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Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Molecular and Cellular Biology
Year Degree Awarded
Month Degree Awarded
Biochemistry | Biophysics | Biotechnology | Molecular Biology
Pore forming proteins are typically the proteins that form channels in membranes. They have several roles ranging from molecule transport to triggering the death of a cell. This work focuses on two E. coli pore forming proteins that have vastly differing roles in nature. Outer membrane protein G (OmpG) is an innocuous β-barrel porin while Cytolysin A (ClyA) is an α-helical pore forming toxin. For OmpG we probed its potential to be a nanopore sensor for protein detection and quantification. A small high affinity ligand, biotin, was covalently attached to loop 6 of OmpG and used to capture biotin-binding proteins. OmpG specifically interacted with target proteins and with a high sensitivity. In addition, we found that OmpG could discriminate among eight target proteins, four avidin homologues and four antibody homologues. Our work was the first revelation that a “noisy” nanopore sensor could not only discriminate among homologous proteins but could maintain this sensitivity and specificity in a serum sample. Unlike OmpG which is a monomeric pore, the ClyA pore is an oligomer which allowed us to probe its assembly. We found an alternative mechanism of assembly of the ClyA pore from its soluble monomer. Our results revealed an off-pathway oligomerization of ClyA. These oligomers are stable in solution but are less efficient at converting into transmembrane pores in the membrane than monomeric ClyA.
Fahie, Monifa, "Pore forming protein assembly and the use in nanopore sensing: a study on E. coli proteins ClyA and OmpG" (2017). Doctoral Dissertations. 1076.