Membranes and Active Transport of Amino Acids

描述（由申请人提供）：ABC蛋白或交通atp酶是一个大型易位子超家族的成员，在原核和真核生物中都有。真核成员包括囊性纤维化跨膜调节因子（CFTR）、多药耐药p糖蛋白（MDR）、肾上腺白质失调（ALD）基因、与抗原加工相关的异二聚体转运蛋白（Tap1/Tap2）、与Stargard黄斑营养不良有关的转运蛋白，以及许多其他具有医学重要性的转运蛋白。原核生物成员中有细菌质周透膜，已被广泛表征。考虑到所有这些蛋白质之间广泛的同源性，真核系统和原核系统在其作用机制上可能也有很强的相似性。因此，原核渗透为实现对这些易位子的活性和功能的一般理解提供了很好的模型。提出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，高效液相色谱分离多肽和质谱分析。比较从交联产物和未经处理的配合物中得到的图案将表明接触部位。实验还将对渗透酶在其活动周期的各个阶段进行。2)。我们也会尝试得到整个渗透酶的晶体结构。如果成功，这将提供对结构的最佳描述。3)。将获得分离的ATP结合域（HisP）的各种突变体的晶体结构，这将为启动ATP水解所需的信号和信号传导机制提供信息。对组氨酸周质渗透酶的这些和其他方面的研究将有助于理解这些转运体的一般机制问题，并有助于揭示它们的几个子结构域是如何起作用的。了解这些系统的结构对于开发研究真核转运体的新方法至关重要。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.