FTIR SPECTROSCOPY OF ISOTOPE LABELED MEMBRANE PROTEINS

描述：FTIR差分光谱可以提供详细的 关于氢键、质子化状态和 膜蛋白中特定化学基团的定向， 微秒时间分辨率 然而，这种技术受到 缺乏将同位素标记置于特定位置的通用方法 在蛋白质中的位置。 在该项目的初始阶段， 标记膜蛋白的方法，称为定点同位素 标记（SDIL）。 这种方法基于表达 含有抑制剂tRNA的体外反应混合物中的蛋白质， 用同位素标记的氨基酸氨酰化。 上 应用，产生细菌视紫红质（bR）的SDIL类似物 含有2 H和13 C标记物，选择性地掺入到两种特异性 酪氨酸残基和骨架肽羰基。 FTIR分析 这些SDIL类似物的鉴定导致了结构活性 可能参与质子运输和偶联的基团 发色团异构化导致蛋白质构象变化。最近， 用于低成本生产SDIL类似物的按比例放大的程序已经被 证明了这一点，为固态NMR研究打开了大门。 研究员建议继续发展指引 方法，以便它可以与FTIR和NMR沿着常规使用 光谱研究膜蛋白。 新方法将 开发用于：i）抑制性tRNA的酶促和化学充电; ii）在体外表达、分离、重折叠和重构a 各种新生蛋白和iii）SDIL的常规生产 类似物用于固态NMR分析。 这些方法将首先 应用于细菌视紫红质，以分配FTIR差异带 由单个残基的结构变化引起， bR光循环。 SDIL方法也将应用于传感器ry 视紫红质I（SRI），一种参与趋光性的受体蛋白。 在 除了进一步了解这些膜蛋白 功能，拟议的研究将产生重要影响，在其他 生物物理学和生物学领域，包括酶催化和蛋白质 折页.

DESCRIPTION: FTIR difference spectroscopy can provide detailed information about changes in the hydrogen bonding, protonation state and orientation of specific chemical groups in a membrane protein with sub- microsecond time resolution. However, this technique is limited by the absence of a general method for placing isotope labels at specific positions in a protein. During the initial phase of this project, a new approach for labeling membrane proteins, termed site-directed isotope labeling (SDIL), was demonstrated. This approach is based on expressing a protein in an in vitro reaction mixture containing a suppressor tRNA, aminoacylated with isotopically labeled amino acid. In the first application, SDIL analogs of bacteriorhodopsin (bR) were produced containing 2H and 13C labels selectively incorporated into both specific tyrosine residues and backbone peptide carbonyl groups. FTIR analysis of these SDIL analogs led to the identification of structurally active groups which may be involved in proton transport and the coupling of chromophobe isomerization to protein conformational changes. Recently, scaled-up procedures for the low-cost production of SDIL analogs has been demonstrated, opening the door for solid-state NMR studies. The Investigator proposes to continue the development of the SDIL approach so that it can be routinely used along with both FTIR and NMR spectroscopy to investigate membrane proteins. New methods will be developed for: i) enzymatic and chemical charging of suppressor tRNAs; ii) in vitro expression, isolation, refolding and reconstitution of a variety of nascent proteins and iii) the routine production of SDIL analogs for solid-state NMR analysis. These methods will first be applied to bacteriorhodosin, in order to assign FTIR difference bands arising from the structural changes of individual residues during the bR photocycle. The SDIL approach will be also applied to sensor ry rhodopsin I (SRI), a receptor protein involved in phototaxis. In addition to an increased understanding of how these membrane proteins function, the proposed research will have an important impact in other areas of biophysics and biology, including enzyme catalysis and protein folding.