STRUCTURE OF P-GLYCOPROTEIN BY ELECTRON MICROSCOPY

描述（由申请方提供）：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水解驱动的疏水分子输出。在动物中，Pgp在环境毒素的排泄和保护中起着重要作用。当Pgp在人癌细胞的质膜中表达时，其可通过阻止疏水性化疗药物到达其在细胞内的靶标而导致化疗失败。Pgp是ATP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，两个位于细胞质中的核苷酸结合结构域（NBD）和两个负责药物结合和转运的跨膜结构域（TMD）。尽管Pgp在人类疾病中起着重要作用，但对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。该提议的直接目标是1）可视化Pgp在催化循环期间经历的结构变化，2）计算在ATP水解和药物转运期间的不同步骤处捕获的Pgp的三维模型，以及3）优化我们目前生成Pgp的二维晶体的条件。结构研究将使用在其天然环境（脂质双层）中结晶的Pgp进行。一个三维模型的Pgp将作为一个基础，解决的结构变化，Pgp预计将在药物转运周期。了解这些结构变化可能最终有助于设计蛋白质的特异性抑制剂，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.