Thermochemistry of Protein-DNA Interaction Studied with Temperature-Controlled Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures - Analytical Methods & Tools for Lyophilization

Thermochemistry of Protein-DNA Interaction Studied with Temperature-Controlled Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures
Received for review September 24, 2004. Accepted December 3, 2004.
Web Release Date: January 22, 2005
Maxim Berezovski and Sergey N. Krylov*
Anal. Chem.
ACS Publications
Copyright ? 2005 American Chemical Society
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada
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 (koff), and the rate constant of complex formation (kon). 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 kon and koff provided insights into how the conformational changes affected two opposite processes: binding and dissociation. Finally, thermodynamic parameters, H and S, 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.
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