Visualization, Kinetics, and Thermodynamics of DNA-Protein Interactions
type: CreativeWork
description: In this work the two spectroscopic techniques surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) as well as the imaging technique cryo-transmission electron microscopy (cryo-TEM) were used to gain kinetic, thermodynamic and structural information about DNA–protein interactions. Furthermore, the micrographs obtained by cryo-TEM were compared to AFM images taken in a previous work. The main goal of this work was to investigate the influence of surfaces on DNA–protein interactions, therefore the methods mentioned above were chosen. Both SPR and AFM deal with molecules attached to a surface, whereas FCS and cryo-TEM monitor the molecules in free solution. As a suitable model system the well characterized interaction between the human replication protein A (RPA) and DNA was chosen. The application of SPR and FCS to analysing the binding of RPA to ssDNA yields information about the kinetics and thermodynamics. No modification of the protein is required and biotinylated and fluorescently labelled DNA strands are available from commercial sources. Salt concentration, pH and temperature can be varied over a wide range. To best of our knowledge, FCS has not been used previously to obtain equilibrium constants at different temperatures. In this work it was demonstrated how temperature dependent SPR and FCS measurements can be performed and evaluated to determine thermodynamic data of DNA–protein interactions. Astonishingly, the equilibrium constant KD for the binding of RPA to ssDNA obtained by FCS is larger than the value obtained by SPR by a factor of 20–25, depending on the temperature. Therefore the values found for the Gibbs free energy were different, whereas the values for the reaction enthalpy were nearly the same for the two methods used. There are clear evidences that the difference in KD and therefore in Gibbs free energy measured by the two methods is due to different reaction entropies. In SPR the reaction is restricted to two dimensio…
abstract: In this work the two spectroscopic techniques surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) as well as the imaging technique cryo-transmission electron microscopy (cryo-TEM) were used to gain kinetic, thermodynamic and structural information about DNA–protein interactions. Furthermore, the micrographs obtained by cryo-TEM were compared to AFM images taken in a previous work. The main goal of this work was to investigate the influence of surfaces on DNA–protein interactions, therefore the methods mentioned above were chosen. Both SPR and AFM deal with molecules attached to a surface, whereas FCS and cryo-TEM monitor the molecules in free solution. As a suitable model system the well characterized interaction between the human replication protein A (RPA) and DNA was chosen. The application of SPR and FCS to analysing the binding of RPA to ssDNA yields information about the kinetics and thermodynamics. No modification of the protein is required and biotinylated and fluorescently labelled DNA strands are available from commercial sources. Salt concentration, pH and temperature can be varied over a wide range. To best of our knowledge, FCS has not been used previously to obtain equilibrium constants at different temperatures. In this work it was demonstrated how temperature dependent SPR and FCS measurements can be performed and evaluated to determine thermodynamic data of DNA–protein interactions. Astonishingly, the equilibrium constant KD for the binding of RPA to ssDNA obtained by FCS is larger than the value obtained by SPR by a factor of 20–25, depending on the temperature. Therefore the values found for the Gibbs free energy were different, whereas the values for the reaction enthalpy were nearly the same for the two methods used. There are clear evidences that the difference in KD and therefore in Gibbs free energy measured by the two methods is due to different reaction entropies. In SPR the reaction is restricted to two dimensio…
author: Schubert, Frank
date published: 2005-07-25
date modified: 2026-07-27T18:49:29Z
provider: Universität Bayreuth
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Universität Bayreuth