Solution NMR Structure and Function of the Integral Membrane Protein DsbB

整体膜蛋白 DsbB 的溶液 NMR 结构和功能

• 批准号: 7133489
• 负责人: JOHN Hackett BUSHWELLER
• 金额: $ 31.46万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-25 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7133489
• 关键词: Escherichia coli; acidity /alkalinity; bacterial proteins; binding sites; detergents; disulfide bond; electron spin resonance spectroscopy; lipid bilayer membrane; membrane proteins; micelles; molecular dynamics; nuclear magnetic resonance spectroscopy; oxidoreductase; protein isoforms; protein protein interaction; protein structure function; quinones

DESCRIPTION (provided by applicant): Integral membrane proteins constitute approximately 30% of all genes, making them a very important class of proteins. In addition, more than half of all current drug targets are membrane proteins. This class of proteins is involved in a variety of functions including transport to create electrical and chemical gradients, channel function to enable action potentials, receptors that mediate extracellular signaling, and cell-cell 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. While the rate of structure determinations of soluble proteins has dramatically accelerated over the last decade, that of membrane proteins has lagged far behind, reflecting the technical challenges associated with working with this class of proteins. The primary focus of this grant is to utilize solution NMR approaches to study the structure and function of the integral membrane protein DsbB. The proteins DsbA and DsbB work in concert to mediate the formation of disulfide bonds in the periplasm of E. coli. DsbA is a soluble protein that functions as the primary oxidant for proteins in the periplasm whereas DsbB is an integral membrane protein that reoxidizes DsbA and is itself reoxidized by various quinones. Mutations of DsbA in pathogenic bacteria are avirulent since many virulence components contain disulfide bonds, suggesting this pathway may be a useful target for development of novel antibiotics. Four Specific Aims using NMR and EPR spectroscopy to characterize the structure and function of DsbB in detergent micelles and in the bilayer are proposed: Aim 1: Determination of the solution structure of DsbB, an integral membrane protein essential for disulfide formation in the periplasm of E. coli. Aim 2: Structural and functional characterization of the quinone binding site on DsbB. Aim 3: Characterization of DsbB dynamics in detergent and in the bilayer. Aim 4: Characterization of the DsbB-DsbA interaction.

描述（由申请人提供）：整合膜蛋白约占所有基因的 30%，使它们成为一类非常重要的蛋白质。此外，目前所有药物靶标中有一半以上是膜蛋白。这类蛋白质涉及多种功能，包括产生电和化学梯度的运输、实现动作电位的通道功能、介导细胞外信号传导的受体以及细胞间识别。这些蛋白质的关键作用进一步体现在识别导致疾病状态的各种膜蛋白突变，例如囊性纤维化中的 CFTR、色素性视网膜炎中的视紫红质和糖尿病中的胰岛素受体。虽然可溶性蛋白质的结构测定速度在过去十年中急剧加快，但膜蛋白的结构测定速度却远远落后，反映出与此类蛋白质相关的技术挑战。该资助的主要重点是利用溶液核磁共振方法来研究整合膜蛋白 DsbB 的结构和功能。蛋白质 DsbA 和 DsbB 协同作用，介导大肠杆菌周质中二硫键的形成。 DsbA 是一种可溶性蛋白质，充当周质中蛋白质的主要氧化剂，而 DsbB 是一种整合膜蛋白，可再氧化 DsbA，并且其本身被各种醌再氧化。病原菌中的 DsbA 突变是无毒的，因为许多毒力成分含有二硫键，这表明该途径可能是开发新型抗生素的有用靶标。提出了使用 NMR 和 EPR 光谱来表征洗涤剂胶束和双层中 DsbB 的结构和功能的四个具体目标： 目标 1：确定 DsbB 的溶液结构，DsbB 是大肠杆菌周质中二硫键形成所必需的完整膜蛋白. 大肠杆菌。目标 2：DsbB 上醌结合位点的结构和功能表征。目标 3：表征去垢剂和双层中的 DsbB 动力学。目标 4：DsbB-DsbA 相互作用的表征。