Year: 2016
Type: dissertation
Abstract: Unlike the inner-membrane, the outer-membrane of Gram-negative bacteria cannot be energised by a proton gradient and the periplasmic space is absent of ATP. This poses a significant problem for active processes such as transport against concentration gradients. TonB-dependent transporters (TBDT) are a class of outer-membrane proteins responsible for the scavenging and import of scarce metallo-organic complexes from the environment. They are structurally characterized by a 22-stranded β-barrel with the lumen occluded by an N-terminal globular ‘plug’ domain which contains a conserved binding motif known as the Ton box. Upon substrate binding, the Ton box becomes exposed to the periplasmic space where it forms a non-covalent complex with the C-terminus of a cytoplasmic membrane protein, TonB. The transport process requires energy from a proton motive force coupled with an inner membrane complex TonB-ExbB-ExbD. Regardless of a wealth of structural information, current models of the TonB-dependent transport mechanism are speculative. Conversely, despite no current experimental evidence, it is generally accepted that the plug domain must undergo a large conformational change facilitated by mechanical force exerted onto the Ton box tether by TonB. \nIn this thesis, force spectroscopy, protein engineering, molecular dynamics and bacterial growth assays are used to investigate the effects of force on TonB:TBDT complexes from E. coli. These experiments demonstrate that the channel of the vitamin B12 transporter (BtuB), reconstituted into synthetic liposome, can be opened by the application of force onto the plug domain via the non-covalent binding partner, TonB. Using wild-type BtuB and several of its mutants together with a related receptor (FhuA), the extent of plug remodelling is found to be highly controlled and determined by the cargo the receptor has evolved to transport. For both receptors, the plug domain can be regarded as comprising a mechanically weak channel forming sub-domain, and a mechanically strong sub-domain used both for allosteric signalling and to limit the size of the channel to allow passage of molecules no larger than its cargo. \nAlongside these findings, structural and biophysical analysis of the periplasmic spanning protein TonB reveals conformation within the proline-rich linker domain, which allows speculation of the origin of the pulling force used for plug remodelling.
Author: Samuel J. Hickman
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2622224823