Photochemical Sciences Ph.D. Dissertations

Single-Molecule Force Manipulation and Nanoscopic Imaging of Protein Structure-Dynamics-Function Relationship

Date of Award

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Photochemical Sciences

First Advisor

H. Peter Lu (Advisor)

Second Advisor

Virginia Dubasik (Other)

Third Advisor

Liangfeng Sun (Committee Member)

Fourth Advisor

Mikhail Zamkov (Committee Member)

Abstract

The anisotropic nature of the force fluctuation inside cells makes it very biologically relevant to study the dynamics of the structure-function relationship of protein molecules under force. The ability to manipulate individual molecules under near-physiological conditions makes the Atomic force microscope a very widely used technique. Force applied by AFM can be either compressive force or pulling force. Here in the thesis, we have explored the compressive force response of the protein molecules and the impact of the compressive force using AFM.

The abrupt ruptures of protein native structures under compressive force were demonstrated by single-molecule AFM-FRET spectroscopic nanoscopy. The simultaneous measurement of the force curve and the FRET efficiency showed a temporal correlation between the compressive force drop and the FRET efficiency drop which indicated a spontaneous and abrupt rupture of the protein native tertiary structure.

Furthermore, a similar compressive force experiment was done on targeted calmodulin molecules to characterize two different forms of CaM, the Ca2+-ligated activated form, and the Ca2+ free non-activated form (Apo-Calmodulin). A sudden and spontaneous rupture of Apo-CaM molecules was observed under the compressive force applied by an AFM tip, though no such events were recorded in the case of the Ca2+-ligated form.

To further prove that this kind of compressive force rupture of Apo-CaM can trigger new chemistry inside the cell, a similar experiment was carried out where we observed compressive force rupture of apo-CaM molecules and successive binding of C28W peptide to the ruptured protein, a typical protein signaling activity that only a Ca2+-activated CaM has. This observation demonstrates that both chemical activation and force activation can play a vital role in biology, such as cell-signaling protein dynamics and function.

Lastly, we further explored the entangled protein state formed following the events of the multiple and simultaneous tau protein ruptures under crowding. Crowded proteins simultaneously rupture and then spontaneously refold to an entangled folding state, different from either folded or unfolded states of the tau protein, which can be a plausible pathway for the tau protein aggregation that is related to several neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease.

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