STRUCTURE OF P-GLYCOPROTEIN BY ELECTRON MICROSCOPY

描述（由申请人提供）：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，在那里它负责atp水解驱动的疏水分子输出。在动物中，Pgp在排泄和保护环境毒素方面起着重要作用。当Pgp在人类癌细胞的质膜中表达时，它可以阻止疏水化疗药物到达细胞内的靶点，从而导致化疗失败。Pgp是ATP结合盒转运蛋白超家族的一员。ABC转运体通常由四个结构域组成，两个核苷酸结合结构域（NBDs）位于细胞质中，两个跨膜结构域（TMDs）负责药物结合和运输。尽管Pgp在人类疾病中发挥着重要作用，但对其结构的了解相对较少。我们利用二维晶体的电子显微镜来研究Pgp的结构。本研究的直接目标是：1)可视化Pgp在催化循环过程中发生的结构变化；2)计算ATP水解和药物运输过程中不同步骤捕获的Pgp的三维模型；3)优化我们目前生成Pgp二维晶体的条件。结构研究将在Pgp的天然环境中进行，即脂质双分子层。Pgp的三维模型将作为解决Pgp在药物运输周期中预期经历的结构变化的基础。了解这些结构变化可能最终有助于设计特定的蛋白质抑制剂，以便能够调节Pgp的活性，从而实现更有效的化疗。

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.