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 the above steps for the QEstrips and assign QE. Note that for His, the assignment is often complicated by the presence of strong signals from the His-tag introduced to facilitate protein purification. The QE strip in the basic spectra is expected to exhibit peaks at 13CD, 13Main.CE, 13CZ, ..., 13CD±QD, 13Main.CE±QE and possibly 13CZ±1HZ (for Phe only).

TRP

• In XEASY, 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.