Aromatic Side Chain Assignment with CARA

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Aromatic Side-chain Resonance Assignment

Overall, aromatic side-chain assignment is very similar to aliphatic side-chain assignment, only one uses aromatic (H)CCH-COSY and aromatic 13C-resolved NOESY.

Tyrosine residues are the easiest to assign, since they only have HD/CD and HE/CE spin pairs, and the chemical shifts of CD and CE are quite different. Histidine residues are relatively easy, while tryptophan residues have a very complex side chain. Phenylalanines can be especially bad, since all HD/CD, HE/CE and HZ/CZ spin pairs have very similar chemical shifts, causing so-called strong coupling effects in the 13C dimensions, and overlap of NOE peaks.

Therefore, the recommended order would be Tyr -> His -> Trp -> Phe.

For aromatic side chain assignment you would need 7 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.
  2. Open aromatic 2D [13C, 1H]-HSQC with Open PolyScope and select aromatic 13C-resolved NOESY for the strip display.
  3. Open aromatic 3D (H)CCH-COSY with Open SystemScope (rotated).... In the rotation matrix pop-up window choose the following orientation: Hinept dimension must be Z*, *Ccosy must be Y*, and *Cinept must be *X*. Thus, the left panel of SystemScope will contain a C-C plane (with Ccosy dimension vertical), and the right panel will contain the H-C plane. This issue is addressed in detail in this FAQ question: http://www.cara.ethz.ch/Wiki/FAQ#III10
  4. Open 2D [15N, 1H]-HSQC with Open PolyScope and select 15N-resolved NOESY for the strip display.
  5. Open long-range 2D [15N, 1H]-HSQC with Open PolyScope (optional - for His assignment).
  6. Open 15N-resolved NOESY with Open SystemScope
  7. Open 13C-resolved NOESY with Open SystemScope

Tyr

For Tyr you have to assign HD/CD and HE/CD spin pairs.

  1. Display HB2/CB or HB3/CB strip in window 1.
  2. Pick an HD spin candidate - this should be a low-to-medium strength peak in both strips. Make sure that you don't mistake it for a long-range amide NOE peak. Note down the chemical shift of this HD spin.
  3. In the HSQC plane of window 2 find a peak at the chemical shift of HD. Since there are usually not that many aromatic residues in a given protein, it will likely be a unique choice. Place the cursor on the peak and pick the CD1 candidate: right-click and select Extend Vertically, then select the HD spin of the residue you are trying to assign.
  4. If the HD/CD1 pair is correctly assigned you should see HB2-HD and HB3-HD NOE peaks in the strip panels of window 2. Adjust the HD1/CD1 position to center the peaks in the strip panels.
  5. In window 3 display the HD/CD1 strip and use Show Depth. Pick the CE1 spin candidate.
  6. Select the CE1 spin and use right-click -> Show Orthogonal to display the orthogonal plane. In the orthogonal plane pick the HE spin.
  7. in window 2 verify and adjust HE/CE1 position. You should see a strong HD-HE NOE peak.

Under normal conditions Tyr rings are almost always flipping fast enough to make HD1/HD2, HE1/HE2, CD1/CD2 and CE1/CE2 chemical shifts degenerate. Therefore, pseudoatom labels HD and HE should be used. The CD and CE labels are not compatible with CYANA, therefore, CD1 and CE1 should be used instead.

Very rarely a stalled Tyr ring is encountered, which flips so slowly that the degeneracy breaks down. In this case, separate HD1/CD1, HD2/CD2, HE1/CE1 and HE2/CE2 spin pairs are observed.

Ring flipping at an intermediate rate leads to line broadening - in the worst case no peaks are observed.

Note that HD and HE can have very similar chemical shifts, even though CD and CE shifts are different.


His

For His you have to assign HD2/CD2 spins pairs. If you have a long-range 2D [15N, 1H]-HSQC spectrum, you can also determine protonation states and assign HE1/CE1 spin pairs.

  1. Display HB2/CB or HB3/CB strip in window 1.
  2. Pick an HD2 spin candidate - this should be a low-to-medium strength peak in both strips. Make sure that you don't mistake it for a long-range amide NOE peak. Note down the chemical shift of this HD2 spin.
  3. In the HSQC plane of window 2 find a peak at the chemical shift of HD2. Since there are usually not that many aromatic residues in a given protein, it will likely be a unique choice. Place the cursor on the peak and pick the CD2 candidate: right-click and select Extend Vertically, then select the HD2 spin of the residue you are trying to assign.
  4. If the HD2/CD2 pair is correctly assigned you should see HB2-HD2 and HB3-HD2 NOE peaks in the strip panels of window 2. Adjust the HD2/CD2 position to center the peaks in the strip panels.

If you have recorded a long-range 2D [15N, 1H]-HSQC you can assign more spins in a histidine side-chain. (Picture needed).

  1. Note the chemical shift of the HD2 spin.
  2. In long-range 2D [15N, 1H]-HSQC (window 5) find the spin system that with the matching HD2 shift. Depending on the protonation state you will encounter one of the following:
     a. For a charged His form you should see two cross-peaks from HD2 at around 170-190 ppm. Use Extend Vertically to pick them as ND1 and NE2.
     a. For an ε-protonated neutral form you should see a cross-peak to NE2 at about 160-180 ppm. Use Extend Vertically to pick it.
     a. For a δ-protonated neutral form you should see a cross-peak to ND1 at about 200-260 ppm. Use Extend Vertically to pick it.
  1. Use Extend Horizontally to pick the HE1 spin
  2. Pick the missing ND1 or NE2 if it is a neutral form.
  3. Note the chemical shift of HE1.
  4. In aromatic 2D [13C, 1H]-HSQC plane (window 2) pick the CE1 spin using Extend Vertically.
  5. Adjust the position of the HE1/CE1 pair according to the aromatic 13C-resolved NOESY spectrum.

Note that the His-tag gives rise to the strongest HD2/CD2 and HE1/CE1 in aromatic 2D [13C, 1H]-HSQC, which overlap closely.

The HD1/ND1 and HE2/NE2 peaks are rarely observed in 2D [15N, 1H]-HSQC because the protons exchange with the solvent. If they are visible, then usually in the neutral forms. The proton chemical shifts are unique at about 11-12 ppm.

Trp

  1. Display HB2/CB or HB3/CB strip in window 1.
  2. Pick an HD1 spin candidate - this should be a low-to-medium strength peak in both strips. Make sure that you don't mistake it for a long-range amide NOE peak. Note down the chemical shift of this HD2 spin.
  3. In the aromatic 2D [13C, 1H]-HSQC plane of window 2 find a peak at the chemical shift of HD1. Since there are usually not that many aromatic residues in a given protein, it will likely be a unique choice. In constant-time HSQC this will be on of the peaks in the middle with a different sign than most. Place the cursor on the peak and pick the CD1 candidate: right-click and select Extend Vertically, then select the HD1 spin of the Trp residue you are trying to assign.
  4. If the HD1/CD1 pair is correctly assigned you should see HB2-HD1 and HB3-HD1 NOE peaks in the strip panels of window 2. Adjust the HD1/CD1 position to center the peaks in the strip panels.
  5. In the same strip pick an HE1 candidate. This spin has a distinctive chemical shift of about 10 ppm.
  6. In window 6 select the H-N strip of the Trp residue. Select the HE1 spin, right-click and use Show Orthogonal to display the orthogonal plane. Pick the NE1 candidate in the orthogonal plane. Look for the HE1-HB2 and HE1-HB3 peaks at the NE1 position. (You can also pick NE1 in the 2D [15N, 1H]-HSQC plane of window 4).
  7. In window 4 adjust the position of the HE1/NE1 pair according to the 15N-resolved NOESY.
  8. In the strip of window panel pick an HZ2 candidate.
  9. Display the HD1/CD1 strip in window 7. Select the HZ2 spin, right-click and select Show Orthogonal from the pop-up menu. In the orthogonal plane pick the CZ2 spin.
  10. Adjust the HZ2/CZ2 position to center the peaks in the strip panels of window 2. The correct HZ2/CZ2 pair must exhibit the HZ2-HE1 peak.
  11. Display the HZ2/CZ2 strip in window 3 and show the depth plane. Pick a CH2 spin candidate.
  12. Select the CH2 spin, right-click and select Show Orthogonal. In the orthogonal plane pick the HH2 candidate.
  13. Adjust the HH2/CH2 position to center the peaks in the strip panels of window 2.
  14. Display the HH2/CH2 strip in window 3 and show the depth plane. Pick a CZ3 spin candidate.
  15. Select the CZ3 spin, right-click and select Show Orthogonal. In the orthogonal plane pick the HZ3 candidate.
  16. Adjust the HZ3/CZ3 position to center the peaks in the strip panels of window 2.
  17. Display the HZ3/CZ3 strip in window 3 and show the depth plane. Pick a CE3 spin candidate.
  18. Select the CE3 spin, right-click and select Show Orthogonal. In the orthogonal plane pick the HE3 candidate.
  19. Adjust the HE3/CE3 position to center the peaks in the strip panels of window 2. Verify that you see weak HE3-HB2 and HE3-HB3 peaks in the strip panels.


Phe

  1. Display HB2/CB or HB3/CB strip in window 1.
  2. Pick an HD spin candidate - this should be a low-to-medium strength peak in both strips. Make sure that you don't mistake it for a long-range amide NOE peak. Note down the chemical shift of this HD2 spin.
  3. In the aromatic 2D [13C, 1H]-HSQC plane of window 2 find a peak at the chemical shift of HD1. Since there are usually not that many aromatic residues in a given protein, it will likely be a unique choice. Place the cursor on the peak and pick the CD1 candidate: right-click and select Extend Vertically, then select the HD1 spin of the Phe residue you are trying to assign.
  4. If the HD/CD1 pair is correctly assigned you should see HB2-HD1 and HB3-HD1 NOE peaks in the strip panels of window 2. Adjust the HD/CD1 position to center the peaks in the strip panels.
  5. Display the HD/CD1 strip in window 3 and show the depth plane. Pick a CE1 spin candidate.
  6. Select the CE1 spin, right-click and select Show Orthogonal. In the orthogonal plane pick the HE candidate.
  7. Adjust the HE/CE position to center the peaks in the strip panels of window 2.
  8. Display the HE/CE1 strip in window 3 and show the depth plane. Pick a CZ spin candidate.
  9. Select the CZ spin, right-click and select Show Orthogonal. In the orthogonal plane pick the HZ candidate.
  10. Adjust the HZ/CZ position to center the peaks in the strip panels of window 2.

The above procedure works only for ideal case. Very often it would be difficult to pick 13C spins, since they will all have similar chemical shifts. In this case it may be easier picking 1H spins based on NOE peaks.


The starting point is to assgin HD spins of Phe and Tyr and HD2 of His in aliphatic 13C-resolved NOESY from the cross-peaks to HB2/3. For Trp you have to follow weaker correlations HB2/3 -> HD2 -> HE1 -> HZ2 or HB2/3 -> HE3.

Then directly attached 13C spins are assigned in aromatic [13C, 1H]-HSQC and aromatic 13C-resolved NOESY using PolyScope.

The procedure to assign remaining spins is similar to that of aliphatic side-chain assignment. The LUA script to calculate chemical shifts is GFT_aroHCCH_calc.

The assignment of other His spins is normally not pursued at this stage. The exchngeable HD1 and HE2 protons are not normally seen in NMR spectra. The HE1 spins can often be assigned during structure refinement based on preliminary structure. It is also possible to use HCN experiments (developed for RNA/DNA) or HMBC and long-lange HSQC experiments to assign HE1.

Important! Normally CD/HD and CE/HE spin pairs of Tyr and Phe are completely degenerate due to fast ring flippping. They should ultimately be labeled as CD1/HD and CE1/HE, respectively. Even though in this case CD and CE are valid labels for Phe and Tyr in CARA (as 13C atom groups aka. pseudoatoms), they are not compatible with CYANA 2.1. Also see the related issue with CD and CG spins of Leu and Val.


-- Main.AlexEletski - 20 Jul 2007

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