Judith Jiménez-Guzmán

β-Lactoglobulin's Conformational Requirements for Ligand Binding at the Calyx and the Dimer Interphase: a Flexible Docking Study

β-lactoglobulin (BLG) is an abundant milk protein relevant for industry and biotechnology, due significantly to its ability to bind a wide range of polar and apolar ligands. While hydrophobic ligand sites are known, sites for hydrophilic ligands such as the prevalent milk sugar, lactose, remain undetermined. Through the use of molecular docking we first, analyzed the known fatty acid binding sites in order to dissect their atomistic determinants and second, predicted the interaction sites for lactose with monomeric and dimeric BLG. We validated our approach against BLG structures co-crystallized with ligands and report a computational setup with a reduced number of flexible residues that is able to reproduce experimental results with high precision. Blind dockings with and without flexible side chains on BLG showed that: i) 13 experimentally-determined ligands fit the calyx requiring minimal movement of up to 7 residues out of the 23 that constitute this binding site. ii) Lactose does not bind the calyx despite conformational flexibility, but binds the dimer interface and an alternate Site C. iii) Results point to a probable lactolation site in the BLG dimer interface, at K141, consistent with previous biochemical findings. In contrast, no accessible lysines are found near Site C. iv) lactose forms hydrogen bonds with residues from both monomers stabilizing the dimer through a claw-like structure. Overall, these results improve our understanding of BLGs binding sites, importantly narrowing down the calyx residues that control ligand binding. Moreover, our results emphasize the importance of the dimer interface as an insufficiently explored, biologically relevant binding site of particular importance for hydrophilic ligands. Furthermore our analyses suggest that BLG is a robust scaffold for multiple ligand-binding, suitable for protein design, and advance our molecular understanding of its ligand sites to a point that allows manipulation to control binding.

Effect of an Exopolysaccharide-Producing Strain of Streptococcus Thermophilus on the Yield and Texture of Mexican Manchego-Type Cheese

An exopolysaccharide-producing strain of Streptococcus thermophilus was evaluated in the production of Mexican manchego-type cheese. This ropy strain improved water and fat retention, and significantly increased cheese yield. Furthermore, the ropy strain cheese retained more moisture than control cheese during ripening, suggesting that exopolysaccharide strongly bound water within the protein matrix of the cheese. Scanning electron microscopy confirmed that exopolysaccharide bound to the protein matrix of the cheese, producing a dense network that helped to increase water and fat retention and leading to a more open structure of the cheese that gave a softer product, as confirmed by instrumental texture profile analysis and sensory evaluation. Comparison of scanning electron microscopy micrographs of the different sections of the cheese showed higher concentration of exopolysaccharide in the centre than in the outer sections, indicating that exopolysaccharide production continued during ripening and that the environment at the centre of the cheese (moisture and/or oxygen concentration) favoured exopolysaccharide production. Instrumental texture profile analysis also demonstrated that the ropy strain cheese was more cohesive and less elastic than the control; in contrast, exopolysaccharide did not affect chewiness. The changes in texture could be correlated to composition: hardness increased as water and fat decreased, while springiness decreased with increasing fat. The interactions of exopolysaccharide with the cheese protein matrix had an affect on the increase in cohesiveness of the ropy strain cheese.

Antihypertensive and antithrombotic activities of a commercial fermented milk product made with Lactobacillus casei Shirota and Streptococcus thermophilus

Peptides which inhibit the human angiotensin-converting enzyme (ACE) may be released during milk fermentation, and the micro-organisms or fermentation conditions influence the specific peptides produced. The aim of this study was to evaluate the ACE inhibitory and antithrombotic activities of a fermented milk product commercially available in Mexico. Viable cell numbers, protein hydrolysis and the pH remained constant during refrigerated storage. The IC50 of ACE inhibitory activity was 31.38 mg/mL. Eight peptide fractions exhibited ACE inhibitory activity and six showed antithrombotic activity. Two fractions showed both. This is the first time that both activities have been reported in a commercial probiotic product.

Role of Lysine ε-Amino Groups of β-Lactoglobulin on Its Activating Effect of Kluyveromyces lactis β-Galactosidase

Native beta-lactoglobulin binds and increases the activity of Kluyveromyces lactis beta-galactosidase. Construction of a three-dimensional (3D) model of beta-lactoglobulin showed that lysine residues 15, 47, 69, and 138 are the most exposed ones, thus the ones more likely to interact with beta-galactosidase. Molecular docking estimated the interaction energies of amino acid residues with either lactose or succinic anhydride, showing that Lys(138) is the most likely to react with both. Affinity chromatography demonstrated that succinylated beta-lactoglobulin diminished its ability to bind to the enzyme. Furthermore, when activity was measured in the presence of succinylated beta-lactoglobulin, its activating effect was lost. Since succinylation specifically blocks Lys epsilon-amino groups, their loss very likely causes the disappearance of the activating effect. Results show that the activating effect of beta-lactoglobulin on beta-galactosidase activity is due to the interaction between both proteins and that this interaction is very likely to occur through the Lys epsilon-amino groups of beta-lactoglobulin.