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: 6298091
• 负责人: FRANCIS C NEUHAUS
• 金额: $ 0.75万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6298091
• 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.

将 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 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）。 理解结构-功能 载体蛋白的关系，解决方案的分析 该载体蛋白的结构已经确定。 这个结构 需要了解受体和供体反应 D-丙氨酸掺入中载体蛋白的特异性 系统。 确定该载体结构的所有尝试 通过晶体学方法鉴定蛋白质尚未成功。 随着 来自大肠杆菌的酰基载体蛋白，使用溶液 NMR 来建立 参与脂肪酸的载体蛋白的结构 生物合成。 由于 D-丙氨酰载体蛋白 (Dcp) 是同源物 对于该 ACP，我们决定使用 NMR 来测定 溶液构象。 同核二维核磁共振实验提供了 Dcp 的部分顺序分配。统一标记15N的研究 已进行 DCP（质谱分析 96%）。 15N 同核单量子相干（HSQC）实验获得于 芝加哥大学表明Dcp适合NMR分析 使用 3D 双共振光谱。 优异的抑水性能 是借助梯度和形状脉冲技术实现的 600 MHz 和浓度 0.5 mM。 预计可观测15N-1H 共振与氨基酸含量有很好的相关性 蛋白质。 为了实现 85-95% 序列分配的目标，我们将 需要 3D NOESY-HSQC 并优化混合时间以获得最大 顺序分配和具有最大各向同性的 3D TOCSY-HSQC 混合时间。 这些实验需要使用梯度来运行 以及用于抑制水的水翻转技术。 两者都不是 这些技术可在生物分子 NMR 设施中使用，网址为 西北大学。