Studying protein unfolding and translocation by the protease FtsH with optical tweezers
C.M.M. Overtoom-van Aartrijk (TU Delft - BN/Marie-Eve Aubin-Tam Lab)
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Abstract
The protease FtsH is a membrane anchored protease that degrades misfolded (membrane) proteins. To study the mechanism behind unfolding and translocation with optical tweezers, the protease had to be purified. However, full-length FtsH has hardly been studied as solubilizing this membrane anchored protease is challenging. Our group (Dr. V. Carvalho and Dr. I. Prabudiansyah) managed to solubilize a thermophilic variant that is found in Aquifex aeolicus. Creating new challenges as it is only active at high temperatures, and optical tweezers usually perform measurements at room temperature. We constructed a new technique to heat the optical tweezers sample to high temperatures (70 ◦C) and developed a new method to locally determine the temperature. With this heating system an attempt was made to study the mechanisms of FtsH, but unfortunately no successful unfolding and degradation patterns were obtained.