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Title:Direct real-time correlation of protein conformation and substrate recognition
Author(s):Ghoneim, Mohamed Karem
Director of Research:Spies, Maria
Doctoral Committee Chair(s):Chemla, Yann
Doctoral Committee Member(s):Gruebele, Martin; Ha, Taekjip
Department / Program:School of Molecular & Cell Bio
Discipline:Biophysics & Computnl Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):multi-color single-molecule imaging
protein domain motion
DNA damage recognition
Abstract:Conformational transitions in a protein and its interaction with the cognate substrate exemplify two important biomolecular processes that may be correlated, uncorrelated, or partially correlated. While the degree to which these processes are correlated may bear heavily on the mechanism and regulation of the said protein, an experimental design which follows only one reaction coordinate, such as monitoring and comparing the kinetics of only one process in the absence and the presence of another process, is often hindered by the lack of simple scheme to interpret the experimental results. I developed a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an iron-sulfur (FeS) cluster-containing DNA repair protein. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by co-localization of the DNA- and protein-derived signals. I show that DNA binding does not strictly enforce XPD to assume a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA.
Issue Date:2015-03-24
Rights Information:Copyright 2015 Mohamed Ghoneim. Portions of this dissertation have been previously published. Chapter III reproduced with permission from Ghoneim & Spies 2014. Nano Letters 14 (10), pp. 5920–5931 Copyright © 2014 American Chemical Society
Date Available in IDEALS:2015-07-22
Date Deposited:May 2015

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