New rapid method of protein crystal structure analyzes making use of protein engineering techniques

目的是开发一种新的方法，既可以解决相问题，又可以利用蛋白质工程技术解释所得的电子密度图。具体而言，在本研究中，利用蛋白质工程技术制备了含有硒代甲硫基残基代替天然甲硫基残基的蛋白质样品。利用这些技术，我们解决了两种蛋白质的三维结构，BphC酶(250K道尔顿；Biol. 255,735-752(1996))和BphD酶（250K道尔顿；手稿正在准备中）都在联苯及其衍生物的代谢途径中工作，包括臭名昭着的环境污染物，多氯联苯。对本方法作了以下观察。1)就x射线衍射强度测量而言，使用传统的实验室（而不是同步加速器）x射线（没有异常色散数据），仅基于选定位点（所谓的SIR方法）的相位测定不足以导致最终的蛋白质结构解决方案。2)尽管如此，上述得到的相信息仍然可以提高其他重原子衍生物提供的粗略相信息的质量。此外，对于以这种方式获得的改进相信息，各种现代电子密度改进技术（如溶剂平坦化方法）可以有效地导致最终的蛋白质结构溶液。

The aim was to develop a new method useful for both the solution of the phase problem and interpretation of the resultant electron density map making use of protein engineering techniques. Specifically, in this study, the protein engineering techniques were used to prepare potein specimens containing selenomethionyl residues in place of natural methionyl residues.Making use of these techniques, we solved three-dimensional structures of two proteins, the BphC enzyme (250K dalton ; J.Mol. Biol. 255,735-752 (1996)) and the BphD enzyme (250K dalton ; manuscript in preparation) both working in a metabolic pathway for biphenyl and its derivatives including the notorious environmental pollutant, PCB.The following observation as to the present method have been made.1) In so far as the X-ray diffraction intensity measurement is done using conventional laboratory (rather than synchrotron) X-rays (without anomalous dipersion data), the phase determination solely based on the selen sites (the so-called SIR method) is not strong eough to lead to a final protein structure solution.2) Still, the phase information derived as above can improve the quality of the rough phase information provided by other heavy-atom derivatives. Furthermore, for improved phase information obtained in this way, various modern techniques of electron density improvement (such as the solvent-flattening method) can be as effective as to lead to a fianl protein structure solution.