CAREER: Residue Specific Probes of the Cytochrome c Folding Mechanism

这个职业项目的目标是开发一个详细的模型，将细胞色素c折叠成其天然的三维结构。PI的方法是基于开发一种对折叠过程敏感的探针，因此可以用于表征折叠过程。首先，通过将细胞色素c的C-H键替换为C-D键，在平衡和动力学折叠条件下，将产生伴随细胞色素c折叠的残基特定的结构变化细节。C-D键是非微扰的，对其局部环境很敏感。最重要的是，C-D键在光谱中完全没有其他蛋白质或变性剂吸收的区域吸收红外光，因此很容易观察和表征。为了表征细胞色素c的折叠，C-D谱将被描述为蛋白质折叠，无论是在平衡条件下还是在动力学条件下。将特别关注蛋白质的不同部分在折叠过程中如何相互作用，以及蛋白质如何与结合的铁原子相互作用，众所周知，结合的铁原子不仅对功能重要，而且对折叠也很重要。将进行计算机模拟研究，以促进对实验观察结果的理解。理论和实验相结合的方法应该会以前所未有的清晰度定义细胞色素c的折叠过程。开发的表征细胞色素c折叠的技术将适用于其他蛋白质，因此，这些研究将对生物物理学的其他广泛领域产生影响。这个职业项目的教育活动包括培训学生在化学和生物的界面和扩展基于互联网的学习工具。该项目由分子和细胞生物科学部的分子生物物理学计划和化学部的实验物理化学计划共同资助。

The objective of this CAREER project is to develop a detailed model for cytochrome c folding into its native three dimensional structure. The PI's approach is based on developing a probe that is sensitive to the folding process, and may therefore be used for its characterization. First, residue-specific detail of the structural changes that accompany cytochrome c folding will be generated, both under equilibrium and kinetic folding conditions by replacing cytochrome c C-H bonds with C-D bonds. The C-D bonds are non-perturbative and sensitive to their local environment. Most importantly, the C-D bond absorbs infrared light in a region of the spectrum that is completely free of other protein or denaturant absorptions, and may thus be easily observed and characterized. To characterize the folding of cytochrome c, the C-D spectra will be characterized as the protein folds, both under equilibrium conditions and under kinetic conditions. Specific attention will be given to how different parts of the protein interact with each other during the folding process, but also to how the protein interacts with a bound iron atom which is known to be important not only for function, but for folding as well. Computer simulation studies will be pursued to facilitate the understanding of the experimental observations. The combined theoretical and experimental approach should define the cytochrome c folding process with unprecedented clarity. The techniques developed to characterize cytochrome c folding will be applicable to other proteins, and thus, these studies will have an impact on a broad range of other areas of biophysics. The educational activities in this CAREER project include training students at the interface of Chemistry and biology and expansion of internet based learning tools. This project is jointly funded by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences and the Experimental Physical chemistry Program in the Chemistry Division.