Amide Side Chain assignment with NOESY

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This section describes the procedure to assign the amide side chains of Asn (d2) and Gln (e2) using the simultaneous 15N, 13C-resolved [1H, 1H]-NOESY (or by combining separate 15N resolved and 13C-resolved [1H, 1H]-NOESY spectra). Assuming that you have complete assignments of the Asn/Gln aliphatic side chain, extending the assignment to include the d2 and e2 is straightforward.

  1. Load two copies of the NOESY into XEASY. Be sure to use two different orientations for the two spectra. Spectrum I should be viewed as 13C/15N(x-axis), indirect 1H(y-axis), and direct 1H(z-axis) while spectrum II should be viewed as direct 1H(x-axis), indirect 1H(y-axis), and 13C/15N (z-axis). Additionally, load the appropriate peak list, sequence list, and atom list. Select Asn and Gln peaks with sp (type ASN GLN in the box labeled fragment type and type 0 in the box labeled sequential range). Create strips in both spectra (es, se, rc, rs, se, gs).
  2. Look at the CB strips of Asn or the CG strips of Gln. You should see 2 strong cross peaks near 7ppm in both the HB2(HG2) and HB3(HG3) strips which correspond to HB-HD21/HB-HD22 or HG-HE21/HG-HE22. At this point, it is helpful to create 4 new peaks (ND2/HD22/HD22, ND2, HD21/HD21, ND2/HD22/HD21, and ND2/HD21/HD22 for Asn and NE2/HE22/HE22, NE2, HE21/HE21, NE2/HE22/HE21, and NE2/HE21/HE22 for Gln. The location of these peaks is not important at this time because they will be moved to the proper location.) Once you have identified the 2 amide side chain peaks and created the new peaks, use mr to move the HD22/HD21 (or HE22/HE21) peaks in spectrum II (the one with the direct 1H dimension along the x-axis).
  3. Recreate the strips (es, se, se, gs) to see the updated peak positions and the newly created peaks. Use mr again to move the peaks in spectrum I.
  4. Recreate the strips (es, se, se, gs) to verify the new assignments.
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