EXAMINATION OF SUBUNIT INTERACTIONS IN THE LACTOSE REPRESSOR BY PHYSICAL AND CHEMICAL METHODS (CYSTEINE, METHYL METHANETHIOSULFONATE, QUATERNARY STRUCTURE, ALLOSTERISM, MONOMER)
DALY, THOMAS JOSEPH
Doctor of Philosophy
The lactose repressor was modified with three sulfhydryl specific reagents which form mixed disulfide adducts. Methyl methanethiosulfonate (MMTS) and 5,5'-dithiobis(nitrobenzoic acid) (DTNB) completely reacted with all three cysteine residues, whereas only partial reaction was observed with didansyl cystine (DDC). Modification of cysteine 281 correlated with decreased operator binding affinity and decreased inducer association and dissociation rate constants. A monomeric mutant lactose repressor protein (T-41), containing serine at position 282 in place of tyrosine was purified. The inducer equilibrium binding constant for this monomeric mutant repressor was comparable to that of the tetrameric wild-type repressor at pH 7.5, while operator binding was not detectable. In contrast to wild-type repressor, equilibrium and kinetic rate constants for inducer binding to the monomer were largely independent of pH; thus, the quaternary structure of the wild-type repressor is required for the pH-associated effects on inducer binding. From the modification pattern with MMTS, cys 281, adjacent to the site of the T-41 mutation, appears to be located on the surface of the monomer in a region crucial for subunit interaction. Cysteine modification and variations in pH elicited alterations in the kinetic and equilibrium parameters for inducer binding to the repressor protein. While operator binding affinity of the repressor and its inducer complex were minimally affected by increased pH, inducer binding was decreased for both unliganded protein and the repressor-operator complex. The cooperativity for inducer binding to the repressor increased with pH. The midpoint for the changes in inducer affinity and cooperativity was pH 8.3; this value correlates well with cysteine ionization. Theoretical patterns for inducer binding under different conditions have been generated using the Monod-Wyman-Changeux model for allosteric regulation. Using this analysis, high pH favors the T (high operator/low inducer affinity) state, while modification of cysteine 281 with MMTS elicits a shift into the R (high inducer/low operator affinity) state. Ionization/protonation or covalent modification of cysteine 281 apparently influences the dynamics and equilibria of subunit interactions and consequently affects the induction process.