Reversible Equilibrium Unfolding of Triosephosphate Isomerase from Trypanosoma cruzi in Guanidinium Hydrochloride Involves Stable Dimeric and Monomeric Intermediates

Abstract

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.

Identification of a Dimeric Intermediate in the Unfolding Pathway for the Calcium-Binding Protein S100B

“…However, this result is consistent with that of several other dimeric proteins including factor for inversion stimulation protein, 50 the ATPase SecA, 72 organophosphorus hydrolase, 73 triosephosphate isomerase 74,75 and glutathione reductase 76 where a dimeric intermediate has been observed. Like apo-S100B, each of these proteins has the appearance of a transition corresponding to a F 2 ⇄ I 2 step at relatively low GuHCl concentrations (b 2 M), indicative that the dissociation of the dimer is a less favourable process.…”

Bile salt hydrolase, the member of Ntn‐hydrolase family: Differential modes of structural and functional transitions during denaturation

“…Multiple unfolded states have been reported in the case of glutathione transferase (14). Reversible equilibrium of unfolding of triophosphate isomerase from Trypanosoma cruzi in Gdn-HCl has been reported (15). Irreversible unfolding of a-amylase was analysed by unfolding kinetics.…”

The Oligomeric Nature of Triosephosphate Isomerase. Studies of Monomerization

“…Regarding protein folding studies with homologous proteins, in the early reports it was reported that the folding pattern is conserved trough evolution; however, in the literature different folding pathways for homologous proteins with the same denaturant condition have been reported. In this context, TIM is one of the most studied proteins (3). The oligomeric nature of TIM has been analyzed on enzymes mainly from mesophile organisms, where it is always a homodimeric protein, being the simplest model of oligomerization.…”

“…Sometimes the denaturation reaction is reversible, while in others, unspecific irreversible aggregation appears. For some species, the unfolding model is described by a two-state transition (24,(35)(36)(37)(38), while in others, more complex processes, including intermediates, have been observed (3,32,(39)(40)(41)(42)(43). These intermediates are responsible for the aggregation and irreversibility observed during unfolding experiments in vitro.…”

“…These intermediates are responsible for the aggregation and irreversibility observed during unfolding experiments in vitro. The intermediates reported in the folding pathway of TbTIM and TcTIM show two states of oligomerization: a dimeric intermediate, which is catalytically competent and less frequently found that the other state, a monomeric intermediate, always inactive (3,32). Notwithstanding that both enzymes share the same folding pathway induced by Gdn-HCl, TbTIM is irreversible (32) while TcTIM is fully reversible (3).…”