Investigations in the lactose repressor core domain: Characterization of the tryptophans and the inducer binding site
Gardner, Joseph Andrew
Matthews, Kathleen S.
Doctor of Philosophy
The lactose repressor protein, the prototypic genetic control element, contains two tryptophan residues at positions 201 and 220. We have utilized site-specific mutagenesis techniques to generate mutant lac repressors with single tryptophans. Fluorescence emission quenching studies and inducer difference spectra determination show that the two tryptophans reside in very different environments within the protein. Trp201 is buried within the subunit and is not in contact with the solvent. Trp220, however, appears to be much more exposed to the solvent, and its inducer difference spectrum indicates a direct involvement in the binding of inducer. Several carbohydrate-binding proteins whose structures have been determined by x-ray analysis reveal that tryptophan residues can be involved in a stacking interaction with the ligand and may even confer some specificity or selectivity for different ligands. Specifically, arabinose-binding protein and galactose-binding protein contain tryptophan residues which stack with the sugar ligand and preclude binding of some anomers of the natural ligands. We postulate that the lac repressor may utilize a similar binding interaction involving Trp220. Previous studies of energy transfer between dansyl and tryptophan in repressor have generated inconclusive results. The single-Trp mutant repressors were specifically modified at Cys140 with 1,5-IAEDANS in an effort to elucidate the energy transfer interactions between each Trp residue and the dansyl moiety. Based on the efficiency of energy transfer observed in these mutants and wild-type protein, as well as a mutant which does not form tetramers (T-41), we have shown that both Trp residues can transfer energy to an acceptor bound to Cys140. It appears that Trp201 is involved in intrasubunit transfer only, while Trp220 is able to transfer both within the subunit as well as between subunits. The energy transfer efficiency was utilized to determine molecular distances between the tryptophans and the specific dansyl group bound at position 140. These measurements support the hypothesis that the lac core domain may be structurally similar to some sugar-binding proteins.
Biochemistry; Cell biology