Lübke Markus, Guichard Elisabeth, Tromelin Anne, Le Quéré Jean Luc
INRA-UMRA, 17 rue Sully, 21065 Dijon Cedex, France.
J Agric Food Chem. 2002 Nov 20;50(24):7094-9. doi: 10.1021/jf020513k.
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.
利用二维核磁共振光谱研究了一种特性明确的蛋白质——β-乳球蛋白与两种风味化合物——β-紫罗兰酮和γ-癸内酯之间的相互作用。在β-乳球蛋白以单体状态存在的条件下，于水溶液（pH 2.0，12 mM氯化钠，10% D₂O）中记录核磁共振光谱。记录了蛋白质以及蛋白质与配体之间复合物的TOCSY和NOESY光谱。NH-CH(α)区域的光谱显示了由于多肽主链中N键合质子与C键合质子之间的耦合而产生的交叉信号。在配体存在下观察到的化学位移变化可归因于蛋白质构象的改变。分子可视化结果表明，γ-癸内酯的结合似乎影响了几个氨基酸的侧链，这些氨基酸指向中心腔（Leu46、Ile56、Met107和Gln120），而β-紫罗兰酮的结合则影响了位于蛋白质外表面附近凹槽中的氨基酸（Leu104、Tyr120和Asp129）。这项核磁共振研究提供了结合位置的精确信息，并证实了β-乳球蛋白上存在两个不同的香气化合物结合位点，这在先前通过荧光测定法或亲和色谱法进行的竞争研究以及从红外光谱获得的结构信息中已有暗示。
