In bacteria, proteins are synthesized at ribosomes in the cytosol. A substantial share of the proteins, however, function outside the cytosol. For instance, in Escherichia coli, they either need to be translocated across the cytoplasmic membrane to reach the periplasm or outer membrane, or they need to be integrated into the cytoplasmic membrane. The Sec pathway is the major route for protein transport across and into the cytoplasmic membrane. This pathway is formed by the Sec translocase, which consists of a protein-conducting channel in the cytoplasmic membrane formed by the SecYEG heterotrimeric membrane protein complex, the motor protein SecA which utilizes ATP to drive the translocation process and the accessory heterotrimeric membrane protein complex SecDFyajC that couples translocation to the proton motive force. A major advance in our understanding of the Sec-translocase was provided by the crystal structure of the SecA-SecYEG complex of Thermotoga maritima. The structure indicates that SecA enters the SecYEG channel by means of the so-called two helix finger (2HF) domain which contacts the cytoplasmic loop 4 of SecY. Here, we examined the importance of the 2HF SecA in protein translocation by extending and shortening the length of the 2HF for up to about two helical turns. The dynamics of the interaction between the 2HF of SecA and the SecYEG pore was also investigated. Further, we also examined the permissiveness of the Sec translocase for the translocation of chemically modified derivatives of proOmpA with a focus on the site of chemical modification.
|Qualification||Doctor of Philosophy|
|Place of Publication||[Groningen]|
|Publication status||Published - 2019|