Abstract
Heavy metals can cause damage to biomolecules such as proteins and DNA in multiple ways. Cells therefore strive for keeping intracellular (heavy) metal ions bound to specific proteins that are capable of handling detoxification, export or integration as cofactors. Metal binding proteins usually provide specific coordination sites that bind certain ions with ultrahigh affinity, with the thermodynamic driving force being the stability of organometallic complexes. However, the metal binding properties of these proteins can be highly variable. Therefore the transfer of specific ions between separate proteins or even between distinct binding sites located on one and the same protein does not always follow affinity gradients, but depends on particular protein interactions that are difficult to predict. We established a method suitable to probe metal transfer between two proteins, provided the proteins are amenable to purification and in vitro handling. It consists of the loading with metals, the co-incubation and the separation of metal-exchanging proteins with subsequent determination of bound metal content. The method is exemplified by experimental data of ours probing the transfer of copper(I) between the membrane-extrinsic metal binding domain MBD2 and the transmembrane domain of CopA, a copper export ATPase from Escherichia coli (Drees et al., 2015).
Keywords: Copper-binding protein, Copper chaperone, Heavy metal associated domain, Copper transporter, P-type ATPase
Materials and Reagents
Equipment
Procedure
Representative data
A typical example of the separation of copper-loaded MBD2 and of lipid-reconstituted CopA is shown in Figure 2. Full exemplary experimental data, i.e., the concentrations of protein (calculated from spectrophotometric read of OD280) and of copper (determined colorimetrically as outlined above) after the transfer experiment are displayed in Figure 4 of Reference 1.
Notes
Recipes
Acknowledgments
We are grateful to the Protein Research Department of the Ruhr-Universität Bochum for financial support.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.