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- Worms in space |
Researchers in the laboratory of Dr. Linda Jen-Jacobson have been using the highly sequence specific type II restriction endonucleases EcoRI, EcoRV, and BamHI as model systems for the study of protein-DNA interactions. Recent work with these proteins has provided a thermodynamic framework for understanding the unique signatures of specific and non-specific protein-DNA interactions.
Lisa Engler and Arabela Grigorescu have employed the techniques of isothermal titration calorimetry and van't Hoff analysis to dissect the enthalpic and entropic contributions to the binding free energy for both specific and nonspecific EcoRI-DNA, EcoRV-DNA, and BamHI-DNA interactions. Nonspecific complex formation is enthalpy-driven, and shows little to no heat capacity change (DCP0 ~ 0). Specific binding, however, is always characterized by a large negative heat capacity change, and can be either enthalpy or entropy-driven. A strongly negative heat capacity change is the "thermodynamic signature" of specific protein-DNA complex formation, and reflects the highly complementary recognition interface. The intimacy of the BamHI-DNA interface is shown in Figure 1 to the right (protein is magenta, DNA is yellow, and water molecules are represented as white spheres).
The sequence context surrounding the recognition site affects the detailed thermodynamic parameters for formation of the specific protein-DNA interface. Flanking sequences that improve the binding free energy (labeled "better" in Figure 2) show a more negative heat capacity change, and more negative enthalpy and entropy contributions. It has been shown that context variations do not change the direct protein-base or protein-phosphate contacts [Dr. John Rosenberg's (left) group], the hydrophobic contribution (see work described below), or the number of trapped water molecules at the recognition interface. We conclude that the improved binding derives from a better (more intimate) fit at the recognition interface: optimized protein-base contacts and loss of strain energy in the complex leading to a more negative (favorable) enthalpy. This optimized fit comes at the expense of an increase in the magnitude of the negative configurational-vibrational contribution: more tightly constrained contacts lead to a more negative, "less favorable" entropy.
Dr. Deng Feng Cao (left) has recently demonstrated the significant role of water release upon specific protein-DNA complex formation. The effect of co-solutes on the binding and cleavage of DNA by each endonuclease was used to probe the stoichiometry of water release. Formation of the site-specific protein-DNA complex was characterized by the release of a large number of water molecules (ranging from 350 for BamHI to 450 for EcoRV). This number was unaffected by the sequence context, demonstrating that context effects are not driven by a difference in the hydrophobic contribution to the binding free energy. The nonspecific endonuclease-DNA interactions, however, showed very little water release (range of 15 for BamHI to 80 for EcoRV). This is consistent with the finding that there is no heat capacity change (DCP0 ~ 0) for nonspecific complex formation, and predicts a loose, less intimate interface. Recent crystal structures of the specific and nonspecific BamHI-DNA and EcoRV-DNA interactions confirm these results. Figure 3 (right) depicts the different levels of surface complementarity for the BamHI interaction with specific (top) and nonspecific (bottom) DNA sites. Regions shaded blue represent points of very high surface intimacy, while those in red indicate points of steric clash. |
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