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Abstract
Proteins that bind preferentially to specific recognition sites on DNA also bind more weakly to nonspecific DNA. We have studied both specific and non-specific binding of the EcoRI and BamHI restriction endonucleases, and determined enthalpic and entropic contributions to binding free energy (ΔG°bind) using both the van't Hoff method and isothermal titration calorimetry. Specific binding is characterized by a strongly negative ΔC°p and can be either enthalpy-driven or entropy-driven, depending on temperature. Nonspecific binding has ΔC°p ≈ 0 and is enthalpy-driven. A strongly negative ΔC°p is the ‚thermodynamic signature’ of site-specific binding, because it reflects the characteristics of a tight complementary recognition interface: the burial of previously hydrated nonpolar surface and restriction of configurational-vibrational freedoms of protein, DNA, and water molecules trapped at the protein-DNA interface. These factors are absent in nonspecific complexes. We probed the contributions to ΔC°p by varying the sequence context surrounding the recognition site. As ΔG°bind improves, ΔC°p' ΔH° and ΔS° all become more negative, and there is a linear correlation between ΔH° and ΔS° (enthalpy-entropy compensation). Because these context variations do not change the protein-base or protein-phosphate contacts, the hydrophobic contribution or the number of trapped water molecules at the interface, we conclude that a better sequence context improves the ‚goodness of fit’ in the interface and and thus increases the magnitude of the negative configurational-vibrational contribution to ΔC°p.