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Spies, Maria Chemla, Yann Gruebele, Martin Ha, Taekjip Ghoneim, Mohamed Karem 2015-07-22T22:16:16Z 2015-07-22T22:16:16Z 2015-05 2015-03-24 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. Submission original under an indefinite embargo labeled 'Open Access'. The submission was exported from vireo on 2015-07-22 without embargo terms The student, Mohamed Ghoneim, accepted the attached license on 2015-03-19 at 15:30. The student, Mohamed Ghoneim, submitted this Dissertation for approval on 2015-03-19 at 15:42. This Dissertation was approved for publication on 2015-03-24 at 14:48. DSpace SAF Submission Ingestion Package generated from Vireo submission #7764 on 2015-07-22 at 10:30:47 Made available in DSpace on 2015-07-22T22:16:16Z (GMT). No. of bitstreams: 2 GHONEIM-DISSERTATION-2015.pdf: 1776388 bytes, checksum: 0f537c202911ba7f07ad69c9c9e2ed94 (MD5) LICENSE.txt: 4212 bytes, checksum: 324d0894db48367ea747b35022014def (MD5) Previous issue date: 2015-03-24 application/pdf http://hdl.handle.net/2142/78339 en 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 multi-color single-molecule imaging protein domain motion DNA damage recognition Direct real-time correlation of protein conformation and substrate recognition text text 2015-5 School of Molecular & Cell Bio Biophysics & Computnl Biology University of Illinois at Urbana-Champaign Dissertation Ph.D.