Comparative thermodynamic analysis of DNA--protein interactions using surface plasmon resonance and fluorescence correlation spectroscopy.
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.
Schubert
Frank
F
Physikalische Chemie II, Universität Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany. frank.schubert@uni-bayreuth.de
Zettl
Heiko
H
Häfner
Wolfgang
W
Krauss
Gerhard
G
Krausch
Georg
G
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
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051716:Replication Protein A