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Stereospecific assignments with a 13C Fractionally Labeled Sample

Isopropyl groups of Val and Leu can be sterospecifically assigned with the help of a 5-10% fractionally 13C-labeled sample. In biosynthesis of amino acids from glucose the pro-R methyl carbon and the quaternary carbon come from one glucose molecule and the pro-S carbon is taken from another. With low-level fractional 13C-labeleing it is extremely unlikely that both glucose molelules are 13C-labeled. Therefore, pro-R 13C carbons have a 13C neighbor, but pro-S do not.

Determining stereospecific assignments

Experimental procedure:

  1. Record constant-time [13C,1H] HSQCs with constant-time delays 28 ms (1/J), 42 ms (1.5/J) and 56 ms (2/J)
  2. Process all HSQCs with identical phase parameters
  3. In XEASY or CARA identify pro-R (QD1/QG1) and pro-S (*QD2/QG2) groups by observing the following
    • QD2/QG2 have the same sign in 28 ms and 56 ms spectra.
    • QD1/QG1 change sign in 28 ms and 56 ms spectra.
    • Only QD2/QG2 are oberved in 42 ms spectrum.
  • 42 ms HSQC is rather insensitive and is optional. It is useful under conditions of severe overlap of methyl groups, when peaks of opposite sign tend to cancel each other in the 28 ms HSQC.
  • 56 ms could also be skipped if the target protein has assigned Met methyl groups. Since they do not have any directly bonded carbon spins, their peaks have the same sign in the 28 ms HSQC as the pro-S (QD2/QG2) groups.


Neri et al. Biochemistry 28 (19): 7510-7516 SEP 19 1989

Applying stereospecific assignments of methyl groups

It is recommended to use stereospecific assignments of methyl groups before running the FOUND module or prior to doing automated structure calculation. This improves accuracy and precision of the initial structure calculation.

It is also possible to apply them later at the refinement stage prior to using GLOMSA if spectra of the fractionally labeled sample were not recorded in time for automated structure calculation.

You can create CYANA scripts as described in the topic about applying stereospecific assignments. Use a swapped atom list and a separate stereo.cya file. For better transparency in a given project it is better to swap assignments in the original file (XEASY atom list or CARA repository), rather than relying on swapping them in CYANA.

To swap methyl spin labels in CARA do the following for Leu (or Val):

  1. In the [13C,1H]-HSQC plane in PolyScope select HD1/CD1 (or HG1/CG1) spins from the list.
  2. Right-click and select Label Spins
  3. In the pup-up Edit Spin Label window change the labels to HD/CD (or HG/CG).
  4. Repeat steps 1-3 to change HD2/CD2 to HD1/CD1 (or HG2/CG2 to HG1/CG1).
  5. Repeat steps 1-3 to change HD/CD to HD2/CD2 (or HG/CG to HG2/CG2).

To swap methyl spin labels in XEASY do the following for Leu (or Val):

  1. Open the current .prot list in a text editor
  2. Change the atom numbers of the QD1/QD2 and CD1/CD2 (or QG1/QG2 and CG1/CG2)
   646   0.831   0.000 QD1    39    #swap 646 and 647
   647   0.724   0.000 QD2    39    #swap 646 and 647
   648  25.020   0.000 CD1    39    #swap 648 and 652
   649 999.000   0.000 HD11   39
   650 999.000   0.000 HD12   39
   651 999.000   0.000 HD13   39
   652  22.667   0.000 CD2    39    #swap 648 and 652
  1. Reload the .prot list in all open spectra


-- Main.AlexEletski - 13 Jun 2007

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