Amide Side Chain assignment with NOESY

Amide Side-chain Resonance Assignment Using The Simultaneous 15N, 13C-resolved (1H,1H) NOESY

The amide side-chain resonance assignment here means the assignment of amide side-chain of residues Asn and Gln, which can be done while doing the aliphatic side-chain assignment if the NOESY is used. The approach described below focuses on the simultaneous 15N, 13C-resolved (1H,1H) NOESY, please refer to Side Chain Assignments Using Simultaneous 15N,13C-resolved(1H,1H)NOESY for addtional information. This approach can also be applied to a combined use of 15N-(1H,1H) NOESY and 13C-resolved (1H,1H) NOESY.
NOTE: It is recommended to obtain aliphatic side-chain assignment before pursuing the following approach.

1. Process the simultaneous NOESY as a single 13C NOESY spectra. In this case, the 15N chemical shift is calibrated as 13C shift. The real chemical shift can be easily calculate back based on the aquisition parameters. It is important to analyse the simultaneous NOESY as a real single NOESY.
2. In UBNMR, run makeNoePeaks to generate a starting simultaneous NOESY PeakList including intra- and sequential NOEs, considering previously assigned chemical shifts and also averaged chemical shifts from BMRB for all assignable unassigned amide side-chain resonances. The 15N chemical shift has already been converted to pseudo-13C chemical shift which will exacltly fit the spectra.
3. In XEASY, use ns to load two copies of the Simultaneous 15N,13C-resolved (1H,1H) NOESY and display the two copies in different views (X-axis :w1(13C/15N), Y-axis: w2(1H), Z-axis: w3(1HC/HN) & X-axis: w3(1HC/HN), Y-axis: w2(1H), Z-axis: w1(13C/15N) use ls, lc, and lp, respectively, to load noe.seq, bbsc.prot and simnoesyI1.peaks; use sp to select peaks of residues Asn; use se and gs to display [w2(1H),w3(1H)]-strips and [w2(1H),w1(13C)]-strips residue by residue.
4. In XEASY, go to the CB-strips of each Asn residue. Two strong peaks corresponding to the HB-HD21 and HB-HD22 NOEs are expected at around 7 ppm along w2(1H, indirect dimension); use mr to move peaks to the identified peaks. Move to the most possible (strongest) two if there are many candidates.
5. In XEASY, use es, se, gs to resort and re-display the [w2(1H),w3(1H)]-strips and [w2(1H),w1(13C)]-strips for residues Asn. Since the chemical shift of HD21 & HD22 are very likely correct, identify the correct ND2 chemical shift in the corresponding ND2/HD21 and ND2/HD22 strips with view (X-axis: w3(15N), Y-axis:w2(1H); z: w3(1H)) based on the expected peak pattern and expected ND2 chemical shifts. Use zd to duplicate the ND2 strips if the expect position of ND2 is out of the strip view. Use gl or gr to change the dulplicate strip view to find the expected ND2; use mr to move the peak positions. Please also see Aliphatic Side-chain Resonance Assignment Using The Aliphatic HCCHCOSY Spectrum for addition information. Go back to the the CB-strips and identify the possible HD21/HD22 if no expected peak pattern cound be found for the initially identified HD21/HD22.
6. In XEASY, use es, se, gs to resort and re-display the [w2(1H),w3(1H)]-strips and [w2(1H),w1(13C)]-strips, double check the assignment obtained.
7. In XEASY, assign the amide side-chain of Gln residues in the same manner. Go to the CG-strips of each Gln residue indead of CB-strips for Asn residue. Strong peaks corresponding to the HG-HE21 and HG-HE22 NOEs are expected at around 7 ppm along w2(1H); use mr to move peaks to the identified peaks. Assign the Gln sidechain NE2 and the HE21 and HE22 peaks as described for Asp.
8. In XEASY, use wp to write an updated peaklist as noesyNO1.peaks.

Met Methyl Resonance Assignment

Usually, the Met methyl resonance assignment can be obtained from NOESY, however, in a different manner as described above. The Met methyl group has unique chemical shifts with 1H around 2.0ppm and 13C as 17ppm. Therefore, the best way to identify Met methyl is starting from the 13C aliphatic HSQC. It is also recommended to obtain aliphatic side-chain assignment before pursuing the following approach.

1. Same as above, process the simultaneous NOESY, make a 2D 13C HSQC project plane for the NOESY, generate a starting simultaneous NOESY PeakList from a AtomList with Met methy chemical shifts.
2. In XEASY, load the 3D NOESY PeakList to the 2D 13C aliphatic HSQC or the 2D 13C HSQC projection plane, type in the dimention names as below in the pop-up window.
   <img src="%ATTACHURLPATH%/3d2d.jpg" alt="3d2d.jpg" width='389' height='147' />
   use pp to manually pick the possible Met methyl peaks in the 2D spectrum, use wr write the update NOESY PeakList.
3. In XEASY, load the two copies of 3D NOESY and the updated 3D PeakList; use sp, es, se, gs to create and display strips from Met residues in two orthogonal views; use sp, se to create addtional strips for these manually unassigned peaks. Note: All the other peaks from the NOESY are simulated and assigned; type in n= and =n when the window below pop up.
   <img src="%ATTACHURLPATH%/strip1.jpg" alt="strip1.jpg" width='388' height='120' />
   
4. In XEASY, use cd, cc to identify these unassigned peaks to the corresponding Met methyl group, use mr to adjust the simulated Met methyl peaks to the assign position, use dp to delete the manually picked peaks that are aready indentified.
5. In XEASY, use aa, ac, wc and wp to save updated AtomList and PeakList.

-- Main.GaohuaLiu - 25 Mar 2007