Collini Maddalena, D'Alfonso Laura, Molinari Henriette, Ragona Laura, Catalano Maddalena, Baldini Giancarlo
Dipartimento di Fisica, Università degli Studi di Milano-Bicocca, Piazza della Scienza 3, 20126 Milan, Italy.
Protein Sci. 2003 Aug;12(8):1596-603. doi: 10.1110/ps.0304403.
The use of spectroscopy in the study of fatty acids binding to bovine beta-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS-BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.
在研究脂肪酸与牛β-乳球蛋白（BLG）结合时，使用光谱学似乎是一项艰巨的任务，因为这些被认为是蛋白质天然配体的酸性化合物，并未表现出有利的光学响应，例如吸收或荧光。因此，通过利用脂肪酸与ANS（一种广泛使用的荧光疏水探针）之间的竞争来解决BLG脂肪酸平衡问题，该探针在蛋白质上的结合位点最近已得到表征。已发现ANS-BLG复合物有两种寿命衰减；较长的一种归因于内部结合位点，较短的一种归因于外部位点。随着脂肪酸浓度的增加，与结合到内部位点的ANS相关的分数权重下降，这与描述染料与脂肪酸竞争的模型一致，而外部位点占有率似乎不受脂肪酸与BLG结合的影响。对接研究支持了该模型。通过用竞争结合模型对结合的ANS强度进行拟合，获得了BLG对酸结合亲和力的估计值。发现在6至8的溶液pH范围内存在相关依赖性，这与由EF环构象调节的花萼可及性相关。发现具有较长脂肪链的脂肪酸（棕榈酸酯和月桂酸酯）对该蛋白质表现出更大的亲和力，并且相互作用自由能与蛋白质花萼内的接触数很好地相关，这与对接模拟一致。
