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

进化的原理是众所周知的，并反映在这个星球上美丽和复杂的生命中。了解生命如何随着时间的推移而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进化背后的思想。也许最著名的驯化例子之一是狗，曾经是野狼，现在是伴侣。虽然很容易理解狗和狼之间的外在差异，但要确定由它们编码的基因和蛋白质的微小变化是如何引起每个品种特征的变化的，就比较困难了。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，生物体对新环境的适应需要比实验室研究中实际可行的时间长得多。通过专注于在30分钟内繁殖的简单的嗜热生物（一种生活在高温下的细菌），在一个实验室实验中可以研究数千代。为了促进这些研究，“薄弱环节”方法是在以前的国家科学基金会的支持下发展起来的。在“薄弱环节”方法中，一种转基因的嗜热生物（嗜热硬脂嗜热地杆菌）的存活依赖于一种仅在较低温度下起作用的基本分子的变化。为了在更高的温度下繁殖，必需基因必须发生突变。通过在低温下培养细菌，然后逐渐提高温度，细菌可以迅速适应高温环境。然后，就有可能确定基本基因的变化是如何产生一种蛋白质的，这种蛋白质很好地适应了高温，并允许细菌在新环境中生存。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：进化的可复制性如何？如果实验进行三次，是否会以同样的顺序发生同样的变化？如果选择的条件改变了会发生什么？适应将如何改变分子作为回应？除了探索所有生命的基本原理外，这些发现还可以应用于蛋白质工程和生物技术，以生产更坚固的蛋白质。更广泛的影响科学教育和研究的整合是研究者实验室的主要关注点。九名本科生和三名研究生的研究人员已经在这个项目上进行了独立研究，并在科学会议上发表了研究成果。该项目从生物到高分辨率结构中的电子密度的广度要求学生在进化，微生物学，生物化学和生物物理学方面受到广泛的教育。他是NSF-IGERT细胞工程项目、休斯顿分子生物物理项目和墨西哥湾沿岸联盟的教师导师，也是莱斯进化中心的联合创始人。PI教授生物化学，并将从这些实验中吸取的经验教训纳入蛋白质折叠和稳定性的讨论，以及进化如何通过耐药病原体的兴起影响我们的世界。在过去的三年里，该实验室已经接待了7个高中小组（包括一个高中AP教师项目），并通过Rice AGEP项目指导来自代表性不足的西班牙裔社区的暑期研究人员。PI还将这个项目作为基于社区的关于进化本质的研讨会（“咖啡壶中的进化”）的基础。

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'').