Doctor en Ciencias con Mención en Biología Molecular, Celular y Neurociencias / No autorizada por el autor para ser publicada a texto completo hasta diciembre de 2013 / Single-molecule manipulation has increasingly become a useful methodology for the study of macromolecular dynamics including unfolding-refolding transitions. In this study we use the optical tweezers to investigate the effect of substrate on the mechanical stability of glucokinase from the hiperthermophilic archeon Thermococcus litoralis. To this end, we derivatize the enzyme with DNA handles, via reactive cysteines and attach them to polystyrene beads. The enzyme with different handle attachments was separated using a native gel electrophoresis assay, and the activity of the enzyme in each gel band was monitored in situ by coupling the formation of glucose-6-phosphate to the formation of formazan and the DNA-protein-DNA construct was purified. Different constructs were pulled to investigate the effect of the ADP substrate on the stability of the protein. At 28 pN, the construct S4C/T57C showed hopping between two conformations with a difference in extension of 5.3 nm. For the unfolding reaction, the distances to the transition state were x‡ = 3.09 ± 0.35 nm in the absence and 2.81 ± 0.62 nm in the presence of ADP, respectively; the corresponding values during refolding were 2.24 ± 0.24 nm and 2.46 ± 0.43 respectively. The rate of unfolding extrapolated to zero force was an order of magnitude smaller in the presence than in absence of ADP, with no changes in the extrapolated refolding rates. These results suggest that the folded state of the protein is stabilized by the substrate with no effect on the distance to the transition state for the unfolding reaction.
| Identifer | oai:union.ndltd.org:UCHILE/oai:repositorio.uchile.cl:2250/114149 |
| Date | January 2011 |
| Creators | Wilson Moya, Christian Andrés Marcelo |
| Contributors | Babul, Jorge, Bustamante Monteverde, Carlos, Facultad de Ciencias, Departamento de Biología |
| Publisher | Universidad de Chile |
| Source Sets | Universidad de Chile |
| Language | Spanish |
| Detected Language | English |
| Type | Tesis |
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