Contribution of Low-Affinity Sites to Strong Multivalent Protein-Membrane Binding: Detection using Single-Molecule TIRF Microscopy

Abstract

Biomolecular interactions such as interfacial protein-membrane binding are often the cumulative effect of multivalent attractions. We have shown that the stoichiometry of peripheral protein-membrane interactions can be measured based on single-molecule diffusion using supported lipid bilayers. Here we apply this technique to granuphilin, a synaptotagmin-like protein containing tandem membrane-targeting C2 domains, C2A and C2B. Granuphilin C2A binds simple lipid membranes containing anionic lipids such as phosphatidylserine (PS), but C2B affinity for PS is undetectable using standard protein-membrane binding approaches. Here, we set out to determine the PS affinity of C2B based on a comparison of the diffusion rates of the C2A domain and the C2AB tandem on supported lipid bilayers composed of phosphatidylcholine (PC) and PS. Total internal reflection florescence (TIRF) microscopy with single particle tracking was used to identify diffusion constants of each individual or tandem C2 domain. Granuphilin C2A displays a lateral diffusion constant of ∼2 μm2/s when bound to 1:1 DOPC:DOPS bilayers, comparable to other C2 domains. However, the diffusion of the granuphilin C2AB tandem on the same membrane is significantly slower; suggesting substantial PS contacts for the C2B domain within the C2AB tandem. This effect represents a weak but potentially physiologically relevant interaction that influences the membrane-bound state of this strong membrane binding protein. In a separate experiment, we also show that the diffusion of the individual C2A domain decreases significantly in the presence of 2% phosphatidylinositol-(4,5)-bisphosphae (PIP2), suggesting separate binding sites for these two lipid ligands. Overall, single-molecule tracking can reveal membrane-binding states that are difficult to detect with traditional ensemble approaches.