| Popis: |
Crystallization of ion channel proteins is a difficult task for several reasons related to the hydrophobic nature of these proteins, and still is a matter of trials and errors. In this work I will present an experimental approach to the crystallization of a group of small potassium channels: the viral Kcv channels. The first part deals with the expression of MA-1D Kcv in the heterologous system Pichia pastoris and its purification by detergent solubilization. Attempts to increase the yield of the protein by modification of the construct at the DNA level are discussed. In parallel the production of Fab fragments from monoclonal antibodies that recognized the tetrameric form of the protein has been established in order to make protein-antibody complexes that can promote an ordered crystallization process by increasing the polar contacts within the crystals. In the second part of this thesis, the planar lipid bilayer technique is applied to study the functional properties of several Kcv channels at the single channel level. In particular I have analyzed the block by barium of the wt PBCV-1 Kcv and of its mutants in the 4th site of the selectivity filter, residue Threonine 63. This mutation affects protein sensitivity to barium, but also alters the open probability and the number of subconductance levels. The mutation of an adjacent aminoacid, Serine 62, recovers the wt functions. The T63 mutation was then moved also to another Kcv channel, MA-1D Kcv, to check if the behavior related to the mutation is conserved. The third part deals with Kesv, a Kcv-like channel founding a related class of viruses, ESV that, differently to Kcv, shows a mitochondrial localization when expressed in heterologous systems. Due to the difficulties encountered in measuring from mitochondria of transfected cells, we have not been able to record currents from this channel in the past. It was therefore decided to produce and purify recombinant protein for functional studies in artificial lipid bilayer. Since all attempts to express it in Pichia pastoris failed, it was decided to express it in a cell-free system in collaboration with the lab of Dr. Bernhard, at the University of Frankfurt. Functional studies on the reconstituted protein channel have revealed that the protein forms a functional, selective K+ channel with overall features of the Kcv-like channels. |
