STRUCTURE OF P-GLYCOPROTEIN BY ELECTRON MICROSCOPY

描述(申请人提供)：P-糖蛋白(Pgp；也称为多药耐药蛋白)存在于高等真核生物的质膜中，负责ATP水解性疏水分子的输出。在动物体内，PGP在排泄和保护环境毒素方面发挥着重要作用。当Pgp表达在人类癌细胞的质膜上时，可以通过阻止疏水性化疗药物到达细胞内的靶点而导致化疗失败。Pgp是三磷酸腺苷结合盒(ABC)转运蛋白超家族的成员。ABC转运蛋白通常由四个结构域组成，两个核苷酸结合域(NBD)位于细胞质中，两个跨膜结构域(TMD)负责药物的结合和转运。尽管Pgp在人类疾病中起着重要作用，但人们对Pgp的结构知之甚少。我们正在使用二维晶体的电子显微镜来研究PGP的结构。这个建议的近期目标是1)可视化PGP在催化循环中正在经历的结构变化，2)计算在ATP水解和药物运输过程中不同步骤捕获的PGP的三维模型，以及3)优化我们目前产生PGP二维晶体的条件。结构研究将与PGP在其天然环境中结晶的脂双层进行。PGP的三维模型将作为解决PGP在药物转运周期中预期经历的结构变化的基础。了解这些结构变化最终可能有助于设计针对该蛋白的特定抑制剂，以便能够调节Pgp的活性，从而更有效地进行化疗。

DESCRIPTION (provided by applicant): P-glycoprotein (Pgp; also called multidrug resistance protein) is found in the plasma membrane of higher eukaryotes where it is responsible for ATP-hydrolysis-driven export of hydrophobic molecules. In animals, Pgp plays an important role in excretion of and protection from environmental toxins. When expressed in the plasma membrane of human cancer cells, Pgp can lead to failure of chemotherapy by preventing the hydrophobic chemotherapeutic drugs from reaching their targets inside the cells. Pgp is a member of the superfamily of ATP binding cassette (ABC) transporter proteins. ABC transporters consist typically of four domains, two nucleotide binding domains (NBDs) located in the cytoplasm and two trans-membrane domains (TMDs) responsible for drug binding and transport. Despite its important role in human disease, relatively little is known about the structure of Pgp. We are using electron microscopy of two-dimensional crystals to study the structure of Pgp. The immediate goals of this proposal are 1) to visualize the structural changes Pgp is undergoing during the catalytic cycle, 2) to calculate a three dimensional model of Pgp trapped at the different steps during ATP hydrolysis and drug transport and 3) to optimize the conditions under which we currently generate two dimensional crystals of Pgp. The structural studies will be conducted with Pgp crystallized in its native environment, the lipid bilayer. A three dimensional model of Pgp will serve as a basis for resolving the structural changes which Pgp is expected to undergo during the drug transport cycle. Understanding these structural changes might ultimately aid in the design for specific inhibitors for the protein in order to be able to regulate the activity of Pgp for a more effective chemotherapy.