BIOSYNTH OF BACTERIAL CELL WALL COMPONENTS: STRUCT OF D ALANYL CARRIER PROTEIN

细菌细胞壁成分的生物合成：D-丙氨酰载体蛋白的结构

• 批准号: 6281603
• 负责人: FRANCIS C NEUHAUS
• 金额: $ 0.37万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6281603
• 关键词: bacteria; biological products; biomedical resource; enzymes; nuclear magnetic resonance spectroscopy; proteins

The incorporation of D-alanine into membrane-associated D-alanyl-lipoteichoic in Lactobacillus casei requires the 56 kDa D-alanine-D-alanyl carrier protein ligase (Dcl) and the 8.9 kDa D-alanyl carrier protein (Dcp). To understand the structure-function relationships of the carrier protein, an analysis of the solution structure of this carrier protein has been undertaken. This structure is required for understanding the acceptor and donor reaction specificities of the carrier protein in the D-alanine incorporation system. All attempts to determine the structure of this carrier protein by crystallographic methods have been unsuccessful. With the acyl carrier protein from E. coli, solution NMR was used to establish the structure of this carrier protein involved in fatty acid biosynthesis. Since the D-alanyl carrier protein (Dcp) is a homolog of this ACP, we have decided to use NMR for the determination of the solution conformation. Homonuclear 2D NMR experiments have provided partial sequential assignment of Dcp. Studies of uniformly labeled 15N Dcp (96% by mass spectrometry) have been undertaken. A 15N homonuclear single quantum coherence (HSQC) experiment obtained at the University of Chicago shows that Dcp is suitable for NMR analysis using 3D double resonance spectroscopy. Excellent water suppression was achieved with the aid of gradients and shape pulse technology at 600 MHz and concentration of 0.5 mM. Expected observable 15N-1H resonances are in good correlation with the amino acid content of th protein. To achieve our goal of 85-95% sequence assignment, we would require a 3D NOESY-HSQC with mixing time optimized for maximum sequential assignment and a 3D TOCSY-HSQC with a maximum isotropic mixing time. These experiments will need to be run using gradients and water flip back techniques for water suppression. Neither of these techniques is available in the biomolecular NMR facility at Northwestern University.

D-丙氨酸掺入膜相关的 干酪乳杆菌中的D-丙氨酰-脂磷壁酸需要56 kDa的 D-丙氨酸-D-丙氨酰载体蛋白连接酶（Dcl）和8.9 kDa D-丙氨酰载体蛋白（Dcp）。 为了理解结构-功能 载体蛋白的关系，分析解决方案 该载体蛋白的结构已经进行。 这种结构 是理解受体和供体反应所必需的 载体蛋白在D-丙氨酸掺入中的特异性 系统 所有试图确定这艘航母结构的努力 蛋白质的结晶方法是不成功的。 与 E.大肠杆菌，溶液NMR用于建立 该载体蛋白质结构涉及脂肪酸 生物合成 由于D-丙氨酰载体蛋白（Dcp）是一种同源物， 在这种ACP中，我们决定使用NMR来确定 溶液构象 Homeland 2D NMR实验提供了 Dcp的部分顺序分配。均匀标记15 N的研究 DCP（96%通过质谱法）已经进行。 A 15 N 单量子相干（HSQC）实验， 芝加哥大学的研究表明，DCP适合于NMR分析 使用3D双共振光谱。 优异的水抑制性能 在梯度和形状脉冲技术的帮助下， 600 MHz，浓度为0.5 mM。预期可观察到15 N-1H 共振峰与氨基酸含量呈良好的相关性， 蛋白 为了实现85-95%序列分配的目标，我们将 需要3D NOESY-HSQC，混合时间最佳， 顺序分配和具有最大各向同性的3D TOCSY-HSQC 混合时间 这些实验将需要使用梯度运行 和用于抑制水的水翻转技术。 都不 这些技术可在生物分子NMR设备中获得， 西北大学。