AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore towards 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 solubilisation 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 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.
The results show that the majority of patients undergoing orthognathic treatment are increasingly looking to social media to gather information on their treatment. They are using this alongside traditional sources of information and extensive discussion with medical professionals but seemingly gain some level of reassurance online.
Cdc42 is a small Rho-type GTPase and the main regulator of cell division in eukaryotes. It is surrounded by a large network of regulatory proteins. To understand the processes around cell division, in-depth understanding of Cdc42 and its regulation is required. In vitro reconstitutions are a suitable tool for such detailed mechanistic studies, as they allow a high level of control over the conditions and components used and. For these Cdc42 and its regulators need to be expressed, purified, and tested for their activity. There are many methods described for this, but their details, possible difficulties, and points of failure are rarely discussed. This makes in vitro studies on Cdc42 less accessible to scientists that have a background different from biochemistry. We here present our experience with working with Cdc42 in vitro. We describe the recombinant expression and purification behaviour of 12 Cdc42, six Cdc42-mNeonGreen and four Cdc42-sfGFP constructs in E. coli. We explore Cdc42 dimerisation in vitro and assess its activity using GTPase Glo assays and Flag-pulldown assays. GTPase Glo assays turn out to be a reliable tool to quantitatively asses GTPase activities, wheareas pulldown experiments are more error prone. We find that most Cdc42 constructs, with the exception of those with an N-terminal Twin-Step-tag, show a similar GTPase activity and interaction with the GDP/GTP exchange factor Cdc24. We close with using enterokinase and TEV protease to generate untagged Cdc42. Enterokinase also cuts Cdc42 in an undesired position. TEV protease leads to the desired product, which retains its GTPase activity but shows a reduced Cdc24 interaction. The work presented here acts as a guide for scientists desiring to work with Cdc42 in vitro through describing Cdc42s properties in detail and examining assays that can be used to study its behaviour or act as activity checks.
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