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

周质结合蛋白(PBPs)根据其三维结构的折叠模式分为1型和2型。已经推测，在PBPS的进化过程中，通过交换核心结构中的β-链，从类型1到类型2的变化只发生过一次。为了找出这种变化的触发因素，我们试图通过人为地引起链交换来创造一种具有类型1功能和类型2折叠的嵌合体蛋白。在比较其三维结构的基础上，将大肠杆菌1型蛋白的两段多肽(MglB或ARAF)和2型蛋白的两段多肽(ArgT)制成人工嵌合体蛋白。虽然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，嵌合体蛋白的配基结合能力明显弱于野生型蛋白。我们试图用噬菌体展示的方法恢复配体的结合，但这一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路径，以重新设计人造蛋白质。我们通过尿素梯度凝胶电泳法、快速蛋白质大小排除法和疏水染料结合实验对MglB和Art的折叠途径进行了表征，发现Art比MglB具有更复杂的折叠途径(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。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(在制备中)所取代。我们现在正试图使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种类型取代。

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.