University of Illinois Urbana-Champaign

Direct real-time correlation of protein conformation and substrate recognition

Ghoneim, Mohamed Karem

Content Files
GHONEIM-DISSERTATION-2015.pdf

Permalink

https://hdl.handle.net/2142/78339

Description

Title
Direct real-time correlation of protein conformation and substrate recognition
Author(s)
Ghoneim, Mohamed Karem
Issue Date
2015-03-24
Director of Research (if dissertation) or Advisor (if thesis)
Spies, Maria
Doctoral Committee Chair(s)
Chemla, Yann
Committee Member(s)
  • Gruebele, Martin
  • Ha, Taekjip
Department of Study
School of Molecular & Cell Bio
Discipline
Biophysics & Computnl Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2015-07-22T22:16:16Z
Keyword(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.
Graduation Semester
2015-5
Type of Resource
text
Permalink
http://hdl.handle.net/2142/78339
Copyright and License 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

Owning Collections

Graduate Dissertations and Theses at Illinois PRIMARY
Graduate Theses and Dissertations at Illinois
Dissertations and Theses - Biophysics and Quantitative Biology