Date of Award
Doctor of Philosophy (PhD)
Chemistry & Biochemistry
Marcelo C. Sousa
The cellular envelope of bacterial cells protects it from the extracellular environment it experiences. A key component of this envelope is the outer membrane along with its associated integral membrane proteins. Outer membrane protein biogenesis is a complicated process involving many steps. This process starts in the cystosol with the synthesis of the apo form of outer membrane proteins that have a signal sequence that directs their transport by the cytosolic chaperone SecB to the SecYEG translocon. They are translocated across the inner membrane and upon reaching the periplasm this signal sequence is cleaved.
The steps in the biogenesis process from this point forward are not well characterized. There are two periplasmic chaperones, Skp and SurA, which bind nascent outer membrane proteins after they are translocated and transport them to the β-barrel assembly machine located at the outer membrane. The β-barrel assembly machine is a five-member protein complex that is responsible for the folding and insertion of the nascent outer membrane proteins into the outer membrane.
In this work I focus on two areas of this process occurring within the periplasm. First, I solved the structure of BamD: the essential lipoprotein within the β-barrel assembly machine. The structure consists of five tetratricopeptide repeat domains of which the typical function of each is to act as a scaffolding protein. Based on the structure and known genetic information of BamD, I performed a series of biochemical experiments to elucidate the nature of binding between BamD and the associated lipoprotein BamC and determine if BamD is involved in recognition of putative outer membrane biogenesis signals. While I did not observe binding of BamD to peptides containing these signals I did observe binding by BamD to the regions corresponding to β-strands within two model outer membrane proteins. This suggests BamD plays a role in binding outer membrane proteins during the processing of the β-barrel assembly machine. Finally, I performed biochemical experiments to compare the differences of the two periplasmic chaperones (Skp and SurA), specifically their ability to protect outer membrane proteins from aggregation.
Sandoval, Cristina Marie, "Structural and Biochemical Characterization of Proteins Involved in the Biogenesis of Outer Membrane Proteins" (2014). Chemistry & Biochemistry Graduate Theses & Dissertations. 120.