The twin-arginine protein transport (Tat) system is one of two general protein transport systems in the cytoplasmic membranes of bacteria, and is conserved in the thylakoid membranes of plants. The Tat system transports fully folded proteins of varying sizes across the membrane. This feat is achieved by the use of just three conserved integral membrane proteins, TatA, TatB and TatC. Passenger proteins are designated for transport by the system by the use of an N-terminal extension, termed a signal peptide which contains an invariant twin arginine motif. The TatC component of the transport system is responsible for recognising the signal peptide, as well as binding to TatB and the TatA components. TatC therefore emerges as the functional and organisational ‘core’ of the Tat system. There is a wealth of functional data relating to the TatC protein, but no high resolution structural information on TatC was available to interpret this data. In order to gain an understanding of the Tat system at the molecular level, a structure was required of TatC. Preliminary expression screening using green fluorescent protein had previously identified the TatC protein from the thermophilic bacterium Aquifex aeolicus as being amenable for structural work. This study purified the A. aeolicus TatC construct in a variety of detergents, used native mass spectrometry and light scattering techniques to assay the sample homogeneity and set up extensive crystallisation trials. Following optimisation of a selenomethionine minimal media growth protocol, crystals grown from selenomethionine-substituted TatC purified in lauryl maltose neopentyl glycol diffracted to 3.5Å resolution, and allowed the structure of TatC to be solved by single wavelength anomalous dispersion. Analysis of the novel structure of TatC, in combination with an isothermal titration calorimetric binding assay, gave information on how TatC binds to signal peptides. Further structural analysis combined with additional experimental data from collaborators allowed a model to be proposed for how TatC binds to the two additional integral membrane components of the system, TatB and TatA, as part of the transport mechanism. Subsequent work was undertaken in this study to attempt to gain a co-crystal structure of TatC with the signal peptide. Construct and detergent screening was carried out in order to aim towards high resolution structural characterisation of the TatBC complex.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595985 |
| Date | January 2013 |
| Creators | Rollauer, Sarah Elizabeth |
| Contributors | Lea, Susan M. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:caf1615c-5dfc-42e4-8d96-c3e3ab4deb52 |
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