Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering
CHAPTER 1.3: Real-time Detection of Thiol Chemistry in Single Proteins
Published:27 Jul 2018
E. C. Eckels, D. J. Echelman, J. A. Rivas-Pardo, and J. M. Fernández, in Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering, ed. M. J. Feige, The Royal Society of Chemistry, 2018, pp. 52-80.
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Thiol chemistry provides a way for proteins to alter their form and function rapidly and reversibly. Although a variety of bulk techniques have been developed to ascertain the oxidation state and bonding of cysteine thiols, these methods may destroy the sample or lead to unwanted side reactions. Single-molecule force spectroscopy harnesses the ability to track protein folding and unfolding pathways with ångstrom precision to detect changes in thiol chemistry in a real-time and non-destructive manner. As the oxidation state of the thiol changes, owing to intramolecular disulfide bonding or post-translational modification, changes to the protein topology and stability can be detected by unfolding of single-protein domains using the atomic force microscope. Not only does this provide a means to probe the mechanism of covalent bond scission by small nucleophiles and enzymes, but also a tool by which to monitor the activity of single oxidoreductase molecules as they introduce and rearrange disulfide bonds while protein substrates fold. Although a carnivore's bite damages tissue by tearing apart molecular bonds, nature has provided enzymatic machinery to repair the bonds, a process that can be directly observed using single-molecule techniques.