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 respond to calcium by 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 domains. Proteins were genetically modified for SMFS to have terminal amino acid tags recognized by the enzyme Sortase A, for both enzyme-mediated polyprotein conjugation and specific molecular tethering between the AFM tip and substrate. We characterize the mechanical response of tethered RTX polyproteins 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.