Aromatic side chain assignment with Aro-HCCH-COSY in XEASY

Aromatic (4,3)D HCCH shows signals for aromatic H-C-C-H moeties. See aliphatic (4,3)D HCCH Analysis for comparison.

Before proceeding, it is helpful to look for the QD (Phe and Tyr), HD2 (His), and HD1 (Trp) resonances in the simultaneous [1H, 1H]-NOESY. Look for a consistent peak that is visible in the amide, alpha, and beta strips near 7ppm.

Phe, Tyr, and His

• Run the following macro in UBNMR to generate a peaklist and atom list:
  

 init
 read seq myprot.seq    #change filename to something appropriate
 read prot noe.prot     #change filename to your most current prot list

 add GFTatom COSY_C ATTACHED_H

 simulate 3D CG COSY_H 0 1
 simulate 3D CG COSY_C 0 1
 simulate 3D CG1 COSY_CH 0 1
 simulate 3D CG1 COSY_C 0 1
 simulate 3D CG2 COSY_CH 0 1
 simulate 3D CG2 COSY_C 0 1

 simulate 3D CD COSY_CH 0 2
 simulate 3D CD COSY_C 0 2
 simulate 3D CD1 COSY_CH 0 2
 simulate 3D CD1 COSY_C 0 2
 simulate 3D CD2 COSY_CH 0 2
 simulate 3D CD2 COSY_C 0 2

simulate 3D CZ2 COSY_CH 0 3
 simulate 3D CZ2 COSY_C 0 3
 simulate 3D CZ3 COSY_CH 0 3
 simulate 3D CZ3 COSY_C 0 3
 simulate 3D CE3 COSY_CH 0 4
 simulate 3D CE3 COSY_C 0 4
 simulate 3D CH2 COSY_CH 0 4
 simulate 3D CH2 COSY_C 0 4

 write prot hcchAroI1.prot    #the newly created starting prot list
 write peaks hcchAroI1.peaks  #the newly created starting peak list 

• In XEASY, use ns to load the three sub-spectra of aromatic (4,3)D HCCH; use ls, lp and lc to load the sequence, peak list, and chemical shift list, respectively and se, gs to sort and display [w1(13C;1H);w3(1H)]-strips. In XEASY, use pm, es, se and gs (or sf) to display [w1(13C;1H);w2(13C)]-planes, and sort and [w1(13C;1H);w2(13CD)]-planes; use mr to accurately adjust peak positions to assign 13CD chemical shifts; use pm and gs to re-display [w1(13C;1H); w3(1H)]-planes and [w1(13C;1H); w3(1H)]-strips; use mr to accurately adjust peak positions to confirm QD chemical shifts. The strips in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, ..., 13CD±QD and 13CD±QE. Use mr to accurately position peaks along w1; use ac, wp and wc to save updated PeakList and AtomList. In UBNMR, run updatacosyGFT to calculate SQ shifts from updated AtomList. Repeat 1.- 3. and 4. for QE strips and assign HE. Note that for His, assignment is often complicated by the presence of strong signals from HIS-Tags introduced to facilitate protein purification. The QE strip in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, 13CZ, ..., 13CD±QD, 13Main.CE±QE and possible 13CZ±1HZ (for Phe only). .See also: Assign Aromatic Side-chain Resonances using the NOESY and HCCH.

• TRP

1. 1. In XEASY, for residue Trp, repeat steps 1.-3. for strips in the order HH2 > HZ2 > HZ3 > HE3 (instead of the strip order QD > QE for Tyr and Phe). In HH2-strips, assign 13CH2±1HH2, and 13CZ2±1HZ2. In HZ2-strips assign 13CZ2± 1HH2, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3-strips, assign 13CZ3±1HZ3, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3 strip, assign 13CZ3±1HZ3, and 13CE3±1HE3. See also Assign Aromatic Side-chain Resonances using the NOESY and HCCH.
   2. In XEASY, use ac, wp and wc to save the final PeakList and AtomList

-- Main.GaohuaLiu - 01 Feb 2007