PROBING PEPTIDE CONFORMATION USING IR SPECTROSCOPY

确定多肽链折叠成 稳定的三维构象(“蛋白质折叠问题”)是一种 生物化学和生物医学科学最重要的挑战之一。不 只有这个问题的解决方案才是迪凯特博士 对生物功能的分子基础的理解，也是 由蛋白质错误折叠引起的几种重要疾病。这个项目 通过发展一种研究方法来解决蛋白质折叠问题 结合特定同位素标记和红外的蛋白质构象和动力学 光谱学和快速混合动力学技术。 首先用傅立叶变换红外光谱研究富含丙氨酸的多肽的构象 光谱学。这些多肽是研究蛋白质的经典模型系统。 α螺旋的构象和动力学。在这个项目中，关系 主链溶剂化和螺旋稳定性之间的关系将通过红外进行研究 光谱学。因为无论是溶剂-主链相互作用还是次要的 结构影响酰胺I振动的多肽，IR是非常敏感的 探讨了溶剂化作用与构象的关系。FTIR 将对特定标记的多肽进行光谱测量，以获得 作为残基位置的函数的构象。 接下来，我们将对这些多肽进行停流红外光谱分析。红外 在生物分子的研究中，快速混合的检测不太常见 圆二色体或荧光检测，但当应用该方法时 到特定同位素标记的多肽和蛋白质，构象变化 可以分解到残留量水平。这些实验的数据结合在一起 通过平衡FTIR和温度跳跃测量，将用于 描述螺旋形成的成核和传播步骤。 最后，这种方法将扩展到对完整蛋白质的研究。 脱脂肌红蛋白是一种研究较多的单区球状折叠模型。 蛋白质。低pH值无肌红蛋白折叠中间体中的螺旋 将通过表达的蛋白连接用13C进行特异性标记。这 一系列标记的蛋白质可以通过稳态和停流进行研究。 IR方法获得折叠途径的描述。

Determining the mechanism by which a polypeptide chain folds into a stable three dimensional conformation ("the protein folding problem") is one of the most important challenges of biochemistry and biomedical science. Not only is the solution to this problem prerequisite for Dr. Decatur's understanding of the molecular basis of biological function, it also underlies several important diseases which are caused by protein misfolding. This project addresses the protein folding problem by developing a method of studying protein conformation and dynamics combining specific isotope labeling, infrared spectroscopy, and rapid mixing kinetics techniques. First the conformation of alanine-rich peptides will be studied by FTIR spectroscopy. These peptides are classic model systems for investigating the conformation and dynamics of alpha helices. In this project, the relationship between backbone solvation and helix stability will be investigated by infrared spectroscopy. Because both solvent-backbone interactions and secondary structure affect the amide I vibrations of peptides, IR is a very sensitive probe for dissecting the relationship between solvation and conformation. FTIR spectra will be measured on specifically-labeled peptides in order to obtain conformation as a function of residue position. Next, these peptides will be studied by stopped-flow infrared spectroscopy. IR detection of rapid mixing is less common for the study of biomolecules than circular dichrosim or fluorescence detection, but, when this method is applied to specifically isotope-labeled peptides and proteins, conformational changes can be resolved to the residue level. The data from these experiments, combined with the equilibrium FTIR and temperature jump measurements, will be used to characterize the nucleation and propagation steps of helix formation. Finally, this method will be expanded to the study of a full protein. Apomyoglobin is a well-studied model for folding in single domain globular proteins. Helices involved in the low pH folding intermediate of apomyoglobin will be specifically labeled with 13C using expressed protein ligation. This series of labeled proteins can then be studied by steady state and stopped-flow IR methods to obtain descriptions of the folding pathway.