Co-translational insertion and assembly of a multi-spanning membrane protein from Escherichia coli monitored in vivoKrč, Ajda (Avtor) Novinec, Marko (Mentor) polytopic membrane proteinspulling forcestransmembrane α helixco-translational insertionMost membrane proteins are inserted into the membrane co-translationally. In bacteria, such as the gram-negative Escherichia coli (E. coli), the insertion usually follows the secretory (Sec) pathway with the Sec translocon as a key protein complex that enables partitioning of membrane proteins into the lipid bilayer. To monitor the process of co- translational insertion and folding of membrane proteins, an in vivo translation technique that adopts translational arrest peptides as “force sensors” has been developed recently. Using this technique, the “pulling forces” acting on the nascent polypeptide chain can be measured during integration of transmembrane helices into the membrane. In this study, the method was used to follow the insertion of the multi-spanning membrane protein BtuC, the transmembrane domain of the vitamin B12 translocase in E. coli. Previous work on BtuC suggests that the transmembrane helices (TMHs) insert more or less sequentially, one after the other, following the predicted insertion pattern based on ΔG calculations. However, there are discrepancies in the second part of the generated force profile which imply that apart from hydrophobicity, other forces might play a role in insertion of downstream TMHs of BtuC. Therefore, the aim was to see (1) whether upstream TMHs have an effect on insertion of downstream TMHs and (2) whether they can insert efficiently by themselves. Surprisingly, the results of the first part show no significant difference in pulling forces when the first four upstream TMHs are deleted from the BtuC constructs. This suggests that downstream TMHs of BtuC insert for the most part independently regardless of the presence or absence of the upstream TMHs. The only discrepancy is seen in TMH5, which seems to insert later. For that reason, we decided to test whether the N-terminally engineered hydrophobic Lep segment (TMH1) plays a role in insertion of BtuC constructs. Unfortunately, the results show very poor expression of BtuC constructs without the Lep segment. That indicates that upstream TMHs of BtuC cannot efficiently insert and/or are not stable enough by themselves, at least not in conditions used in the force assay in vivo. In conclusion, hydrophobicity represents the major force contributing to insertion of the multi-spanning membrane protein BtuC. Any other force that might act on transmembrane helices during the process cannot yet be detected using the force measurement assay in vivo.20202020-08-31 15:00:00Diplomsko delo/naloga118722VisID: 9761COBISS_ID: 27356163sl