Stepwise unfolding of titin under force-clamp atomic force microscopy

Here we demonstrate the implementation of a single-molecule force damp adapted for use with an atomic farce microscope. We show that under force-clamp conditions, an engineered titin protein elongates in steps because of the unfolding of its modules and that the waiting times to unfold are exponentially distributed. Force-clamp measurements directly measure the force dependence of the unfolding probability and readily captures the different mechanical stability of the 127 and 128 modules of human cardiac titin, Force-clamp spectroscopy promises to be a direct way to probe the mechanical stability of elastic proteins such as those found in muscle, the extracellular matrix and cell adhesion.