Membranes and Active Transport of Amino Acids

描述(由申请人提供)：ABC蛋白或交通ATPase是原核生物和真核生物转位蛋白超家族的成员。真核细胞成员包括囊性纤维化跨膜调节蛋白(CFTR)、多药耐药的P-糖蛋白(MDR)、肾上腺脑白质营养不良(ALD)相关基因、与抗原处理相关的异源二聚体转运蛋白(TAP1/TAP2)、参与Stargard黄斑营养不良的转运蛋白等。细菌周质透膜是原核生物的成员之一，已被广泛研究。考虑到所有这些蛋白质之间的广泛同源性，很可能真核和原核系统在作用机制上也会有很大的相似之处。因此，原核膜为全面了解这些转运子的活性和功能提供了极好的模型。这项拟议的工作旨在详细了解模型系统的三维结构：组氨酸渗透酶。我们的计划：1)。渗透酶的空间组织将通过化学交联、核磁共振和自旋标记研究来评估，这些研究将通过定义单个组分之间的最近邻接触来提供关于三维结构的信息。我们将对单个交联物进行提纯，进行蛋白质降解、多肽的高效液相色谱分离和质谱分析。将从交联物和未经处理的复合体获得的模式进行比较，将表明接触的位置。还将在其活性周期的不同阶段对渗透酶进行实验。2)。我们还将尝试获得整个渗透酶的晶体结构。如果成功，这将是对该结构的最佳描述。3)。将获得分离的ATP结合域(Hisp)的各种突变体的晶体结构，这将提供关于启动ATP水解所需的信号和信号机制的信息。对组氨酸周质通透酶的这些和其他方面的研究将有助于理解这些转运蛋白的一般机制问题，并有助于揭示它们的几个亚域是如何发挥作用的。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是至关重要的。然后，这些知识可以用来帮助开发治疗人类疾病的工具。

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.