EQUILIBRIUM DNA-BINDING OF SAC7D PROTEIN FROM THE HYPERTHERMOPHILE SULFOLOBUS-ACIDOCALDARIUS - FLUORESCENCE AND CIRCULAR-DICHROISM STUDIES

The thermodynamics of the binding of the Sac7d protein of Sulfolobus a cidocaldarius to double-stranded DNA has been characterized using spec troscopic signals arising from both the protein and the DNA. Ligand bi nding density function analysis has been used to demonstrate that the fractional change in protein intrinsic tryptophan fluorescence quenchi ng that occurs upon DNA binding is equal to the fraction of protein bo und. Reverse titration data have been fit directly to the McGhee-von H ippel model [McGhee, J., & von Hippel, P. (1974) J. Mol. Biol. 86, 469 -489] using nonlinear regression. Sac7d binds noncooperatively to poly (dGdC). poly(dGdC) with an intrinsic affinity of 6.5 x 10(6) M(-1) and a site size of 4 base pairs in 1 mM KH2PO4 and 50 mM KCI (pH 6.8). So me binding sequence preference is noted, with the binding to poly(dIdC ). poly(dIdC) over 10-fold stronger than to poly(dAdT). poly(dAdT). Th e binding is largely driven by the polyelectrolyte effect and is consi stent with a release of 4.4 monovalent cations from DNA upon complex f ormation or the formation of 5 ion pairs at the protein-DNA interface. Extrapolation of salt back-titration data to 1 M KCI indicates a -2.2 kcal/mol nonelectrostatic contribution to the binding free energy. A van't Hoff analysis of poly(dGdC). poly(dGdC) binding shows that the b inding enthalpy is approximately zero and the process is entropically driven. The affinity decreases slightly between pH 5.4 and 8.0. There is no significant difference between the binding parameters of recombi nant Sac7d and native Sac7 proteins, indicating that methylation of th e native protein has no effect on the DNA binding function. The bindin g of Sac7d to various DNAs leads to a significant increase in the DNA long-wavelength circular dichroism (CD) band, the intensity of which s hows a sigmoidal dependence on Sac7d concentration. The sigmoidal CD b inding isotherm can be quantitatively modeled by a conformational tran sition in the DNA that is cooperatively induced when protein monomers are bound within a given number of base pairs, ranging from zero for p oly(dIdC). poly(dIdC) to 8 or less for poly(dAdG). poly(dCdT).