Lactose repressor protein modified with fluorescein mercuric acetate.

The reaction of lac repressor protein with fluorescein mercuric acetate has been studied. A maximum of 1.5 cysteine residues/monomer was modified with this reagent; the presence of ligands (inducer, anti-inducer, nonspecific DNA) did not affect ‘the reaction. Modification of the cysteines increased the affinity of the protein for inducer, but did not affect binding to nonspecific DNA. The operator binding activity, in contrast, was abolished at excesses of reagent 20.75 mercurials/monomer. Reversal of the effects on operator and inducer binding activity could be obtained by addition of dithiothreitol to the modified repressor protein. Mapping studies to determine the extent of reaction at each of the cysteines indicated that with excesses of fluorescein mercuric acetate below 0.5 molecules/monomer, cysteine 268 was less reactive than either cysteine 107 or cysteine 140; at the point of maximum reaction, each of the cysteines was approximately 50% reacted. Using 2-bromoacetamido-4-nitrophenol as a selective blocking agent for cysteines 107 and 140 prior to reaction with fluorescein mercuric acetate, the loss of operator binding activity was decreased, while no effect was observed on the increased affinity for inducer noted for mercurial modified protein. The loss of operator DNA binding activity with simultaneous maintenance of nonspecific DNA binding activity suggests that, while the determinants for binding may overlap, there are also independent determinants. It is of interest to note that loss of operator binding occurred with modification of residues in the core region of the molecule rather than in the NH2 terminus. Perturbations of the protein structure by inducer, anti-inducer, and nonspecific DNA were reflected in the fluorescein absorbance spectrum. Inducer difference spectra and anti-inducer difference spectra exhibited opposite characteristics; this is consistent with their different effects on the function of the protein molecule. Nonspecific DNA also perturbed the fluorescein spectrum; part of this difference spectrum involves direct interaction of the fluorescein mercuric acetate with the nucleic acid, but the spectrum also contained components attributable to perturbation of the fluorescein spectrum indirectly through the protein. This constitutes the first direct spectral evidence for the effect of nonspecific DNA on the protein structure. It is interesting to note that the spectral changes arise from residues which are in the core region of the molecule