Dresden 2014 – scientific programme

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BP: Fachverband Biologische Physik

BP 14: Posters: Protein Structure and dynamics

BP 14.11: Poster

Tuesday, April 1, 2014, 09:30–12:30, P1

Resolving conformational switching of AAA+ protease FtsH in real time using single-molecule FRET — •Martine Ruer, Philip Gröger, Nadine Bölke, and Michael Schlierf — B CUBE - Center for Molecular Bioengineering, TU Dresden, 01307 Dresden

FtsH is a highly conserved, homo-hexameric AAA+ protease embedded in the bacterial membrane, where it recognizes, unfolds, translocates and degrades protein substrates to be degraded. Previous crystal structure data of Thermotoga maritima FtsH in an ADP and an ATP-bound state show an ATPase and a protease domain linked by a flexible hinge, facilitating a large conformational change event upon ADP/ATP binding. Based on these structural data, a model was presented where the unfolding and proteolytic mechanism are tightly coupled. However, in protease assays FtsH shows an ATP independent mechanism. How is the chemical energy converted into mechanical work for protein unfolding and translocation? We are using single-molecule Förster Resonance Energy Transfer (smFRET) experiments to resolve the conformational changes of FtsH upon ATPase and protease activities. Therefore, we have developed an in vitro assay with vesicle encapsulated, self-assembled FtsH hexamers in absence or presence of degradation substrates. In absence of a degradation substrate, the labeled FtsH protease shows 2 or 3 conformational states and the conformational switching is strongly dependent on the ATP concentration. In further experiments, we study the ATP dependent kinetics of the different conformational states in dependence of various protein substrates.