Biophysical Analyses of Intermolecular Ion-Pairs in Protein-DNA Complexes
type: CreativeWork
description: For biological processes, one of the most fundamental interactions is the formation of the ion pair. In protein-DNA complexes, ion pairs form between basic side chains, such as lysine and arginine, and the phosphate backbone of DNA. Moreover, ion pairs are found in dynamic equilibrium between two major states. While great strides have been made to improve the understanding of ion pair in protein-DNA interactions, much remains to be understood on the role of ion pair dynamics in protein-DNA interactions. Thus, in this work we investigate the role of ion pair dynamics in protein-DNA interactions. First, we investigate side chain dynamics of the Antennapedia homeodomain (Antp) for both lysine and arginine side chains. Using NMR spectroscopy, we determine the mobility of lysine and arginine side chains in the free and DNA-bound state of Anp through 15N relaxation experiments. We show that side chain mobility is retained for both Arg and Lys side chains that form an ion pair with the phosphate backbone. Second, we investigate the role of ion pair dynamics of the Antp homeodomain through the oxygen-to-sulfur substitution (dithioation) of a single phosphate that forms an ion pair with a lysine side chain. We show an enhancement in affinity for the dithioated DNA compared to the unmodified DNA by the Antp homeodomain using fluorescence assays. Using ITC, we show that the enhancement in affinity is due to an entropic contribution. Through NMR spectroscopy, we show that the lysine side chain interacting with the dithioated phosphate has a higher mobility compared to the unmodified phosphate and confirm these observations using X-ray crystallography. We further investigate the stereospecific effects of this modification. Lastly, we establish a system to study kinetic parameters of the Antp homeodomain using Ficoll, as a macromolecular crowder, to investigate the role of ion-pair dynamics in DNA target search. Using fluorescence-based stopped flow kinetics experiments, we show …
abstract: For biological processes, one of the most fundamental interactions is the formation of the ion pair. In protein-DNA complexes, ion pairs form between basic side chains, such as lysine and arginine, and the phosphate backbone of DNA. Moreover, ion pairs are found in dynamic equilibrium between two major states. While great strides have been made to improve the understanding of ion pair in protein-DNA interactions, much remains to be understood on the role of ion pair dynamics in protein-DNA interactions. Thus, in this work we investigate the role of ion pair dynamics in protein-DNA interactions. First, we investigate side chain dynamics of the Antennapedia homeodomain (Antp) for both lysine and arginine side chains. Using NMR spectroscopy, we determine the mobility of lysine and arginine side chains in the free and DNA-bound state of Anp through 15N relaxation experiments. We show that side chain mobility is retained for both Arg and Lys side chains that form an ion pair with the phosphate backbone. Second, we investigate the role of ion pair dynamics of the Antp homeodomain through the oxygen-to-sulfur substitution (dithioation) of a single phosphate that forms an ion pair with a lysine side chain. We show an enhancement in affinity for the dithioated DNA compared to the unmodified DNA by the Antp homeodomain using fluorescence assays. Using ITC, we show that the enhancement in affinity is due to an entropic contribution. Through NMR spectroscopy, we show that the lysine side chain interacting with the dithioated phosphate has a higher mobility compared to the unmodified phosphate and confirm these observations using X-ray crystallography. We further investigate the stereospecific effects of this modification. Lastly, we establish a system to study kinetic parameters of the Antp homeodomain using Ficoll, as a macromolecular crowder, to investigate the role of ion-pair dynamics in DNA target search. Using fluorescence-based stopped flow kinetics experiments, we show …
author: Nguyen, Dan
keywords: Ion-Pair Dynamics, Protein, Antennapedia, DNA
date modified: 2026-07-24T05:50:59Z
provider: University of Texas Medical Branch
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: The University of Texas Medical Branch at Galveston

PropertyValue
property ID: Handle
value: https://hdl.handle.net/2152.3/10719