I am wondering if there is an effective way to trap ATP in NBD by addition of high concentration of EDTA? I read papers that used EDTA to inhibit ATP hydrolysis. But I would like to know if there is other way trapping ATP or inhibiting in some other way.
Your thoughts are greatly appreciated.
Hello Sung,
EDTA is a very effective Mg ion scavenger. If you add it to an ATP/Mg solution, the Mg ions will bind to the EDTA and can no long complex with the ATP molecules. As a result, ATP cannot bind to your NBDs (this is the basis of the ATP hydrolysis inhibitory effect of EDTA) and the result will be the opposite of what you are trying to achieve.
If you don't want to use vanadate to trap ATP in the NBDs, you could consider using another transition state analog-like inhibitor like AMP-PNP.
It seem clear that Mg ion helps to hydrolysis of ATP in NBD of ABC transporter and promote inward facing conformation.
Then, is there clear evidence regrading magnesium ion for binding ATP to NBD? I read a paper (http://www.jbc.org/content/288/29/20785.long) that present ATP EDTA has similar effect of ATP(ADP) vanadate. Would you recommend other paper about this?
Thank you,
The effects of EDTA and Vanadate are similar (both are inhibitors) but their mechanisms are different. EDTA chelates Mg preventing ATP binding. Vanadate is a phosphate mimic that locks the protein into a transition state with bound ADP. http://www.sciencedirect.com/science/article/pii/S0014579304012529
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