Isotope labelling schemes

Introduction

We will now have a look at isotope labelling patterns. Select M: Molecules: Isotopomer Schemes. You will see that Analysis already comes with some ready made prepared isotope labelling patterns, such as "2-13C Glycerol" and "GAFY" under |Name|.

Labelling schemes

We will have a look at one of these labelling schemes, so under |Code| select the "13Glycerol" row and then select the {Isotopomers} tab. Select the Ala row in the top table and you will see that the bottom table is filled to show that the carbonyl "C" and "CB" have a 19:1 13C:12C labelling (i.e. 95% 13C 5% 12C). Note that the "CA" atom remains at natural abundance ratios (98.93% 12C). The setup for Ala within this scheme is relatively simple, as it only has one pattern. Returning to the top table again you can see that whereas Ala has only one labelling variant Arg has 8 variants (8 isotopomers):

Click on each of the Arg variant rows in turn and you will see that in each case a different combination of its carbon atoms is labelled with 95% 13C, this is because the labelling scheme represents the pattern that results from feeding bacteria 1,3 13C labelled glycerol and in the case of arginine there are multiple ways that the carbon atoms from glycerol can find their way into the amino acid. All of these residue labelling patterns must be considered when we do NMR because overall they tell us which atoms will be visible (labelled) in an experiment and individually they specify which correlations are observable; e.g. can we see a CA to CB peak. - In the case of Ala with 1,3 13C Glycerol, the answer is no.

If we wanted to we could define our own isotope labelling schemes, but for now we will leave the default set and see how such schemes are used at various points within Analysis. First have a look at M: Molecules: Atom Browser, choose the "Chain:" to be "Default:A" and select only the [C] button (turning other element types off as required). Now in the labelling scheme pulldown on the {Options} tab select "13Glycerol". You will see that selection of the labelling scheme has removed some of the atom options; these are now deemed to be unlabelled and hence invisible. Referring back to what we discovered about alanine in this labelling scheme, note that there is no Ala CA labelled:

Now change the labelling scheme to "GAFY"; which represents a situation where only five kinds of residues have 13C labelling (i.e. alanine, glycine, phenylalanine, serine & tyrosine). Looking at the carbon atoms in the sequence you will see that most are not shown, indeed only those for Gly, Ala, Phe, Ser & Tyr are shown, consistent with the labelling pattern. So you see that overall if you have a labelled molecule in your NMR sample as long as you select the correct scheme you will be presented only with those atoms which it ought to be possible to assign to.

Labelling schemes can also affect assignment options, as well as atom options. To illustrate this go to window1 and toggle the [HSQC:115] spectrum on (you may need to select [Spectra] to do this). With the mouse over one of the assigned peaks press the 'a' key to bring up the Assignment: Assignment Panel popup. You will see that the F1 (1H) dimension and F2 (15N) dimension both have resonances assigned to them, as indicated in the left hand tables. In the right hand tables note that both dimensions have one or more resonance possibilities (and one of these will be chosen for the assignment). In the "Labelling Scheme:" pulldown select "uni_15N13C2H" to pretend that we have a triple-labelled sample with deuterium instead of 1H. Note how with this scheme selected the earlier hydrogen resonance possibilities to disappear from the top right table; under such a labelling scheme it ought not be possible to assign 1H resonances (the peak at 1H:8,6 15N:114):

Finally we will look at how a labelling scheme can also be used to filter out impossible combinations when generating distance restraints. Select M: Structure: Make Distance Restraints and then set the "Peak Lists:" pulldown menu in the resulting popup to be "C-NOESY". Now press [Make Shift Match Restraints], after a while you will see that this generates around 1107 distance restraints for our carbon NOESY experiment. And clicking on the {Restraints} table on the Structure: Restraints & Violations popup you will see that these restraints are highly ambiguous. Now return to Structure: Make Distance Restraints and change the labelling scheme to "2Glycerol", set the "Minimum Isotope Fraction:" to 0.25 and then click [Make Shift Match Restraints] once again. Once the restraint generation is complete (it takes longer this time because of the labelling scheme), return to the restraint table and select the second restraint list in the pulldown menu. Note that this time we have generated fewer restraints (and that the restraints are less ambiguous). This is because we have only pairs of protons where the bound carbon is labelled in the scheme.

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