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

分子伴侣在所有生物中都是高度保守的，在许多物种中都可以发现同源蛋白。因此，它们完成一般的家务任务，但也允许细胞在压力条件下（主要是热）保持活力。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并不高度专门化。由于伴侣蛋白是高度保守的，必须相互作用来执行某些任务的成分可以来自不同的物种（同源系统），但在体外仍具有功能。我们想利用的情况下，同源系统似乎没有功能，以了解具体的性质，可能已经适应了相关的生物体。首先，来自嗜热热菌DnaK系统的伴侣应该进化，以赋予缺乏相应成分的大肠杆菌菌株耐热性。其次，这些伴侣应该量身定制，以改善对选定目标底物的折叠辅助。所期望的变化的潜在功能和结构原因将用光谱、动力学和结构方法进行详细分析。

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.