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This is an excerpt from an email conversation that Walter contributed, with advice on the subject of how best to work with EDTA or calcium in your NMR sample:
From: Eric To: Walter Subject: calbindin Hi Walter, [...] If I'm titrating EGTA into a calbindin sample and collecting NMR spectra at various EGTA concentrations, what is the best way in which to add the EGTA? Is it necessary to take the sample out of the NMR tube each time EGTA is added? And if not, then how do I titrate such a small volume into the sample? [...] Best regards, Eric
From: Eric To: Walter Subject: calbindin Hi Walter, I have some unlabeled, lyophilized calbindin. I was going to bring it up in a 2.1 molar excess of Ca and collect a 1D spectrum for the calcium-loaded state. Then I was going to titrate EGTA into the sample and collect additional 1D spectra in order to observe intermediate states of Ca binding and ultimately the apo-form of the protein. We wanted to demonstrate to ourselves that we can successfully control the Ca-binding state of calbindin Best, Eric
From: Walter To: Eric Subject: calbindin The basic idea is sound. The intermediate states are not really accessible due to cooperativity of Ca binding and intermediate exchange phenomena between states. What is important is that you can prepare pure apo and Ca loaded states of the protein with confidence. Here is what I recommend: The key to everything is following the highest field methyl peak in the spectrum. In the apo state it is a Val 70 gamma and in the Ca-loaded state it is the Ile 73 delta-methyl. The resonance frequencies are something like 0.23 and 0.18, respectively, but key is that the former is a doublet and the latter is a triplet. Though it requires great shimming to clearly see the full splitting, you should always be able to tell a doublet shape from a triplet shape. At intermediate Ca loadings, you can see both siganals simultaneously, even at 500 MHz. 1. Learn what state your preps leave your samples in. Usually this is pure Ca-loaded or mostly apo with 1-10% Ca, depending on the prep. This means just put your protein in the NMR tube and take a spectrum. 2A. If you are fully Ca loaded. Do an EDTA or EGTA titration. Make sure pH is adjusted to 7.5 or higher to get full chelation effect of chelator! Add 1 molar equivalent, then 5, then 10, then 20. Only the last point is likely to leave you fully apofied. 2B. If you are nearly fully Ca loaded, add Ca and bring to fully Ca loaded state, then carry out 2A. 2C. If you are nearly apo, titrate in aliquots of 1 molar equivalent of chelator (check pH) until you see pure apo protein, then carry out a Ca titration. Remember now that you have to fill up all of the EDTA/EGTA sites as well as the protein sites before reaching the Ca-loaded state. 3. The standard approach to carefully control Ca content is to first prepare apo protein in the NMR tube by adding chelator. Then try to remove all chelator either via ultra-filtration or a similar approach, or by running down a desalting column. Check by NMR, and then carry out your Ca titration. Remember it is hard to get the protein concentration exact. - EDTA and EGTA have NMR signals so you should make standards in the absence and presence of Ca so you have markers. Of course, they are easy to find amongst the spectrum of the protein because they have very sharp lines because they are small molecules. I have copied Mark on this e-mail, and Jonathan Sheehan so he puts this on our wisdom pages. Best, Walter