Dear all,
We are studying a protein which contains a coordinated bivalent cation (Mn2+/Fe2+/Zn2+). My first questions is how to determine whether the metal ion is present in the protein we expressed in E.Coli. and purified with Ni2+ affinity chromatography. What kind of specific Mass spectrum or other techique can identify whether the metal ion exits in the protein and what ion it is?
I tend to believe that a metal ion is present in the protein because all crystal structures for this protein published in PDB contain a metal ion.
We want to remove the metal ion from the protein to examine the binding dynamics of the metal ion to the protein. So the second question is how to remove the metal ion. Some references claim that Ni2+-free NTA gel can remove metal ion from metal-coordinated proteins ( Anal Bioanal Chem 2006;385(8):1409-13. doi: 10.1007/s00216-006-0603-2); Some people believe that EDTA of high concentrations can remove the metal ions; and some think that PMIB (polyvalent metal ion binding resin) can do this.
However, I think it might not be that esay because the metal ion is chelated by four residues in the proteins (two histidines, a glutamate and an asparate) and should be very stably coordinated. I think that denaturing the protein with urea, dialysis and then refolding this protein can definitely remove the metal ions.
Do you have any experience of working on the metal protein or have you read some conviencing references for this?
Best regard
Yeping Sun
For proteins, mild ionization mass spectrometry and tandem mass spectrometry are recommended.
https://cyberleninka.ru/article/n/mass-spektrometriya-s-myagkimi-metodami-ionizatsii-v-proteomnom-analize-obzor/viewer
On the second question, you should agree with your plan. Urea reduces hydrophobic interaction (unfolds globules) and will compete for the coordination of the protein with the nickel ion.
Dear Yeping
if you have a protein sample that bind an unknown metal in an unknown amount probably the best technique to determine and quantify the metal is probably the ICP-MS is the most sensitive and accurate approach.
Some metals can be also quantified through a titration with chelating agents able to form coloured complexes (as PAR for Zinc,c BCS for Copper (I)) but some of those they are not very specific and therefore you can use it to evaluate the number or metal bounded to the protein if you metallate the apo protein with a single specific metal but not to determine wich is the unkown metal binded in a sample.
However is it possible that if you purify the protein from E.coli using a Ni-NTa, some Nickel 2+ will bind the protein instead its native metal.
You can try to produce the apo protein from the IMAC eluted by incubating the protein with EDTA at acidic pH and then (if your protein is stable in this conditions) you can remove the metal-EDTA complex by desalting and re-metallate the protein with the different metals (Mn2+/Fe2+/Zn2+) and compare their binding ability.
One tecnique that you can use in this case is the DSF. Generally the binding of the metal induce a protein stabilization and therefore an increase in the Tm. Of couse if your protein is an enzime, the best way to check the metal specificity is perform an enzimatic assay in the presence of the different metals.
You can find an example of protein demetallation, remetallation and analisys by DSF on the attached paper.
More information abour DSF are also available on my blog: PRoteoCool (https://proteocool.blogspot.com/) in the ProteoCool n°24(Differential Scanning Fluorimetry - DSF: A simple and powerful technology for protein QC) available at PAGE3.
good luck
Manuele
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