FTIR SPECTROSCOPY OF ISOTOPE LABELED MEMBRANE PROTEINS

描述：FTIR差示光谱可提供详细的 关于氢键、质子化状态和氢键变化的信息 膜蛋白中特定化学基团的亚基定位 微秒时间分辨率。但是，此技术受到 缺乏将同位素标记放置在特定位置的通用方法 蛋白质中的位置。在这个项目的初始阶段，一个新的 标记膜蛋白的方法，称为定点同位素 标记法(SDIL)。这种方法是基于表达 含有抑制性tRNA的体外反应混合物中的蛋白质， 用同位素标记的氨基酸进行氨基酰化。在第一个 应用，生产了细菌视紫红质(BR)的SDIL类似物 含有2H和13C标签，选择性地结合到两种特定的 酪氨酸残基和主链多肽的羰基。FTIR分析 这些SDIL类似物导致了结构活性的鉴定 可能参与质子输运和偶合的基团 嫌色异构化对蛋白质构象的影响。最近， 已经扩大了SDIL类似物低成本生产的程序 证明了这一点，为固体核磁共振研究打开了大门。 调查员建议继续发展SDIL 方法，以便它可以与FTIR和核磁共振一起常规使用 光谱学研究膜蛋白。新的方法将是 开发用于：i)抑制tRNAs的酶和化学充电； Ii)体外表达、分离、复性和重组 新生蛋白质的种类和III)SDIL的常规生产 用于固态核磁共振分析的类似物。这些方法将首先是 应用于细菌素，以便指定FTIR差异谱带 产生于单个残基的结构变化 Br光循环。Sdil方法也将应用于传感器研究。 视紫红质I(SRI)，一种参与趋光性的受体蛋白。在……里面 除了进一步了解这些膜蛋白是如何 功能，拟议的研究将在其他方面产生重要影响 生物物理学和生物学领域，包括酶催化和蛋白质 折叠。

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.