Investigations on mercury sensitivity and molecular determinants of selectivity in two plant aquaporins.
Technische Universität, Darmstadt
[Ph.D. Thesis], (2011)
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|Item Type:||Ph.D. Thesis|
|Title:||Investigations on mercury sensitivity and molecular determinants of selectivity in two plant aquaporins.|
Aquaporins (AQPs) are members of major intrinsic proteins and form water conducting channels in the biological membranes. These may also transport small solutes as well as gases. The latter are, however, considered to be transported through a separate tetrameric pore. Not much is known about the structure of plant AQPs, particularly in case of TIP sub-family, no structural data is so far available. In the present study, detailed point mutational as well as transmembrane domain swapping analyses were performed to understand the molecular determinants of selectivity in AQPs. The mutational analysis involved substitutions of the native Cys residues in TaTIP2;2 and introduction of an already known Hg-sensitive site belonging to another TIP isoform (gamma-TIP). The results revealed that the presence of a Cys at a particular site in TM3 of the plant TIPs would render them Hg-sensitive. This lead to proposing a consensus region of metal sensitivity in plant AQPs (similar to the one in mammalian AQPs) for accommodating a Hg-sensitive Cys. A significant part of the study involved identification of molecular signatures of selectivity located both inside and outside the selectivity regions (ar/R region and NPA motif) of AQPs. The sequence alignment including predominantly water conducting AQPs in comparison to the ammonia conducting ones suggested a couple of positions with the residues distinguishable between the two AQP sub-families. The amino acid substitutions at the corresponding positions of TaTIP2;2 and NtPIP2 couldn’t turn the PIP into an ammonia-conducting channel however, a drastic effect of the said mutations on water permeability of PIP (and not of TIP) was observed suggesting their role in the water transport function of the PIPs sub-family. As TIP mutants retained their ammonia transport function similar to the wild type, the varying effects of the given residues may be attributed to the difference in structure between TIPs and PIPs besides pointing towards the presence of a central pore in AQPs. As regards the mutations inside the selectivity regions, none of the mutants was functional for water and ammonia transport. Same was the case with a number of chimeric proteins containing different transmembrane domains swapped between TIP and PIP. However, the leakage of protons could be demonstrated in some of these AQP variants. For future studies, the joint mutations combining the molecular signatures suggested in the present study with those located inside the selectivity regions may help to further understand the molecular mechanism of selectivity.
|Place of Publication:||Darmstadt|
|Classification DDC:||500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie|
|Divisions:||10 Department of Biology|
|Date Deposited:||09 Nov 2011 11:36|
|Last Modified:||07 Dec 2012 12:01|
|Referees:||Kaldenhoff, Prof. Dr. Ralf and Thiel, Prof. Dr. Gerhard|
|Refereed:||18 October 2011|