Molecular-scale mechanical properties of calcium-responsive proteins

Chemically responsive proteins are responsible for many biological processes, but the molecular-scale mechanics of ion-driven protein folding remain elusive. Repeats-in-Toxin (RTX) proteins are calcium-responsive, undergoing conformational changes from random coils to folded beta-roll structures upon binding to calcium ions. We have generated fusion proteins containing RTX domains that replicate the calcium-responsive behavior of naturally occurring RTX proteins. Using atomic force microscopy (AFM), we demonstrate single-molecule force spectroscopy (SMFS) of RTX and model fusion proteins. Proteins were genetically modified for SMFS to have terminal thiol and amine groups for molecular tethering between the AFM tip and substrate. We characterize the mechanical response of tethered RTX proteins in both their disordered calcium-free state and folded calcium-bound state, and we compare AFM force–extension curves to the worm-like chain model. Understanding the molecular-scale mechanical behavior of RTX proteins will enable the development of tunable biomaterials and other chemically responsive proteins.