Nuclear Magnetic Resonance Spectroscopic Study of β-Lactoglobulin Interactions with Two Flavor Compounds, γ-Decalactone and β-Ionone

Abstract

Interactions between a well-characterized protein, beta-lactoglobulin, and two flavor compounds, beta-ionone and gamma-decalactone, were studied by 2D NMR spectroscopy. NMR spectra were recorded in aqueous solution (pH 2.0, 12 mM NaCl, 10% D(2)O) under conditions such that beta-lactoglobulin is present in a monomeric state. TOCSY and NOESY spectra were recorded on the protein and the complexes between protein and ligands. The spectra of the NH-CH(alpha) region showed the cross-signals due to the coupling between N- and C-bonded protons in the polypeptide backbone. The observed chemical shift variations in the presence of ligands can be assigned to changes in the protein conformation. It appears that the side chains of several amino acids are affected by binding of gamma-decalactone point into the central cavity (Leu46, Ile56, Met107, and Gln120), whereas binding of beta-ionone affects amino acids located in a groove near the outer surface of the protein (Leu104, Tyr120, and Asp129), as illustrated by molecular visualization. This NMR study provides precise information of the location of binding and confirms the existence of two different binding sites for aroma compounds on beta-lactoglobulin, which was suggested in previous competition studies by fluorometry or affinity chromatography and by structural information obtained from infrared spectroscopy.

Binding Studies and Computer‐Aided Modelling of Macromolecule/Odorant Interactions

“…Based on the literature results stating that fatty acids bind in the central hydrophobic pocket of BLG [18], it was concluded that the above-mentioned substances have the same binding position. Different behavior was found for the binding of b-ionone to BLG and this was confirmed by the 2D-NMR study of Lübke et al [19]. The 2D-NMR results suggested binding of g-decalactone (pH 2.0) in the central hydrophobic pocket of BLG and, for b-ionone, an external binding site in a groove on the outer surface of BLG that is built up by the helix and the external part of the b-barrel.…”

“…From these results, one can expect a binding position of g-decalactone to BLG, which is different from that of the central hydrophobic pocket. In contrast, 2D-NMR studies at pH 2.0 [19] and molecular-modelling investigations [13] suggested binding of g-decalactone in the central hydrophobic pocket of BLG.…”

Interaction between flavour compounds andβ-lactoglobulin: approach by NMR and 2D/3D-QSAR studies of ligands

“…Based on estimated affinity values, the semi-empirical classifying procedure led to obtaining three subsets: Mapping of compounds from group S21 on the best significant hypothesis of group S21 surprisingly showed that β-ionone 27 and γ -decalactone 33 were in good alignment, even when NMR observation pointed out two different binding regions on the BLG. 10 We noted an underestimation of S21 compounds by the best significant hypothesis generated by group P24 and an overestimation of P24 compounds by the best significant hypothesis generated by group S21. There is an assumption that this ensemble of compounds is related to the same binding site, which is able to undergo conformational changes according to experimental conditions.…”

“…[4][5][6][7][8]10 Thus, Catalyst confirms the existence of at least two binding sites on the BLG. It strongly suggests that some ligands recognize specifically the same binding site.…”

“…9 In our laboratory, interactions between BLG and two flavour compounds, β-ionone and γ -decalactone, were studied by 2D-NMR spectroscopy. 10 NMR spectra were recorded in aqueous solution under conditions such that BLG is present in a monomeric state (pH 2.0). TOCSY and NOESY spectra were recorded on the protein and protein-ligand complexes.…”