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					NMR Scalar and Dipolar Coupling

Through                                         Through
 Space                                           Bonds

   Coupling of nuclei gives information on structure
         Resonance Assignment


                          OH         CH2 CH3

          Which signal from which H atoms?

The key attribute: use the scalar and dipolar couplings to
  match the set of signals with the molecular structure
Proteins Have Many Signals
    1H NMR Spectrum of Ubiquitin
    ~500 resonances

 A large number of signals are overlapped
       Protein NMR:
   Overlapped Sub-Spectra
  Each residue in the sequence gives rise to an
independent NMR sub-spectrum, which is much
  simpler than the complete protein spectrum

                                  Methods have been
                                  developed to extract
                                  each sub-spectrum
                                  from the whole
  Simplifies Strategy to Assign
    Resonances in a Protein
1. Identify resonances for each amino
   acid         T G L S       R G

2. Put amino acids in order (2 steps)
   - Find next neighbors (R-G-S,T-L-G-S)
   - Sequence-specific assignment
            1   2   3   4   5   6   7
Homonuclear 1H Assignment Strategy
• Scalar coupling to identify resonances, dipolar
  couplings to place in sequence
• Based on backbone NH
• Concept: build out from the backbone to
  identify the side chain resonances
• 2nd dimension resolves overlaps, 3D rare
                 1H    1H     1H
COSY: One J coupling
      H   H
  A   N—C

      H   H
  B   N—C

      H   H
  C   N—C
R-COSY: Add A 2nd J Coupling
               H   H H
           A   N—C—C

               H   H H
           B   N—C—C

               H   H
           C   N—C—CH3
DR-COSY: Add A 3rd J Coupling
                      H   H H
                  A   N—C—C
                      H   H H
                  B   N—C—C
                      H   H
                  C   N—C—CH3
TOCSY: All Coupled Spins
           H   H H   H
       A   N—C—C—C—COOH
                   H H
           H   H H   H   H   H
       B   N—C—C—C—C—C—NH3
                   H H   H   H
           H   H
       C   N—C—CH3
Heteronuclear (1H,13C,15N) Strategy
• One bond at a time - all atoms (except O)
• Even handles backbone 15N1H overlaps
     disperse with backbone
• Works on bigger proteins because one
  bond scalar couplings are larger

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