Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, UK.
Biochemistry. 2010 Mar 16;49(10):2130-9. doi: 10.1021/bi901939z.
The structure and stability of the allergenic nonspecific lipid transfer protein (LTP) of peach were compared with the homologous LTP1 of barley and its liganded form LTP1b. All three proteins were resistant to gastric pepsinolysis and were only slowly digested at 1 to 2 out of 14 potential tryptic and chymotryptic cleavage sites under duodenal conditions. Peach LTP was initially cleaved at Tyr79-Lys80 and then at Arg39-Thr40 (a site lost in barley LTP1). Molecular dynamics simulations of the proteins under folded conditions showed that the backbone flexibility is limited, explaining the resistance to duodenal proteolysis. Arg39 and Lys80 side chains were more flexible in simulations of peach compared with barley LTP1. This may explain differences in the rates of cleavage observed experimentally for the two proteins and suggests that the flexibility of individual amino acid side chains could be important in determining preferred proteolytic cleavage sites. In order to understand resistance to pepsinolysis, proteins were characterized by NMR spectroscopy at pH 1.8. This showed that the helical regions of both proteins remain folded at this pH. NMR hydrogen exchange studies confirmed the rigidity of the structures at acidic pH, with barley LTP1 showing some regions with greater protection. Collectively, these data suggest that the rigidity of the LTP scaffold is responsible for their resistance to proteolysis. Gastroduodenal digestion conditions do not disrupt the 3D structure of peach LTP, explaining why LTPs retain their ability to bind IgE after digestion and hence their allergenic potential.
桃的变应原非特异性脂质转移蛋白（LTP）的结构和稳定性与大麦同源的 LTP1 及其配体形式 LTP1b 进行了比较。这三种蛋白质均能抵抗胃胃蛋白酶水解，仅在十二指肠条件下 14 个潜在胰蛋白酶和糜蛋白酶裂解位点中的 1 到 2 个缓慢消化。桃 LTP 最初在 Tyr79-Lys80 处裂解，然后在 Arg39-Thr40 处裂解（在大麦 LTP1 中丢失的位点）。蛋白质在折叠条件下的分子动力学模拟表明，骨架柔韧性有限，这解释了对十二指肠蛋白水解的抗性。与大麦 LTP1 相比，Arg39 和 Lys80 侧链在桃的模拟中更具柔韧性。这可能解释了这两种蛋白质在实验中观察到的裂解速率差异，并表明单个氨基酸侧链的柔韧性可能对确定首选蛋白水解裂解位点很重要。为了了解抗胃蛋白酶水解的机制，在 pH 1.8 下通过 NMR 光谱对蛋白质进行了表征。这表明两种蛋白质的螺旋区在该 pH 下仍保持折叠状态。NMR 氢交换研究证实了酸性 pH 下结构的刚性，大麦 LTP1 显示出一些具有更大保护作用的区域。总的来说，这些数据表明 LTP 支架的刚性是其抗蛋白水解的原因。胃十二指肠消化条件不会破坏桃 LTP 的 3D 结构，这解释了为什么 LTP 在消化后仍保留与 IgE 结合的能力，从而保留其变应原性。
