Mutational Analysis of Residues in the TonB Protein of Escherichia coli

Open Access
- Author:
- Swayne, Cheryl Dianne
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- December 11, 2009
- Committee Members:
- Kathleen Postle, Thesis Advisor/Co-Advisor
Kathleen Postle, Thesis Advisor/Co-Advisor - Keywords:
- E. coli
energy transduction
TonB - Abstract:
- The TonB system in Escherichia coli is responsible for providing chemical energy to its specific outer membrane transporters to bring scarce or large nutrients into the cell. In these studies, we examine both the important residues in the C-terminus of the TonB protein and the essential residue, His20, in the sole transmembrane domain. Previous to these works, it was found that there are five aromatic amino acids in the C-terminus of TonB participating in an aromatic cluster. In the first study, discussed in chapter 2, a double mutant cycle analysis is performed on two additional aromatic residues that had been previously uncharacterized. It was found that F125 is not participating in this aromatic cluster, but Y163 is participating with these other aromatic amino acids. The relative importance of serine and threonine residues in the C-terminus was examined in this study as well by making alanine substitutions at each of these positions. None of the serines or threonines examined were important for TonB function and all alanine substitutions at these positions had full wild-type activity. In the N-terminal transmembrane domain, it has previously been shown that His20 has the sole functionally significant side chain. To examine the function of this residue further, two mutagenesis studies were performed. In the first study, His20 was substituted by each of the other amino acids. In this case, only H20N had wild type activity and this suggested that the proton pathway in this system may not be operating as previously thought. In the second study, the His20 residue was moved to each of the other transmembrane positions on an otherwise inactive H20A template. The only His relocation mutant to regain activity was V24H H20A. Because this residue is predicted to be on the same face of the helix as His20, it suggested that His20 was active along a face of the transmembrane domain. However, because none of the other residues on this face regained activity when substituted to histidines, the His20 residue is only able to function in a narrow depth. The transmembrane mutants described above were also coexpressed with a known suppressor of TonB transmembrane mutations, ExbB A39E. In these studies, it was evident that this suppressor is not allele specific and that a wide range of these histidine mutations could be suppressed to some extent.