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

Amino acid alterations were designed at the C terminus of the hinge segment (amino acids similar to 51-59) that links two functional domains within lactose repressor protein (LacI), Gly was introduced between Gly(58) and Lys(59) to generate Gly(58+1); Gln(60) was changed to Gly or Pro, and up to three additional glycines were inserted following Gln(60) --> 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 similar to 100-fold in affinity for O-1 operator, and sequential Gly insertion C-terminal to Gln(60) --> Gly resulted in progressively decreased affinity for O-1 operator, approaching nonspecific levels for insertion of greater than or equal to 2 glycines. Where sufficient affinity for O-1 operator existed, decreased binding to O-1 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.