Membranes and Active Transport of Amino Acids

描述（由申请人提供）：ABC蛋白或运输ATP酶是原核和真核易位子大超家族的成员。真核生物成员包括囊性纤维化跨膜调节因子（CFTR）、多药耐药（MDR）的P-糖蛋白、肾上腺脑白质营养不良（ALD）的基因、与抗原加工相关的异二聚体转运蛋白（Tap 1/Tap 2）、与Stargard黄斑营养不良相关的转运蛋白以及许多其他具有医学重要性的基因。在原核成员中有细菌周质透酶，其已被广泛表征。考虑到所有这些蛋白质之间的广泛同源性，真核生物和原核生物系统在其作用机制方面也可能存在很强的相似性。因此，原核生物的渗透酶提供了很好的模型，实现了这些translocators的活动和功能的一般理解。 拟议的工作旨在详细了解的三维结构的模型系统：组氨酸通透酶。我们计划：1）.将通过化学交联、NMR和自旋标记研究来评估通透酶的空间组织，这些研究将通过定义各个组分之间的最近邻接触来提供关于三维结构的信息。我们将纯化单个交联产物，进行蛋白水解消化，肽的HPLC分离和质谱分析。从交联产品和未处理的复合物中获得的图案之间的比较将指示接触部位。还将对渗透酶在其活性循环的各个阶段进行实验。2）。我们还将尝试获得整个通透酶的晶体结构。如果成功，这将提供对结构的最佳描述。3）。将获得分离的ATP结合结构域（HisP）的各种突变体的晶体结构，这将提供关于启动ATP水解所需的信号和关于信号传导机制的信息。组氨酸周质通透酶的这些和其他方面的考试将有助于了解这些转运蛋白的一般机制问题，并将有助于解开他们的几个子域功能。了解这些系统的结构对于开发研究真核转运蛋白的新方法至关重要。然后，这些知识可以应用于帮助开发人类疾病的治疗工具。

DESCRIPTION (provided by applicant): ABC proteins or traffic ATPases are members of a large superfamily of translocators, both prokaryotic and eukaryotic. The eukaryotic members include the cystic fibrosis transmembrane regulator (CFTR), the P-glycoprotein of multidrug resistance (MDR), the gene responsible for adrenoleukodistrophy (ALD), the heterodimeric transporter associated with antigen processing (Tap1/Tap2), the transporter involved in Stargard macular dystrophy, and many others of medical importance. Among the prokaryotic members are bacterial periplasmic permeases, which have been extensively characterized. Considering the extensive homology between all of these proteins it is likely that there will also be strong similarities between eukaryotic and prokaryotic systems in their mechanism of action. Thus, prokaryotic permeases provide excellent models for achieving a general understanding of the activity and function of these translocators. The proposed work aims at understanding in detail the 3-dimensional structure of a model system: the histidine permease. We plan: 1). The spatial organization of the permease will be assessed by chemical cross-linking, NMR, and spin-labeling studies which will provide information on the three-dimensional structure by defining the nearest neighbor contact between the individual components. We will purify the individual cross-linked products, perform proteolytic digestion, HPLC separation of the peptides and mass spectrometry analysis. Comparison between patterns obtained from cross-linked products and from untreated complex will indicate the sites of contact. Experiments will also be performed on the permease in various stages of its activity cycle. 2). We will also try and obtain the crystal structure of the entire permease. If successful, this would provide the best description of the structure. 3). Crystal structures will be obtained of various mutants of the separated ATP-binding domains (HisP), which will provide information on which signals are needed to initiate ATP hydrolysis and on signaling mechanisms. Examination of these and other aspects of the histidine periplasmic permease will contribute to the understanding of general mechanistic questions for these transporters and will help unravel how their several sub-domains function. An understanding of the structure of these systems is essential for developing new approaches for investigating eukaryotic transporters. This knowledge can then be applied to help develop therapeutic tools for human diseases.