Amino Acid Sequence; Animals; Base Sequence; Chickens/genetics; Enzyme Stability; Escherichia coli/enzymology/genetics; Models, Chemical; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Engineering; Protein Folding; Recombinant Fusion Proteins/chemistry/metabolism; Triose-Phosphate Isomerase/*chemistry/genetics/metabolism
Abstract :
[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Mainfroid, Véronique
Goraj, Karine
Rentier-Delrue, Françoise ; Université de Liège - ULiège > Département des sciences de la vie > Biologie et génétique moléculaire - GIGA-R : Coordination scientifique
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