CAREER: Developing Site-Specific Photocrosslinkers Using Unnatural Amino Acids for Studies on in vivo Complexes

生物化学家面临的一个主要挑战是解剖，重建和操纵生物体中存在的数千种亚稳态蛋白质复合物的能力。 对蛋白质信号组装体、膜结合转运体和蛋白质机器的结构/功能分析受到许多仅在体内组装和正常发挥功能的事实的限制。 在天然条件下工作的交联工具，与所有氨基酸交联，不需要化学引发，并导致可预测的结构变化，将彻底改变研究蛋白质复合物的方法。 这项工作将确定是否位点特异性掺入光交联氨基酸到蛋白质在体内可以克服目前的局限性，研究蛋白质复合物的功能如何与它们的组装和结构变化。 虽然这项研究的重点是细菌化学感受器和二聚体模型系统的光交联，但它将定义位点特异性光交联氨基酸的实验参数，因此它们将在生物化学和生物材料中具有广泛的应用。在课程中，该项目将用于强调如何将化学和生物学的基本原理结合起来研究复合物的结构和功能生物系统。 入门学生将看到化学反应，动力学和热力学的基础知识如何与未来的理解和解决生物学问题。 相反，生物化学专业的学生将体验到生物问题的化学基础如何有助于破译生物过程。这项研究在教学和研究实验室中连接生物学和化学，并在实验设计，数据解释，科学方法以及特定生物化学和有机化学技术的使用方面对本科生进行培训。

A major challenge confronting biochemists is the ability to dissect, reconstruct, and manipulate each of the thousands of metastable protein complexes that exist in organisms. Structure/function analyses on protein signaling assemblies, membrane-bound transporters, and protein machines are limited by the fact that many only assemble and function properly in vivo. A crosslinking tool that works under native conditions, crosslinks with all amino acids, needs no chemical initiation, and results in predictable structural changes would revolutionize the methods for studying protein complexes. This work will determine if site-specific incorporation of photocrosslinking amino acids into proteins in vivo can overcome the current limitations for studying how the function of protein complexes relates to their assembly and structural changes. While this research focuses on photocrosslinking of bacterial chemoreceptors and dimeric model systems, it will define the experimental parameters for site-specific photocrosslinking amino acids so they will have broad application in biochemistry and biological materials.Broader impacts: Within the curriculum, this project will be used to stress how the fundamental principals of chemistry and biology are combined to study the structure and function of complex biological systems. Introductory students will see how the basics of chemical reactions, kinetics and thermodynamics relate to future understanding and solving of biological problems. Conversely, biochemistry students will experience how the chemical basis of biological problems is instrumental in deciphering a biological process. This research connects biology and chemistry in the teaching and research laboratories, and trains undergraduates in experimental design, data interpretation, the scientific method, and in the use of specific biochemistry and organic chemistry techniques.