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Li, Peiyun

University of British Columbia

Optical tweezers (OT) are a powerful single-molecule technique that utilizes highly focused laser beams to trap and manipulate microscopic particles, allowing precise measurement of forces and movements at the molecular level. In this thesis, OT were employed to investigate the mechanical unfolding and refolding processes of the copper-containing protein azurin, known for its role in bacterial electron transfer. Azurin, with significant implications for understanding protein folding mechanisms, was studied in both its apo- (copper-free) and holo- (copper-bound) forms. Our research revealed that apo-azurin unfolds and refolds through both 2-state and 3-state processes. In contrast, holo-azurin displayed more complex behaviors, undergoing 2-state, 3-state, and even 4-state pathways. Interestingly, holo-azurin refolds in the opposite direction of its unfolding, with the copper coordination site reconstituting first, thus acting as a refolding nucleus. This highlights the intricate role of copper ions in the folding dynamics of azurin. Kinetic analysis showed that holo-azurin has faster unfolding and refolding rates compared to apo-azurin. Additionally, we successfully observed the apo-to-holo transition in real-time, confirming that copper binding lowers the unfolding force required. These findings not only confirm previous insights into azurin's mechanical stability but also advance our understanding by exploring the refolding mechanisms and direct observation of metal ion influence at the single-molecule level. This research contributes to a deeper comprehension of metalloprotein mechanics and the critical role of metal ions in protein folding, with potential applications in the development of protein- based nanotechnologies.

Text

2024-08-21

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88983

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/