Thermochemistry of protein-DNA interaction studied with temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures.

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.