Direct real-time correlation of protein conformation and substrate recognition
type: CreativeWork
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.
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.
author: Ghoneim, Mohamed Karem
language: en
keywords: multi-color single-molecule imaging, protein domain motion, DNA damage recognition
date published: 2015-07-22T22:16:16Z
date modified: 2026-07-22T22:26:11Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: http://hdl.handle.net/2142/78339