Photochemical Sciences Ph.D. Dissertations


Single Molecule Optical Magnetic Tweezers Microscopy Studies of Protein Dynamics

Date of Award


Document Type


Degree Name

Doctor of Philosophy (Ph.D.)


Photochemical Sciences

First Advisor

H. Peter Lu (Advisor)

Second Advisor

Neocles Leontis (Committee Member)

Third Advisor

John Cable (Committee Member)

Fourth Advisor

John Farver (Committee Member)


This dissertation presents our research work aiming at conformational manipulation of single enzyme protein molecules, performed by single molecule magnetic tweezers correlated with optical fluorescence spectroscopy. To experimentally investigate the enzyme-substrate interactions and the related conformational fluctuations, we have developed a new approach to manipulate the enzymatic conformation and enzyme-substrate interaction at the single-molecule level by using a combined magnetic tweezers and simultaneous fluorescence resonance energy transfer (FRET) spectroscopic microscopy. By a repetitive pulling-releasing manipulation of a Cy3-Cy5 dye labeled 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) molecule under the conditions with and without enzymatic substrates, we have probed and analyzed the enzymatic conformational dynamics. Our results indicate that the enzymatic conformational flexibility can be regulated by enzyme-substrate interactions: (1) the enzyme at its conformation-perturbed state has less flexibility when binding substrates, and (2) substrate binding to the enzyme significantly changes the enzyme conformational flexibility, experimental evidence of so called entropy trapping in and enzyme-substrate reactive transition state. Furthermore, our results provide significant experimental analysis of folding-binding interactions of the enzyme-substrate interactions, and reveal the dynamic nature of the enzyme-substrate interactions. We also find supportive results from Steered Molecular Dynamics (SMD) Simulation, showing that in our studies, conformational manipulation by magnetic tweezers is able to distort the active domain of the enzyme molecules to an extent that significantly beyond thermal conformational fluctuations.

Furthermore, we have also revealed the impact of partially unfolding the enzyme molecules on their activity by using single-molecule TIRF-magnetic tweezers spectroscopy to manipulate conformation of the enzyme molecules to a partially unfolded, yet not fully denatured condition. By conformationally distorting horseradish peroxidase (HRP) molecules via magnetic tweezers at the single molecule level, we successfully manipulated and examined the activity changes of the HRP catalyzed H2O2-Amplex Red reaction. We have observed significant tolerance of the enzyme activity to the enzyme conformation in its deformed or partially-unfolded states. We have identified that (1) enzymatic activity can be manipulated by our TIRF-magnetic tweezers at single molecule level; and (2) enzyme molecules in partially unfolded conformation are still capable of showing significant activity, although at a lower but measurable level, due to the enzymatic active site conformational fluctuation and substrate binding induced folding-binding conformational changes. We further provide our understanding of the enzyme behavior based on enzymatic conformational fluctuation, enzyme-substrate interactions, enzyme-substrate active complex formation, and protein folding-binding interactions.