Single-Molecule Analysis of Lipid–Protein Interactions in Crude Cell Lysates

Recognition of signaling phospholipids by proteins is a critical requirement for the targeting and initiation of many signaling cascades. Most biophysical methods for measuring protein interactions with signaling phospholipids use purified proteins, which do not take into account the effect of post-translational modifications and other cellular components on these interactions. To potentially circumvent these problems, we have developed a single-molecule fluorescence approach to analyzing lipid–protein interactions in crude cell extracts. As a proof of principle for this assay, we show that a variety of lipid-binding domains (LBDs) can be recruited from cell lysates specifically onto their target phospholipids. With single-molecule analysis in real-time, our assay allows direct determination of binding kinetics for transient lipid–protein interactions and has revealed unique assembly properties and multiple binding modes of different LBDs. Whereas single-copy LBDs display transient interaction with lipid ...