Directionality

When you have found two neighbours for a residue via the NH NOEs you do not initially know which one is the N-terminal neighbour and which the C-terminal neighbour. An easy trick is the fact that the Hα of residue i is usually much closer to the NH of residue i+1, than the NH of residue i-1. If you overlay your 15N-TOCSY-HSQC onto your 15N-NOESY-HSQC you can identify the Hα within the central strip (it will be the first peak above the diagonal in the 15N-TOCSY-HSQC spectrum, usually between about 4 and 6 ppm) and then you can see a direction-dependent pattern: the strip belonging to the C-terminal neighbour (i+1) should have a strong NOE to the Hα while the strip belonging to the N-terminal neighbour (i-1) will have no or only a very week NOE the Hα. This is particularly marked in β-sheet sections as you can see below.

Determining Directionality