An interface point‐mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations

[摘要]

Wild-type trypanosomal triosephosphate isomerase (wtTIM) is a very tight dimer. The interface residue His-47 of wtTIM has been mutated into an asparagine. Ultracentrifugation data show that this variant (H47N) only dimerises at protein concentrations above 3 mg/ml. H47N has been characterised at a protein concentration where it is predominantly a monomer. Circular dichroism measurements in the near-UV and far-UV show that this monomer is a compactly folded protein with secondary structure similar as in wtTIM. The thermal stability of the monomeric H47N is decreased compared to wtTIM: temperature gradient gel electrophoresis (TGGE) measurements give T _m-values of 41°C for wtTIM, whereas the T _m-value for the monomeric form of H47N is approximately 7°C lower.

[学科分类] 生物化学/生物物理

[关键词] Dimer;Triosephosphate isomerase;Interface;Ultracentrifugation;Monomer;Point mutation;Bis-Tris;bis[2-hydroxyethyl]imino-tris[hydroxymethyl]-methane;CD;circular dichroism;DHAP;dihydroxyacetone phosphate;DTT;dithiothreitol;EDTA;ethylenediamine tetraacetic acid;GAP;d-glyceraldehyde-3-phosphate;TGGE;temperature gradient gel electrophoresis;TIM;triosephosphate isomerase (EC 5.3.1.1);wtTIM;wild-type trypanosomal triosephosphate isomerase;2PG;2-phosphoglycollate