用鸡源同源物替换大肠杆菌TIM的（β-α）-单元8会产生一种稳定且有活性的杂合酶。

Replacing the (beta alpha)-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme.

作者信息

Mainfroid V, Goraj K, Rentier-Delrue F, Houbrechts A, Loiseau A, Gohimont A C, Noble M E, Borchert T V, Wierenga R K, Martial J A

机构信息

Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Sart Tilman, Belgium.

出版信息

Protein Eng. 1993 Nov;6(8):893-900. doi: 10.1093/protein/6.8.893.

DOI:10.1093/protein/6.8.893

PMID:8309937

Abstract

In order to investigate how structural modifications interfere with protein stability, we modified a (beta alpha)-unit in E.coli triosephosphate isomerase (TIM), a typical (beta alpha)-barrel protein, assuming that the pseudosymmetrical beta-barrel can be divided into eight successive loop/beta-strand/loop/alpha-helix motifs. We replaced the eighth (beta alpha)-unit of E.coli TIM with the corresponding chicken (beta alpha)-unit. The substitution, involving the replacement of 10 of the 23 residues of this (beta alpha)-unit, was evaluated first by modelling, then experimentally. Modelling by homology suggests how the amino acid replacements might be accommodated in the hybrid E.coli/chicken TIM (ETIM8CHI). Both natural and hybrid recombinant TIMs, overproduced in E.coli, were purified to homogeneity and characterized as to their stability and kinetics. Our kinetic studies show that the modification performed here leads to an active enzyme. The stability studies indicate that the stability of ETIM8CHI is comparable to that of the wild type TIM.

摘要

为了研究结构修饰如何影响蛋白质稳定性，我们对大肠杆菌磷酸丙糖异构酶（TIM）中的一个（β-α）单元进行了修饰，TIM是一种典型的（β-α）桶状蛋白，我们假定这种假对称的β桶可分为八个连续的环/β链/环/α螺旋基序。我们用相应的鸡源（β-α）单元替换了大肠杆菌TIM的第八个（β-α）单元。首先通过建模，然后通过实验评估了这种替换，该替换涉及此（β-α）单元23个残基中的10个残基的替换。同源性建模表明氨基酸替换在大肠杆菌/鸡源杂交TIM（ETIM8CHI）中可能如何被容纳。在大肠杆菌中过量表达的天然和杂交重组TIM均被纯化至同质，并对其稳定性和动力学进行了表征。我们的动力学研究表明，此处进行的修饰产生了一种活性酶。稳定性研究表明，ETIM8CHI的稳定性与野生型TIM相当。