Titre : Measurement of Exothermic Enzyme Activity-Induced Structural Destabilization
Endroit : Pavillon Roger-Gaudry, salle G-415 à 11 h 00.
Cette conférence sera prononcée par Monsieur Scott Harroun, étudiant au doctorat du laboratoire de Alexis Vallée-Belisle, professeur au Département de chimie de l'Université de Montréal.
RÉSUMÉ: Despite much research into the evolution of enzymes, some fundamental questions remain unanswered. Recently, several studies have reported enhanced diffusion of enzymes during exothermic catalysis, but explaining this phenomenon has proven to be controversial. How does heat produced at the active site diffuse into the surrounding medium? Can the heat released by an enzyme destabilize its structure; in other words, can the enzyme “overheat”? At present, these questions are a matter of debate. In this project, we propose to use programmable DNA switches to measure structural destabilization, and possibly local temperature rise in the vicinity of an enzyme. The stability and melting temperature of these DNA switches can be readily tuned by varying the ratio of G-C to A-T base pairs in the stem. Structures with more G-C base pairs in the stem have higher stability, and thus unfold at higher temperatures. By attaching a fluorophore and quencher pair at both extremities of the stem loop, one can obtain a library of fluorescent switches. These DNA switches can also act as DNA nanothermometers, with a specific linear dynamic range spanning 12°C. Accordingly, the DNA switch is anchored onto an enzyme to measure structural destabilization and/or local rise in temperature due to heat released during the enzymatic reaction, by monitoring fluorescence variation. Initial results have found that the while the DNA switches attached to the enzyme are opened during catalysis, control DNA switches that are free in the solution, but otherwise identical, do not undergo such destabilization. The two principle designs used herein involve coupling of biotinylated DNA switches to streptavidin-modified alkaline phosphatase (AP), or to biotin-modified AP with free streptavidin. AP was chosen because its enzymatic conversion of para-nitrophenylphosphate to para-nitrophenol is highly exothermic, and this enzyme has been reported elsewhere to undergo enhanced diffusion during this reaction. This project proposes to measure local change in temperature to experimentally validate the rate of temperature diffusion, and to determine if enzymes “overheat” and destabilize when functioning at high rates. As a future application, by using these thermosensitive nanoswitches attached to enzymes, it may be possible to trigger drug release via local temperature rise around an enzyme.
