Abstract
Beta-lactoglobulins, belonging to the lipocalin family, are a widely studied group of proteins, characterized by the ability to solubilize and transport hydrophobic ligands, especially fatty acids. Despite many reports, the mechanism of ligand binding and the functional role of these proteins is still unclear, and many contradicting concepts are often encountered in the literature. In the present paper the comparative analysis of the binding properties of beta-lactoglobulins has been performed using sequence-derived information, structure-based electrostatic calculations, docking simulations, and NMR experiments. Our results reveal for the first time the mechanism of beta-lactoglobulin ligand binding, which is completely determined by the opening-closing of EF loop, triggered by Glu89 protonation. The alkaline shift observed for Glu89 pKa in porcine beta-lactoglobulin (pKa 9.7) with respect to the bovine species (pKa 5.5) depends upon the interplay of electrostatic effects of few nearby key residues. Porcine protein is therefore able to bind fatty acids provided that the appropriate pH solution conditions are met (pH > 8.6), where the EF loop conformational change can take place. The unusually high pH of binding detected for porcine beta-lactoglobulin seems to be functional to lipases activity. Theoretical pKa calculations extended to representative beta-lactoglobulins allowed the identification of key residues involved in structurally and functionally important electrostatic interactions. The results presented here provide a strong indication that the described conformational change is a common feature of all beta-lactoglobulins.
Highlights
The physicochemical and biological characteristics of -lactoglobulins, which belong to the lipocalin family, have been extensively studied in the last 30 years, but despite the wealth of data, the biological function of these extracellular proteins is still undefined (Ref. 1 and references therein). -Lactoglobulins isolated from cow, goat, and sheep milk samples, under nondenaturing conditions, showed endogenously bound fatty acids [2]
Titration experiments of bovine -lactoglobulin (BLG) with palmitic acid (PA) [4] have clearly shown that: (i) at neutral pH the primary site for palmitic acid binding is within the protein calyx; (ii) the amount of bound PA is drastically reduced upon decreasing pH and the ligand is completely released at pH 2; (iii) in the pH range 7.3– 6.4, a conformational equilibrium was observed for the bound ligand reflecting the dynamics of EF loop (Fig. 1), triggered by the titration of Glu89 at anomalously high pKa (ϳ6.5) [6]
Our results reveal for the first time the mechanism of -lactoglobulin ligand binding, which is fully determined by the conformational change involving the opening-closing of the EF loop [11]
Summary
Materials—PLG was purified from milk at NIZO Food Research (The Netherlands) as previously described [8]. Palmitic acid-protein complexes were prepared as described previously [4]. NMR titrations at different pH levels were performed adding a few microliters of 0.25 N H3PO4 or 1 M NaOH to the PA-protein complex prepared at pH 7.0. One-dimensional proton decoupled 13C spectra were recorded on the complex prepared with carboxyl-enriched PA (13C1 PA) in the pH range 7.0 –10.0. Two-dimensional 1H-13C HSQC experiments were recorded on 13C-PA and on PLG1⁄713C-PA complex with 512 and 2048 data points in the t1 and t2 dimensions, respectively, and a spectral width of 7002 (t2) and 5040 (t1) Hz. The spectra were processed and analyzed with the programs XWINNMR (Bruker) and XEASY [12]. The target proteins were considered rigid, and hydrogens were added with the program GRIN (part of the GRID package).
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