Directed Evolution of the Dnak/ClpB chaperone system - towards substrate specific optimization and complementation with orthologous systems

Dnak/ClpB 伴侣系统的定向进化 - 实现底物特异性优化和与直系同源系统的互补

• 批准号: 5427654
• 负责人: Privatdozent Dr. Joachim Reinstein
• 金额: --
• 依托单位: Max-Planck-Institut für medizinische Forschung
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5427654?language=en
• 关键词: Directed; Evolution; Dnak; ClpB; chaperone

Molecular chaperones are highly conserved among all organisms and orthologous proteins may be found among a wide range of species. Accordingly they fulfill general housekeeping tasks but also allow cells to remain viable at stress conditions (mainly heat). Their principle tasks apply to a wide range of substrate proteins and as a consequence they are not highly specialized towards certain substrates. Since chaperones are so highly conserved, components that have to interact to perform certain tasks can originate from different species (orthologous systems) and yet be functional in vitro. We want to exploit cases where orthologous systems do not appear to be functional to learn about specific properties that may have been adapted for the relevant organism. Firstly, chaperones from the DnaK system of Thermus thermophilus should be evolved to confer heat resistance to Escherichia coli strains that lack the corresponding component. Secondly, these chaperones should be tailored for improved folding assistance towards selcted target substrates. The underlying functional and structural reasons for the desired changes will the be analysed in detail with spectroscopic, kinetic and structural methods.

分子伴侣在所有生物中高度保守，同源蛋白可能存在于广泛的物种中。相应地，它们完成一般的家务任务，但也允许细胞在应激条件下(主要是高温)保持存活。它们的主要任务适用于广泛的底物蛋白质，因此它们对某些底物不是高度专业化的。由于伴侣蛋白高度保守，必须相互作用才能完成某些任务的成分可能来自不同的物种(同源系统)，但在体外仍具有功能。我们想要利用同源系统似乎不起作用的情况，来了解可能已经适应相关有机体的特定性质。首先，应该进化出嗜热嗜热菌DNAK系统中的伴侣，使其对缺乏相应成分的大肠杆菌菌株具有耐热性。其次，这些伴侣分子应该量身定做，以改进对选定目标底物的折叠辅助。将用光谱、动力学和结构方法详细分析所需变化的潜在功能和结构原因。