Structure determination of bovine B-lactoglobulin variants A and B : the structural consequences of point mutations, the structural basis of the Tanford transition, the structural influence of ligand on bovine BLGA : [a thesis] submitted to Massey University as partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry

Abstract

From the structure determination of bovine β-lactoglobulin variants A and B, the structural consequences of point mutations, the structural basis of the Tanford transition and mode of ligand binding by fatty acids have been elucidated. Bovine β-lactoglobulin was isolated first in 1934 by Palmer from skim milk. Bovine β-lactoglobulin contains 162 amino acid residues with 8 major β-strands which fold forth and back to form a β-barrel -creating a cup-shaped molecule or calyx. As a member of the lipocalin superfamily, this protein demonstrates the ability to bind a variety of small hydrophobic molecules, the most notable of which is retinol. Bovine β-lactoglobulin has at least six variants and undergoes pH-dependent conformational changes. Around pH 7, the conformational change is named as the Tanford transition and is characterized by the exposure of a buried COOH group above pH 7. Even though bovine β-lactoglobulin has a long history of structural studies, several regions of this protein molecule have remained poorly characterized. We applied X-ray diffraction techniques and successfully determined the structures of bovine β-lactoglobulin for variant A in lattice Z at pH 6.2, 7.1 and 8.2, for variant B in lattice Z at pH 7.1, and for variant A in lattice Z at pH 7.3 with the ligand 12-bromododecanoic acid (BrC12) bound. The structures have resolutions of 2.56, 2.24, 2.46, 2.22, and 2.23 Å, respectively. The corresponding values of R (R f) are 19.19% (24.03%), 23.35% (27.93%), 23.16% (27.69%), 23.93% (26.62)%, and 23.23% (27.93%). The C and N termini, as well as two disulfide bonds, are clearly defined in these models. Bovine β-lactoglobulin can be divided into several portions: the flexible top region which involves loops AB, CD, EF, and GH; the more rigid bottom region comprising loops BC, DE and FG; the calyx handle region which partially covers the β-barrel; and the β-barrel which is formed by β-sheets I and II. While the major portions of bovine β-lactoglobulin remain unchanged over the pH range 6.2 to 8.2, the loop EF, which contains Glu89, experiences a critical conformational change. This transition causes the side chain of Glu89, which is buried at pH 6.2, to become exposed at pH 7.1 and 8.2. This conformational change provides a structural basis for a variety of pH-dependent phenomena which are collectively known as the Tanford transition. Bovine β-lactoglobulin variant A differs from variant B at two point mutation sites: D64G and V118A. The first point mutation occurs in a rather mobile region of bovine β-lactoglobulin, loop CD, and results in the side chain of Glu65 adopting a different orientation. The second point mutation occurs in a very rigid region of bovine β-lactoglobulin, β-sheet II, and results in no significant conformational difference between two variants. While the major portions of the structures of variants A and B at pH 7.1 remain very similar, the loop EF adopts a different conformation. At pH 7.1, loop EF of variant B is closed, whereas that of variant A is opened. The crystal structure of the complex of bovine β-lactoglobulin variant A with 12-bromododecanoic acid (BrC12) reveals that the primary binding site for an aliphatic acid is in the interior of the calyx. The ligand (BrC12) has limited influence on the structure of bovine β-lactoglobulin A. Compared with unliganded BLGA at pH 7.1, one hundred and fifty six Cα atoms (96.3% out of total of 162 Cα atoms) have a displacement smaller than 0.5 Å among these Cβ atoms, the average displacement is only 0.169 Å. The region of major disturbance is loop GH, which has the maximum displacement of Cα, 1.38 Å. Comparison of the structures of bovine β-lactoglobulin (BLG) which crystallised in lattices X (triclinic), Y (orthorhombic), z (trigonal) reveals that the core portion of bovine β-lactoglobulin, which includes the "bottom" region, the β-sheets I and II, is relatively more rigid than other portions of bovine β-lactoglobulin molecule. The Cα atom trace of the structure of bovine β-lactoglobulin in lattice X is closer to that in lattice Z than that in lattice Y. The "lock and key" interface of bovine BLG uniquely exists in lattice Z, whereas the dimer interfaces of bovine BLG are found in all three lattices. Bovine BLG in lattice X and in lattice Y have similar "bottom:bottom" interface. Comparison of the structure of bovine β-lactoglobulin with retinol-binding protein, odorant-binding protein and bilin-binding protein reveals a similar fold, typical of the lipocalin family: an eight β-stranded β-barrel to which is attached a three-turn α-helix. However, the β-barrel of bovine β-lactoglobulin is potentially relatively flexible, as it is comprised of two β-sheets. This flexibility may reflect the fact that bovine β-lactoglobulin is able to bind a variety of small and not-so-small hydrophobic molecules. The conformational transition of loop EF may be relevant to the physiological function of β-lactoglobulin. The acid- and proteinase-resistant bovine β-lactoglobulin may hold and protect its ligand inside the calyx in the acidic conditions of stomach. After the holo bovine β-lactoglobulin passes the stomach, the basic conditions of the intestine cause the conformational change of loop EF, which opens the "door" to release the ligand. This property of bovine β-lactoglobulin suggests a potential pharmaceutical application - as a shuttle to convey acid-sensitive medicines