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-半醛氨基交换酶（GSAM）和（ii）海洋葡萄球菌右旋盘状螺旋（RHCC）。目的是获得这两个系统的机制和化学信息，以揭示GSAM负协同作用中的亚基间信号传导，并研究古菌蛋白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-功能研究中出现或尚未回答的原子水平机制问题。这项研究不仅将增强我们对GSAM中与催化转化相关的变构通讯的认识，而且将为基于结构的高选择性除草剂设计提供见解。由于该酶在动物体内不存在对应物，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目标。了解极低pH下热稳定性的复杂机制，以及RHCC与枯草杆菌家族蛋白酶的复杂形成，将为极端条件下蛋白质稳定性的本质提供进一步的信息。RHCC独特的吸收簇状排列水分子的能力使其适合于一个新的研究方向，即卷绕线圈的存储功能。所有这些研究都涉及结构（x射线晶体学和溶液核磁共振）和光谱（UV-Vis， CD， DSC和FRET）技术的结合，并辅以体外功能分析。

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.