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Direct Monitoring of Equilibrium Protein Folding-Unfolding by Atomic Force Microscopy: Pushing the Limit

Abstract

The direct observation of protein folding and unfolding at/near equilibrium by atomic force microscopy (AFM) has been challenging due to the technological burden of limited force precision and resolution of AFM. Using chemically etched low-drift cantilevers and protein handles that are easy to pick up, here we demonstrate on a commercial AFM, the direct observation of the at/near equilibrium folding-unfolding dynamics of protein G-related albumin-binding domain (GA), an all-α protein consisting of a three-helix bundle. Unfolding and refolding of GA occurred under a low-force regime (<10 pN) previously difficult to observe using AFM. GA unfolded/refolded at/near equilibrium as evidenced by rapid hopping between folded and unfolded states. The high stability of our experimental platform allowed for constant separation experiments to probe at (near)-equilibrium kinetics of GA in real-time. Our results indicate GA unfolding occurs by two-state fashion at low force (~10 pN) and displays high malleability, a large distance to the unfolded transition state. Finally, binding of human serum albumin (HSA) brought GA unfolding/folding out of equilibrium, where a near two-fold increase in unfolding force was observed relative to unbound GA. Taken together, these techniques further extend the capability of commercial AFMs towards studying protein unfolding/refolding at equilibrium as well as increasingly complex biomolecular systems in the context of biological function.

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Publication details

The article was received on 13 Aug 2019, accepted on 07 Oct 2019 and first published on 07 Oct 2019


Article type: Communication
DOI: 10.1039/C9CC06293A
Chem. Commun., 2019, Accepted Manuscript

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    Direct Monitoring of Equilibrium Protein Folding-Unfolding by Atomic Force Microscopy: Pushing the Limit

    A. Xiao and H. Li, Chem. Commun., 2019, Accepted Manuscript , DOI: 10.1039/C9CC06293A

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