Repressor proteins: Ligand binding, thermodynamics and assembly
Matthews, Kathleen S.
Doctor of Philosophy
LacI and PurR are members of the extended LacI/GalR family of bacterial proteins that regulate genetic expression. Detailed kinetic and thermodynamic studies on PurR were compared with results for homologous LacI. Operator binding affinity was increased by the presence of guanine as demonstrated previously, and conversely guanine binding affinity was increased by the presence of operator. Guanine enhanced operator affinity by increasing the association rate constant and decreasing the dissociation rate constant for binding. Operator had minimal effect on the association rate constant for guanine binding; however, this DNA decreased the dissociation rate constant for corepressor by ∼10-fold. Despite significant sequence and structural similarity between PurR and LacI proteins, PurR binds to its corepressor ligand with a lower association rate constant than LacI binds to its inducer ligand. However, the rate constant for PurR-guanine binding to operator is ∼3-fold higher than for LacI binding to its cognate operator under the same solution conditions. The distinct metabolic roles of the enzymes under regulation by these two repressor proteins provide a rationale for the observed functional differences. Addition of the LacI C-terminal tetramerization domain to the C-terminus of PurR resulted in the tetramerization of PurR. This tetrameric mutant of PurR exhibited very similar corepressor and operator binding affinity to wild-type PurR. These results establish that the C-terminal assembly motif from LacI can elicit tetramer formation in a naturally folded homologous dimer without interference with its biological function. Studies of the N-terminal DNA binding domain of LacI with variant hinge regions linked by a disulfide bond confirmed that the hinge region is important for operator recognition and interaction. DNA binding domains containing the hinge region from LacI (LH and VH) have similar affinity to lacO 1 operator, even though they form the disulfide bond at different positions in the hinge region. In addition, they bind to lacO 1 operator with ∼10-fold tighter affinity than that derived from the LacI DNA binding domain with the hinge region from PurR (PH). Deletion of the hinge region from the DNA binding domain (nH) resulted in a mutant without detectable operator binding even when linked by a disulfide bond.