The bacterial secretory pathway (Sec) is the main process responsible for the localization of a large number of different essential proteins that need to be translocated across the cytoplasmic membrane into periplasm to be active. However, Sec substrate proteins are capable of assuming misfolded conformations or aggregate in stress conditions, compromising the cell viability. Therefore, bacteria have evolved a complex network of various accessory proteins(molecular chaperones(MCs)) that cooperate to maintain in unfolded state the nascent proteins synthetized by the ribosome prior their translocation ( A. Tsirigotaki et al, Nat Rev Microbiol. 2017 15(1):21-36; 2) T. Cranford-smith et al. J. Biol. Chem. 295 (2020) 7516-7528).. . In 2020, a new MC related to Sec machinery, called YecA, has been identified in E.coli revealing a role in membrane biogenesis. This MC promiscuously binds various proteins while they are still nascent polypeptides and collaborates with other MCs to maintain the translocation activity. The stress conditions required for its overexpression are currently not well understood, even if the iron availability seems to be one of the principal factors2. Due to its recent discovery, YecA is yet poorly investigated and essential information are missing. In particular, the molecular mechanism for the recognition and the release of Sec-substrates, as well as the interaction with other MCs and components of Sec still need to be investigated.
In this project, a structural and functional characterization of E.coli YecA will be carried out through the development of an innovative SDSL-EPR approach. Specifically, novel experiments based on SDSL-EPR spectroscopy will be carried out to decipher the interactions of YecA with other MCs and components of Sec pathway, focusing on the role of protein dynamics in the complexes formation. Combining EPR results with other biophysical techniques (ITC, DLC, CD, NMR and X-ray cryst.), the role of this MC in the E.coli Sec pathway and in the maintenance of bacterial proteome will be investigated.