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

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Abstract
Interactions between a well-characterized protein, β-lactoglobulin, and two flavor compounds, β-ionone and γ-decalactone, were studied by 2D NMR spectroscopy. NMR spectra were recorded in aqueous solution (pH 2.0, 12 mM NaCl, 10% D2O) under conditions such that β-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−CHα 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 γ-decalactone point into the central cavity (Leu46, Ile56, Met107, and Gln120), whereas binding of β-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 β-lactoglobulin, which was suggested in previous competition studies by fluorometry or affinity chromatography and by structural information obtained from infrared spectroscopy. Keywords: Aroma; binding site; protein; β-lactoglobulin; NMR spectroscopy; ionone; decalactone

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