FTIR SPECTROSCOPY OF ISOTOPE LABELLED MEMBRANE PROTEINS

傅里叶变换红外(FTIR)差分光谱是一种强大的 膜蛋白结构变化的探测。它可以揭示详细的信息 关于氢键、质子化状态和氢键变化的信息 微秒时间定位特定氨基酸侧链 决议。但是，需要一种通用的方法来分配 单个氨基酸振动的红外差分光谱 蛋白质中的残基。我们建议通过结合FTIR实现这一点 点定向同位素标记(SDIL)差示光谱技术 蛋白质中的单个原子。这种方法将提供详细的 关于单个氨基酸残基在 蛋白质的功能。两种细菌膜蛋白-- 细菌视紫红质和粘菌素EL已被选为我们的初始 研究。这些蛋白质代表了广泛的 包括能量传递、主动和被动的生物膜现象 传输、电压通道选通和信号转导。真核生物 完整的膜蛋白，白介素8受体，也被 被选中用于本项目后期的FTIR-SDIL研究。这 蛋白质是七种α-螺旋G蛋白的代表 偶联受体。为本项目的初步研究提供了依据 用于工程ANA生产新的tRNA，以满足 对SDIL的要求。此外，我们已经证明了FTIR的差异 光谱学可以探测微克量的蛋白质，检测 微秒分辨率的构象变化及其研究 水环境中的生物膜。除了膜之外 生物物理学，提出的研究将对其他领域产生重要影响 包括蛋白质折叠、酶催化及其应用 生物系统的二维核磁共振。

Fourier transform infrared (FTIR) difference spectroscopy is a powerful probe of structural changes in membrane proteins. It can reveal detailed information about changes in the hydrogen bonding, protonation state and orientation of specific amino acid side chains with microsecond time resolution. However, a general method is needed to assign bands in the infrared difference spectrum to the vibrations of individual amino acid residues in a protein. We propose to do this by combining FTIR difference spectroscopy with site directed isotope labelling (SDIL) of individual atoms in a protein. This approach will provide detailed information about the role of individual amino acid residues in the functioning of a protein. Two bacterial membrane proteins-- bacteriorhodopsin and colicin El have been selected for our initial research. These proteins are representative of a wide range of biomembrane phenomena including energy transduction, active and passive transport, voltage channel gating and signal transduction. A eucaryotic integral membrane protein, the interleukin-8 receptor, has also been selected for FTIR-SDIL studies in the later stages of this project. This protein is representative of the class of seven alpha-helix G-protein coupled receptors. Initial research for this project provides the basis for the engineering ana production of novel tRNAs which will satisfy the requirements for SDIL. Further, we have shown that FTIR difference spectroscopy can probe microgram quantities of protein, detect conformational changes with microsecond resolution and investigate biomembranes in an aqueous environment. In addition to membrane biophysics, the proposed research will have an important impact in other areas including protein folding, enzyme catalysis and the application of 2-D NMR to biological systems.