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

周围质结合蛋白（pbp）根据其三维结构的折叠模式分为1型和2型。据推测，在PBPs的进化过程中，通过交换核心结构中的β链，从1型转变为2型只发生了一次。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造一种具有1型功能和2型折叠的嵌合体蛋白。在三维结构比较的基础上，用大肠杆菌1型蛋白（MglB或AraF）和2型蛋白（ArgT）的两条肽段合成了人工嵌合体蛋白。虽然在CD光谱分析中观察到部分二级结构的形成，但在平衡透析中嵌合体蛋白的配体结合明显弱于野生型蛋白。我们尝试用噬菌体展示法恢复配体结合，但这种方法被非特异性吸附所阻碍。因此，我们研究了这两种类型的折叠途径来重新设计人工蛋白。我们利用尿素梯度凝胶电泳、快速蛋白尺寸排除液相色谱和疏水染料ANS结合实验对MglB和ArgT的折叠途径进行了表征，发现ArgT的折叠途径比MglB更复杂（J. Biochem133: 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所取代。协同折叠显示在一些蛋白质中，一个结构上保守的区域被另一个类型（在准备中）取代。我们现在正在尝试用体外进化系统对它们进行修饰，并将它们结合起来构建嵌合体蛋白，其中两个区域被另一个类型取代。

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.