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Experimental study to verify structural changes of proteins taken place in the evolutionary process

验证蛋白质在进化过程中发生结构变化的实验研究

基本信息

批准号：
12480204
负责人：
NISHIKAWA Ken
金额：
$ 9.41万
依托单位：
National Institute of Genetics
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480204/
关键词：
periplasmic binding protein molecular evolution design of artificial protein evolutionary protein engineering folding pattern chimera protein assembly PCR method protein three-dimensional structure アセンフーリーPCR法

项目摘要

Periplasmic binding proteins (PBPs) are classified into type 1 and type 2 groups according to the folding pattern of their three-dimensional structure. It has been inferred that the change from type 1 into type 2 occurred only once in the evolution of PBPs by exchanging beta-strands in the core structure. To find out the trigger of the change, we attempted to create a chimera protein that have type 1 function and type 2 folding, by artificially causing the strand exchange. Based on the comparison of the three-dimensional structures, an artificial chimera protein was made out of two pieces of peptides from E.coli type 1 protein (MglB or AraF) and two pieces of peptides from type 2 protein (ArgT). Although partial formation of the secondary structures was observed in the CD spectroscopic analysis, the chimera protein showed substantially weaker ligand binding than the wild type proteins in the equilibrium dialysis. We tried to recover the ligand binding using the phage display method, which was obstructed by non-specific adsorption. We thus investigate the folding pathways of the two types to re-design the artificial protein. We characterized the folding pathways of MglB and ArgT by using urea gradient gel electophoresis, fast protein size-exclusion liquid chromatography and hydrophobic dye ANS binding assay, and found that ArgT has more complicated folding pathway than MglB (J. Biochem133 : 371). We then constructed chimera proteins where only one region was replaced with the corresponding region of another type. Cooperative folding was shown in some of the proteins where a structurally conserved region was replaced with another type (in preparation). We are now trying to modify them using in vitro evolution system and combine them to build chimera proteins where two regions are replaced with another type.

周质结合蛋白（Periplasmic binding proteins，PBPs）根据其三维结构的折叠模式分为1型和2型。据推测，从1型到2型的变化只发生在一次的PBPs的核心结构中的β-链交换的演变。为了找出这种变化的触发因素，我们试图通过人工引起链交换来创建具有1型功能和2型折叠的嵌合蛋白。基于三维结构的比较，由来自大肠杆菌1型蛋白的两段肽（MglB或AraF）和来自2型蛋白的两段肽（ArgT）制成人工嵌合蛋白。虽然在CD光谱分析中观察到二级结构的部分形成，但在平衡透析中，嵌合体蛋白质显示出比野生型蛋白质弱得多的配体结合。我们尝试用噬菌体展示方法恢复配体结合，但这种方法受到非特异性吸附的阻碍。因此，我们研究这两种类型的折叠途径，以重新设计人工蛋白。我们利用尿素梯度凝胶电泳、快速蛋白质尺寸排阻液相色谱和疏水染料ANS结合试验对MglB和ArgT的折叠途径进行了表征，发现ArgT的折叠途径比MglB复杂（J. Biochem 133：371）。然后，我们构建了嵌合体蛋白，其中只有一个区域被另一种类型的相应区域取代。在一些蛋白质中显示了协同折叠，其中结构保守的区域被另一种类型取代（制备中）。我们现在正尝试使用体外进化系统对它们进行修饰，并将它们联合收割机构建嵌合体蛋白，其中两个区域被另一种类型取代。