CAREER: Understanding the Role of Amino Acid Radicals in Electron Transfer

氨基酸自由基对许多基本生物过程的催化机制至关重要，包括核酸代谢、DNA修复、光合作用和有氧呼吸。氨基酸自由基在氧化过程中可以作为正电荷的“汇”，通过多步隧穿促进蛋白质中的极长距离（25埃）电子转移。然而，尽管氨基酸自由基在生物学中具有重要意义，但它们的电化学和电子转移性质并没有得到很好的定义。在这个项目中，这些属性将直接使用β-折叠蛋白天青蛋白和4-螺旋束蛋白细胞色素c'作为模型系统进行测量。为了研究电化学电势对电子转移机制的影响，将在多肽链的特定位点掺入非天然色氨酸和酪氨酸氨基酸。与天然氨基酸相比，这扩展了电化学电势的范围~ +/-0.4 V。此外，几种Tyr类似物具有在生物pH范围内的pKa值。这些提供了一个独特的机会，动力学分离电子转移从伴随的质子转移步骤，这是在生物电子转移的强制性。类似物还提供了独特的光谱处理定义本地氨基酸environments.Broader影响：再加上拟议的研究是一个面向研究的上层生物化学课程，将讲座纳入实验室时间的发展。该课程的目标是让更多的本科生接触科学研究，并鼓励他们追求科学事业。课程评价和成果评估的准则也包括在内。该课程还为研究生或博士后学者提供了探索教学和呈现课程材料的创新方式所需的资源。特别努力招募美国本土的本科生和研究生从事该项目。与蒙大拿州部落学院的现有联系极大地促进了这一点。

Amino acid radicals are vital to the catalytic mechanisms of many fundamental biological processes, including nucleic acid metabolism, DNA repair, photosynthesis, and aerobic respiration. Amino acid radicals may serve as "sinks" for positive charges during oxidation, to facilitate very long-range (25 angstrom) electron transfer in proteins via multi-step tunneling. Yet despite the significance of amino acid radicals in biology, their electrochemical and electron transfer properties are not well defined. In this project, these properties will be measured directly using the beta-sheet protein azurin and the 4-helix bundle protein cytochrome c' as model systems. To investigate the effects of electrochemical potentials on electron transfer mechanisms, non-natural Trp and Tyr amino acids will be incorporated at specific sites of the polypeptide chains. This extends the range of electrochemical potentials ~ +/- 0.4 V compared with the natural amino acids. Moreover, several Tyr analogs have pKa values within the biological pH range. These offer a unique opportunity to kinetically separate electron transfer from the accompanying proton transfer step that is obligatory in biological electron transfer. The analogs also provide distinctive spectroscopic handles for defining local amino acid environments.Broader impacts: Coupled with the proposed research is the development of a research-oriented upper level biochemistry course that integrates lectures into laboratory time. The goals of the course are to expose more undergraduates to scientific research and encourage them to pursue careers in science. Guidelines for course evaluation and outcomes assessment are also included. The course also provides a graduate student or postdoctoral scholar with the resources needed to explore innovative ways of teaching and presenting course material. Special efforts are made to recruit native American undergraduate and graduate students to work on the project. This is greatly facilitated by existing contacts with tribal colleges in Montana.