Numerous proteins directly or indirectly bind membranes to exert their roles in a wide variety of biological processes. Such membrane binding often occurs in the presence of an external mechanical force. It remains challenging to quantify these interactions using traditional experimental approaches based on a large number of molecules, due to ensemble averaging or the lack of mechanical force. Here we described a new single-molecule approach based on high-resolution optical tweezers to characterize protein-membrane interactions. A single membrane binding protein is attached to the lipid bilayer coated on a silica bead via a flexible polypeptide linker, tethered to another bead via a long DNA handle, and pulled away from the bilayer using optical tweezers. Dynamic protein binding and unbinding is detected by the corresponding changes in the extension of the protein-DNA tether with high spatiotemporal resolution, which reveals the membrane binding affinity, kinetics, and intermediates. We demonstrated the method using C2 domains of extended synaptotagmin 2 (E-Syt2) with a detailed protocol. The method can be widely applied to investigate complex protein-membrane interactions under well-controlled experimental conditions.
Keywords: C2 domain; Extended synaptotagmins; Optical tweezers; Protein–membrane interactions; Supported bilayer.
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