Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Poster Abstracts

15-POS Board 8 Biochemical and Structural Studies of FtsH, a Membrane Anchored Degradation Machine Vanessa Carvalho 1 , Mohamed Chami 2 , Roland Kieffer 1 , Marie-Eve Aubin-Tam 1 , Henning Stahlberg 2 , Andreas Engel 1 . 1 Delft University of Technology, Delft, Zuid Holland, Netherlands, 2 University of Basel, Basel, Switzerland. Proteases are responsible for elimination of non-functional proteins, controlling protein levels, or modification of protein function. An important group of proteases are part of the ATPases associated with various cellular activities proteases (AAA+) family. AAA+ proteases are degradation machines, which exploit energy from ATP hydrolysis to unfold protein substrates and translocate unfolded polypeptides through a central pore, down towards a degradation chamber. In particular, FtsH is a membrane-anchored AAA+ protease, which play crucial roles in membrane protein quality control, protein transport across the membrane and dislocation of specific transmembrane segments. Although cytoplasmic structures are described, the full-length structure and the route by which soluble or integral membrane proteins translocate into the FtsH central pore to be unfolded, remains unclear. Structural characterization of full-length FtsH solubilized with either detergent or styrene maleic acid (SMA) nanodiscs provides insights on this mechanism and on FtsH integration in the lipid bilayer. We optimise expression and purification protocols, using the full-length sequence that encodes Aquifex Aeolicus FtsH. The results of the detergent solubilized FtsH, in negative staining and cryo-electron microscopy single particle analysis, show the first structure of the full-length FtsH. Proteolytic and ATPase activities are also measured. We also use SMA as solubilisation agent, which enables the formation of SMA-FtsH-nanodiscs such that FtsH remains in its native membrane environment.

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