Insight into signaling linked to catalytic turnover (GSAM) and understanding of protein stability and storage properties (RHCC)

该研究的主要目标是详细了解蛋白质作为具有酶活性或储存和递送功能的动态系统的结构-功能关系。研究将集中在（i）叶绿素生物合成中依赖维生素B6的关键酶，谷氨酸-1-半醛氨基变位酶（GSAM）和（ii）海洋葡萄球菌右手卷曲螺旋（RHCC）。目的是获得有关这两个系统的机制和化学信息，以阐明GSAM负协同性中的亚基间信号传导，并研究古细菌蛋白RHCC的极端稳定性和储存特性。研究将解决在正在进行的结构-功能研究中已经出现或尚未回答的原子水平的机械问题。这项研究不仅将增强我们对GSAM中与催化周转相关的变构通信的了解，而且将深入了解基于结构的高选择性除草剂设计。由于这种酶在动物中不存在对应物，因此它是安全、选择性除草剂和合理设计此类化合物的一个有希望的靶点。了解在极低pH下的热稳定性的复杂机制，以及RHCC与枯草杆菌蛋白酶家族的蛋白酶的复合物形成，将提供关于在极端条件下蛋白质稳定性的性质的进一步信息。RHCC的不寻常的能力，将集群状排列的水分子，使其适合一个新的研究方向，存储功能的卷曲的线圈。所有这些研究都涉及结构（X射线晶体学和溶液NMR）和光谱（UV-Vis，CD，DSC和FRET）技术的组合，并辅以体外功能测定。

The primary goal of the proposed research is to understand in detail the structure-function relationship of proteins as dynamic systems with either enzymatic activity or storage and delivery functions. The research will be focused (i) on the vitamin B6-dependent key enzyme in chlorophyll biosynthesis, glutamate-1-semialdehyde aminomutase (GSAM) and (ii) the Staphylothermus marinus right-handed coiled coil (RHCC). The purpose is to obtain mechanistic and chemical information about the two systems to shed light on the intersubunit signaling in the negative cooperativity of GSAM and to investigate the extreme stability and storage properties of the archaeal protein RHCC. Research will address atomic level mechanistic questions that have arisen or have not yet been answered in the on-going structure-function studies. Not only will the research enhance our knowledge of the allosteric communication in GSAM linked to catalytic turnover, but insights will be gained into structure-based design of highly selective herbicides. Because no counterpart of this enzyme exists in animals, it is a promising target for safe, selective herbicides and the rational design of such compounds. Understanding the intricate mechanisms of thermostability at extreme low pH, together with the complex formation of RHCC with a protease of the subtilisin family, will provide further information about the nature of protein stability under extreme conditions. The unusual capability of RHCC to incorporate cluster-like arranged water molecules makes it suitable for a new direction of research, the storage function of coiled coils. All these studies involve a combination of structural (X-ray crystallography and solution NMR) and spectroscopic (UV-Vis, CD, DSC and FRET) techniques complemented by in-vitro functional assays.