Reversible equilibrium unfolding of triosephosphate isomerase from Trypanosoma cruzi in guanidinium hydrochloride involves stable dimeric and monomeric intermediates.
The reversible guanidinium hydrochloride-induced unfolding of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was characterized under equilibrium conditions. The catalytic activity was followed as a native homodimeric functional probe. Circular dichroism, intrinsic fluorescence, and size-exclusion chromatography were used as secondary, tertiary, and quaternary structural probes, respectively. The change in ANS fluorescence intensity with increasing denaturant concentrations was also determined. The results show that two stable intermediates exist in the transition from the homodimeric native enzyme to the unfolded monomers: one (N(2*)) is a slightly more expanded, non-native, and active dimer, and the other is a partially expanded monomer (M) that binds ANS. Spectroscopic and activity data were used to reach a thermodynamic characterization. The results indicate that the Gibbs free energies for the partial reactions are 4.5 (N(2) <==> N(2*)), 65.8 (N(2*) <==> 2M), and 17.8 kJ/mol (M <==> U). It appears that TcTIM monomers are more stable than those found for other TIM species (except yeast TIM), where monomer stability is only marginal. These results are compared with those for the guanidinium hydrochloride-induced denaturation of TIM from different species, where despite the functional and three-dimensional similarities, a remarkable heterogeneity exists in the unfolding pathways.
Chánez-Cárdenas
María Elena
ME
Laboratorio de Patología Vascular Cerebral, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, México, DF, Mexico.
Pérez-Hernández
Gerardo
G
Sánchez-Rebollar
Brenda Guadalupe
BG
Costas
Miguel
M
Vázquez-Contreras
Edgar
E
D000818:Animals; D019791:Guanidine; D011489:Protein Denaturation; D020673:Protein Renaturation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology*
D014305:Triose-Phosphate Isomerase; D019791:Guanidine