Thermodynamic, crystallographic and computational studies of non-mammalian fatty acid binding to bovine β-Lactoglobulin (original) (raw)
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Bovine β-lactoglobulin/fatty acid complexes: binding, structural, and biological properties
Dairy Science & Technology, 2014
Ligand-binding properties of β-lactoglobulin (β-lg) are well documented, but the subsequent biological functions are still unclear. Focusing on fatty acids/β-lg complexes, the structure-function relationships are reviewed in the light of the structural state of the protein (native versus non-native aggregated proteins). After a brief description of β-lg native structure, the review takes an interest in the binding properties of native β-lg (localization of binding sites, stoichiometry, and affinity) and the way the interaction affects the biological properties of the protein and the ligand. The binding properties of non-native aggregated forms of β-lg that are classically generated during industrial processing are also related. Structural changes modify the stoichiometry and the affinity of β-lg for fatty acids and consequently the biological functions of the complex. Finally, the fatty acid-binding properties of other whey proteins (αlactalbumin, bovine serum albumin) and some biological properties of the complexes are also addressed. These proteins affect β-lg/fatty acids complex in whey given their competition with β-lg for fatty acids.
Invited Review: β-Lactoglobulin: Binding Properties, Structure, and Function
Journal of Dairy Science, 2004
β-Lactoglobulin (β-LG) is the major whey protein of ruminant species and is also present in the milks of many, but not all, other species. Its amino-acid sequence and 3-dimensional structure show that it is a 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.
Ligand binding and self-association cooperativity of β-lactoglobulin
Journal of Molecular Recognition, 2013
Unlike most small globular proteins, lipocalins lack a compact hydrophobic core. Instead, they present a large central cavity that functions as the primary binding site for hydrophobic molecules. Not surprisingly, these proteins typically exhibit complex structural dynamics in solution, which is intricately modified by intermolecular recognition events. Although many lipocalins are monomeric, an increasing number of them have been proven to form oligomers. The coupling effects between self-association and ligand binding in these proteins are largely unknown. To address this issue, we have calorimetrically characterized the recognition of dodecyl sulfate by bovine b-lactoglobulin, which forms weak homodimers at neutral pH. A thermodynamic analysis based on coupled-equilibria revealed that dimerization exerts disparate effects on the ligand-binding capacity of b-lactoglobulin. Protein dimerization decreases ligand affinity (or, reciprocally, ligand binding promotes dimer dissociation). The two subunits in the dimer exhibit a positive, entropically driven cooperativity. To investigate the structural determinants of the interaction, the crystal structure of b-lactoglobulin bound to dodecyl sulfate was solved at 1.64 Å resolution.
A novel pH-dependent dimerization motif in β-lactoglobulin from pig ( Sus scrofa )
Acta Crystallographica Section D Biological Crystallography, 2002
Lactoglobulin (BLG) is a lipocalin and is the major protein in the whey of the milk of cows and other ruminants, but not in all mammalian species. The biological function of BLG is not clear, but a potential role in carrying fatty acids through the digestive tract has been proposed. The capability of BLG to aggregate and form gels is often used to thicken foodstuffs. The structure of the porcine form is suf®ciently different from other known BLG structures that SIRAS phases had to be measured in order to solve the crystal structure to 2.4 A Ê resolution. The r.m.s. deviation of C atoms is 2.8 A Ê between porcine and bovine BLG. Nevertheless, the typical lipocalin fold is conserved. Compared with bovine BLG, the tilted -helix alters the arrangement of surface residues of the porcine form, completely changing the dimerization behaviour. Through a unique pH-dependent domain-swapping mechanism involving the ®rst ten residues, a novel dimer interface is formed at the N-terminus of porcine BLG. The existence of this novel dimer at low pH is supported by gel-®ltration experiments. These results provide a rationale for the difference in physicochemical behaviour between bovine and porcine BLG and point the way towards engineering such dimerization motifs into other members of the lipocalin family.
A novel pH-dependent dimerization motif in beta-lactoglobulin from pig (Sus scrofa)
Acta Crystallographica Section D Biological Crystallography
beta-Lactoglobulin (BLG) is a lipocalin and is the major protein in the whey of the milk of cows and other ruminants, but not in all mammalian species. The biological function of BLG is not clear, but a potential role in carrying fatty acids through the digestive tract has been proposed. The capability of BLG to aggregate and form gels is often used to thicken foodstuffs. The structure of the porcine form is sufficiently different from other known BLG structures that SIRAS phases had to be measured in order to solve the crystal structure to 2.4 A resolution. The r.m.s. deviation of C(alpha) atoms is 2.8 A between porcine and bovine BLG. Nevertheless, the typical lipocalin fold is conserved. Compared with bovine BLG, the tilted alpha-helix alters the arrangement of surface residues of the porcine form, completely changing the dimerization behaviour. Through a unique pH-dependent domain-swapping mechanism involving the first ten residues, a novel dimer interface is formed at the N-ter...
Protein Science, 2003
The use of spectroscopy in the study of fatty acids binding to bovine β-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS–BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.
Journal of Molecular Structure, 2015
Bovine β-lactoglobulin is able to interact with different bioactive compounds, thus being an important candidate in the development of delivery systems with improved functionality. The heat induced changes in the β-lactoglobulin-oleic acid complex were examined by means of fluorescence spectroscopy and molecular modeling techniques. Fluorescence spectroscopy results indicated a rigid protein structure in the temperature range 25-70°C, whereas at temperatures over 75°C, the rearrangements of the polypeptide chains led to higher exposure of hydrophobic residues. The most significant increase of the accessible surface area with temperature increase was identified in case of Tyr 99 and Tyr 102. The phase diagram method indicated an all or none transition between two conformations. Due to conformational changes, no contact between Ile 56 or Lys 60 and the fatty acid could be identified at 85°C, but new non-bonding interaction were established with Ile 12 and Val 15. The results obtained in this study provide important details about thermal induced changes in the conformation of β-lactoglobulin-oleic acid complex. Significant conformational changes were registered above 75°C, suggesting the possibility of obtaining highly functional complexes between whey proteins and natural unsaturated fatty acids.
Ultra-high resolution crystal structure of recombinant caprine beta-lactoglobulin
FEBS Letters, 2014
b-Lactoglobulin (blg) is the most abundant whey protein in the milks of ruminant animals. While bovine blg has been subjected to a vast array of studies, little is known about the caprine ortholog. We present an ultra-high resolution crystal structure of caprine blg complemented by analytical ultracentrifugation and small-angle X-ray scattering data. In both solution and crystalline states caprine blg is dimeric (K D < 5 lM); however, our data suggest a flexible quaternary arrangement of subunits within the dimer. These structural findings will provide insight into relationships among structural, processing, nutritional and immunological characteristics that distinguish cow's and goat's milk. Structured summary of protein interactions: cBlg and cBlg bind by cosedimentation in solution (View interaction) bBlg and bBlg bind by cosedimentation in solution (View interaction) cBlg and cBlg bind by X ray scattering (View interaction) cBlg and cBlg bind by X-ray crystallography (View interaction)
Ovine β-lactoglobulin at atomic resolution
Acta crystallographica. Section F, Structural biology communications, 2014
The crystal structure of the triclinic form of the milk protein β-lactoglobulin from sheep (Ovis aries) at 1.1 Å resolution is described together with a comparison of the triclinic structures of the low-pH bovine and high-pH ovine proteins. All three structures are remarkably similar, despite the well known pH-dependent conformational transition described for the bovine and porcine proteins that occurs in solution. The high resolution of the present structure determination has allowed a more accurate description of the protein than has hitherto been possible, but it is still not clear whether flexibility changes in the external loops can compensate for the presence of a significant void in the unliganded interior of the structure.