1. Assignment Strategies -N-C  -CO-N-C  -CO- H H-C-H H O-C-O HH H-C-H O-H -N-C  -CO-N-C  -CO- H H-C-H H O-C-O HH H-C-H O-H Homonuclear  Two steps needed  Scalar coupling within each amino acid  Dipolar coupling between amino acids Heteronuclear  Build up one atom at a time  Scalar coupling within and between each amino acid; some experiments provide intra- and inter-residue peaks

2. Pairs of Experiments Distinguishes Intra-residue from Inter-residue -N-C  -CO-N-C  -CO- H H-C-H H H HH H HNCA  H(t 1 ), N(t 2 ), CA(t 3 )  2 peaks- Intra + Inter  Mixing from N  C  occurs over 1 bond to same C  and over 2 bonds to adjacent C  -N-C  -CO-N-C  -CO- H H-C-H H H HH H HN(CO)CA  H(t 1 ), N(t 2 ), CA(t 3 )  1 peak- Inter only  Mixing from N  C  occurs ONLY over 2 bonds to adjacent C 

3. -N-C  -CO-N-C  -CO- H H-C-H H H HH H HNCACB  H(t 1 ), N(t 2 ), CA and CB(t 3 )  4 peaks- intra + inter  Mixing from N  C  occurs over 1 bond to same C  and over 2 bonds to adjacent C  -N-C  -CO-N-C  -CO- H H-C-H H H HH H HN(CO)CACB  H(t 1 ), N(t 2 ), CA and CB(t 3 )  2 peaks- inter only  Mixing from N  C  occurs ONLY over 2 bonds to adjacent C  Pairs of Experiments Distinguishes Intra-residue from Inter-residue

4. -N-C  -CO-N-C  -CO- H H-C-H H H HH H CBCANH  CB and CA(t 1 ), N(t 2 ), H(t 3 )  Mixing from N  C  occurs over 1 bond to same C  and over 2 bonds to adjacent C  -N-C  -CO-N-C  -CO- H H-C-H H H HH H HNCACB  H(t 1 ), N(t 2 ), CA and CB(t 3 )  Mixing from N  C  occurs over 1 bond to same C  and over 2 bonds to adjacent C  Equivalent Experiments Can be run in either direction