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

D-丙氨酸掺入膜相关蛋白的研究 干酪乳杆菌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)及其8.9 kDa D-丙氨酰载体蛋白(DCP)。理解结构--功能 载体蛋白的相互关系，解决方案的分析 对该载体蛋白的结构进行了研究。这个结构 是理解受体和供体反应所必需的 D-丙氨酸掺入过程中载体蛋白的特异性 系统。所有试图确定该载体结构的尝试 用结晶学方法研究蛋白质的方法并不成功。与 利用溶液核磁共振技术从大肠杆菌中提取酰基载体蛋白 这种与脂肪酸有关的载体蛋白的结构 生物合成。由于D-丙氨酰载体蛋白(DCP)是同系物 对于这种ACP，我们决定使用核磁共振来确定 溶液构象。同核2D核磁共振实验提供了 DCP的部分顺序分配。均匀标记~(15)N的研究 已经进行了DCP(96%通过质谱分析)。A 15N 同核单量子相干(HSQC)实验在北京获得 芝加哥大学的研究表明，DCP适合于核磁共振分析 使用三维双共振谱。优良的防水性能 在梯度和形状脉冲技术的帮助下实现 600 MHz，浓度0.5 mm。预期可观测的15N-1H 波谱与氨基酸含量有很好的相关性。 蛋白。为了实现85%-95%的序列分配目标，我们将 需要3D NOESY-HSQC，混合时间优化为最大 序列分配和具有最大各向同性的三维TOCSY-HSQC 混合时间。这些实验需要使用渐变来运行 和水回喷技术，以抑制水。两个都不是 这些技术可在生物分子核磁共振设施中获得，网址为 西北大学。

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.