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

深入了解与催化周转相关的信号传导 (GSAM) 并了解蛋白质稳定性和储存特性 (RHCC)

• 批准号: 342077-2007
• 负责人: Stetefeld, Jörg
• 金额: $ 2.55万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2008
• 资助国家: 加拿大
• 起止时间: 2008-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=399764
• 关键词: Insight; into; signaling; linked; catalytic

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.

这项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(1)依赖维生素B6的叶绿素生物合成关键酶--谷氨酸-1-半醛氨基变位酶(GSAM)和(2)海洋葡萄球菌右手螺旋螺旋(RHCC)。其目的是获得关于这两个系统的机制和化学信息，以阐明GSAM负协同作用中的亚基间信号转导，并研究古细菌蛋白RHCC的极端稳定性和储存特性。研究将解决正在进行的结构-功能研究中已经出现或尚未得到回答的原子水平的机制问题。这项研究不仅将加强我们对GSAM中与催化周转有关的变构通讯的了解，还将对高选择性除草剂的基于结构的设计获得见解。由于这种酶在动物中不存在对应的酶，因此它是安全、选择性除草剂和此类化合物的合理设计的有希望的靶标。了解极端低pH条件下热稳定性的复杂机制，以及RCC与枯草杆菌蛋白家族的一种蛋白酶的复合体的形成，将提供关于极端条件下蛋白质稳定性本质的进一步信息。RHC独特的结合簇状排列水分子的能力使其适合于一个新的研究方向，即盘绕线圈的存储功能。所有这些研究都涉及结构(X射线结晶学和溶液核磁共振)和光谱(UV-Vis、CD、DSC和FRET)技术的结合，并辅之以体外功能分析。