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 lac repressor, with natural DNA and alternating polydeoxynucleotides has been studied by means of fluorescence and circular dichroism. The important quenching of the intrinsic tyrosine fluorescence of the headpiece upon complexation has been used for the determination of the binding isotherms under various environmental conditions. By comparison with theoretical binding curves, we have determined a physical site size of three base pairs. The "perturbated" site size as determined from circular dichroism measurements (about four 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, we determine a number of three electrostatic interactions, assuming no anion release from the protein upon complexation. This value is independent of pH. On the contrary, 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 histidine-29. The finding is in favor of the idea that the lac repressor interacts with DNA via two headpieces as earlier work has shown that the interaction of the lac repressor with DNA requires the protonation of two groups of the protein.