Structural Changes Accompanying pH-Induced Dissociation of the β-Lactoglobulin Dimer

We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and to characterize the backbone dynamics, of a recombinant version of bovine β-lactoglobulin (variant A) at pH 2.6, where the protein is a monomer. The structure of this low-pH form of β-lactoglobulin is very similar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2 (crystal) structure, calculated for backbone atoms of residues 6−160, is ∼1.3 Å. Differences arise from the orientation, with respect to the calyx, of the A−B and C−D loops, and of the flanking three-turn α-helix. The hydrophobic cavity within the calyx is retained at low pH. The E−F loop (residues 85−90), which moves to occlude the opening of the cavity over the pH range 7.2−6.2, is in the “closed” position at pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of 15N T 1s and T 2s and 1H−15N heteronuclear NOEs at pH 2.6 and 37 °C. Although the residues of the E−F loop (residues 86−89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well defined by the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Several residues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T 1 to T 2, consistent with conformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3D structure of β-lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity.