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

进化的原则是众所周知的，并反映在这个星球上生命的美丽和复杂性中。了解生命如何随时间进化和变化是现代生物学中的一个重要问题。甚至在DNA被发现是生命的遗传物质之前，通过对植物和动物的驯化就可以很容易地观察到潜在的进化思想。也许最广为人知的驯化例子之一是狗，它曾经是野狼，现在是同伴。虽然很容易理解狗和狼之间的外表差异，但更难确定基因及其编码的蛋白质的微小变化如何导致每个品种的特征发生变化。PI研究分子的进化，以及温度等环境条件的变化如何选择使这些分子更好地适应的变化(突变)。通常情况下，生物体适应新环境的时间比实验室研究中可行的时间要长得多。通过专注于一种简单的嗜热有机体(一种生活在高温下的细菌)，它可以在30分钟内繁殖，在一次实验室实验中可以研究数千代。为了促进这些研究，在以前的国家科学基金会的支持下，发展了“薄弱环节”方法。在“薄弱环节”的方法中，转基因嗜热有机体(嗜热脂肪杆菌)的生存依赖于一种基本分子的变化，这种分子只在较低的温度下起作用。为了在较高温度下繁殖，必需基因必须发生突变。通过在低温下培养细菌，然后逐渐升高温度，细菌可以很快适应成能够在高温下生存的细菌。然后就可以确定基本基因的变化是如何产生一种蛋白质，这种蛋白质能够很好地适应高温下的工作，并允许细菌在新的环境中生存。单基因的实验进化也为研究自然选择过程中蛋白质适应性进化的功能中间体提供了机会。通过这项工作可以解决几个基本问题。例如：进化的可重复性有多强？如果实验执行三次，是否会发生相同的变化并以相同的顺序进行？如果选择的条件发生变化会发生什么？适应将如何改变分子的反应？除了探索所有生命的基本原理外，这些发现还应用于蛋白质工程和生物技术，以生产更粗糙的蛋白质。影响更广的是科教研结合是S研究员实验室的首要着力点。9名本科生和3名研究生作为独立研究人员开展了这一项目的工作，并在科学会议和已发表的研究中提出了研究结果。该项目的范围从生物体到高分辨率结构中的电子密度，要求学生在进化、微生物学、生物化学和生物物理学方面获得广泛的教育。PI是NSF-IGERT细胞工程计划、休斯顿分子生物物理计划和墨西哥湾沿岸财团的教师导师，也是水稻进化中心的联合创始人。该研究所教授生物化学，并将从这些实验中学到的经验教训纳入到蛋白质折叠和稳定性的讨论中，以及进化如何通过耐药病原体的崛起影响我们的世界。在过去的三年里，该实验室接待了7个高中小组(包括高中AP教师计划)，并通过大米AGEP计划指导来自代表性不足的西班牙裔社区的暑期研究人员。该协会还利用这一项目作为以社区为基础的关于进化论本质的研讨会(“咖啡壶中的进化论”)。

The principles of evolution are well known and are reflected in the beauty and complexity of life on this planet. Understanding how life evolves and changes through time is an important question in modern biology. Even before DNA was discovered to be the hereditary material of life, the ideas underlying evolution could be observed readily by the domestication of plants and animals. Perhaps one of the most well known examples of domestication is the dog, once a wild wolf and now a companion. Though it is easy to appreciate the outward differences between a dog and a wolf, it is more difficult to determine how small changes in the genes and the proteins encoded by them give rise to changes in the characteristics of each breed. The PI studies the evolution of molecules and how changes in environmental conditions such as temperature select for changes (mutations) that make those molecules better adapted. Typically, the adaptation of organisms to new conditions takes much longer than is practicable in lab studies. By focusing on a simple thermophilic organism (a bacterium that lives at high temperatures) that reproduces in 30 minutes, thousands of generations can be studied during a single laboratory experiment. To facilitate these studies the ''weak link'' approach was developed with previous NSF support. In the ''weak link'' approach, survival of a genetically modified thermophilic organism (Geobacillus stearothermophilus) is dependent on changes to an essential molecule that only functions at lower temperature. In order to reproduce at higher temperature, mutations must occur to the essential gene. By growing the bacteria at low temperature and then gradually increasing the temperature the bacteria can be quickly adapted into one that can live at high temperatures. It is then possible to determine how changes to the essential gene produced a protein that was well adapted to work at high temperature and permit the bacteria to survive in the new environment. Experimental evolution of a single gene also provides an opportunity to investigate the functional intermediates of protein adaptive evolution during the course of natural selection. Several fundamental questions can be addressed through this work. For example: How reproducible is evolution? If the experiment is performed three times do the same changes occur and in the same order? What happens if the conditions of the selection change? How will adaptation change the molecule in response? In addition to exploring the basic principles for all life, these findings have application to protein engineering and biotechnology for the production of more rugged proteins. Broader Impact The integration of science education and research is a primary focus of the investigator''s laboratory. Nine undergraduate and three graduate researchers have carried out work on this project as independent research and have presented findings at scientific conferences and in published research. The breadth of the project from organism to electron density in high resolution structures requires students to become broadly educated in evolution, microbiology, biochemistry and biophysics. The PI is faculty mentor for the NSF-IGERT Program for Cellular Engineering, Houston Molecular Biophysics Program and the Gulf Coast Consortia and is a co-founder of the Rice Center for Evolution. The PI teaches Biochemistry and incorporates the lessons learned from these experiments into the discussion of protein folding and stability as well as how evolution impacts our world through the rise of drug resistant pathogens. In the last three years, the lab has hosted 7 High School groups (including a High School AP teachers program) as well as mentoring summer researchers from the under represented Hispanic community through the Rice AGEP program. The PI has also used this project as the basis for community based seminars on the nature of evolution (''Evolution in a coffee pot'').