I have a small bacterial protein (7kDa) potentially involved in stress response which has the capacity to interact with different kind of metals such as Ni2+, Cu2+, Fe2+, Zn2+, Co2+ and Cd2+. No interaction was detected with other metal ions. I have demonstrated the interaction by X-ray crystallography, Circular Dichroïsm, Isothermal Titration Calorimetry and Thermal Shift Assay but I have no idea of its physiological role.
Something to think abut is whether the protein will have metal bound at physiologically relevant metal ion concentrations. Your ITC experiments should have given you the dissociation constants for the metal ions, so now you need to find out what the environmental metal ion concentrations are, and use those and the Kds to calculate the metal occupancy of the protein.
To demonstrate a biological relevance of observed in vitro effects, one usually turns to genetic experiments which are fairly straightforward in bacteria. Basically, in your in vivo experiments you are looking for changes in phenotype in bacteria with specific genotype.
Since not much details provided, just from the top of head:
I would make a whole list of mutants for this protein (e.g. mutate those AAs you think are involved in metal binding - you likely already did it, etc.), proteins it interacts with (if known and if relevant to your question), and I would also use mutants for metals intake and metabolism (in E. coli, for example, there are mutants available for copper and iron metabolism; not sure about other metals, I did not work with them), etc. There are really a lot of things you could do :))
Something to think abut is whether the protein will have metal bound at physiologically relevant metal ion concentrations. Your ITC experiments should have given you the dissociation constants for the metal ions, so now you need to find out what the environmental metal ion concentrations are, and use those and the Kds to calculate the metal occupancy of the protein.
The experiment to see the physiological relevance could be designed after knowing what kind of domains and motifs this protein posses. As you have described that you have X-ray crystal structure of the metal-protein complex. You may see what kind of active site it may have? From sequence and structure one can guess potential activity of the protein and that could be tested in vitro in presence and absence of these particular metal ions. Testing activities in presence of different metals may also tell which metal is preferred as a co-factor among these.
Thank you for your help. I can try some possibilities. I'll keep you posted if I find something
As you have performed X-ray assays, to try to think of something else I have a question: Is just one metal ion bound per protein molecule? Is it water accessible, or is it deep in the structure? WOuld be it possible for the protein to have a hemo or some porphyrin-like heterocycle (as in heme, which is in cytochromes, myoglobin, etc). I say this because ions often (for physiological purposes) need many points of coordination (apart from electrostiatic interactions with negatively charged amino acid side chains) and nitrogens are good for "coordinations"
In my case, only one metal ion is coordinated by two histidines and four water molecules on the surface of the protein. Both histidine residues are highly conserved among the family. The Kd for such coordination is between 10 and 35µM for all metal ions.
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