Abstract
Many biological processes rely on protein-membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach, single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2-7 pN and had binding energies of 4-14 kBT per C2 domain. Regulation by bilayer composition or Ca2+ recapitulated known properties of both proteins. The method can be widely applied to study protein-membrane interactions.
Highlights
Protein–membrane interactions play pivotal roles in numerous biological processes, including membrane protein folding (Yu et al, 2017; Popot and Engelman, 2016; Min et al, 2015), lipid metabolism and transport
Reversible protein–membrane binding is detected based on the associated extension changes with high spatiotemporal resolution, allowing us to derive binding affinity and kinetics as a function of force, soluble factors, and lipid compositions
We chose to apply this new approach to C2 domains of Extended Synaptotagmin-2 (E-Syt2) and Syt1, since previous bulk and some single-molecule measurements exist for comparison
Summary
Protein–membrane interactions play pivotal roles in numerous biological processes, including membrane protein folding (Yu et al, 2017; Popot and Engelman, 2016; Min et al, 2015), lipid metabolism and transport 2015; Wong et al, 2017), membrane trafficking (Zhou et al, 2017; Perez-Lara et al, 2016; Wu et al, 2017; Hurley, 2006; Shen et al, 2012; McMahon and Gallop, 2005), signal transduction (Dong et al, 2017; Aggarwal and Ha, 2016; Lemmon, 2008; Das et al, 2015), and cell motility (Wang and Ha, 2013; Tsujita and Itoh, 2015) Studying these interactions is often difficult, especially when they involve multiple intermediates, multiple ligands, mechanical force, large energy changes, or protein aggregation (Dong et al, 2017; Perez-Lara et al, 2016; Arauz et al, 2016). High resolution single-molecule methods to probe protein–membrane interactions in the presence of force are lacking
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