(218b) Molecular-Scale Mechanics of Calcium-Responsive Repeat Proteins

Ion-responsive proteins drive numerous biological processes, but the molecular-scale mechanics of ion-driven protein folding remain elusive. A class of repetitive proteins exhibit interesting ion-driven responses by undergoing conformational changes from random coils to folded beta-roll structures upon binding to calcium ions. Using atomic force microscopy (AFM), we demonstrate single-molecule force spectroscopy (SMFS) of repetitive calcium-responsive proteins. Proteins were genetically modified for SMFS to have terminal amino acid tags recognized by the enzyme Sortase A, which enables enzyme-mediated polyprotein conjugation and specific molecular tethering between the AFM tip and substrate. Polyproteins comprising multiple, covalently linked proteins often improve the statistics of single-molecule AFM measurements and provide fingerprints to filter force–extension curves from nonspecific interactions. Polyprotein reaction progress was confirmed using polyacrylamide gel electrophoresis, ion-exchange chromatography, and mass spectrometry. Then, we measured the single-molecule mechanical responses of tethered polyproteins while varying calcium concentration. We observe the broadest distribution of unfolding behavior at an intermediate calcium concentration, suggesting a transition from disordered calcium-free states and folded calcium-bound states. We also compare AFM force–extension curves to the worm-like chain model. Overall, understanding the molecular-scale mechanical behavior of ion-responsive proteins will enable the development of tunable biomimetic and biofunctional materials.