Metallothioneins (MTs) are small cysteine-rich proteins that play key roles in metal ion homeostasis and detoxification. Understanding their metalation pathways is essential for elucidating their biological functions and their interactions with both essential and toxic metal ions. Potentiometric titration has proven to be a valuable tool in studying these pathways, as it allows for the determination of metal binding affinities and stepwise coordination events. However, the technique also presents limitations, particularly when dealing with dynamic binding events and overlapping deprotonation constants, which are common in MTs.
In this presentation, we will discuss our findings from potentiometric investigations on MT metalation, emphasizing the method’s strengths in capturing equilibrium constants and identifying binding trends. For instance, our approach has enabled us to dissect stepwise metal binding events in two fungal MTs and to compare metal binding stabilities in both truncated constructs and amino acid mutants.1,2
Despite its strengths, potentiometry faces several challenges when applied to MT systems due to the high number of residues with overlapping pKa values. Moreover, distinguishing between metal-thiolate bond formation and coordination to histidine residues has proven surprisingly difficult. To overcome these challenges, we complement our potentiometric studies with spectroscopic methods - including UV-Vis, circular dichroism (CD), and NMR spectroscopy. These techniques not only confirm ligand coordination modes but also provide structural context that potentiometric titration alone cannot deliver, Together, this integrated, multimodal approach serves as a powerful tool for deciphering the metalation pathways in MTs.
Financial support from the Swiss National Science Foundation, the Faculty of Science at UZH, and the Polish National Agency for Academic Exchange (NAWA, grant no. PPN/BEK/2020/1/00268 to AH) is gratefully acknowledged.