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β-Lactoglobulin (β-LG) is the primary component of whey protein of cow’s milk with a concentration of 0.3 g/100 mL <ref>PMID:14254409</ref> and was first isolated in 1934 <ref>http://www.jbc.org/content/104/2/359.citation</ref>. | β-Lactoglobulin (β-LG) is the primary component of whey protein of cow’s milk with a concentration of 0.3 g/100 mL <ref>PMID:14254409</ref> and was first isolated in 1934 <ref>http://www.jbc.org/content/104/2/359.citation</ref>. | ||
Under physiological conditions | Under physiological conditions β-lactoglobulin exists as an equilibrium mixture of monomeric and dimeric forms (Fig. 1) | ||
Its amino-acid sequence and 3-dimensional structure show that it is a member of lipocalin, a widely diverse family, most of which bind small hydrophobic ligands and thus may act as specific transporters, as does serum retinol binding protein. <ref>PMID:15259212</ref> β-Lactoglobulin is synthesized in mammary gland and secreted in milk. It causes an allergic reaction in human and is one of the causes of cow's milk allergy. | Its amino-acid sequence and 3-dimensional structure show that it is a member of lipocalin, a widely diverse family, most of which bind small hydrophobic ligands and thus may act as specific transporters, as does serum retinol binding protein. <ref>PMID:15259212</ref> β-Lactoglobulin is synthesized in mammary gland and secreted in milk. It causes an allergic reaction in human and is one of the causes of cow's milk allergy. | ||
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<ref>http://www.sciencedirect.com/science/article/pii/S0958694698000211</ref> | <ref>http://www.sciencedirect.com/science/article/pii/S0958694698000211</ref> | ||
β-Lactoglobulin is a small protein, soluble in dilute salt solution as befits a globulin, with 162 amino acid residues (Mr ∼18,400) that fold up into an 8-stranded, antiparallel β-barrel with a 3-turn α-helix on the outer surface and a ninth β-strand flanking the first strand. | β-Lactoglobulin is a small protein, soluble in dilute salt solution as befits a globulin, with 162 amino acid residues (Mr ∼18,400)for each monomer that fold up into an 8-stranded, antiparallel β-barrel with a 3-turn α-helix on the outer surface and a ninth β-strand flanking the first strand. | ||
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Retinol and Palmitate Binding | Retinol and Palmitate Binding | ||
β-lactoglobulin (β-Lg) is a lipocalin, like plasma retinol-binding protein, so that ligand association was expected to make use of the central cavity in the protein.<ref>PMID:12054801</ref> | |||
A cocrystallized β-Lg with palmitic acid, and the refined structure (R = 0.204, R free = 0.240 for 6,888 reflections to 2.5-Å resolution) reveals that the ligand binds in the central cavity in a manner similar to the binding of retinol to the related lipocalin, serum retinol-binding protein.<ref>PMID:9867826</ref> It is probably also involved in the transport of that molecule.<ref>PMID:15259212</ref>. | A cocrystallized β-Lg with palmitic acid, and the refined structure (R = 0.204, R free = 0.240 for 6,888 reflections to 2.5-Å resolution) reveals that the ligand binds in the central cavity in a manner similar to the binding of retinol to the related lipocalin, serum retinol-binding protein.<ref>PMID:9867826</ref> It is probably also involved in the transport of that molecule.<ref>PMID:15259212</ref>. | ||
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The loops that connect the β-strands at the closed end of the calyx, BC, DE, and FG,are generally quite short, whereas those at the open end, AB, CD, EF,and GH, are significantly longer and more flexible [19]. In the calyx,there is a large central cavity which is surrounded by hydrophobic residues and is accessible to solvent. This cavity provides the principal ligand-binding site. βLG contains two tryptophan residues, Trp 19 on the A strand and Trp 61 on the C strand. The former is buried in the hydrophobic core whereas the latter is exposed to the solvent in the native structure, making them useful probes for monitoring site-specific conformational changes.<ref>PMID:19362581</ref> | The loops that connect the β-strands at the closed end of the calyx, BC, DE, and FG,are generally quite short, whereas those at the open end, AB, CD, EF,and GH, are significantly longer and more flexible [19]. In the calyx,there is a large central cavity which is surrounded by hydrophobic residues and is accessible to solvent. This cavity provides the principal ligand-binding site. βLG contains two tryptophan residues, Trp 19 on the A strand and Trp 61 on the C strand. The former is buried in the hydrophobic core whereas the latter is exposed to the solvent in the native structure, making them useful probes for monitoring site-specific conformational changes.<ref>PMID:19362581</ref> | ||
In addition, studies on the monomer–dimer equilibrium [30,32,42,43] and the reactivity of the thiol group of Cys121 which deeply buried between the α-helix and H strand [44–48] revealed other important properties of | In addition, studies on the monomer–dimer equilibrium [30,32,42,43] and the reactivity of the thiol group of Cys121 which deeply buried between the α-helix and H strand [44–48] revealed other important properties of β-LG.<ref>PMID:19362581</ref> the stability of the structure also depend so heavily upon the external loop around residue 64 or the β strand with the free thiol.<ref>PMID:9115437</ref> | ||
At physiological conditions, bovine b-lactoglobulin forms a dimer (Fig. 1), with each monomer consisting of 162 amino acid residues and characterized by a molecular mass of 18,350. Below pH 3, the dimer dissociates into <scene name='56/564055/ | At physiological conditions, bovine b-lactoglobulin forms a dimer (Fig. 1), with each monomer consisting of 162 amino acid residues and characterized by a molecular mass of 18,350. Below pH 3, the dimer dissociates into <scene name='56/564055/Native_β-lactoglobulin/1'>monomers (click here)</scene> which preserve their native conformation.<ref>PMID:11734004</ref> | ||
Genetically, b-lactoglobulin may exist as one of several variants, among which the variants A and B are the most abundant. The A and B variants of the protein differ from each other by amino acid residues at positions Asp64 (Gly64 in variant B) and Val118 (Ala118 in variant B). These differences in primary structure render the two variants slightly different with respect to isoelectric point, solubility, self-association properties, as well as pressure and temperature stability.<ref>PMID:11734004</ref> | Genetically, b-lactoglobulin may exist as one of several variants, among which the variants A and B are the most abundant. The A and B variants of the protein differ from each other by amino acid residues at positions Asp64 (Gly64 in variant B) and Val118 (Ala118 in variant B). These differences in primary structure render the two variants slightly different with respect to isoelectric point, solubility, self-association properties, as well as pressure and temperature stability.<ref>PMID:11734004</ref> | ||
However, the structural characteristics of the A and B variants of bovine b-lactoglobulin are virtually indistinguishable. In its native state, | However, the structural characteristics of the A and B variants of bovine b-lactoglobulin are virtually indistinguishable. In its native state, β-lactoglobulin is a predominantly β-sheet protein containing nine b-strands and three a-helices. The core of the protein is formed by a flattened b-barrel (a calyx) composed of eight antiparallel b-strands (A to H).<ref>PMID:11734004</ref> | ||
Ligands | Ligands | ||
Most lipocalins bind small hydrophobic molecules within the central cup or calyx. The true function of | Most lipocalins bind small hydrophobic molecules within the central cup or calyx. The true function of β-Lg is unknown, but it has been suggested that it is involved in | ||
the transport of retinol and/or fatty acids [8,50]. It binds retinol with a higher affinity than does RBP [51] and, as with RBP, specific binding of retinol to | the transport of retinol and/or fatty acids [8,50]. It binds retinol with a higher affinity than does RBP [51] and, as with RBP, specific binding of retinol to β-Lg has been observed in the small intestine of the neonatal calf [3]. The structure of RBP with retinol bound within the hydrophobic calyx has been solved [2] and retinol was successfully modelled into our previous β-Lg structure [3]. β-Lg contains two tryptophans, Trp19 and Trp61, and their fluorescence is altered when retinol is bound [51].<ref>PMID:9115437</ref> | ||
Co-crystallized β-Lg with palmitic acid, and the refined structure (R = 0.204, R free = 0.240 for 6,888 reflections to 2.5-Å resolution) reveals that the ligand binds in the central cavity in a manner similar to the binding of retinol to the related lipocalin, serum retinol-binding protein. The carboxyl group binds to both Lys-60 and Lys-69 at the entrance to the cavity. The hydrophobic tail stretches in an almost fully extended conformation into the center of the protein. | |||
==Molecular mechanism of the Tanford transition== | ==Molecular mechanism of the Tanford transition== | ||
Above pH 6.5, b-lactoglobulin undergoes the so-called Tanford transition which is triggered by protonation of Glu89 exhibiting an anomalously | Above pH 6.5, b-lactoglobulin undergoes the so-called Tanford transition which is triggered by protonation of Glu89 exhibiting an anomalously | ||
high pKa value. The Tanford transition involves displacement of the loop EF (residues 85 to 90) that acts as a lid which closes the protein interior/binding site below pH 7.3 and opens it at higher pH. The Tanford transition may involve some other structural changes as well. For example, the transition is accompanied by a change in the microenvironment of Tyr428 and causes an alteration in the relative orientation of <scene name='56/564055/ | high pKa value. The Tanford transition involves displacement of the loop EF (residues 85 to 90) that acts as a lid which closes the protein interior/binding site below pH 7.3 and opens it at higher pH. The Tanford transition may involve some other structural changes as well. For example, the transition is accompanied by a change in the microenvironment of Tyr428 and causes an alteration in the relative orientation of <scene name='56/564055/Native_β-lactoglobulin/1'>monomers (click here)</scene> in the dimer by as much as 5 degrees, which breaks a number of intersubunit hydrogen bonds. It should be noted that all transitions that take place between pH 2 and pH 9 do not cause any appreciable changes in the nativelike β-barrel conformation of β-lactoglobulin. | ||
<ref>PMID:11734004</ref> | <ref>PMID:11734004</ref> | ||
The structures of the trigonal crystal form of bovine | The structures of the trigonal crystal form of bovine β-lactoglobulin variant A at pH 6.2, 7.1, and 8.2 have been determined by X-ray diffraction methods at a resolution of 2.56, 2. 24, and 2.49 A, respectively. The glutamate side chain of residue 89 is buried at pH 6.2 and becomes exposed at pH 7.1 and 8.2. This conformational change, involving the loop 85-90, provides a structural basis for a variety of pH-dependent chemical, physical, and spectroscopic phenomena, collectively known as the Tanford transition.<ref>PMID:9760236</ref> | ||
It was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue.<ref>PMID:11168385</ref> | It was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue.<ref>PMID:11168385</ref> | ||
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Dimeric Lactoglobulin molecules exist in the open conformation at basic pH, whereas they exist in the closed conformation at acidic pH, after undergoing Tanford transition around neutral pH.<ref>PMID:17932936</ref> | Dimeric Lactoglobulin molecules exist in the open conformation at basic pH, whereas they exist in the closed conformation at acidic pH, after undergoing Tanford transition around neutral pH.<ref>PMID:17932936</ref> | ||
The Tanford transition is a conformational change of bovine | The Tanford transition is a conformational change of bovine β-lactoglobulin occurring at around pH 7, identified originally on the basis of optical rotatory dispersion and the accessibility of a thiol group. X-ray analysis has suggested that a conformational change to the EF-loop is responsible for the Tanford transition, with the loop closing the hydrophobic cavity of the β-barrel of the β-LG molecule below pH 7 and flipping to open the cavity above pH 7.<ref>PMID:16368109</ref> | ||
==Implications or possible application== | ==Implications or possible application== | ||
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In the dairy industry, bovine milk is frequently heated for pasteurization (62.5°C for 30 min) and sterilization. This heating process may induce oxidative losses of proteins, unsaturated lipids, vitamins, active | In the dairy industry, bovine milk is frequently heated for pasteurization (62.5°C for 30 min) and sterilization. This heating process may induce oxidative losses of proteins, unsaturated lipids, vitamins, active | ||
enzymes, and immunological factors. Cross-linking the free thiol groups of | enzymes, and immunological factors. Cross-linking the free thiol groups of β-LG by heating (100 degrees C for 2 min), or chemically modifying the β-LG by carboxymethylation to block the thiol groups resulted in a substantial loss of antioxidant activity. The data suggest that Cys-121 plays an essential role in the antioxidant nature of β-LG.Because β-LG is extremely sensitive to thermal denaturation, to maintain | ||
its antioxidant nature, dairy products consumed daily should not be overheated in order to maintain its antioxidant nature.<ref>PMID:17235131</ref> | |||
[[Image: | [[Image:β_lac_2Q2M.png|thumb|right|320px|Fig. 2 Bovine β-Lactoglobulin Native]] | ||
==Other β-Lactoglobulin related 3D Structures and complexes== | ==Other β-Lactoglobulin related 3D Structures and complexes== | ||
[[2q2m]] - Bovine | [[2q2m]] - Bovine β-Lactoglobulin Native (Fig. 2) | ||
[[1b8e]] - Crystal structure of the Bovine | [[1b8e]] - Crystal structure of the Bovine β-Lactoglobulin (Isoforms A and B) in orthorombic space group | ||
[[1qg5]] - Crystal structure of the Bovine | [[1qg5]] - Crystal structure of the Bovine β-Lactoglobulin (Isoforms A) | ||
[[1beb]] - Bovine | [[1beb]] - Bovine β-Lactoglobulin, Lattice X | ||
[[1cj5]] - Bovine | [[1cj5]] - Bovine β-Lactoglobulin A | ||
[[1gx8]] - Bovine | [[1gx8]] - Bovine β-Lactoglobulin complexed with Retinol, Trigonal Lattice Z | ||
[[1gx9]] - Bovine | [[1gx9]] - Bovine β-Lactoglobulin complexed with Retinoic acid, Trigonal Lattice Z | ||
[[1gxa]] - Bovine | [[1gxa]] - Bovine β-Lactoglobulin complexed with Retinol and Palmitic acid, Trigonal Lattice Z | ||
[[1b0o]] - Bovine | [[1b0o]] - Bovine β-Lactoglobulin complexed with Palmitate, Lattice Z | ||
[[1bsy]] [[2blg]] [[3blg]] - Structural Basis of the Tanford Transitioon of Bovine | [[1bsy]] [[2blg]] [[3blg]] - Structural Basis of the Tanford Transitioon of Bovine β-Lactoglobulin from crystal structures at 3 pH values | ||
