When I searched I got a protocol to remove bound calcium introduced by  Haschke and Friedhoff in 1978. They reported as " Removal of the bound calcium from the isoenzymes was achieved by exposing the peroxidase to 6 M guanidine hydrochloride - 10 mM EDTA, pH 7-8 for approximately 4 hours at room temperature. The solution was then dialyzed overnight in 5 mM EDTA, pH 7.0 followed by extensive dialysis in water".  Is this a good method ?

But I am worried that use of guanidine hydrochloride will cause denaturing of protein.

You can apply different strategies. Probably your protein is better bound to calcium (e.g., higher stability, and more structured), and, therefore, a posible strategy is to express it bound to calcium and later on remove calcium by adding EDTA and a dialysis step. Of course, your protein must show lower affinity for calcium than that of EDTA. Then, you can perform the NMR calcium titration and see what structural changes occur along with calcium saturation.

Another possibility is to express your protein with calcium and perform a dialysis in a buffer with controlled saturating calcium concentration. Then, you can perform an NMR EDTA titration and see what structural changes occur along calcium dissociation. Of course, your protein must show lower affinity for calcium than that of EDTA.

Calcium interaction with proteins, similar to EDTA-calcium interaction, is highly dependent on pH. Thus, you may play with the pH in order to raise/lower binding affinities.

Calcium chelating resins represent another option for removing calcium.

Hi Sujeesh,

I would be very wary of denaturing your protein using gdn-HCl unless you are certain you know how to refold it.

I agree with Adrian that probably the easiest thing to try would be to express and purify the protein in its calcium bound state and then perform an EDTA titration in the magnet.  If this succeeds in removing the calcium (which you'd expect to see as changes in the HSQC spectrum) you could then perform another titration with Ca or possibly with another ion such as Mg to determine the specificity of the metal ion binding.

Hope this helps

Andy

You may benefit from reading details in my paper:

http://www.ncbi.nlm.nih.gov/pubmed/12034713

1) Express protein with Ca2+

2) Collect 1D NMR or far-UV-CD spectrum

3) Add excess EDTA to remove Ca2+ (dialyse)

4) Collect 1D NMR or CD spectrum

5) Comparing (2) and (4) you should have a good idea about Ca2+'s role in folding.

6) If the protein is stable (inspite of not being folded) in the absence of Ca2+, then dialyse to get all EDTA out and then titrate in Ca2+ and follow folding by 1DNMR or CD.

At the end of this exercise you will have a good idea of how your protein behaves in the presence and absence of Ca2+. You can use your CD or NMR spectra as signatures for specific state of the protein, before you use it for any other experiment.

It is very likely that your protein samples between many states in the Ca2+ absent state and Ca2+ binding moves it to the stable bound conformation. So if you play with 2DNMR, dont be surprised to see some broad peaks in the absence of Ca2+ along with the sharp peaks from unstructured residues. Be cautious with your analysis.

Exciting problem - Good luck!