Functional implications of structural differences between variants A and B of bovine β‐lactoglobulin

Abstract

The structure of the trigonal crystal form of bovine b-lactoglobulin variant B at pH 7.1 has been determined by X-ray diffraction methods at a resolution of 2.22 Å and refined to values for R and R free of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine b-lactoglobulin variant A at pH 7.1, which was determined previously @Qin BY et al., 1998, Biochemistry 37:14014-14023#, the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD~residues 61-67!, there are small changes in main-chain conformation, whereas the substitution V118A on b-strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH-dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.Keywords: bovine b-lactoglobulin; crystal structure; genetic variants; hydrophobic stabilization Bovine b-lactoglobulin~BLG!, the major whey protein of cow's milk at a concentration of 0.3 g0100 mL~Bell & McKenzie, 1964!, was first isolated by Palmer~1934!. Mature bovine b-lactoglobulin has 162 residues, and is a member of the lipocalin protein superfamily~Flower, 1994, 1996!. Members of this family have a distinctive eight-stranded b-barrel structure, the central cavity of which binds a variety of hydrophobic molecules~Banaszak et al., 1994!. In the case of BLG, the primary site for fatty-acid binding has recently been established crystallographically to be inside this cavity~Qin et al., 1998b; Wu et al., 1999!. The structure of BLG is now reliably known following two independent redeterminations of the structure in the triclinic~lattice X!~Brownlow et al., 1997! and orthorhombic~lattice Y! forms~Bewley et al., 1997!. The latter structure has led to our redetermination of the structure in the trigonal~lattice Z! form~Qin et al., 1998a!. In lattice Z, in contrast to lattices X and Y, the entire molecule is well defined from electron density maps.Three variants of BLG, labeled as A, B, and C, commonly occur in cow's milk. Variants A and B~BLGA and BLGB! differ at two sites: Asp64 in A is changed to Gly in B, and Val118 in A is changed to Ala in B. Variants B and C differ at one site: Gln59 in B is changed to His in C. Thus, the isoelectric points for variants A, B, and C differ slightly: pI ϭ 5.26, 5.34, and 5.33, respectively, in 0.1 M KCl at room temperature~McKenzie, 1971!. Of technological significance, bovine BLG variants have different effects on the industrial processing of milk and on the characteristics of milk products~Hill et al., 1996!. Thermal stability is in the order B ...

Two modes of fatty acid binding to bovine β‐lactoglobulin—crystallographic and spectroscopic studies

“…The structure of liganded and unliganded LGB is similar to other trigonal structures of lactoglobulin reported previously (Brownlow et al 1997;Qin et al, 1999;. Nine stranded antiparallel b-sheet (strands A-I) is a core of a protein molecule (Figure 2A).…”

“…The only polar residues Lys60, Glu62 and Lys69 are located on the CD loop at the entrance to the calyx. Similarly to other trigonal LGB structures determined at pH higher than 7.1, the flexible loop EF is in the open conformation allowing ligand to enter the central calyx (Qin et al, 1999;Kontopidis et al, 2002;. The LGB molecule contains two tryptophan residues.…”

The influence of kappa-casein and beta-lactoglobulin genotypes on milk coagulation properties in Latvia dairy breed

“…Main alleles are A and B for coding this protein and only two amino acids are different. Aspartic acid is substituted with glycine at position 64 and valine with alanine at position 118 (Qin et al, 1999). Casein is milk protein which contains four casein fractions -α s1 -casein, α s2 -casein, β-casein and κ-casein.…”

Structure and stability of Gyuba, a β‐lactoglobulin chimera

“…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. Eight of the bstrands (A-H) form an up-and-down b-barrel, and the two I-strands form an intermolecular b-sheet containing four intermolecular hydrogen bonds.…”