Double Resonance Backbone Assignment

Assigning proteins which are only 15N-labelled is harder than assigning 15N,13C-labelled proteins. But for proteins of up to about 130 residues in length this is not an unreasonable task, especially, if the secondary structure and topology are known from a crystal structure or a homologous protein. The general principle of assignment is outlined in the theory section. In addition to your HSQC spectrum you will need a 3D 15N-NOESY-HSQC and a 3D 15N-TOCSY-HSQC. There is no set way of doing double resonance backbone assignment in Analysis, but several useful moves and strategies are outlined below. It is assumed that you have already initialised your HSQC and peakpicked your 3D spectra.

The overall workflow is as follows:
(a) Find the sequential neighbours to each residue using the NOEs between their NH groups. You can do this relatively easily using the Match Peaks function.
(b) Determine the correct sequential order, or directionality, of the neighbouring residues using NOEs between the NH group of one residue and the side-chain of the other.
(c) Once you have strung several residues together sequentially, identify (or exclude) the amino acid type of some of the residues and match this to your sequence in order to obtain the sequence-specific assignment.

If you know the secondary structure and topology of your protein, you can use this to help you. α-helices and β-sheets have slightly different patterns of NOEs which can help with the assignment – residues with α-helical or β-sheet NOE patterns will necessarily be grouped together in the sequence. If you have a β-sheet, then the cross-strand NOEs can help you assign and build up two neighbouring strands in parallel.