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

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 p...

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Bibliographic Details
Published in:FEBS letters 1995-07, Vol.367 (3), p.315-318
Main Authors: Borchert, T.V., Zeelen, J.Ph, Schliebs, W., Callens, M., Minke, W., Jaenicke, R., Wierenga, R.K.
Format: Article
Language:English
Subjects:
2-phosphoglycollate
2PG
Animals
Bis-Tris
bis[2-hydroxyethyl]imino-tris[hydroxymethyl]-methane
Circular Dichroism
d-glyceraldehyde-3-phosphate
DHAP
dihydroxyacetone phosphate
Dimer
dithiothreitol
DTT
EDTA
ethylenediamine tetraacetic acid
GAP
Interface
Kinetics
Macromolecular Substances
Monomer
Mutagenesis, Site-Directed
Point mutation
Protein Structure, Secondary
Protozoan Proteins - chemistry
Protozoan Proteins - metabolism
Protozoan Proteins - ultrastructure
Structure-Activity Relationship
Temperature
temperature gradient gel electrophoresis
TGGE
TIM
Triose-Phosphate Isomerase - chemistry
Triose-Phosphate Isomerase - metabolism
Triose-Phosphate Isomerase - ultrastructure
Triosephosphate isomerase
triosephosphate isomerase (EC 5.3.1.1)
Trypanosoma brucei brucei - enzymology
Ultracentrifugation
wild-type trypanosomal triosephosphate isomerase
wtTIM
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Description
Summary: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.
ISSN:0014-5793
1873-3468
DOI:10.1016/0014-5793(95)00586-X