Photochemical Sciences Ph.D. Dissertations

Title

Single Molecular Spectroscopy and Atomic Force Manipulation of Protein Conformation and Dynamics

Date of Award

2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Photochemical Sciences

First Advisor

H. Peter Lu (Advisor)

Second Advisor

Massimo Olivucci (Committee Member)

Third Advisor

John Cable (Committee Member)

Fourth Advisor

Ray Larson (Other)

Abstract

The present thesis describes the study of protein conformation dynamics probed by single molecule techniques. Protein structural dynamic have been studied for many years. Interesting topics such as protein folding/unfolding, protein denature, and protein-protein interactions had been widely studied. Scientists are still interested in questions like: what exact forces contribute to the stability of protein secondary and tertiary structure; why different protein has different function based on their structure. Single molecular spectroscopy has the advantage to observe inhomogeneous behavior of protein molecule compared to ensemble measurement. The typical approach of single molecular spectroscopy is to record one single molecule at a time and repeat this process at one or multiple molecules that are chemically identical. Then use statistics way to analyze these data.

The binding dynamics of CaM-C28W complex was studied using single molecule FRET experiment. The statistics results indicate the inhomogeneous nature of the process and the evidence of a slower process gauging the binding dynamics has been claimed.

Furthermore, the responding mechanical dynamics of CaM molecule under AFM tip manipulation has been investigated by AFM-FRET correlated measurement. The repeated “collapsing” pattern in the force curve leads us to deeper study on the process. The nature of the process and the dynamics of the underlying conformation change have also been detailed described.

Lastly, a similar AFM manipulation experiment has been performed on EGFR dimer complex that has more biological potential in terms of dimer formation and aggregation dynamics. In order to fit the nature of the molecule, we used two identical dyes to label either monomer. Instead of single molecule FRET, photo-stamping measurement has been used to build an AFM-optical correlated measurement. The statistics results shows the presents of multiple intermediate states. With the help of known crystal structure and the knowledge of EGFR dimer complex, we managed to find clue from our correlated results and assign possible conformation of each intermediate states. A possible conjugated state upon collapsing has been claimed.

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