Estructura residual en el desplegamiento de una proteína dimérica tipo ᵦ/a. influencia en la eficiencia de replegamiento
Saccharomyces cerevisiae triosephosphate isomerase (yTIM) is a dimeric protein comprised by two identical monomers of 248 residues each. This enzyme shows noncoincident unfolding and refolding transitions (hysteresis) in temperature scans, a phenomenon indicative of the slow forward and backward rea...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2018 |
| País: | México |
| Institución: | Universidad Autónoma Metropolitana |
| Repositorio: | Repositorio Institucional de la UAM Iztapalapa |
| Idioma: | español |
| OAI Identifier: | oai:bindani.izt.uam.mx:t148fh21h |
| Acceso en línea: | https://doi.org/10.24275/uami.t148fh21h |
| Access Level: | acceso abierto |
| Palabra clave: | info:eu-repo/classification/LEM/Química info:eu-repo/classification/LEM/Bioquímica física info:eu-repo/classification/LEM/Bioquímica info:eu-repo/classification/LEM/Chemistry info:eu-repo/classification/LEM/Biochemistry info:eu-repo/classification/LEM/Physical biochemistry info:eu-repo/classification/cti/2 |
| Sumario: | Saccharomyces cerevisiae triosephosphate isomerase (yTIM) is a dimeric protein comprised by two identical monomers of 248 residues each. This enzyme shows noncoincident unfolding and refolding transitions (hysteresis) in temperature scans, a phenomenon indicative of the slow forward and backward reactions of the native-unfolded process. Thermal unfolding scans suggest that no stable intermediates appear in the unfolding of yTIM. However, reported evidence points to the presence of residual structure in the denatured monomer at high temperature. In this work, it was found that thermally-denatured yTIM displays a clear trend to form aggregation-prone, β-strand-like residual structure when pH decreased from 8.0 to 6.0, even though thermal unfolding profiles retained a simple monophasic appearance regardless of pH. However, kinetic studies performed over a relatively wide temperature range (54−64 °C) revealed a complex unfolding mechanism comprising up to three observable phases, with largely different time constants, each accompanied by changes in secondary structure. Furthermore, a simple sequential mechanism is unlikely to explain the observed variation of amplitudes and rate constants with temperature. Rather than a simple sequential pathway, a complex mechanism involving off-pathway intermediates or even parallel pathways may be operating. This kinetic complexity is, however, not linked to the appearance of residual structure. On the other hand, the kinetic stability of yTIM (as reflected in the value of the rate constant for the main unfolding phase) showed a significant decrease in the pH range from 7.5 to 9.5, a range in which the conformation of the unfolded protein is typical of thermallyunfolded small proteins. That is, the appearance of β-strand residual structure is not related to significant changes in kinetic stability. In contrast, the presence of residual structure in denatured yTIM does apparently accelerate refolding at lower temperatures (42 °C), though it is clearly associated with increased irreversibility when the protein is brought back to 25 °C. |
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