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

Aromatic Side-chain Resonance Assignment Using the Aromatic HCCHCOSY Spectrum

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

• Phe, Tyr, His

1. 1. In UBNMR, run makeAroPeaks and use the most recent version of bbsc.prot to generate starting lists hcchcosyaroI1.peaks and hcchcosyaroI1.prot.
   2. In XEASY, use ns to load the three sub-spectra of aromatic (4,3)D HCCH; use ls, lp and lc to load, respectively, noe.seq, hcchcosyaroI1.peaks (with 1HD (QD) assigned in NOESY) and hcchcosyaroI1.prot; use se and gs to sort and display [w1(13C;1H);w3(1H)]-strips.
   3. 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.
   4. In UBNMR, run updatacosyGFT to calculate SQ shifts from updated AtomList.
   5. 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