CAREER: Site-specific vibrational probes of membrane binding and protonation in proteins

在这个由化学系化学结构、动力学和机制项目资助的CAREER项目中，哈弗福德学院的凯西·隆德根教授和一群本科生研究助理将利用红外和拉曼光谱，结合特定位点的化学标记方案，开发几种新的方法，用于光谱分离特定的蛋白质残基，并观察与功能相关的局部环境的变化。重大的结构性和动态性变化。具有独特振动频率的官能团需要利用振动光谱的快速固有时间尺度来报告具有复杂自组装和结合活性的蛋白质中特定位点周围的整个动态构象分布。 该项目将开发三个这样的功能组用于特定的结构背景，同时强调理解其环境依赖性的基本物理机制，并使用已证明的依赖性来解决特别困难的基于蛋白质的结构问题。在某些情况下，人工氨基酸是最有用的，而在其他情况下，保持天然结构的残基是非常重要的。膜结合蛋白质的界面取向将使用氰基化半胱氨酸的CN伸缩带和叠氮基取代的氨基酸的NNN不对称伸缩振动进行研究。2-氘代组氨酸的C-D伸缩振动将用于揭示pH依赖性结构变化的蛋白质中特定组氨酸残基的质子化状态和结构作用，随着一组物理上清晰的基线结果的掌握，这组新的振动探针组将用于研究与神经退行性疾病和病毒感染性相关的蛋白质的动态结构。参与这项研究的各级本科生将在光谱学，化学操作，蛋白质表达和纯化方面进行广泛的培训。 综合研究方法将伴随着努力开发实验设计和探究为基础的模块，与费城地区的高中教师合作本科教学实验室。

In this CAREER project funded by the Chemical Structure, Dynamics and Mechanisms Program of the Chemistry Division, Prof. Casey Londergan and a group of undergraduate research assistants at Haverford College will use infrared and Raman spectroscopy, coupled with site-specific chemical labeling schemes, to develop several new methods for spectroscopically isolating specific protein residues and observing changes in their local environments associated with functionally important structural and dynamic changes. Functional groups with unique vibrational frequencies are needed to harness the fast intrinsic time scale of vibrational spectroscopy to report on the entire dynamic conformational distribution around particular sites in proteins with complicated self-assembly and binding activity. This project will develop three such functional groups for use in particular structural contexts, with a dual emphasis on understanding the fundamental physical mechanisms of their environmental dependence and using demonstrated dependences to solve particularly difficult protein-based structural problems. In some contexts, artificial amino acids are most useful, and in others, maintaining natively structured residues is centrally important. The interfacial orientation of membrane-bound proteins will be investigated using the CN stretching band of cyanylated cysteine and the NNN asymmetric stretching vibration of azido-substituted amino acids. The C-D stretching vibration of 2-deuterated histidine will be used to reveal the protonation state and structural role of specific histidine residues in proteins with pH-dependent structural changes.With a set of physically clear baseline results in hand, this group of new vibrational probe groups will be used to investigate the dynamic structures of proteins associated with neurodegenerative disease and viral infectivity. Undergraduate students of all levels involved in this research will be trained broadly in spectroscopy, chemical manipulation, and protein expression and purification. The integrative research approach will be accompanied by an effort to develop experimental design- and inquiry-based modules for undergraduate teaching laboratories in collaboration with Philadelphia-area high school teachers.