Ligand interactions with lactose repressor protein and the repressor-operator complex: the effects of ionization and oligomerization on binding.
Specific interactions between proteins and ligands that modify their functions are crucial in biology. Here, we examine sugars that bind the lactose repressor protein (LacI) and modify repressor affinity for operator DNA using isothermal titration calorimetry and equilibrium DNA binding experiments. High affinity binding of the commonly-used inducer isopropyl-beta,D-thiogalactoside is strongly driven by enthalpic forces, whereas inducer 2-phenylethyl-beta,D-galactoside has weaker affinity with low enthalpic contributions. Perturbing the dimer interface with either pH or oligomeric state shows that weak inducer binding is sensitive to changes in this distant region. Effects of the neutral compound o-nitrophenyl-beta,D-galactoside are sensitive to oligomerization, and at elevated pH this compound converts to an anti-inducer ligand with slightly enhanced enthalpic contributions to the binding energy. Anti-inducer o-nitrophenyl-beta,D-fucoside exhibits slightly enhanced affinity and increased enthalpic contributions at elevated pH. Collectively, these results both demonstrate the range of energetic consequences that occur with LacI binding to structurally-similar ligands and expand our insight into the link between effector binding and structural changes at the subunit interface.
Wilson
Corey J
CJ
Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street, Houston, TX 77005, USA.
Zhan
Hongli
H
Swint-Kruse
Liskin
L
Matthews
Kathleen S
KS
D001426:Bacterial Proteins / Q000737:chemistry*; D002151:Calorimetry; D004247:DNA / Q000737:chemistry*; D019281:Dimerization; D006027:Glycosides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D056968:Lac Repressors; D008024:Ligands; D009598:Nitrophenylgalactosides / Q000737:chemistry; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry*
D001426:Bacterial Proteins; D006027:Glycosides; D056968:Lac Repressors; D008024:Ligands; D012097:Repressor Proteins; C047034:2-nitrophenyl-beta-fucoside; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D007544:Isopropyl Thiogalactoside; D004247:DNA