Characterization of novel potassium transport proteins from Chlorella viruses.
[Ph.D. Thesis], (2011)
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|Item Type:||Ph.D. Thesis|
|Title:||Characterization of novel potassium transport proteins from Chlorella viruses|
Large DNA viruses are a source of transport proteins with minimal structure and robust function. In the first part I characterized two new variants of the K+ channel Kcv. The two Kcvs, namely KcvSmith and KcvNext-to-Smith, have a monomer size of only 82 amino acids, but exhibit most of the hallmarks of K+ channel pores, i.e. two transmembrane domains (TMs), a pore region and a selectivity filter, with the conserved sequence TxxTxGYGD. The two channels differ in 11 amino acids with most of the differences in the outer and the inner TM. Both channels are fully functional with selectivity for K+; they also conduct Rb+ and NH4+, but no Na+ and Li+. The selectivity of the two channels differs, even though they have an identical filter region. Mutational studies revealed that an exchange of a single amino acid in the outer TM already changed in KcvNext-to-Smith the selectivity for K+ and Rb+ towards the selectivity of KcvSmith, implying a relevance of the outer TM for selectivity. The two channels are sensitive to Ba2+ and Cs+, but not to TEA or amantadine. Both channels are fully blocked by Ba2+ and the responsible binding site for Ba2+ in the filter region could be identified by site-directed mutagenesis. Most notable is the difference in the mode of Cs+ block. While KcvSmith was blocked is a moderate voltage-dependent manner, KcvNext-to-Smith showed an extremely steep block with complex kinetics. Again, mutational studies revealed the relevance and sensitivity of the amino acid composition in the outer TM for the block.
The second protein of interest is a putative K+ transporter encoded by virus Fr483. Analysis of the sequence revealed high similarity to plant potassium transporters including a conserved putative signature sequence; topology prediction algorithms revealed structural similarity with known K+ transporters. Yeast complementation assays and Rb+ uptake experiments have shown that the transporter protein is functional. Northern blot hybridization confirmed that the transporter gene is expressed during the infection cycle. The function of the protein is yet unclear. Overall, this is the first functional potassium transporter encoded by a virus.
In the third part, we made phylogenetic analyses together with the group of Prof. Hamacher (TU Darmstadt). The generated trees show that K+ channels from the alga Chlorella NC64A are most distantly related to channels of viruses, which infect it, thus, they were most likely not acquired from the hosts. By generating a consensus sequence of the viral channels we also found a bacterial protein, which has high sequence similarity to KcvATCV-1, but is probably not a K+ channel. Overall, it is not clear from were the chlorella viruses acquired their channel proteins, and it is possible that they are even the architects of these.
|Classification DDC:||500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie|
|Date Deposited:||27 Jul 2011 07:39|
|Last Modified:||07 Dec 2012 12:00|
|Referees:||Thiel, Professor Gerhard and Bertl, Professor Adam|
|Refereed:||15 July 2011|
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