The core lipocalin, bovine β-lactoglobulin

Sawyer, Lindsay; Kontopidis, George

doi:10.1016/s0167-4838(00)00160-6

Cited by 340 publications

(301 citation statements)

References 90 publications

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“…In native-like solution conditions (10 mM ammonium acetate buffer pH 5.0), BLG mainly existed in its dimeric form. However, at the lowest concentrations tested for BLG, peaks representing free monomers were also present, consistent with the reported K d value ($7.0 mM) for the B variant (Sawyer & Kontopidis, 2000). The mass spectrum of the D1 Fab fragment measured in a 10 mM ammonium acetate buffer exhibited a distribution of charge states of a single protein form (Fig.…”

Section: Resultssupporting

confidence: 87%

Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen

et al. 2007

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A D1 Fab fragment containing the allergen-binding variable domains of the IgE antibody was characterized by ESI FT-ICR mass spectrometry and crystallized with bovine -lactoglobulin (BLG) using the hanging-drop vapour-diffusion method at 293 K. X-ray data suitable for structure determination were collected to 2.8 Å resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 67.0, b = 100.6, c = 168.1 Å . The three-dimensional structure of the D1 Fab fragment-BLG complex will provide the first insight into IgE antibody-allergen interactions at the molecular level.

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Section: Resultssupporting

confidence: 87%

Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen

et al. 2007

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“…Since LG is a molten globule with a heating for 16 min (Table 1). 26 To confirm it further, we monitored the binding of heated LG (100°C for 16 min) with various amounts of vitamin D 3 , and a stoichiometry of 1:1 was observed between heat-denatured LG and vitamin D 3 using the same fluorescence quenching analysis. 26 Taking the previous pH and thermal experiments together (Table 1), we concluded that a thermally stable site (defined as an exosite) beyond the calyx exists for vitamin D 3 binding.…”

Section: Resultsmentioning

confidence: 92%

“…Crystals grew in larger quantity when the complex was prepared at pH 8 with a vitamin D 3 /LG ratio of 3 (data not shown). Although some precipitation was observed while the ratio of vitamin D 3 /LG increased, this was probably due to an excess of water-insoluble vitamin D 3 . Analysis of the diffraction pattern indicated that the crystals exhibited trigonal symmetry and systematic absences suggested that the space group was P3 2 21, with unit-cell parameters a ) b ) 53.78 Å and c ) 111.57 Å.…”

Section: Crystallization and Diffraction Of Lg-vitamin D 3 Complex Prmentioning

confidence: 93%

“…The final refined structure determined using the LG-vitamin D 3 complex prepared at pH 8 with a vitamin D 3 /LG ratio of 3:1 was given in detail and discussed elsewhere. 26 The surface model of the LG-vitamin D 3 complex reveals that vitamin D 3 almost perpendicularly inserts into the calyx cavity, while the other vitamin D 3 is located near the carboxyl terminus of LG (residues 136-149 including part of the R-helix and -strand I) ( Figure 6). It shows the exosite containing most hydrophobic residues fitted parallel with one vitamin D 3 molecule (Figure 6).…”

Section: Crystallization and Diffraction Of Lg-vitamin D 3 Complex Prmentioning

confidence: 99%

“…10 In a previous study, we first conducted a ligand binding assay using the fluorescence changes by retinol, palmitic LG was 2:1, whereas that of retinol or palmitic acid was 1:1 (Table 1), 26 suggesting that there was another binding site for the vitamin D 3 molecule. Second, we manipulated the binding capability by switching off the gate of calyx at low pH (2)(3)(4)(5)(6) 15 to further substantiate the "two site hypothesis". As expected, the binding capability of retinol and palmitic acid diminished under this condition, but it retained the vitamin D binding with a vitamin D 3 to LG stoichiometry of 1:1 (Table 1).…”

Section: Introductionmentioning

confidence: 99%

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Rational Design for Crystallization of β-Lactoglobulin and Vitamin D₃ Complex: Revealing a Secondary Binding Site

Yang

Guan

Yang

et al. 2008

Crystal Growth & Design

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-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D 3 binding, although the existence of another site beyond the calyx is controversial. Using fluorescence spectral analyses in the previous study, we showed the binding stoichiometry for vitamin D 3 to LG to be 2:1 and a stoichiometry of 1:1 when the calyx was "disrupted" by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. To help localize this secondary site using X-ray crystallography in the present study, we used bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) to identify the potential location of this site. We then optimized the occupancy and enhanced the electron density of vitamin D 3 in the complex by altering the pH and initial ratios of vitamin D 3 /LG in the cocrystal preparation. We conclude that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. Both pH (8) and initial ratio of vitamin D 3 /LG (3:1) are crucial to optimize the occupancy and enhance the electron density of vitamin D 3 in the complex for rational-designed crystallization. The strategy in practice may be useful for future identification of a ligand-binding site in a given protein.

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Oxidative folding pathways of bovine milk β‐lactoglobulin with odd cysteine residues

2019

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Bovine β‐lactoglobulin (BLG) is a major whey protein with unique structural characteristics: it possesses a free Cys thiol (SH) and two disulfide (SS) bonds and consists of a β‐barrel core surrounded by one long and several short α helices. Although SS‐intact conformational folding has been studied in depth, the oxidative folding pathways and accompanying SS formation/rearrangement are poorly understood. In this study, we used trans‐3,4‐dihydroxyselenolane oxide, a water‐soluble selenoxide reagent which undergoes rapid and quantitative SS formation, to determine the oxidative folding pathways of BLG variant A (BLGA) at pH 8.0 and 25 °C. This was done by characterizing two key one‐SS intermediates, a particular folding intermediate having a Cys66–Cys160 SS bond (I‐1) and a particular folding intermediate having a Cys106–Cys119 SS bond (I‐2), which have a native Cys66–Cys160 and Cys106–Cys119 SS bond, respectively. In the major folding pathway, the reduced protein (R) with abundant α helices was oxidized to I‐1, which was then transformed to I‐2 through SS rearrangement. The native protein (N) was formed by oxidation of I‐2. The redundant Cys121 thiol facilitates SS rearrangement. N is also generated from an ensemble of folding intermediates having two SS bonds (2SS) intermediates with scrambled SS bonds through SS rearrangement, but this minor pathway is deteriorative due to aggregation or overoxidation of 2SS. During oxidative folding of BLGA, α→β conformational transition occurred as previously observed in SS‐intact folding. These findings are informative not only for elucidating oxidative folding pathways of other members of the β‐lactoglobulin family, but also for understanding the roles of a redundant Cys thiol in the oxidative folding process of a protein with odd Cys residues.

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The core lipocalin, bovine β-lactoglobulin

Cited by 340 publications

References 90 publications

Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen

Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen

Rational Design for Crystallization of β-Lactoglobulin and Vitamin D₃ Complex: Revealing a Secondary Binding Site

Oxidative folding pathways of bovine milk β‐lactoglobulin with odd cysteine residues

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The core lipocalin, bovine β-lactoglobulin

Cited by 340 publications

References 90 publications

Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen

Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen

Rational Design for Crystallization of β-Lactoglobulin and Vitamin D3 Complex: Revealing a Secondary Binding Site

Oxidative folding pathways of bovine milk β‐lactoglobulin with odd cysteine residues

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Rational Design for Crystallization of β-Lactoglobulin and Vitamin D₃ Complex: Revealing a Secondary Binding Site