Date of Award
Doctor of Philosophy (PhD)
Thomas T. Perkins
Single molecule force spectroscopy (SMFS) is a technique by which a biomolecule is unfolded by the application of force. Analysis of the forces applied to a protein and the distance the protein consequently extends reveal the configurations the protein adopts during the unfolding process, known as intermediates. By looking at a single molecule at a time, SFMS offers the ability to resolve rarely or transiently occupied intermediates that are obscured by averaging in ensemble techniques. However, detection of such intermediates has been limited by the spatiotemporal resolution of the force probe. Historically, atomic force microscopy-based SMFS has used long cantilevers (>100 μm in length). We utilized modified ultrashort cantilevers (9 μm in length), which were optimized for 1-microsecond temporal resolution and improved spatial resolution, to reexamine the unfolding of the model membrane protein Bacteriorhodopsin within its native lipid bilayer. Numerous new intermediates were detected, with many spaced by as little as two amino acids. The pathways by which the protein unfolded exhibited complex dynamics, including frequent unfolding and refolding, and intermediate occupancies shorter than 10 μs. For a particular fast folding transition, we deduced the folding free-energy landscape. Further, by unfolding the protein from both the C-terminal and N-terminal ends, we obtained complementary sets of intermediates that help identify some of the interactions that fold and stabilize the protein. Lastly, we removed the retinal cofactor and examined the change in the unfolding behavior in an attempt to discern the role of the retinal in the stability of the protein. Surprisingly, for this last experiment, we observed no significant change, although more study is needed to confirm this conclusion. These results sharpen the picture of the mechanical unfolding of membrane proteins and provide details into the interactions that are responsible for the folded structure of membrane proteins. These ultrashort cantilevers can be applied to SMFS studies on other biomolecules, to reveal dynamics previously obscured by a lack of spatiotemporal resolution of the force probe.
Siewny, Matthew G.W., "Hidden Dynamics in the Unfolding of Membrane Protein Revealed by Ultrafast Cantilevers" (2018). Physics Graduate Theses & Dissertations. 234.
Available for download on Sunday, July 26, 2020