Pauline Bentz

Abstract

Ammonium transport (Amt) proteins are integral membrane proteins found in all kingdoms of life, where they are in charge of selectively transporting ammonium (NH₄⁺) across biological membranes. Several high-resolution crystal structures of members of the Amt/Rh family have been solved, revealing that they share a high structural similarity. Our group could recently show that Amt1 from the archaeon Archeoglobus fulgidus is a slow electrogenic transporter of NH₄⁺, thus rejecting the controversial hypothesis that Amt proteins are rigid NH₃ gas channels. Nevertheless, there is still a lot to discover about the detailed transport mechanism of these proteins. For example, it is still unclear whether Amt proteins undergo conformational changes during transport.

In order to address this question, a fluorescent reporter was recently created by fusing the Arabidopsis thaliana Amt1;3 to a GFP. This construct was used to report fluorescent changes which could directly be correlated with NH₄⁺ transport through the Amt domain of the chimera in live S. cerevisiae cells.

We now seek to further characterize this construct using X-ray crystallography to gain more insights into the transport mechanism of Amt proteins on an atomic level.

Methods

protein expression and purification, detergents, protein crystallization, x-ray structure determination, liposome and proteoliposome handling, solid-supported membrane based electrophysiology.