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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >The conserved disulfide bond of human tear lipocalin modulates conformation and lipid binding in a ligand selective manner.
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The conserved disulfide bond of human tear lipocalin modulates conformation and lipid binding in a ligand selective manner.

机译：人眼泪脂蛋白的保守二硫键以配体选择性方式调节构象和脂质结合。

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摘要

The primary aim of this study is the elucidation of the mechanism of disulfide induced alteration of ligand binding in human tear lipocalin (TL). Disulfide bonds may act as dynamic scaffolds to regulate conformational changes that alter protein function including receptor-ligand interactions. A single disulfide bond, (Cys61-Cys153), exists in TL that is highly conserved in the lipocalin superfamily. Circular dichroism and fluorescence spectroscopies were applied to investigate the mechanism by which disulfide bond removal effects protein stability, dynamics and ligand binding properties. Although the secondary structure is not altered by disulfide elimination, TL shows decreased stability against urea denaturation. Free energy change (DeltaG(0)) decreases from 4.9+/-0.2 to 2.1+/-0.3kcal/mol with removal of the disulfide bond. Furthermore, ligand binding properties of TL without the disulfide vary according to the type of ligand. The binding of a bulky ligand, NBD-cholesterol, has a decreased time constant (from 11.8+/-0.2 to 3.3s). In contrast, the NBD-labeled phospholipid shows a moderate decrease in the time constant for binding, from 33.2+/-0.2 to 22.2+/-0.4s. FRET experiments indicate that the hairpin CD is directly involved in modulation of both ligand binding and flexibility of TL. In TL complexed with palmitic acid (PA-TL), the distance between the residues 62 of strand D and 81 of loop EF is decreased by disulfide bond reduction. Consequently, removal of the disulfide bond boosts flexibility of the protein to reach a CD-EF loop distance (24.3A, between residues 62 and 81), which is not accessible for the protein with an intact disulfide bond (26.2A). The results suggest that enhanced flexibility of the protein promotes a faster accommodation of the ligand inside the cavity and an energetically favorable ligand-protein complex.
机译：这项研究的主要目的是阐明人眼泪脂蛋白（TL）中二硫键诱导的配体结合改变的机制。二硫键可以充当动态支架，以调节构象变化，从而改变蛋白质功能，包括受体-配体相互作用。 TL中存在单个二硫键（Cys61-Cys153），在脂环蛋白超家族中高度保守。圆二色性和荧光光谱用于研究二硫键去除影响蛋白质稳定性，动力学和配体结合特性的机理。尽管二级结构不会因二硫键消除而改变，但TL显示出降低的抗尿素变性稳定性。随着二硫键的去除，自由能变化（DeltaG（0））从4.9 +/- 0.2降至2.1 +/- 0.3kcal / mol。此外，不含二硫键的TL的配体结合特性根据配体的类型而变化。庞大的配体NBD-胆固醇的结合具有降低的时间常数（从11.8 +/- 0.2到3.3s）。相反，NBD标记的磷脂的结合时间常数从33.2 +/- 0.2到22.2 +/- 0.4s适度降低。 FRET实验表明，发夹CD直接参与了配体结合和TL柔韧性的调节。在与棕榈酸络合的TL（PA-TL）中，D链残基62和环EF 81之间的距离通过二硫键的还原而减小。因此，去除二硫键可增强蛋白质的柔韧性，以达到CD-EF环距离（残基62和81之间为24.3A），而具有完整二硫键的蛋白质（26.2A）则无法接近。结果表明，增强的蛋白质柔韧性促进了腔体内配体的更快容纳以及能量上有利的配体-蛋白质复合物。

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