Exploring The Interaction Between Colicin B And The TonB Dependent Transporter FepA Using Colicin B Mutants
Open Access
- Author:
- Bhimwal, Rijul
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Kathleen Postle, Thesis Supervisor
David Scott Gilmour, Thesis Honors Advisor - Keywords:
- TonB
Colicin B
FepA
Outer Membrane
Bacteriocin
Escherichia coli
Gram-negative - Abstract:
- In order to design new drugs against Gram-negative pathogens it is vital to understand the transport mechanisms across these bacteria. One particular system of interest is the TonB system. This system is used to transport nutrients such as iron, vitamin B12, and even bacteriocins across the outer membrane of Gram-negative bacteria. In this study, we investigated the means of translocation of the bacteriocin Colicin B (ColB) across the outer membrane embedded TonB-dependent transporter FepA, which is not well understood. FepA is a 22-stranded β-barrel with a globular cork domain inside the lumen of the barrel. ColB is a large, dumbbell shaped protein with a molecular weight of 55 kDa, which is larger than the lumen of FepA. This has led to the hypotheses that either the globular cork domain of FepA completely exits the barrel allowing ColB translocation, or that the cork does not exit the barrel completely or at all and ColB denatures to traverse the outer membrane. In this study, we introduced dicysteine substitutions in the receptor domain of ColB to facilitate the formation of disulfide bonds to try and trap some of the colicin mutants in their native structure and observe whether translocation was affected by comparing pore forming activity of the mutants to wild-type ColB. Our results indicate that introducing a disulfide bond in the native structure of ColB still causes lethality, suggesting that ColB does not completely denature during translocation. However, more work is needed to investigate the denaturing hypothesis, especially to rule out partial denaturation. Understanding this mechanism of translocation of ColB through FepA could lead to the creation of novel antibiotics, such as those that can block translocation through FepA and ‘Trojan Horse’ drugs that mimic ColB to traverse FepA.