I have been working on a protein which have multiple sites for binding magnesium ions. These sites are present at different domains of the same protein.
How would I measure the binding affinity of Mg2+ ions to these different sites without mutating their binding sites?
What kind of techniques will distinguish the binding stoichiometry of these domains to Mg2+?
Isothermal titration calorimetry would be a good method to learn about the energetics and stoichiometry of Mg2+ binding. I doubt it would have sufficient resolution to measure each of the affinities of several binding sites at once, however. 2-D protein-observe NMR might offer additional information about the affinities of individual binding sites and, if the peaks were assigned to individual residues, NMR would also be informative about the locations of the binding sites. X-ray crystallography would show you the locations of the binding sites.
Isothermal titration calorimetry would be a good method to learn about the energetics and stoichiometry of Mg2+ binding. I doubt it would have sufficient resolution to measure each of the affinities of several binding sites at once, however. 2-D protein-observe NMR might offer additional information about the affinities of individual binding sites and, if the peaks were assigned to individual residues, NMR would also be informative about the locations of the binding sites. X-ray crystallography would show you the locations of the binding sites.
Is there any way to study the binding of Mg2+ at one particular site by saturating the other binding site of the protein?
This could be done if there is a large difference between the affinities of the sites, such that one site or set of sites was saturated with low concentrations of Mg2+ that were too low to begin to fill the other site. The affinity differential would have to be quite large, at least 100-fold, maybe more, to make this work.
Another approach might be to use metal ions other than Mg2+, especially Mn2+ or Ca2+, to try to differentiate between sites.
My interest of protein have multiple low affinity binding sites and one high affinity binding site. I want to study the stoichiometry of low affinity binding sites in the presence of high affinity binding site.
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