CAREER: Using Laboratory Evolution to Study Protein Dynamics In Vivo

智力价值为了更好地了解蛋白质中哪些原子对支持酶功能的关键性质起决定性作用，该项目将生成包含不同类别突变(随机替代、甘氨酸替代、骨架分裂、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蛋白质折叠和细胞内的功能。腺苷酸激酶将用于这些研究，因为可以利用大量先前存在的生物物理数据来解释系统突变实验的结果。这项研究将确定蛋白质对不同类别突变的功能耐受模式是否包含有关其初级序列中不同的天然位置如何对结构、稳定性、折叠、动力学和功能做出贡献的信息。这一实验策略将潜在地用于研究任何可以进行体内筛选的蛋白质，包括不容易用体外物理方法检测的表现不佳的蛋白质。更广泛的影响这个项目的更广泛的影响将是为对科学职业感兴趣的研究生和本科生提供培训。执行这项研究的PI和研究生将为附近社区大学录取的本科生进行年度圆桌讨论。这些社区大学的学生从讲座课程开始他们的教育，并不一定暴露在一个类似研究实验室的环境中。圆桌讨论将被用来增加学生的自信心，向初学者展示他们可以阅读同行评议的研究文章，向这些学生展示研究培训的好处，并促进社区大学生招募到研究经验。这些努力将垂直整合本科生和研究生教育，为研究生提供有益的培训，建立从研究型大学到服务于社区学院的少数族裔的自我维持的联系，并为社区学院的学生提供研究经验。

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.