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Fabian Frank

Fabian Frank

Functional and structural investigation of the Archaeglobus fulgidus Amt-1

Principal investigator: Prof. Dr. Susana Andrade

Institut für Biochemie
Albertstr. 21
79104 Freiburg

Phone: +49 (761) 203-6054
frank@bio.chemie.uni-freiburg.de

Abstract

Ammonium transport proteins (Amt) mediate the selective transport of ammonium ions across membranes. Together with their mammalian homologs, the Rhesus proteins, they form the Amt/Rh family. High-resolution crystal structures of different Amt/Rh family members have been solved, revealing highly conserved residues in potentially functional relevant positions. The lack of an adequate functional assay to selectively detect NH4+/NH3 led to a discrepancy of published results and raised a long-standing debate in the field; Amt and Rh proteins have been defined as electrogenic transporters (NH4+ uniporters or NH3/H+ symporters), electroneutral (NH4+/H+) antiporters or passive NH3 gas channels.

We have optimized and established an in vitro electrophysiology assay that makes use of pure protein and proved that the archaeal Amt1 and Amt3 proteins from Archaeoglobus fulgidus are electrogenic NH4+ transporters.

Based on this data, we are investigating potential functionally relevant residues in these proteins to help us decipher the mechanistic details of Amt/Rh transport at atomic resolution. For that we use site-directed mutagenesis to produce variants that are then characterized by solid-supported membrane (SSM)-based electrophysiology and protein crystallography.

 

Methods

Site-directed mutagenesis, membraneprotein expression and purification, detergents, protein crystallization, X-ray structure determination, liposome and proteoliposome preparation, solid-supported membrane based electrophysiology.