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

在这个由化学系化学结构、动力学和机制计划资助的职业项目中，哈弗福德学院的Casey Londergan教授和一群本科生研究助理将使用红外和拉曼光谱，结合现场特定的化学标记计划，开发几种新的方法来以光谱方式分离特定的蛋白质残基，并观察与功能重要的结构和动态变化相关的当地环境的变化。需要具有独特振动频率的官能团来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置周围的整个动态构象分布。这个项目将开发三个这样的官能团，用于特定的结构背景，同时双重强调了解它们对环境依赖的基本物理机制，并利用已证明的依赖关系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，而在另一些情况下，保持天然结构残基是至关重要的。用氰化半胱氨酸的CN伸缩带和叠氮取代氨基酸的NNN不对称伸缩振动研究膜结合蛋白的界面取向。这组新的振动探针组将被用来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。参与这项研究的所有级别的本科生都将接受光谱学、化学操作以及蛋白质表达和纯化方面的广泛培训。在综合研究方法的同时，将努力与费城地区的高中教师合作，为本科教学实验室开发基于实验设计和探究的模块。

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.