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Development of the protein structure analysis system -Use of Se-protein and third-generation synchrotron radiation-

蛋白质结构分析系统的开发 -利用Se-蛋白质和第3代同步辐射线-

基本信息

批准号：
09044047
负责人：
TANAKA Isao
金额：
$ 1.92万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

For the last 40 years, almost all new protein structures have been solved by the multiple isomorphous replacement (MIR) method. To apply this method for the phase problem we need to prepare good heavy-atom derivatives, which is one of the most time-consuming process for the protein structure analysis, because it entails the trial-and-error experiments. Recently, an alternative approach has been devised for de novo phasing of protein crystals. This is the multiwavelengh anomalous diffraction(MAD). Especially important is the use of selenium atom for the anomalous scatterer, since it is easily incorporated into protein crystal by the protein engineering as a form of selenomethionine(Se-Met). In this project we applied this method to the analyses of several new proteins for further improvement of the method and showed that this method is very useful.Ribosomal protein 57 is located at the head of the small subunit facing to the decoding center. It is one of the primary 165 rRNA binding pro … More teins responsible for initiating the assembly of the head of the 30S subunit. The structure of ribosomal protein S7 from Bacillus stearothermophilus has been solved at 2.5A resolution by the multiwavelength anomalous diffraction method using Se-Met substituted proteins. The molecule consists of a helical hydrophobic core domain and a beta-ribbon arm extending from the hydrophobic core. The helical core domain is composed of a pair of entangled helix-turn-helix motifs similar to a DNA architectural factor. Highly conserved basic and aromatic residues are clustered on one face of the S7 molecule and create a 165S rRNA contact surface. The molecular feature, together with former cross-link experiments, suggests how S7 binds to the 3' major domain of 16S rRNA and mediates the folding of 165 rRNA to create the ribosome decoding center.Ribosomal protein L2 is the largest protein component in the ribosome. It is located at or near the peptidyl transferase center and has been a prime candidate for the peptidyl transferase activity. It binds directly to 23 S rRNA and plays a crucial role in its assembly. The three dimensional structure of the RNA-binding domain of L2 from Bacillus stearothermophilus has been determined at 2.3A resolution by X-ray crystallography using the Se-Met MAD method. The RNA-binding domain of L2 consists of two recurring motifs of about 70 residues each. The residues Arg86 and Arg155, which have been identified by mutation experiments to be involved in the 23S rRNA binding, are located at the gate of the interface region between the two domains.The molecular architecture suggests how this important protein has evolved from the ancient nucleic acid binding proteins to create a 23S rRNA-binding domain in the very remote past. Less

在过去的40年里，几乎所有新的蛋白质结构都是通过多重同晶置换（MIR）方法来解决的。为了将这种方法应用于相位问题，我们需要制备好的重原子衍生物，这是蛋白质结构分析中最耗时的过程之一，因为它需要反复试验。最近，已经设计了一种替代方法用于蛋白质晶体的从头定相。这就是多波长反常衍射。特别重要的是使用硒原子作为异常散射体，因为它很容易通过蛋白质工程以硒代蛋氨酸（Se-Met）的形式掺入蛋白质晶体中。在本项目中，我们将该方法应用于几种新蛋白质的分析，进一步改进了该方法，并表明该方法是非常有用的。核糖体蛋白57位于面向解码中心的小亚基的头部。它是一种主要的165 rRNA结合蛋白， ...更多信息负责启动30 S亚基头部组装的蛋白。用多波长反常衍射方法，用硒-蛋氨酸取代蛋白质，在2.5A分辨率下解析了嗜热脂肪芽孢杆菌核糖体蛋白S7的结构。该分子由螺旋疏水核心结构域和从疏水核心延伸的β-带状臂组成。螺旋核心结构域由一对缠结的螺旋-转角-螺旋基序组成，类似于DNA结构因子。高度保守的碱性和芳香族残基聚集在S7分子的一面，并产生165 S rRNA接触表面。这一分子特征以及先前的交联实验结果表明，S7是如何与16 SrRNA的3'端结合，并介导165 rRNA的折叠，从而形成核糖体解码中心的。它位于肽基转移酶中心或附近，是肽基转移酶活性的主要候选者。它直接与23 S rRNA结合，并在其组装中发挥关键作用。用X射线晶体学方法用Se-Met MAD法测定了嗜热脂肪芽孢杆菌L2 RNA结合区的三维结构。L2的RNA结合结构域由两个重复的基序组成，每个基序约70个残基。Arg 86和Arg 155位于23 SrRNA结合区的门区，突变实验证实这两个残基与23 SrRNA结合有关，其分子结构表明这一重要的蛋白质是如何从古老的核酸结合蛋白进化到23 SrRNA结合区的。少