title: Comparative thermodynamic analysis of DNA--protein interactions using surface plasmon resonance and fluorescence correlation spectroscopy.
abstract: We report a kinetic and thermodynamic analysis of interactions between ssDNA and replication protein A (RPA) using surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) at variable temperature. The two methods yield different values for the Gibbs free energy but nearly the same value for the reaction enthalpy of ssDNA-RPA complex formation. The Gibbs free energy was determined by SPR and FCS to be -62.6 and -54.7 kJ/mol, respectively. The values for the reaction enthalpy are -64.4 and -66.5 kJ/mol. It is concluded that the difference in Gibbs free energy measured by the two methods is due to different reaction entropies. The entropic contribution to the free energy at 25 degrees C is -1.8 kJ/mol for SPR and -11.8 kJ/mol for FCS. In SPR, the reaction is restricted to two dimensions because of immobilization of the DNA molecules to the sensor surface. In contrast, FCS is able to follow complex formation without spatial restrictions. In consequence, the reaction entropy determined from SPR experiments is lower than for FCS experiments.
authors.affiliation: Physikalische Chemie II, Universität Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany. frank.schubert@uni-bayreuth.de
authors.forename: Frank
authors.initials: F
authors.lastname: Schubert
authors.forename: Heiko
authors.initials: H
authors.lastname: Zettl
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Häfner
authors.forename: Gerhard
authors.initials: G
authors.lastname: Krauss
authors.forename: Georg
authors.initials: G
authors.lastname: Krausch
mesh_terms: D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D051716:Replication Protein A; D013050:Spectrometry, Fluorescence / Q000379:methods; D020349:Surface Plasmon Resonance; D013696:Temperature; D013816:Thermodynamics
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051716:Replication Protein A