Solution NMR Structures of Integral Membrane Proteins

描述（由申请人提供）：整合膜蛋白包括ca.占所有基因的30%，是目前绝大多数药物的靶点。这类蛋白质参与各种关键功能，包括产生电和化学梯度的运输，使动作电位成为可能的通道功能，介导细胞外信号传导的受体和呼叫识别。这些蛋白质的关键作用进一步反映在导致疾病状态的各种膜蛋白的突变的鉴定中，例如囊性纤维化中的CFTR、视网膜色素变性中的视紫红质和糖尿病中的胰岛素受体。与可溶性蛋白质结构测定速率的显著加快相反，膜蛋白的结构数据相对较少，PDB中只有大约30个结构。最近成功地应用溶液NMR方法来确定膜蛋白的结构，包括我们自己的OmpA结构的测定，表明溶液NMR可以在这一领域发挥重要作用。 目的1提出了一种可推广的方法来应用溶液NMR测定膜蛋白，特别是螺旋膜蛋白的高分辨率骨架结构。我们建议采用我们用于OmpA的方法，并增加了来自电子核弛豫的长程距离限制和偶极耦合的细化。我们正在测试各种方法，包括开发适当的媒体，以实现对齐，使用OmpA作为测试案例。 目的2将这些方法应用于21 kDa螺旋膜蛋白DsbB。作为一种据称是单体的小螺旋膜蛋白，这是Aim 1中开发的方法的完美测试。DsbB是Dsb蛋白家族的成员，所有这些蛋白都是大肠杆菌周质中正确形成二硫键所必需的。杆菌致病菌中DsbB同源物的突变使其无毒，这是由于在许多毒力所必需的蛋白质中不适当的二硫键形成，从而使其成为潜在的抗菌靶标。DsbB通过两个氧化还原活性二硫醇/二硫化物位点氧化周质蛋白DsbA，随后将这些还原当量转移至泛醌或甲基萘醌。这种蛋白质的结构对于理解DsbA和醌的识别以及催化机制至关重要。

DESCRIPTION (provided by applicant): Integral membrane proteins comprise ca. 30% of all genes and are the targets of the vast majority of current drugs. This class of proteins is involved in a variety of critical functions including transport to create electrical and chemical gradients, channel function to enable action potentials, receptors that mediate extracellular signaling, and call-call recognition. The critical role of these proteins is further reflected in the identification of mutations in various membrane proteins leading to disease sates such as the CFTR in cystic fibrosis, rhodopsin in retinitis pigmentosa, and the insulin receptor in diabetes. In contrast to the dramatic acceleration in the rate of structure determinations of soluble proteins, there is a relative paucity of structural data on membrane proteins with only approximately 30 structures in the PDB. Recant successes in the application of solution NMR methodology to the structure determination of membrane proteins, including our own determination of the structure of OmpA, suggest solution NMR can can play a significant role in this area. Aim 1 proposes to develop a generalizable approach to the application of solution NMR to the determination of high-resolution backbone structures of membrane proteins, particularly helical membrane proteins. We propose to employ the methods we used for OmpA with the addition of long-range distance restraints from election - nuclear relaxation and refinement with dipolar couplings. We are testing various approaches to each of these, including development of appropriate media to achieve alignment, using OmpA as a test case. Aim 2 proposes to apply these methods to the 21kDa helical membrane protein DsbB. As a purportedly monomeric small helical membrane protein, this is the perfect test for the methods developed in Aim 1. DsbB is a member of the Dsb family of proteins, all of which are essential for proper disulfide bond formation in the periplasm of E. coli. Mutation of DsbB homologs in pathogenic bacteria render them avirulent due to the improper disulfide formation in many proteins that are essential for virulence, thus making this a potential antibacterial target. DsbB oxidizes the periplasmic protein DsbA by means of two redox-active dithiol/disulfide sites, subsequently transferring these reducing equivalents to ubiquinone or menaquinone. A structure of this protein is critical to understand recognition of both DsbA and quinones, and the catalytic mechanism.