Coiled-Coil Probes Capture Mechanical Unfolding Pathways of Individual Proteins

Abstract

Force-induced unfolding of a consensus ankyrin repeat protein NI6C was suggested to follow a vectorial pathway from the C to the N terminus. We further test this vectorial unfolding hypothesis by introducing, at the gene level, a 70 amino acid coiled-coil (CC) polypeptide probe at different positions along the NI10C sequence and determine the unfolding force extension data of the hybrid proteins with single-molecule atomic force spectroscopy (AFM). The basic idea assumes that the coiled-coil folds independently of the host protein and does not significantly disturb its structure, and that the occurrence and the position of the CC unfolding force peak is indicative of the progress of the unfolding process past the location of the CC probe. First, AFM measurements of the CC probe flanked by I27 domains captured a single ∼35pN unfolding peak characteristic of the unzipping of the coiled-coil. Second, the unfolding force-extension data of the NI8CCI2C hybrid indicated that the unfolding of the CC occurs early in the unfolding process, consistent with the unfolding starting at the C terminus. Third, the force-extension data of the NI4CCI6C indicate that the unfolding of the CC occurs late during the unfolding process, corroborating our vectorial unfolding hypothesis. More generally, our studies suggest that coiled-coil probes maybe very useful to examine mechanical unfolding pathways of many proteins, including large proteins that are rarely studied in bulk folding measurements.