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Break in the Heat Capacity Change at 303 K for Complex Binding of Netropsin to AATT Containing Hairpin DNA Constructs

Matthew W. Freyer ^*, Robert Buscaglia ^*, Amy Hollingsworth ^*, Joseph Ramos ^*, Meredith Blynn ^*, Rachael Pratt ^*, W. David Wilson  and Edwin A. Lewis ^*

^* Department of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, Arizona 86011; and Department of Chemistry, Georgia State University, Atlanta, Georgia 30302

Correspondence: Address reprint requests to Professor Edwin A. Lewis, Northern Arizona, University, Dept. of Chemistry & Biochemistry, Building 20, Room 125, Flagstaff, AZ 86011-5698. Tel.: 928-523-7064; Email: edwin.lewis{at}nau.edu.

Studies performed in our laboratory demonstrated the formation of two thermodynamically distinct complexes on binding of netropsin to a number of hairpin-forming DNA sequences containing AATT-binding regions. These two complexes were proposed to differ only by a bridging water molecule between the drug and the DNA in the lower affinity complex. A temperature-dependent isothermal titration calorimetry (ITC)-binding study was performed using one of these constructs (a 20-mer hairpin of sequence 5'-CGAATTCGTCTCCGAATTCG) and netropsin. This study demonstrated a break in the heat capacity change for the formation of the complex containing the bridging water molecule at 303 K. In the plot of the binding enthalpy change versus temperature, the slope (Cp) was –0.67 kcal mol^–1 K^–1 steeper after the break at 303 K. Because of the relatively low melting temperature of the 20-mer hairpin (341 K (68°C)), the enthalpy change for complex formation might have included some energy of refolding of the partially denatured hairpin, giving the suggestion of a larger Cp. Studies done on the binding of netropsin to similar constructs, a 24-mer and a 28-mer, with added GC basepairs in the hairpin stem to increase thermal stability, exhibit the same nonlinearity in Cp over the temperature range of from 275 to 333 K. The slopes (Cp) were –0.69 and –0.64 kcal mol^–1 K^–1 steeper after 303 K for the 24-mer and 28-mer, respectively. This observation strengthens the argument regarding the presence of a bridging water molecule in the lower affinity netropsin/DNA complex. The Cp data seem to infer that because the break in the heat capacity change function for the lower affinity binding occurs at the isoequilibrium temperature for water, water may be included or trapped in the complex. The fact that this break does not occur in the heat capacity change function for formation of the higher affinity complex can similarly be taken as evidence that water is not included in the higher affinity complex.

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