociated, it is difficult to determine if dissociation is driven by unfolding of one of the binding partners and loss of the binding epitope structure, or by global loss of contact between two well-folded proteins. 61 Given that disulfide bonding supports overall VHH fold stability, if VHH/mCherry mechanical dissociation were driven by loss of VHH tertiary structure, then removal of the disulfide bond could potentially lower the forces at which the VHH/mCherry complex dissociates. We performed AFM-SMFS on VHH(C24A), VHH(C98A), and VHH(C24A, C98A) using identical expression, purification and surface chemical procedures as for VHH(WT).…”

Dronpa: A Light-Switchable Fluorescent Protein for Opto-Biomechanics

“…In order to characterize individual pdDronpa1.2 homodimers by SMFS, we designed a protein construct, where we linked two Dronpa domains with a flexible linker . The dimer was further fused to a pulling handle, a strategy that has already been successfully applied to probe the unfolding of individual proteins. , The linker was made out of 73 amino acids, which corresponds to a contour length increment of ca.…”

Next Generation Methods for Single-Molecule Force Spectroscopy on Polyproteins and Receptor-Ligand Complexes

“…Since receptor-ligand rupture typically results in loss of the tether between the cantilever and the surface, calculation of L c upon rupture does not have the same physical meaning for receptor-ligand rupture experiments as for domain unfolding experiments, however, L c calculations can be incorporated for fingerprinting of receptor-ligand interaction curves as well. Also, tethered protein receptor-ligand (Bertz et al, 2009;Kim et al, 2010;Pernigo et al, 2010;Berkemeier et al, 2011;Vera and Carrión-Vázquez, 2016;Milles and Gaub, 2019) and DNA systems (Halvorsen et al, 2011;Yang et al, 2016) have been reported where the rupture of a molecular interaction results in extension of a flexible tether providing a known contour length increment. Therefore, L c analysis can be highly applicable not only to domain unfolding studies but also to receptor-ligand rupture experiments.…”