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The structure of bovine ?-lactoglobulin in crystals grown at pH 3.8 exhibiting novel threefold twinning.

ABSTRACT: Bovine ?-lactoglobulin was crystallized from 3?M NaCl buffered at pH 3.8 with sodium citrate as thick hexagonal prisms of greater than 1?mm in edge length. Analyses of the X-ray diffraction intensities using three different current algorithms were unanimous in specifying the space group to be P6322, with unit-cell dimensions a = b = 75.47, c = 140.79?Å. No progress could be made, however, towards an acceptable solution by molecular replacement using this symmetry. In the end, it was found that the true space group was C2221, a subgroup of P6322, with a = 65.89, b = 114.12, c = 140.51?Å, with the apparent 622 symmetry arising from an unusual threefold or tritohedral twinning. An assembly based on a model of the protein in another crystal form (PDB entry 1beb) containing three molecules in the asymmetric unit was refined to 2.3?Å resolution with a final R factor of 0.23 and Rfree of 0.26. NCS restraints were maintained throughout. For the most part, the molecules found in this crystal form are virtually the same as in PDB entry 1beb, although there are numerous local variations, particularly in loop elements, rotamer conformation differences and some alterations, including additions, at the termini.

SUBMITTER: Yeates TO

PROVIDER: S-EPMC6777136 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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The structure of bovine β-lactoglobulin in crystals grown at pH 3.8 exhibiting novel threefold twinning.

Yeates Todd O TO McPherson Alexander A

Acta crystallographica. Section F, Structural biology communications 20190920 Pt 10

Bovine β-lactoglobulin was crystallized from 3 M NaCl buffered at pH 3.8 with sodium citrate as thick hexagonal prisms of greater than 1 mm in edge length. Analyses of the X-ray diffraction intensities using three different current algorithms were unanimous in specifying the space group to be P6<sub>3</sub>22, with unit-cell dimensions a = b = 75.47, c = 140.79 Å. No progress could be made, however, towards an acceptable solution by molecular replacement using this symmetry. In the end, it was f ...[more]

PMID: 31584012

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Project description:Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.

Dataset Information

The structure of bovine ?-lactoglobulin in crystals grown at pH 3.8 exhibiting novel threefold twinning.

Publications

The structure of bovine β-lactoglobulin in crystals grown at pH 3.8 exhibiting novel threefold twinning.

Similar Datasets

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