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Thermodynamic characterization of yeast triosephosphate isomerase refolding: insights into the interplay between function and stability as reasons for the oligomeric nature of the enzyme.

ABSTRACT: The reasons underlying the oligomeric nature of some proteins such as triosephosphate isomerase (TIM) are unclear. It has been proposed that this enzyme is an oligomer, mainly because of its stability rather than for functional reasons. To address this issue, the reversible denaturation and renaturation of the homodimeric TIM from baker's yeast ( Saccharomyces cerevisiae ) induced by guanidinium chloride and urea have been characterized by spectroscopic, functional and hydrodynamic techniques. The unfolding and refolding of this enzyme are not coincident after 'conventional' equilibrium times. Unfolding experiments did not reach equilibrium, owing to a very slow dissociation and/or unfolding process. By contrast, equilibrium was reached in the refolding direction. The simplest equilibrium pathway compatible with the obtained data was found to be a three-state process involving an inactive and expanded monomer. The Gibbs energy changes for monomer folding (delta G (0)(fold) = -16.6+/-0.7 kJ x mol(-1)) and monomer association (delta G (0)(assoc) = -70.3+/-1.1 kJ x mol(-1)) were calculated from data obtained in the two denaturants. From an analysis of the present data and data from the literature on the stability of TIM from different species and for other beta/alpha barrels, and model simulations on the effect of stability in the catalytic activity of the enzyme, it is concluded that the low stability of the monomers is neither the only, nor the main, cause for the dimeric nature of TIM. There is interplay between function and stability.

SUBMITTER: Najera H

PROVIDER: S-EPMC1223230 | biostudies-other | 2003 Mar

REPOSITORIES: biostudies-other

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Project description:Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology. Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation. Results. We identified four fibrillogenic regions in the HsTPI corresponding to the ?3, ?6, ?7 y ?8 of the TIM barrel. From these, the ?3-strand region (residues 59-66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-? structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis. Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where ?-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting ?-aggregation, as well as the formation of hydrophobic disordered aggregates. Being this last pathway kinetically favored over the thermodynamically more stable fibril aggregation pathway.

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Thermodynamic characterization of yeast triosephosphate isomerase refolding: insights into the interplay between function and stability as reasons for the oligomeric nature of the enzyme.

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