IDENTIFICATION AND CHARACTERIZATION OF ESSENTIAL RESIDUES IN THE TONB CARBOXY TERMINUS
Open Access
- Author:
- Kastead, Kyle A
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- None
- Committee Members:
- Kathleen Postle, Thesis Advisor/Co-Advisor
- Keywords:
- TonB
- Abstract:
- In gram-negative bacteria, the difficulty of iron acquisition is overcome by the secretion of siderophores and the active transport of these iron-siderophore complexes across the cell envelope. TonB is the dynamic central protein in this system, responding to energy from the proton motive force (pmf) at the cytoplasmic membrane and transducing that energy to outer membrane for transport through the TonB-gated transporters (TGTs) which bind the iron-siderophores. Determining how this protein achieves that has proven to be a difficult task with several discrepancies between the in vivo and in vitro data. TonB possesses two functional domains: an N-terminal á-helix which is required for cytoplasmic membrane association and receiving energy; and a semi-structured carboxy-terminal domain which responds to energization, interacts with the TGTs, and transduces the necessary energy for transport. This study shows that the only residue in the N-terminal helix to provide an important side-chain is the histidine at position 20. The Q160 region in the carboxy-terminus is known to interact directly with the TGTs and although the arginines at positions 158 and 166 have been proposed to form contacts essential for function, neither shows a significant loss of activity when substituted by alanine. In fact very few residues exhibit any loss of activity when substituted by cysteines and none are completely inactive. The ones that do have phenotypes are the aromatic residues, the Q160 region from positions 161-164, and the glycine at position 186. This glycine, as well as a phenylalanine at position 125 form strong disulfide linked dimers when substituted by cysteines as seen previously for the aromatic amino acids at positions 202, 213, 215, and 230. These data suggest that TonB is a protein that functions largely through the conformational changes is its backbone as opposed to having a few catalytically important side-chains.