Lactose repressor protein modified with dansyl chloride: activity effects and fluorescence properties

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Abstract
Chemical modification using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) was used to explore the importance of lysine residues involved in the binding activities of the lactose repressor in Escherichia coli and to introduce a fluorescent probe into the protein. Dansyl chloride modification of lac repressor resulted in loss of operator DNA binding at low molar ratios at reagent/monomer. Loss of nonspecific DNA binding was observed only at higher molar ratios, while isopropyl .beta.-D-thiogalactoside binding was not affected at any of the reagent levels studied. Lysine residues were the only modified amino acids detected. Protection of lysines-33 and -37 from modification by the presence of nonspecific DNA correlated with maintenance of operator DNA binding activity, and reaction of lysine-37 paralleled operator binding activity loss. Energy transfer between dansyl incorporated in the core region of the repressor protein and tryptophan-201 was observed, with an approximate distance of 23 .ANG. calculated between these 2 moieties.