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

利用蛋白质工程技术进行蛋白质晶体结构分析的新方法

• 批准号: 07559006
• 负责人: MITSUI Yukio
• 金额: $ 4.03万
• 依托单位: Nagaoka University of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07559006/
• 关键词: protein engineering method; protein engineering; X-ray analysis; crystal structure analysis; three-dimensional structure protein; selenomethionine

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.

目的是开发一种新的方法，用于解决相位问题和解释所得的电子密度图，利用蛋白质工程技术。本研究利用蛋白质工程技术制备了含硒代甲硫氨酰残基的蛋白质样品，并利用这些技术解析了两种蛋白质的三维结构：BphC酶（250 K道尔顿; J.Mol. 255，735 -752（1996））和BphD酶（250 K道尔顿;手稿在准备中）都在联苯及其衍生物（包括臭名昭著的环境污染物）的代谢途径中工作，对本方法进行了以下观察：1）就X射线衍射强度测量而言，使用常规实验室进行（而不是同步加速器）X射线（没有异常色散数据），仅基于硒位点的相位确定（所谓的SIR方法）不足以导致最终的蛋白质结构解决方案。2）尽管如此，如上导出的相位信息可以改善由其他重原子衍生物提供的粗略相位信息的质量。此外，对于以这种方式获得的改进的相位信息，电子密度改进的各种现代技术（例如溶剂平坦化方法）可以有效地导致最终的蛋白质结构解决方案。

项目成果

Tanaka,N.,Nonaka,T.,Nakanishi,M.,Deyashiki,Y.,Hara,A. & Mitsui,Y.: "Crystal structure of the ternary comnplex of mouse lung carbonyl reductase at 1.8 A resolution : The structural origin of distinct coenzyme specificities among the enzymes of the short-ch

田中，N.，野中，T.，中西，M.，出屋敷，Y.，原，A.

Tanaka, N., Nonaka, T., Nakanishi, M., Deyashiki, Y., Hara, A. & Mitsui, Y.: "Crystal structure of the ternary comnplex of mouse lung carbonyl reductase at 1.8 A resolution : The structural origin of distinct coenzyme specificities among the enzymes of th

田中，N.，野中，T.，中西，M.，出屋敷，Y.，原，A.

Tanaka,N.,Nonaka,T.,Tanabe,T.,Yoshimoto,T.,Tsuru,D. & Mitsui.Y.: "Crystal structures of the binary and ternary complexes of 7α-hydroxy-steroid dehydrogenase from E.coli." Biochemistry. 35. 7715-7724 (1996)

Tanaka, N.、Nonaka, T.、Tanabe, T.、Yoshimoto, T.、Tsuru, D. 和 Mitsui Y.：“大肠杆菌 7α-羟基类固醇脱氢酶的二元和三元复合物的晶体结构。 ”生物化学。35。7715-7724（1996）

Kurihara,J.,Mitsui,Y.,Nakamura,K.T. et al.: "The crystal structure of ribonuclease from Rhizopus niveus at 2.0 A resolution." J. Mol. Biol.255. 310-320 (1996)

栗原 J.、三井 Y.、中村 K.T.

Senda,T.& Mitsui: ""Molecular Biology of Pseudomonas"(T.Nakazawa et al.eds.)Chapter 27,pp311-318," ASM Press,Washington D.C., 500 (1996)

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