In Vivo Pathways of Molecular Evolution: Acquisition of Thermostability by Mesophilic Adenylate Kinase

利用体内分子进化，P.I.正在研究生物体如何突变一个基本基因，作为对其环境变化的反应。该模式生物是嗜热硬脂芽孢杆菌的一种转基因菌株，其必需腺苷酸激酶基因（adk）的野生型拷贝被嗜热枯草芽孢杆菌基因所取代。该基金主要关注分子进化的两个关键属性；首先，细菌种群在适应极端环境时的特征是什么，其次，从这些选择中产生的蛋白质的物理化学性质是什么。实验跟踪突变细菌群体，因为它们竞争生存，并将使用x射线晶体学来检查热稳定性增加的立体化学基础。基于序列和结构的中温性和亲热性同源物的比较表明，氨基酸序列的大量变化可以增加热稳定性，而体外定向进化表明，较少数量的变化通常可以带来活性/热稳定性所需的变化。从这些观察中经常得出的一个结论是，在序列上看到的许多差异是进化漂变的结果，而不是进化的必然性。为了确定指导蛋白质进化的原理，一群细菌在进化一种必需蛋白质以获得更高的热稳定性时所采取的突变路径正在被确定。与沿着这条路径的蛋白质中间体的原子分析一起，将探索构成模型蛋白质热稳定性突变路径的复杂序列表面。改变进化实验的条件也可以改变细菌种群使用的“突变路径”，从而进一步了解进化。因此，这项工作的主要目标是阐明体内使用的途径，并应用生物物理学方法来了解详细突变途径的物理基础。

Using in vivo molecular evolution the P.I. is studying how an organism mutates an essential gene as a response to changes in its environment. The model organism is a genetically modified strain of B. stearothermophilus (a thermophile) that has had the wild type copy of the essential adenylate kinase gene (adk) replaced by the mesophilic B. subtilis gene. The grant focuses on two key attributes of molecular evolution; firstly, what are the characteristics of bacterial populations as they adapt to environmental extremes, and secondly, what are the physicochemical properties of the proteins generated from these selections. The experiments follow populations of mutant bacteria as they compete for survival and will use x-ray crystallography to examine the stereochemical basis for increases in thermostability.Sequence and structure based comparisons of mesophilic and thermophilic homologs have suggested that numerous changes in amino acid sequence can increase thermostability, whereas in vitro directed evolution has shown that a smaller number of changes can frequently bring about the required changes in activity/thermostability. One conclusion frequently drawn from these observations is that many of the differences seen in sequence are the result of evolutionary drift, rather than evolutionary necessity. To determine the principles that guide evolution of a protein, the mutational path(s) taken by a population of bacteria as they evolve an essential protein to higher thermostability is being determined. Together with atomic analysis of the protein intermediates along this path, the complex sequence surface that constitutes the mutational path to thermostability for the model protein will be explored. Altering conditions of the evolution experiment can also change the "mutational path" used by the bacterial populations and allow further insights into evolution. Thus the primary goal of the work is to elucidate the pathway(s) used in vivo and to apply biophysical methods to understand the physical basis for the elaborated mutational pathway(s).