Thermodynamics of sequence-specific glucocorticoid receptor-DNA interactions

摘要: The thermodynamics of sequence-specific DNA-protein interactions provide a complement to structural studies when trying understand the molecular basis for sequence specificity. We have used fluorescence spectroscopy study chemical equilibrium between wild-type and triple mutant glucocorticoid receptor DNA-binding domain (GR DBD wt GR DBDEGA, respectively) four related sites (response elements). NMR was confirm that structure two proteins is very similar in uncomplexed state. Binding DNA oligomers containing single half-sites palindromic binding studied obtain separate determinations association constants cooperativity parameters involved dimeric binding. Equilibrium were determined at 10-35 degrees C 85 mM NaCl, 100 KCl, 2 MgCl2, 20 Tris-HCl pH 7.4 (20 C) low concentrations an antioxidant nonionic detergent. DBDwt binds preferentially consensus response element (GRE) with constant (7.6 +/- 0.9) x 10(5) M-1 parameter 10 1 C. DBDEGA has highest affinity estrogen (ERE) (2.2 0.3) 121 17 difference processes, which indicates significant differences modes, confirmed using gel mobility assays. van't Hoff analysis shows all cases entropy driven within investigated temperature range. find delta H0obs S0obs formation DBDwt-GRE versus DBDEGA-ERE complex are significantly different despite G0obs values. A comparison GREs reveals discrimination these (specific) due favorable delta(delta S0obs) overcompensates unfavorable H0obs), i.e., specificity this case driven. Thus, entropic effects decisive importance as well GR-DNA interactions. measured thermodynamic discussed on published structures DBD-GRE ER DBD-ERE complexes.