Assignment methods that use heteronuclear shift correlation for larger proteins (>10-15 kD), assignment methods based on 2D homonuclear 1 H- 1 H correlation methods (COSY/TOCSY/NOESY) don’t work very well because of overlapping resonances and broad linewidths. an alternative (which is now used even for small proteins in most cases) is to use heteronuclear shift correlation experiments on 13 C, 15 N labelled samples. in these experiments, magnetization is transferred from 1 H to 13 C and/or 15 N through large one-bond scalar couplings. Some relevant scalar coupling constants:

15 N- 1 H HSQC based techniques as we have seen, one of the simplest types of heteronuclear shift correlation is the HSQC experiment, which correlates 1 H chemical shift to the chemical shift of a 15 N or 13 C connected by a single bond heteronuclear shift correlation can be combined with homonuclear experiments such as 1 H- 1 H NOESY or TOCSY to yield 3-dimensional spectra

3D HSQC-NOESY and HSQC-TOCSY view of a 3D NOESY experiment these planes can be thought of as a 15 N- 1 H HSQC these planes can be thought of as a 1 H- 1 H NOESY the 15N shift dimension can resolve peaks that would overlap in a 2D NOESY

Triple-resonance experiments there is a whole raft of experiments that use both 13 C and 15 N correlations to 1 H nuclei the beauty of these experiments is that they can connect adjacent residues without requiring any nOe information--it’s all through-bond scalar coupling interactions. Makes sequence-specific assignment more reliable. they also use mostly one-bond couplings, which aren’t very sensitive to the protein conformation (unlike, say, three- bond couplings, which vary significantly with conformation, as we will see) limiting factors: 13 C is expensive and these exp’ts can be tricky