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

在过去的40年里，几乎所有新的蛋白质结构都是用多重同构置换(MIR)方法解决的。为了将这种方法应用于相问题，我们需要制备良好的重原子导数，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，另一种方法被设计用于蛋白质晶体的从头相变。这就是多波长反常衍射。尤其重要的是使用硒原子作为反常的散射体，因为它很容易通过蛋白质工程以硒蛋氨酸(Se-Met)的形式被结合到蛋白质晶体中。在本项目中，我们将该方法应用于几种新的蛋白质的分析，以进一步改进该方法，结果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。它是主要的165rRNA结合原…之一更多负责启动30s亚单位头部组装的teins。利用Se-Met取代蛋白的多波长反常衍射法，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。该分子由一个螺旋疏水核心区和一个从疏水核心区延伸出来的β-带状结构臂组成。螺旋核心区由一对缠绕在一起的螺旋-旋转-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分子的一侧，形成165S rRNA接触面。分子特征结合以往的交联实验表明，S7是如何与16S rRNA的3‘主要结构域结合，并介导165rRNA的折叠形成核糖体解码中心的。核糖体蛋白L2是核糖体中最大的蛋白质组分。它位于多肽转移酶中心或附近，是多肽转移酶活性的主要候选者。它直接与23个S核糖核酸结合，在其组装过程中起着至关重要的作用。采用Se-Met-MAD方法，用X射线结晶学方法在2.3A分辨率下测定了嗜热脂肪芽孢杆菌L2的RNA结合域的三维结构。L2的RNA结合域由两个重复的基序组成，每个基序约70个残基。Arg86和Arg155残基位于这两个结构域之间的界面区的门口，已被突变实验证实与23S rRNA结合有关。这种分子结构表明，这个重要的蛋白质是如何在非常遥远的过去从古老的核酸结合蛋白进化成一个23S rRNA结合域的。较少

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