title: Thermochemistry of protein-DNA interaction studied with temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures.
abstract: We introduce temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and demonstrate its use to study thermochemistry of protein-DNA interactions. Being a homogeneous kinetic method, temperature-controlled NECEEM uniquely allows finding temperature dependencies of equilibrium and kinetic parameters of complex formation without the immobilization of the interacting molecules on the surface of a solid substrate. In this work, we applied temperature-controlled NECEEM to study the thermochemistry of two protein-DNA pairs: (i) Taq DNA polymerase with its DNA aptamer and (ii) E. coli single-stranded DNA binding protein with a 20-base-long single-stranded DNA. We determined temperature dependencies of three parameters: the equilibrium binding constant (Kb), the rate constant of complex dissociation (k(off)), and the rate constant of complex formation (k(on)). The Kb(T) functions for both protein-DNA pairs had phase-transition-like points suggesting temperature-dependent conformational changes in structures of the interacting macromolecules. Temperature dependencies of k(on) and k(off) provided insights into how the conformational changes affected two opposite processes: binding and dissociation. Finally, thermodynamic parameters, DeltaH and DeltaS, for complex formation were found for different conformations. With its unique features and potential applicability to other macromolecular interactions, temperature-controlled NECEEM establishes a valuable addition to the arsenal of analytical methods used to study dynamic molecular complexes.
authors.affiliation: Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.
authors.forename: Maxim
authors.initials: M
authors.lastname: Berezovski
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Krylov
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D019914:Taq Polymerase / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
chemical_list: D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011089:Polydeoxyribonucleotides; D011506:Proteins; C476430:SSB protein, E coli; D004247:DNA; D019914:Taq Polymerase