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Sunidhi Jaiswal Dissertation Final.pdf (2.42 MB)

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Single-Molecule Spectroscopy Studies of Protein Conformational Dynamics in DNA Damage Recognition and Cell Signaling

Jaiswal, Sunidhi
http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1648574708082215

Abstract Details

2022, Doctor of Philosophy (Ph.D.), Bowling Green State University, Photochemical Sciences.
H. Peter Lu, Advisor Single-molecule fluorescence spectroscopy has been developed as a powerful technique that provides details of protein-protein and protein-DNA interactions, enzyme reactions, and conformational dynamics. It is highly informative to study protein’s conformational dynamics during their activity or under the enzymatic condition to understand their function. The real-time monitoring of the enzyme’s active site conformational dynamics and simultaneously activity is informative towards understanding the mechanism of action. Studying how conformational fluctuation dynamics play role in protein or enzyme activity can shed light on the field of enzymology. Particularly in this dissertation, we have employed Förster Resonance Energy Transfer (FRET) as a powerful tool to study the conformational dynamics of Calmodulin (CaM) during its interaction with an autoinhibitory domain (C28W peptide) of the Plasma Membrane Calcium ATPase (PMCA). FRET between donor dye-labeled N-domain of the calmodulin interacting with acceptor dye-labeled peptide reports the real-time conformational dynamics of this interaction which is essential for PMCA activation. Interestingly, by using a unique statistical method, the results provide a mechanistic understanding of CaM signaling interaction and activation of the Ca-ATPase through multiple-state binding to the C28W. The new single-molecule spectroscopic analyses demonstrated in this work can be applied for broad studies of protein functional conformation fluctuation and protein-protein interaction dynamics. In another study, the conformational dynamics of recognition proteins are studied to understand the mechanism of identification of DNA damage by two recognition proteins, Replication Protein A (RPA) and Xeroderma Pigmentosum Protein A (XPA). We use single-molecule fluorescence fluctuation measurements of a dye, labeled at a damaged position on DNA, to understand the interaction of the damage site with RPA14 and XPA. Our results suggest that interactive conformational dynamics of RPA14 with damaged DNA are inhomogeneous due to its low affinity for DNA, whereas binding of XPA with the already formed DNA- RPA14 complex may increase the specificity of damage recognition by controlling the conformational fluctuation dynamics of the complex. Further, we studied the activation of calmodulin by applying compressive force using the AFM technique. Significantly, we found that the picoNewton level compressive force can turn a calcium-free CaM molecule into an active binding form just like the calcium-activated CaM.
Hong Peter Lu, Ph.D. (Advisor)
Vivian J Miller, Ph.D. (Other)
John R Cable, Ph.D. (Committee Member)
Mikhail A Zamkov, Ph.D. (Committee Member)
129 p.
Chemistry
Single-molecule Study; Fluorescence; Imaging; Confocal Microscopy; Atomic Force Microscopy

Recommended Citations

Citations

  • Jaiswal, S. (2022). Single-Molecule Spectroscopy Studies of Protein Conformational Dynamics in DNA Damage Recognition and Cell Signaling [Doctoral dissertation, Bowling Green State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1648574708082215

    APA Style (7th edition)

  • Jaiswal, Sunidhi. Single-Molecule Spectroscopy Studies of Protein Conformational Dynamics in DNA Damage Recognition and Cell Signaling. 2022. Bowling Green State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1648574708082215.

    MLA Style (8th edition)

  • Jaiswal, Sunidhi. "Single-Molecule Spectroscopy Studies of Protein Conformational Dynamics in DNA Damage Recognition and Cell Signaling." Doctoral dissertation, Bowling Green State University, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1648574708082215

    Chicago Manual of Style (17th edition)

bgsu1648574708082215
169
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This open access ETD is published by Bowling Green State University and OhioLINK.