{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/78339"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/78339","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Direct real-time correlation of protein conformation and substrate 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.","abstract_html":"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.","abstract_has_math":false,"creators":["Ghoneim, Mohamed Karem"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biophysics & Computnl Biology","degree_department":null,"school":null,"contributors":["Spies, Maria","Chemla, Yann","Gruebele, Martin","Ha, Taekjip"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-07-22T22:16:16Z","date_published":"2015-07-22T22:16:16Z","updated_at":"2026-07-22T22:26:11Z","subjects":["multi-color single-molecule imaging","protein domain motion","DNA damage recognition"],"languages":["en"],"rights":["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"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/78339","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Spies, Maria","Chemla, Yann","Gruebele, Martin","Ha, Taekjip"]},{"key":"dc:creator","label":"Author","values":["Ghoneim, Mohamed Karem"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-07-22T22:16:16Z","2015-05","2015-03-24","2015-5"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biophysics & Computnl Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["multi-color single-molecule imaging","protein domain motion","DNA damage recognition"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["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"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/78339"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Direct real-time correlation of protein conformation and substrate recognition"]}]}],"canonical_facts":{"dc:contributor":["Spies, Maria","Chemla, Yann","Gruebele, Martin","Ha, Taekjip"],"dc:creator":["Ghoneim, Mohamed Karem"],"dc:date":["2015-07-22T22:16:16Z","2015-05","2015-03-24","2015-5"],"dc:description":["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"],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/2142/78339"],"dc:language":["en"],"dc:rights":["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"],"dc:subject":["multi-color single-molecule imaging","protein domain motion","DNA damage recognition"],"dc:title":["Direct real-time correlation of protein conformation and substrate recognition"],"dc:type":["text"],"thesis:degree_discipline":["Biophysics & Computnl Biology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:26:11Z"}