AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ~20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.

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Proteins are one of the most important constituents of cells, since they are responsible for various processes such as replication, translation, etc. Interestingly, proteins are also responsible for the degradation of proteins that do not assemble well, and/or which are not necessary anymore inside the cell. This process of proteins destruction and recycling of amino acids is called proteolysis. Without this process, the cell would accumulate toxic waste and would not survive for long. This highlights the importance of this process. This process occurs not only in the cell cytoplasm but in the cell lipid bilayer leading to the degradation of soluble and membrane integral proteins. In bacteria, the protein responsible for this mechanism is vital and it is called FtsH. FtsH is part of the AAA+ proteases family, which are deeply investigated since they are responsible for many important biological mechanisms inside the cell. In this thesis, we aimed to answer the question of how can FtsH not only degrade soluble proteins, but also degrade insoluble proteins. In a complex mechanism in which soluble/insoluble proteins are unfolded passing through an ATPase domain, and into a protease domain for degradation. The curiosity about this mechanism is also high, regarding how can this protein coordinate this ATP hydrolysation and coordinate it with the proteolytical process. To answer these questions, this thesis presents a series of purification protocols for E. coli FtsH (Chapter 2) and for an orthologous of FtsH, a thermophile called Aquifex aeolicus (presented in Chapter 3). During this thesis, we show that the movements that the ATPase domain can undergo in relation to the membrane are larger than what was previously described in literature (Chapter 4). The assembling of this protein into dodecamers, in the solubilized form, showed that the intermembrane loops are more flexible than what was thought before. A kinetic characterization of the ATPase and protease activity is also assessed showing that both forms are equally functional. Finally, in Chapter 5, we explore the use of cryo-electron microscopy and tomography to perform an exhaustive single particle study. Although the cryo-TEM results showed in this chapter are preliminary 2D class averages, it is possible to observe the six-fold symmetry structure of this protein, which is an incentive to pursue the studies with this technique. The same is true for the cryo-tomography performed on FtsH in the proteoliposomes which will provide further insights about the protein insertion into the membrane and allow to the study how substrates can access the ATPase domain loops. This thesis describes the efforts made in the FtsH purification protocol optimization to get a sample as pure and stable as possible. This thesis showed that FtsH undergoes much larger conformational changes than previously thought and challenges the currently accepted model for the substrate to access FtsH active site. In the future cryo-electron microscopy of single particles and cryo-tomography of proteoliposomes must be explored too deepen our knowledge of the full-length FtsH structure, and more generally our knowledge about proteolytical mechanisms in cells. @en

Challenges in purification and subsequent functionalization of membrane proteins often complicate their biochemical and biophysical characterization. Purification of membrane proteins generally involves replacing the lipids surrounding the protein with detergent molecules, which can affect protein structure and function. Recently, it was shown that styrene–maleic acid copolymers (SMA) can dissolve integral membrane proteins from biological membranes into nanosized discs. Within these nanoparticles, proteins are embedded in a patch of their native lipid bilayer that is stabilized in solution by the amphipathic polymer that wraps the disc like a bracelet. This approach for detergent-free purification of membrane proteins has the potential to greatly simplify purification but does not facilitate conjugation of functional compounds to the membrane proteins. Often, such functionalization involves laborious preparation of protein variants and optimization of labeling procedures to ensure only minimal perturbation of the protein. Here, we present a strategy that circumvents several of these complications through modifying SMA by grafting the polymer with cysteamine. The reaction results in SMA that has solvent-exposed sulfhydrils (SMA-SH) and allows tuning of the coverage with SH groups. Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy demonstrate that SMA-SH dissolves lipid bilayer membranes into lipid nanodiscs, just like SMA. In addition, we demonstrate that, just like SMA, SMA-SH solubilizes proteoliposomes into protein-loaded nanodiscs. We covalently modify SMA-SH-lipid nanodiscs using thiol-reactive derivatives of Alexa Fluor 488 and biotin. Thus, SMA-SH promises to simultaneously tackle challenges in purification and functionalization of membrane proteins.@en