Manipulating Monomer-Dimer Equilibrium of Bovine β-Lactoglobulin by Amino Acid Substitution

Bovine β-lactoglobulin, a major protein in cow's milk composed of nine β-strands (βA–βI) and one α-helix, exists as a dimer at neutral pH while it dissociates to a native monomer below pH 3.0. It is assumed that the intermolecular β-sheet formed between I-strands and salt bridges at AB-loops play important roles in dimer formation. Several site-directed mutants in which intermolecular interactions stabilizing the dimer would be removed were expressed in the methylotrophic yeast Pichia pastoris, and their monomer-dimer equilibria were studied by analytical ultracentrifugation. Various I-strand mutants showed decreases in Ka , suggesting that the intermolecular β-sheet is essential for dimer formation. By substituting either Asp33 or Arg40 on the AB-loop to oppositely charged residues (i.e. R40D, R40E, and D33R), a large decrease in Ka was observed probably because of the charge repulsion, which is consistent with the role of electrostatic attraction between Arg40 on one monomer and Asp33 on the other monomer in the wild-type dimer. However, when two of these mutants, R40D and D33R, were mixed, a heterodimer was formed by the electrostatic attraction between Arg33 and Asp40 of different molecules. These results suggested that protein-protein interactions of bovine β-lactoglobulin can be manipulated by redesigning the residues on the interface without affecting global folding.