Sandbox Reserved 779: Difference between revisions

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The lipocalin family is a large and diverse family of proteins with functions varying from insect camouflage to small hydrophobic molecule transport typified by the serum retinol-binding protein <ref>http://www.biochemj.org/bj/318/bj3180001.htm</ref> The crystal structures so far determined reveal the typical lipocalin to be an eight-stranded antiparallel β-barrel arranged to form a conical central calyx or cavity in which the hydrophobic ligand is located.<ref>PMID:6540172</ref>

[[Image:Beta_lac_Residues.jpg|thumb|right|~~400px~~|Figure 1. Primary structure of β-LG. β-Lactoglobulin comprises 162 AA, including 5 Cys residues. Two disulfide linkages are located at residues Cys-106 to Cys-119 and Cys-66 to Cys-160. One free Cys is at position 121.(Taken from Liu, et al. 2007)<ref>PMID:17235131</ref>]]

[[Image:Beta_lac_Residues.jpg|thumb|right|320px|Figure 1. Primary structure of β-LG. β-Lactoglobulin comprises 162 AA, including 5 Cys residues. Two disulfide linkages are located at residues Cys-106 to Cys-119 and Cys-66 to Cys-160. One free Cys is at position 121.(Taken from Liu, et al. 2007)<ref>PMID:17235131</ref>]]

[[Image:General_view_of_beta_lactoglobulin.jpg|thumb|left|320px|Figure 2. A general view of b-lactoglobulin, a typical lipocalin. The binding site (filled atoms) is shown in the central calyx, and the putative binding site (open atoms) is indicated on the outer surface of the protein. The structurally conserved regions are at the rear of the molecule on strand A, the FG loop, and the loop before the a-helix.(Wu S et al. J. Biol. Chem. 1999;274:170-174)<ref>PMID:9867826</ref>]]

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===Ligands and Active sites===

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. It binds

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. It binds retinol with a higher affinity than does RBP. β-Lg contains two tryptophans, 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> and their fluorescence is altered when retinol is bound.<ref>PMID:9115437</ref>

Co-crystallized β-Lg with palmitic acid (3D Model 2._1B0O) and the refined structure 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.<ref>PMID:9867826</ref>

In addition, studies on the monomer–dimer equilibrium and the reactivity of the thiol group of Cys121 which deeply buried between the α-helix and H strand revealed other important properties of β-LG. 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>

[[Image:Vitamin D3 binding to the beta-lac calyx and dimer interface.jpg|thumb|left|240px|Figure 4. Vitamin D3 binding to the beta-lac calyx and dimer interface (Dominguez-Ramirez et al, 2013)<ref>PMID:24255705</ref>]]

β-Lg has binding sites for hydrophobic ligands like fatty acids, retinoic acid, and Vitamin D3 (cholecalciferol) and lactose [[ligands]] <ref>PMID:24255705</ref> For hydrophobic ligands two sites have been postulated, one inside the calyx and the other at the dimer interface, on the outer surface of the protein between the α-helix and the β-barrel (Fig. 4). The accessibility to the calyx is pH-dependent.

NMR and Xray analysis showed that the access is mediated by the mobile EF loop. All the structures with ligands bound to the calyx exhibit an open EF loop, suggesting that this site is accessible at neutral pH.

====Biological function====

Besides as binding protein, β-Lg is also assumed to have a transporter function especially for several ligands through the digestive tract in, as the calyx can protect any ligands entered/bound to the cavity.<ref>PMID:15259212</ref>.

=='''Molecular mechanism of the Tanford transition'''==

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 (residues I84 -N90) (Fig. 5) 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>Tanford transition is triggered by protonation of Glu89 exhibiting an anomalously high pKa value.

The Tanford transition may involve some other structural changes as well. For example, the transition is accompanied by a change in the microenvironment of Tyr42 and causes an alteration in the relative orientation of monomers in the dimer by as much as 5 degrees, which breaks a number of intersubunit hydrogen bonds. <ref>PMID:11734004</ref>

[[Image:BLG_monomer_showing_EF_Loop.png|thumb|left|240px|Figure 5. Bovine β-Lactoglobulin Monomer showing EF loop (colored yellow)]]

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. 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 84-90, 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>

=='''Uses / Application'''==

===Food Industry and Pharmacy===

β-Lactoglobulin interaction with hydrophobic molecules and with other proteins, and its sensitivity to chemical, thermal and baric denaturation, all with a view to establishing relationships among structure, properties and functionality and its potential use in food and pharmacy industry.<ref>http://www.sciencedirect.com/science/article/pii/S0958694698000211</ref>

Food Industry has been widely utilize β-LG to increase functionality (sensory, texture, nutritional) in commercial food and beverages application, and also as inexpensive source of protein to increase the overall protein content of their product. Pharmacy Industry is recently investigating and developing design of drugs that utilize β-LG as vehicle to transport antioxidants, drugs and vitamins to the gut.<ref>http://www.faqs.org/patents/app/20110038942</ref>

===Antioxidant Nature===

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 β-LG by heating (100 °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>

Whey Protein whose majority constituent is β-Lactoglobulin, gives abundant source of branched chain amino acids especially Leucine which plays a key role in initiating transcription of protein synthesis that speed recovery and adaptation to stress (exercise) and also a free thiol Cys which is (postulated) utilized in the body to generate antioxidant glutathione.<ref>http://www.kettlercise.com/members-area/supplements/whey-protein.html</ref>

=='''Other β-Lactoglobulin related 3D Structures and complexes'''==

[[2q2m]] - Bovine β-Lactoglobulin Native (Fig. 4)

[[1b8e]] - Crystal structure of the Bovine β-Lactoglobulin (Isoforms A and B) in orthorombic space group

[[1qg5]] - Crystal structure of the Bovine β-Lactoglobulin (Isoforms A)

[[1beb]] - Bovine β-Lactoglobulin, Lattice X

[[1cj5]] - Bovine β-Lactoglobulin A

[[1gx8]] - Bovine β-Lactoglobulin complexed with Retinol, Trigonal Lattice Z

[[1gx9]] - Bovine β-Lactoglobulin complexed with Retinoic acid, Trigonal Lattice Z

[[1gxa]] - Bovine β-Lactoglobulin complexed with Retinol and Palmitic acid, Trigonal Lattice Z

[[1b0o]] - Bovine β-Lactoglobulin complexed with Palmitate, Lattice Z

[[1bsy]] [[2blg]] [[3blg]] - Structural Basis of the Tanford Transitioon of Bovine β-Lactoglobulin from crystal structures at 3 pH values

=='''References'''==

=='''Proteopedia Page Contributors and Editors'''==

[[User:Rini Triani|Rini Triani]]

[[Category:Bos taurus]]

[[Category:Lipocalin]]

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@@ Line 20: / Line 20: @@
 The lipocalin family is a large and diverse family of proteins with functions varying from insect camouflage to small hydrophobic molecule transport typified by the serum retinol-binding protein <ref>http://www.biochemj.org/bj/318/bj3180001.htm</ref> The crystal structures so far determined reveal the typical lipocalin to be an eight-stranded antiparallel β-barrel arranged to form a conical central calyx or cavity in which the hydrophobic ligand is located.<ref>PMID:6540172</ref>
-[[Image:Beta_lac_Residues.jpg|thumb|right|400px|Figure 1. Primary structure of β-LG. β-Lactoglobulin comprises 162 AA, including 5 Cys residues. Two disulfide linkages are located at residues Cys-106 to Cys-119 and Cys-66 to Cys-160. One free Cys is at position 121.(Taken from Liu, et al. 2007)<ref>PMID:17235131</ref>]]
+[[Image:Beta_lac_Residues.jpg|thumb|right|320px|Figure 1. Primary structure of β-LG. β-Lactoglobulin comprises 162 AA, including 5 Cys residues. Two disulfide linkages are located at residues Cys-106 to Cys-119 and Cys-66 to Cys-160. One free Cys is at position 121.(Taken from Liu, et al. 2007)<ref>PMID:17235131</ref>]]
 [[Image:General_view_of_beta_lactoglobulin.jpg|thumb|left|320px|Figure 2. A general view of b-lactoglobulin, a typical lipocalin. The binding site (filled atoms) is shown in the central calyx, and the putative binding site (open atoms) is indicated on the outer surface of the protein. The structurally conserved regions are at the rear of the molecule on strand A, the FG loop, and the loop before the a-helix.(Wu S et al. J. Biol. Chem. 1999;274:170-174)<ref>PMID:9867826</ref>]]
@@ Line 38: / Line 38: @@
 ===Ligands and Active sites===
-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. It binds
+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. It binds retinol with a higher affinity than does RBP. β-Lg contains two tryptophans, 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> and their fluorescence is altered when retinol is bound.<ref>PMID:9115437</ref>
+Co-crystallized β-Lg with palmitic acid (3D Model 2._1B0O) and the refined structure 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.<ref>PMID:9867826</ref>
+In addition, studies on the monomer–dimer equilibrium and the reactivity of the thiol group of Cys121 which deeply buried between the α-helix and H strand revealed other important properties of β-LG. 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>
+[[Image:Vitamin D3 binding to the beta-lac calyx and dimer interface.jpg|thumb|left|240px|Figure 4. Vitamin D3 binding to the beta-lac calyx and dimer interface (Dominguez-Ramirez et al, 2013)<ref>PMID:24255705</ref>]]
+β-Lg has binding sites for hydrophobic ligands like fatty acids, retinoic acid, and Vitamin D3 (cholecalciferol) and lactose [[ligands]] <ref>PMID:24255705</ref> For hydrophobic ligands two sites have been postulated, one inside the calyx and the other at the dimer interface, on the outer surface of the protein between the α-helix and the β-barrel (Fig. 4).  The accessibility to the calyx is pH-dependent.
+NMR and Xray analysis showed that the access is mediated by the mobile EF loop. All the structures with ligands bound to the calyx exhibit an open EF loop, suggesting that this site is accessible at neutral pH.
+<Structure load='1b0o' size='320' frame='true' align='right' caption='3D model 2._β-lactoglobulin complexed with Palmitate, lattice Z_1B0O' scene='Insert optional scene name here' />
+====Biological function====
+Besides as binding protein, β-Lg is also assumed to have a transporter function especially for several ligands through the digestive tract in, as the calyx can protect any ligands entered/bound to the cavity.<ref>PMID:15259212</ref>.
+=='''Molecular mechanism of the Tanford transition'''==
+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 (residues I84 -N90) (Fig. 5) 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>Tanford transition is triggered by protonation of Glu89 exhibiting an anomalously high pKa value.
+The Tanford transition may involve some other structural changes as well. For example, the transition is accompanied by a change in the microenvironment of Tyr42 and causes an alteration in the relative orientation of monomers in the dimer by as much as 5 degrees, which breaks a number of intersubunit hydrogen bonds. <ref>PMID:11734004</ref>
+[[Image:BLG_monomer_showing_EF_Loop.png|thumb|left|240px|Figure 5. Bovine β-Lactoglobulin Monomer showing EF loop (colored yellow)]]
+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. 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 84-90, 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>
+=='''Uses / Application'''==
+===Food Industry and Pharmacy===
+β-Lactoglobulin interaction with hydrophobic molecules and with other proteins, and its sensitivity to chemical, thermal and baric denaturation, all with a view to establishing relationships among structure, properties and functionality and its potential use in food and pharmacy industry.<ref>http://www.sciencedirect.com/science/article/pii/S0958694698000211</ref>
+Food Industry has been widely utilize β-LG to increase functionality (sensory, texture, nutritional) in commercial food and beverages application, and also as inexpensive source of protein to increase the overall protein content of their product. Pharmacy Industry is recently investigating and developing design of drugs that utilize β-LG as vehicle to transport antioxidants, drugs and vitamins to the gut.<ref>http://www.faqs.org/patents/app/20110038942</ref>
+===Antioxidant Nature===
+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 β-LG by heating (100 °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>
+Whey Protein whose majority constituent is β-Lactoglobulin, gives abundant source of branched chain amino acids especially Leucine which plays a key role in initiating transcription of protein synthesis that speed recovery and adaptation to stress (exercise) and also a free thiol Cys which is (postulated) utilized in the body to generate antioxidant glutathione.<ref>http://www.kettlercise.com/members-area/supplements/whey-protein.html</ref>
+=='''Other β-Lactoglobulin related 3D Structures and complexes'''==
+[[2q2m]] - Bovine β-Lactoglobulin Native (Fig. 4)
+[[1b8e]] - Crystal structure of the Bovine β-Lactoglobulin (Isoforms A and B) in orthorombic space group
+[[1qg5]] - Crystal structure of the Bovine β-Lactoglobulin (Isoforms A)
+[[1beb]] - Bovine β-Lactoglobulin, Lattice X
+[[1cj5]] - Bovine β-Lactoglobulin A
+[[1gx8]] - Bovine β-Lactoglobulin complexed with Retinol, Trigonal Lattice Z
+[[1gx9]] - Bovine β-Lactoglobulin complexed with Retinoic acid, Trigonal Lattice Z
+[[1gxa]] - Bovine β-Lactoglobulin complexed with Retinol and Palmitic acid, Trigonal Lattice Z
+[[1b0o]] - Bovine β-Lactoglobulin complexed with Palmitate, Lattice Z
+[[1bsy]] [[2blg]] [[3blg]] - Structural Basis of the Tanford Transitioon of Bovine β-Lactoglobulin from crystal structures at 3 pH values
+=='''References'''==
+<references/>
+=='''Proteopedia Page Contributors and Editors'''==
+[[User:Rini Triani|Rini Triani]]
+[[Category:Bos taurus]]
+[[Category:Lipocalin]]