Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes

Abstract

The reactivation of triosephosphate isomerase (TIM) from unfolded monomers induced by guanidine hydrochloride involves different amino acids of its sequence in different stages of protein refolding. We describe a systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The two similar TIMs of Trypanosoma brucei and Trypanosoma cruzi have different reactivation velocities and efficiencies. We used a small number of chimeric enzymes, additive mutants and planned site-directed mutants to produce an enzyme from T. brucei with 13 mutations in its sequence, which reactivates fast and efficiently like wild-type (WT) TIM from T. cruzi, and another enzyme from T. cruzi, with 13 slightly altered mutations, which reactivated slowly and inefficiently like the WT TIM of T. brucei. Our method is a shorter alternative to random mutagenesis, saturation mutagenesis or directed evolution to find multiple amino acids critical for certain properties of proteins.

Three unrelated and unexpected amino acids determine the susceptibility of the interface cysteine to a sulfhydryl reagent in the triosephosphate isomerases of two trypanosomes

“…However, these extremely similar proteins have some outstanding differences in several functional properties and their behavior to physico-chemical agents and inactivating molecules. For example, their velocity and extent of reactivation from guanidine chloride unfolded monomers [ 5 , 6 ], their susceptibility to digestion with subtilisin [ 7 ], and quantitative differences in their susceptibility to several low molecular weight agents [ 8 ], and in particular to sulfhydryl reagents [ 9 – 13 ].…”

“…This work answers the question by refining the strategy, introducing first “additive mutations” [ 6 ] and then multiple site directed mutagenesis of the wild type (WT) TIMs.…”

“…It is important to point out, that this same strategy has also worked in identifying the residues which are responsible for the differences in reactivation velocity and efficiency of TbTIM and TcTIM [ 6 ], thus broadening the scope of its application.…”

The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes

“…Despite this, they show strikingly different behaviors in several aspects . Recently, a small number of chimeric enzymes, followed by gradually additive mutagenesis and planned site‐directed mutagenesis were used to find the amino acids that are critical for the reactivation of TcTIM and TbTIM from unfolded monomers …”

“…3, [14][15][16] Recently, a small number of chimeric enzymes, followed by gradually additive mutagenesis and planned sitedirected mutagenesis were used to find the amino acids that are critical for the reactivation of TcTIM and TbTIM from unfolded monomers. 17 The main goal of this study is to systematically dissect the effect of specific proline residues to the kinetic stabilities of TcTIM and TbTIM, contrasting with several reports where the thermodynamic stability of proteins has been assessed upon proline introductions. 18-20 Our strategy was, first, to analyze subtle differences between the secondary structure elements of the crystallographic structures.…”