Single Molecule Protein Unfolding Studies
Atomic force microscopy is an excellent tool for studying the folding and unfolding of individual protein domains. We use this technique as a method for understanding the process of mechanical unfolding in molecular detail. We were the first laboratory to get a complete description of the mechanical unfolding of a single elastic protein at the molecular level. A number of proteins experience mechanical force in vivo. To understand the how these proteins withstand mechanical force, we have to look at the forced unfolding pathway of that protein in combination with standard mutational analyses. No other laboratory in the world has combined protein engineering, biophysical and structural techniques and MD simulation with AFM in the way that we have done.
Sample AFM traces (A and B) show results from forced unfolding experiments on wild-type (WT) and 3Pro (P3) PKD, at 300 nm/s. The first unfolding event in each trace is indicated by an arrow. (C) The unfolding forces are significantly lower for 3Pro (red) than WT (black). The dependence of the modal unfolding forces on the pulling speed (D) is the same for wild-type and 3Pro. However, the 3Pro mutant (filled red squares) unfolds at forces that are significantly lower than wild- type (filled black circles).
Selected publications
- Ma, L., Xu, M., Forman, J. R., Clarke, J. & Oberhauser, A. F. (2009). Naturally occurring mutations alter the stability of polycystin-1 PKD domains. J. Biol. Chem. 284, 32942-32949.
- Borgia, A., Steward, A.. & Clarke, J. (2008). An effective strategy for the design of proteins with enhanced mechanical stability. Angew. Chem. Int. Ed. Engl. 47, 6900-6903.
- Randles, L. G., Rounsevell, R. W. & Clarke, J. (2007). Spectrin domains lose cooperativity in forced unfolding. Biophys. J. 92, 571-577.