Glycine insertion in the hinge region of lactose repressor protein alters DNA binding

Abstract

Amino acid alterations were designed at the C terminus of the hinge segment (amino acids ~51-59) that links two functional domains within lactose repressor protein (LacI). Gly was introduced between Gly58 and Lys59 to generate Gly(58+1); Gln60 was changed to Gly or Pro, and up to three additional glycines were inserted following Gln60 → Gly. All mutant proteins exhibited purification behavior, CD spectra, assembly state, and inducer binding properties similar to wild-type LacI and only small differences in trypsin proteolysis patterns. In contrast, significant differences were observed in DNA binding properties. Gly(58+1) exhibited a decrease of ~100-fold in affinity for O1 operator, and sequential Gly insertion C-terminal to Gln60 → Gly resulted in progressively decreased affinity for O1 operator, approaching nonspecific levels for insertion of ≥2 glycines. Where sufficient affinity for O1 operator existed, decreased binding to O1 in the presence of inducer indicated no disruption in the allosteric response for these proteins. Collectively, these results indicate that flexibility and/or spacing between the core and N-terminal domains did not significantly affect folding or assembly, but these alterations in the hinge domain profoundly altered affinity of the lactose repressor protein for its wild-type target sequence.