CAREER: Using Laboratory Evolution to Study Protein Dynamics In Vivo

为了更好地理解蛋白质中哪些原子对支持酶功能的关键特性起决定性作用，该项目将生成包含不同类型突变（随机取代、甘氨酸取代、骨干裂变、环状排列、插入和缺失）的天然蛋白质文库，并研究每种突变类型如何影响细胞内蛋白质折叠和功能。腺苷酸激酶将用于这些研究，因为大量预先存在的生物物理数据可以用来解释系统诱变实验的结果。这项研究将确定蛋白质对不同类型突变的功能耐受性模式是否包含有关其初级序列中不同天然位置如何影响结构、稳定性、折叠、动力学和功能的信息。这种实验策略将潜在地用于研究体内筛选可用的任何蛋白质，包括无法通过体外物理方法检测的不良蛋白质。更广泛的影响这个项目的更广泛的影响将是为对科学事业感兴趣的研究生和本科生提供培训。项目负责人和从事这项研究的研究生将为附近一所社区大学的本科生举行年度圆桌讨论。这些社区大学的学生在课堂上开始他们的教育，并不一定接触到一个反映研究实验室的环境。圆桌讨论将通过向初入大学的学生展示他们可以阅读同行评议的研究文章来增强学生的自信心，向这些学生展示研究训练的好处，并促进社区大学学生的研究经验招募。这些努力将垂直整合本科和研究生教育，为研究生提供有益的培训，建立从研究型大学到少数民族服务社区学院的自我维持的延伸，并为社区学院学生提供研究经验。

Intellectual MeritIn order to better understand which atoms in a protein contribute decisively to the key properties that support enzyme function, this project will generate libraries of natural proteins that contain different classes of mutations (random substitutions, glycine substitutions, backbone fission, circular permutation, insertions, and deletions) and examine how each mutation type affects protein folding and function within cells. Adenylate kinases will be used for these studies, since an abundance of pre-existing biophysical data can be tapped to interpret results from systematic mutagenesis experiments. This research will establish if the pattern of a protein's functional tolerance to different classes of mutation contains information on how different native positions within its primary sequence contribute to structure, stability, folding, dynamics, and function. This experimental strategy will be potentially useful for studying any protein for which an in vivo screen is available, including poorly behaved proteins that cannot readily be examined by in vitro physical methods. Broader ImpactThis broader impact of this project will be to provide training for graduate and undergraduate students interested in science careers. The PI and graduate students performing this research will conduct annual round table discussions for undergraduates enrolled at a nearby community college. These community college students begin their education in lecture courses and are not necessarily exposed to an environment that mirrors a research lab. Round table discussions will be used to increase student self-confidence by showing beginning college students that they can read a peer-reviewed research article, demonstrate to these students the benefits of research training, and facilitate community college student recruitment to research experiences. These efforts will vertically integrate undergraduate and graduate education by providing beneficial training for graduate students, building self-sustaining outreach from a research university to a minority serving community college, and offering research experiences for community college students.