Biochemical and structural studies of FtsH, a membrane anchored degradation machine

Biochemical and structural studies of FtsH, a membrane anchored degradation machine

Dias Ribeiro de Carvalho, V.I. (TU Delft BN/Marie-Eve Aubin-Tam Lab)

Engel, A.H. (promotor)
Aubin-Tam, M.E. (promotor)

Delft University of Technology

2018-11-15

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.

FtsH
AAA proteins
Proteolysis
Structural characterization
Membrane protein

https://doi.org/10.4233/uuid:ccfb250b-48a4-487a-b6b2-10e7392c1f1a

978.90.8593.370.0

Institutional Repository

doctoral thesis

© 2018 V.I. Dias Ribeiro de Carvalho