Amide Side Chain Assignment with CARA

Amide Side-chain Resonance Assignment

You will normally need to have 3 windows open at the same time:

1. Open aliphatic 2D [13C, 1H]-HSQC with Open PolyScope and select aliphatic 13C-resolved NOESY for the strip display. This is similar to displaying 3D HNCO or 3D 15N-resloved NOESY paired with a 2D [15N, 1H]-HSQC.
2. Open 3D 15N-resolved NOESY with Open SystemScope. Rotation is normally not needed, since the default dimension order is Hinept = X*, *Hnoe = Y*, *Ninept = *Z*. Thus, the left panel of SystemScope will contain an H-H plane (with Hnoe dimension vertical), and the right panel will contain the H-N plane.
3. Open 2D [15N, 1H]-HSQC with Open PolyScope and select 15N-resolved NOESY for the strip display.

Asn

1. Open an HB2/CB or HB3/CB strip in window 1.
2. Pick a HD21 candidate. This should be medium peak around 6 - 7 ppm. Make sure the peak is not from the backbone H of the next residue (though, overlap is possible).
3. In window 2 display the H/N strip of the same Asn residue.
4. Select the HD21 candidate. Right-click and choose Show Orthogonal in the pop-up menu.
5. In the orthogonal plane pick an ND2 candidate. The correct position should exhibit HD21-HB2 and HD21-HB3 intra NOE peaks.
6. In window 3 verify and adjust the position of HD21/ND2 spin pair.
7. In window 3 pick the HD22 spin. This is usually the strongest peak in the strip.
8. In window 3 verify and adjust the position of HD22/ND2 spin pair.

Due to rigidity of the -CONH2 group HD21 and HD22 are practically never degenerate. Also, only one of them will display strong NOE peaks to HB2 and HB3.

Gln

1. Open an HG2/CG or HG3/CG strip in window 1.
2. Pick a HE21 candidate. This should be medium peak around 6 - 7 ppm. Make sure the peak is not from the backbone H of the next residue (though, overlap is possible).
3. In window 2 display the H/N strip of the same Gln residue.
4. Select the HE21 candidate. Right-click and choose Show Orthogonal in the pop-up menu.
5. In the orthogonal plane pick an NE2 candidate. The correct position should exhibit HE21-HG2 and HE21-HG3 intra NOE peaks.
6. In window 3 verify and adjust the position of HE21/NE2 spin pair.
7. In window 3 pick the HE22 spin. This is usually the strongest peak in the strip.
8. In window 3 verify and adjust the position of HE22/NE2 spin pair.

Due to rigidity of the -CONH2 group HE21 and HE22 are practically never degenerate. Also, only one of them will display strong NOE peaks to HG2 and HG3.

Arg

1. Open an HD2/CD or HD3/CD strip in window 1.
2. Pick a HE candidate. This should be medium peak around 6 - 7 ppm. Make sure the peak is not from the backbone H of the next residue (though, overlap is possible).
3. In window 2 display the H/N strip of the same Arg residue.
4. Select the HE candidate. Right-click and choose Show Orthogonal in the pop-up menu.
5. In the orthogonal plane pick an NE candidate. The correct position should exhibit HE-HD2, HE-HD3, possibly HE-HG2 and HE-HG3 intra NOE peaks. Remember that NE is most likely folded, since 15N dimension is usually 25-35 ppm wide, centered around 115-120 ppm, while NE is found near 80-100 ppm.
6. In window 3 verify and adjust the position of HE/NE spin pair. The correct folding of NE can be checked in a 2D [15N, 1H] HSQC with a wide spectral width.

HE spins may not be observed for all Arg residues.

-- Main.AlexEletski - 01 Nov 2007