Photochemical Sciences Ph.D. Dissertations

Title

Single-molecule Magnetic Tweezers Development and Application in Studies of Enzyme Dynamics and Cell Manipulation

Date of Award

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Photochemical Sciences

First Advisor

Hong Peter Lu (Advisor)

Second Advisor

Hanfeng Chen (Other)

Third Advisor

John Cable (Committee Member)

Fourth Advisor

Mikhail Zamkov (Committee Member)

Abstract

Single-molecule Magnetic tweezers have been developed as a powerful approach that provides detailed insight into single-molecule studies by mechanically manipulating and simultaneously monitoring the change of the photophysical properties. We have developed a few generations of magnetic tweezers and further utilized them for the studies of the mechanism of the product release during an enzymatic reaction, correlated studies of single-molecule enzymatic reaction dynamics and conformational dynamics, and live-cell motion manipulation. It is highly informative to manipulate the enzyme under the enzymatic conditions and simultaneously real-time monitor the conformational fluctuations of the enzymatic active site as well as the reaction activity change. Understanding how the enzyme dynamics of the conformational fluctuation relates to the enzyme activity can shed light on the field of enzymology. Motion manipulation of living cells and other biological entities is also important in the fields of biological research and tissue engineering.

Particularly, in this dissertation, we have employed a Horseradish Peroxidase (HRP) catalyzed fluorogenic enzymatic reaction as a powerful probe to study the reaction and conformational dynamics of the enzymatic active site under mechanical force manipulation. The typical fluorogenic feature of this reaction makes the nascent-formed product molecule at its perfect fitted position at the enzymatic active site, serving as an in-situ probe to report the real-time active-site configuration and its fluctuations. Interestingly, the product releasing dynamics of HRP show the complex conformational behavior with multiple product-releasing pathways. However, under constant magnetic force manipulation, the complex nature of the multiple product- releasing pathways disappears, and more simplistic conformations of the active site are populated. Under oscillation force manipulation at different frequencies, we have observed that conformational dynamics can be significantly perturbed or altered.

We have developed an integrated double-ring magnetic tweezer imaging microscope to actively manipulate the live-cell motions and selectively pick up the cell with an internalized paramagnetic bead. We have also demonstrated a lock-in amplifier coupled rotating magnetic tweezers approach, allowing synchronization of magnetic force response with the oscillation force.

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