Structural Basis of the Tanford Transition of Bovine β-Lactoglobulin<sup>,</sup>

Qin, Bin; Bewley, Maria C.; Creamer, Lawrence K.; Baker, Heather M.; Baker, Edward N.; Jameson, Geoffrey B.

doi:10.1021/bi981016t

Cited by 465 publications

(581 citation statements)

References 43 publications

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“…Lg, a member of the lipocalin protein family, possesses a large hydrophobic cavity and is known to bind a variety of hydrophobic ligands, including FAs [19,20]. Association constants for the interaction between Lg and several FAs [x=6 (caprylic acid), 10 (lauric acid), and 14 (palmitic acid)], at 25°C and various pH values (6.2, 7.2, and 8.3) have been determined using a competitive fluorescence assay [21].…”

Section: Introductionmentioning

confidence: 99%

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

Liu

Kitova

Klassen

2011

J. Am. Soc. Mass Spectrom.

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The application of the direct electrospray ionization mass spectrometry (ESI-MS) assay to quantify interactions between bovine β-lactoglobulin (Lg) and a series of fatty acids (FA), CH 3 (CH 2 ) x COOH, where x=6 (caprylic acid, CpA), 8 (capric acid, CA), 10 (lauric acid, LA), 12 (myristic acid, MA), 14 (palmitic acid, PA) and 16 (stearic acid, SA), is described. Control ESI-MS binding measurements performed on the Lg-PA interaction revealed that both the protonated and deprotonated gas phase ions of the (Lg + PA) complex are prone to dissociate in the ion source, which leads to artificially small association constants (K a ). The addition of imidazole, a stabilizing solution additive, at high concentration (10 mM) increased the relative abundance of (Lg + PA) complex measured by ESI-MS in both positive and negative ion modes. The K a value measured in negative ion mode and using sampling conditions that minimize insource dissociation is in good agreement with a value determined using a competitive fluorescence assay. The K a values measured by ESI-MS for the Lg interactions with MA and SA are also consistent with values expected based on the fluorescence measurements. However, the K a values measured using optimal sampling conditions in positive ion mode are significantly lower than those measured in negative ion mode for all of the FAs investigated. It is concluded that the protonated gaseous ions of the (Lg + FA) complexes are kinetically less stable than the deprotonated ions. In-source dissociation was significant for the complexes of Lg with the shorter FAs (CpA, CA, and LA) in both modes and, in the case of CpA, no binding could be detected by ESI-MS. The affinities of Lg for CpA, CA, and LA determined using the reference ligand ESI-MS assay, a method for quantifying labile protein-ligand complexes that are prone to in-source dissociation, were found to be in good agreement with reported values.

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Section: Introductionmentioning

confidence: 99%

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

Liu

Kitova

Klassen

2011

J. Am. Soc. Mass Spectrom.

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“…As a result of such delocalization, the electron density of the water molecules may fall below the detection limit. (ii) At a typical resolution limit of Ϸ2 Å, a contaminating nonpolar ligand may be misinterpreted as a water cluster (9,10). (iii) For structures determined at cryogenic temperatures, the hydration status of the cavity may be altered by re-equilibration during the flash cooling process (11).…”

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confidence: 99%

“…Like other members of the lipocalin family, bovine ␤-lactoglobulin (BLG) has a ␤-barrel fold enclosing a cavity that binds nonpolar ligands, including palmitate (9,24,25). This cavity, known as the calyx, has a volume of 315 Å 3 (as gauged by a 1.4 Å probe) with approximately cylindrical (5 ϫ 15 Å) shape.…”

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confidence: 99%

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A dry ligand-binding cavity in a solvated protein

Qvist

Davidovic

Hamelberg

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

129

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Ligands usually bind to proteins by displacing water from the binding site. The affinity and kinetics of binding therefore depend on the hydration characteristics of the site. Here, we show that the extreme case of a completely dehydrated free binding site is realized for the large nonpolar binding cavity in bovine ␤-lactoglobulin. Because spatially delocalized water molecules may escape detection by x-ray diffraction, we use water 17 O and 2 H magnetic relaxation dispersion (MRD), 13 C NMR spectroscopy, molecular dynamics simulations, and free energy calculations to establish the absence of water from the binding cavity. Whereas carbon nanotubes of the same diameter are filled by a hydrogenbonded water chain, the MRD data show that the binding pore in the apo protein is either empty or contains water molecules with subnanosecond residence times. However, the latter possibility is ruled out by the computed hydration free energies, so we conclude that the 315 Å 3 binding pore is completely empty. The apo protein is thus poised for efficient binding of fatty acids and other nonpolar ligands. The qualitatively different hydration of the ␤-lactoglobulin pore and carbon nanotubes is caused by subtle differences in water-wall interactions and water entropy.␤-lactoglobulin ͉ free energy simulation ͉ hydrophobic hydration ͉ magnetic relaxation dispersion G lobular proteins are stabilized by dense atomic packing and by water exclusion from the nonpolar core region. However, the packing density is not uniform and a typical protein contains approximately four cavities per 100 residues of sufficient size to accommodate at least one water molecule (1). Small nonpolar cavities are usually empty and can be regarded as packing defects, whereas small polar cavities tend to be occupied by structural water molecules that stabilize the folded protein by H-bonding to otherwise unsatisfied peptide partners (2). Large cavities are frequently linked to protein functions, such as ligand binding and transport, membrane translocation, and enzyme catalysis. Some of these large cavities are lined exclusively or predominantly by nonpolar sidechains. The question whether such large nonpolar cavities are empty or contain water molecules is of fundamental biological importance (3-8).The principal tool of structural biology, x-ray crystallography, cannot easily resolve this issue. There are three potential problems. (i) Because water molecules interact only weakly with the nonpolar cavity walls, they tend to be positionally disordered. As a result of such delocalization, the electron density of the water molecules may fall below the detection limit. (ii) At a typical resolution limit of Ϸ2 Å, a contaminating nonpolar ligand may be misinterpreted as a water cluster (9, 10). (iii) For structures determined at cryogenic temperatures, the hydration status of the cavity may be altered by re-equilibration during the flash cooling process (11).In favorable cases, water molecules in nonpolar protein cavities can be inferred from intermolecular nuclear O...

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“…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%

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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Structural Basis of the Tanford Transition of Bovine β-Lactoglobulin^,

Cited by 465 publications

References 43 publications

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

A dry ligand-binding cavity in a solvated protein

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

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Structural Basis of the Tanford Transition of Bovine β-Lactoglobulin,

Cited by 465 publications

References 43 publications

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

A dry ligand-binding cavity in a solvated protein

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

Contact Info

Product

Resources

About

Structural Basis of the Tanford Transition of Bovine β-Lactoglobulin^,

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