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

Abstract

Bovine ␤-lactoglobulin, a major protein in cow's milk composed of nine ␤-strands (␤A-␤I) and one ␣-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 ␤-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 K a , suggesting that the intermolecular ␤-sheet is essential for dimer formation. By substituting either Asp 33 or Arg 40 on the AB-loop to oppositely charged residues (i.e. R40D, R40E, and D33R), a large decrease in K a was observed probably because of the charge repulsion, which is consistent with the role of electrostatic attraction between Arg 40 on one monomer and Asp 33 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 Arg 33 and Asp 40 of different molecules. These results suggested that protein-protein interactions of bovine ␤-lactoglobulin can be manipulated by redesigning the residues on the interface without affecting global folding.

Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group

“…As was also shown in our previous papers (12,14), the far-UV CD spectrum of wild-type ␤-lg at pH 7.0 had a minimum at 218 nm with an ellipticity of Ϫ6000 (Fig. 2a), showing that it is a predominantly ␤-sheet protein (Fig.…”

“…The quantity of protein expressed was checked with SDS-PAGE, and one of the strains with the highest level of expression was selected for every mutant and adopted for subsequent mass culture in a 1-liter jar fermenter. For wild-type ␤-lg, we used the strain constructed previously and stored as glycerol stock (12). The broth of mass culture was centrifuged, and the supernatant was filtered with 5.0-m-pore size Millex-SV (Millipore, Bedford, MA).…”

“…Cys-66 and Cys-160 form a disulfide bond near the surface of the protein molecule, Cys-106 and Cys-119 form a disulfide bond inside, and Cys121 retains a free thiol group. Although ␤-lg exists as a homodimer at neutral pH, it dissociates into monomers at acidic pH but still retains the native structure (7)(8)(9)(10)(11)(12). Because of its stability under acidic pH conditions, ␤-lg is resistant to digestion in the stomach and is considered one of the allergens for human infant milk allergy (13).…”

Structure and stability of Gyuba, a β‐lactoglobulin chimera

“…Although BLG is dimeric at neutral pH, it dissociates into monomers at low pH and very low ionic strength condition because of intermolecular electrostatic repulsions. 2,3 However, the monomers retain their tertiary structures at acidic pH. Both crystal [4][5][6][7][8][9] and solution 10,11 structures of BLG show that the monomer contains nine b-strands (strands A-I), an ahelix, and three short 3 10 helices.…”

“…A mutational study has shown that salt bridges between Asp33 and Arg40 of the opposing AB-loops that are important to the stability of the dimer. 3 BLG binds a variety of hydrophobic compounds including retinol, fatty acids, and vitamin D 3 at its hydrophobic cavity inside the barrel. [12][13][14] The loop between the E-and F-strands (EF-loop) obstructs the entrance to the b-barrel in the crystal structure at pH ¼ 6.2, whereas the entrance is open at pH ¼ 7.1.…”