Investigation of Arabidopsis thaliana Tha4 pore formation in planar bilayers using electro-optical methods

  • In the course of evolution, the structure and topology of cells have changed greatly. From simple cells surrounded only by a single membrane to cells containing multiple membrane-enclosed organelles. The advantage of individual reaction compartments is offset by the fact that proteins required there are almost exclusively nuclear-coded. They have to be targeted to and imported into their compartment post-translationally. One of the protein import systems involved, which can even transport large fully folded proteins, is the twin-arginine translocation (TAT) system. It operates both in the thylakoidal membrane and the plasma membrane of a wide range of bacteria. The thesis focuses on Tha4, the potential channel forming subunit of the TAT system of Arabidopsis thaliana. To verify formation of homo-oligomeric membrane channels purified Tha4 protein was used. The oligomeric topology of fluorescence-labelled Tha4 was investigated using fluorescence fluctuation spectroscopy (FFS). The functional properties of the membrane reconstituted Tha4 was investigated using the electrophysiological bilayer technique both in the vertical setup and with the combined fluorescence-opto-electrical horizontal bilayer (HLB) technique. Electrophysiological measurements with artificial planar lipid bilayers revealed cation-selective ion-channels for reconstituted Tha4 with a pore width of 1 nm. Similar results were obtained for co-reconstituted Tha4-Hcf106 proteins in vertical planer bilayers. The investigation of the oligomeric state in solution and in artificial planar lipid bilayers using FFS at the single molecule level with fluorescence-labelled proteins revealed that addition of soluble, NBD-labelled Tha4 showed a strong accumulation in the membrane. Subsequent lifetime measurements disclosed that the NBD-labelled parts are located more close to the polar part of the membrane, in close vicinity of the membrane-water interface.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Philipp Bartsch
Referee:Richard Wagner, Mathias Winterhalter, Wolfgang Schliebs
Advisor:Richard Wagner
Persistent Identifier (URN):urn:nbn:de:gbv:579-opus-1009886
Document Type:PhD Thesis
Date of Successful Oral Defense:2020/12/18
Date of First Publication:2021/07/07
Academic Department:Life Sciences & Chemistry
PhD Degree:Biology
Focus Area:Health
Call No:2020/25

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