Spectroscopic investigation of conformational transitions in copper-transporting P1B-ATPases
Copper serves as a cofactor in redox reactions. However, excessive copper accumulation can be toxic to cells and leads to oxidative damage of biomolecules. Copper ATPases play a key role in the regulation of cellular copper. Cu-ATPases belong to the family of P1B-type ATPases and designated as CPx-type ATPases on the basis of a distinctive intramembraneous CPC (Cys-Pro-Cys) or CPH (Cys-Pro-His) motif.
The inherited mutations in copper human ATPase ATP7A or ATP7B genes cause Menkes or Wilson diseases, respectively.
In my PhD study, we aim to investigate the structure and function of P1B-ATPases (L. pneumophila copA and E. hirae CopB) and their stability using spectroscopic methods e.g. Circular Dichroism (CD), Isothermal Titration Calorimetry (ITC) and Fourier transform infrared spectroscopy (FTIR). Expressing isolated cytosolic domains, we specifically address the question of allosteric coupling between the nucleotide binding pocket and the transmembrane metal transporting channel. Cytoplasmic domain interactions are explored by thermal stability measurements and interference with peptides derived from conserved recognition sites of P1B-type ATPase.
We utilize mutagenesis, truncation and single molecule approaches to further study how the enzymatic activity is regulated by.the interplay of these domains.
Thanks to Marc Solioz (Dept. of Clinical Pharmacology, University of Berne) for providing us with E. hirae CopB.
This work is supported by Dresden International Graduate School for Biomedicine and Bioengineering.