Diploma work in structural biochemistry: Why are aquaporins in spinach leaves sensitive to mercury?

Background
Aquaporins are the membrane proteins that allow water to pass in and out of cells in all living organisms – bacteria, animals and plants. In plants, it is necessary to adjust the water flow very precisely, and therefore, we have studied an aquaporin from spinach leaves – SoPIP2;1. This aquaporin is especially interesting since it has a gating mechanism, which allows the plant to quickly adapt to a changed environment. Our group has previously determined the crystal structure of SoPIP2;1 in an open and closed conformation, and we are now continuing to investigate the mechanism of this regulation. In functional studies, we have seen that mercury activates the channel. This result is puzzling, since aquaporins are normally inactivated after mercury treatment. Now, we have a crystal structure of SoPIP2;1 binding mercury, but to interpret this we need functional and structural studies of mutants.

Your task
... is to overexpress mutants of SoPIP2;1 in the yeast Pichia pastoris where different cysteins (the amino acid that can bind mercury) have been mutated. These will then be functionally assayed to see which cysteine that is responsible for the mercury effect. Presently, we have two techniques for this in the lab – one where the protein is purified and put into artificial membranes (liposomes) and one where the original yeast cells are used (protoplasts). With the stopped-flow technique, the water flow in and out of the cell/liposome can be followed. The mutants showing interesting results can then be used in crystallization trials.
If the results are good there is a strong possibility that the results are published, which means you are one of the co-authors.

Techniques you will learn:
Fermenter growth of yeast, preparation of cell membranes, various chromatography techniques, stopped-flow measurements, preparation of liposomes and protoplasts, plasmid purification and transformation to yeast, SDS-PAGE, Western Blot and crystallization. Probably, there will also be opportunities to participate in experiments at the synchrotron ESRF in Grenoble, France.

The group
The membrane protein group at Gothenburg University consists of around 15 PhD students, post-docs and researchers. The group is very prominent within the field of structural studies of membrane proteins, and has a large experience of aquaporins. The group’s homepage can be found at http://www.csb.gu.se/neutze/

Contact
Please contact Anna Frick if you want to find out more: [email protected] or 031-7862596.