Jill A. Holbrook 1995-2003
Postdoc Fellowship, Hematology/Molecular Biology
University of Heidelberg, Germany
College of Saint Benedict 1995
Biochemistry PhD 2001
M Thomas Record, Jr
Calorimetric studies of DNA helix formation and integration host factor-DNA interactions: Contribution of coupled processes to observed thermodynamics
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The thermodynamics of DNA helix formation and of the interaction of Integration Host Factor (IHF) with specific and nonspecific DNA sites have been investigated by isothermal titration calorimetry (ITC), in conjunction with other methods. For DNA duplex formation, characterization of the temperature-dependent contributions of coupled conformational equilibria in the individual “denatured” strands and in the duplex is essential for understanding the origins of duplex stability. Studies of strand association show that the enthalpy of duplex formation is very strongly temperature-dependent, decreasing with increasing temperature, and resulting in a large, negative heat capacity change. Analysis of the individual strands indicates that the extent of single strand stacking decreases between 0 and 100 degrees Celsius. Since single strand stacking is not complete at any experimentally-accessible temperature, an increase in stacking accompanies duplex formation, contributing to the observed enthalpy change in a temperature-dependent manner and giving rise to the observed large, negative heat capacity change. The thermodynamics of binding IHF to a specific DNA site have also been examined by (ITC). At low potassium ion concentration, competition is observed between specific and nonspecific binding due to a low specificity ratio and a small nonspecific site size. Large negative enthalpy, entropy and heat capacity changes and potassium ion concentration-insensitive binding constants are also observed. At higher potassium ion concentration, binding constants and magnitudes of enthalpy, entropy and heat capacity changes decrease strongly. Along with structural information on the specific complex, the thermodynamics provide compelling evidence for a previously unrecognized design principle by which DNA-binding proteins may control binding constants and effects of temperature and ion concentrations. We propose that in the absence of DNA, many IHF cationic side chains located near DNA phosphates in the complex pair with anionic carboxylate groups in intramolecular salt bridges. To explain the thermodynamics of IHF-DNA interactions, we propose that binding at low potassium ion concentration requires disruption of salt bridges, whereupon unmasked charged groups hydrate and cationic groups interact with DNA. Coupling of protein salt bridge disruption to DNA binding appears to be significant for other DNA-wrapping proteins including the nucleosome, lac repressor, RNA polymerase, HU and SSB. |
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| Thesis Publications |
- Holbrook JA, Tsodikov OV, Saecker RM, Record MT Jr. Specific and non-specific interactions of integration host factor with DNA: thermodynamic evidence for disruption of multiple IHF surface salt-bridges coupled to DNA binding. J Mol Biol 310:379-401, 2001.
- Tsodikov OV, Holbrook JA, Shkel IA, Record MT Jr. Analytic binding isotherms describing competitive interactions of a protein ligand with specific and nonspecific sites on the same DNA oligomer. Biophys J 81:1960-1969, 2001.
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