Nonspecific interaction of the lac repressor headpiece with deoxyribonucleic acid: fluorescence and circular dichroism studies

Abstract

The nonspecific interaction of the short headpiece, the NH2-terminal domain of the [Escherichia coli] lac repressor, with natural DNA and alternating polydeoxynucleotides was studied by means of fluorescence and circular dichroism. The important quenching of the intrinsic tyrosine fluorescence of the headpiece upon complexation was used for the determination of the binding isotherms under various environmental conditions. By comparison with theoretical binding curves, a physical site size of 3 base pairs was determined. The perturbated site size as determined from circular dichroism measurements (about 4 base pairs) is slightly greater. As in the case of the entire lac repressor, the interaction is strongly ionic strength dependent. The plots log Kobsd as a function of log [NaCl] are linear for salt concentrations greater than 50 mM for the interaction with DNA and greater than 25 mM for the interaction with poly[d(G-C)]. From the slopes of the linear parts of these plots, a number of 3 electrostatic interactions was determined, assuming no anion release from the protein upon complexation. This value is independent of pH; the association constant Kobsd depends on pH. Complexation requires the protonation of one titrating group of the headpiece with a pK value of 6.7 .+-. 0.3, probably the residue His-29. The finding is in favor of the idea that the lac repressor interacts with DNA via 2 headpieces, as earlier work has shown that the interaction of the lac repressor with DNA requires the protonation of 2 groups of the protein.