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”）是一个 生物化学和生物医学科学最重要的挑战。不 只有解决这个问题的先决条件迪凯特博士的 了解生物功能的分子基础，它也是 蛋白质错误折叠引起的几种重要疾病。这个项目 解决蛋白质折叠的问题，通过开发一种研究方法， 蛋白质构象和动力学结合特异性同位素标记，红外 光谱和快速混合动力学技术。 首先，我们将利用傅里叶变换红外光谱法研究富含丙氨酸的多肽的构象 谱这些肽是用于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， 主链溶剂化和螺旋稳定性之间的关系将通过红外光谱进行研究。 谱因为溶剂-主链相互作用和二级结构 结构影响肽的酰胺I振动，IR是一种非常灵敏的 探讨溶剂化与构象的关系。FTIR 将在特异性标记的肽上测量光谱，以获得 构象作为残基位置的函数。 接下来，将通过停流红外光谱法研究这些肽。IR 快速混合的检测在生物分子的研究中不太常见， 圆二色谱或荧光检测，但是，当应用这种方法时， 特异性同位素标记的肽和蛋白质，构象变化 可以分解到残留物水平。这些实验的数据 与平衡FTIR和温度跃变测量，将用于 表征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。 脱辅基肌红蛋白是一个研究得很好的单结构域球状折叠模型， proteins.脱辅基肌红蛋白低pH折叠中间体的螺旋结构 将使用表达的蛋白质连接用13 C特异性标记。这 一系列标记的蛋白质，然后可以研究稳态和停止流动 IR方法以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.