Neighbouring Residues

In your 15N-NOESY-HSQC spectrum you should be able to see NOEs between neighbouring NH groups. There are several ways of identifying neighbouring residues. To begin with, select a peak in your HSQC with which you want to start and navigate to that position in the 15N-NOESY-HSQC (right-click the mouse and go to Navigate and windowX where windowX is the window in which you have your 15N-NOESY-HSQC).

Assignment with a 15N-NOESY-HSQC spectrum

With any luck there should be two or more NOEs in this strip to other NH groups. Two should be to the neighbouring residues (i.e. NOEs to NH(i±1)) and additional ones will be medium or long-range NOEs. In α-helical secondary structure you may well see NOEs to NH(i±2) and NH(i±3). These will be weaker and can be helpful in linking up a several sequential residues. In β-sheet secondary structure you will see long-range NOEs to the neighbouring strand which may be of comparable strength to the NH(i±1) NOEs (parallel β-sheet) or considerably stronger than the NH(i±1) NOEs (antiparallel β-sheet).

In principle you want to find the correct spin system that each NOE originates from. In some cases it may be possible to identify the spin system unambiguously because the hydrogen chemical shift is unique to one spin system. However, in many cases there will be several options. One way to know that you have found the correct spin system, is by looking for the symmetrical NOE peak. If spin system i has an NOE to spin system j, then j should also have one to i. When you place the strips alongside one another this makes a nice rectangular pattern.

Identifying neighbouring residues with a 15N-NOESY-HSQC spectrum

Note that in the region between about 6.5 and 7.5 ppm you can not only observe NOEs from NH groups, but also from certain side chains such as Phe, Tyr, Trp, Asn or Gln. If your protein contains Trp residues, you may also be able to observe NOEs to the Trp side-chain Hε1 nuclei between about 9.0 and 11.0 ppm. The Asn, Gln and Trp Hε1 resonances are also visible in your HSQC, however the Phe, Tyr and other Trp resonances are not.

It can be difficult working out which NOEs are from the neighbouring residues and which are from others. And of course when you have found out which are the neighbouring residues, you then still need to determine which is on the N-terminal side and which on the C-terminal side of your central residue. You can do this as outlined here using some of the side-chain NOEs.
On the whole the NOEs to neighbours will be the stronger ones, and they should be of comparable strength to one another. The main exception to this, is the cross-strand NOE in an antiparallel β-sheet which is stronger than the N(i±1) NOEs. NOE intensity scales with the distance between the hydrogen atoms (as r-6). Below are some typical distances found in seconary structure (taken from Wüthrich (1986) NMR of Proteins and Nucleic Acids, WileyBlackwell).

i, i ± 1 i, i ± 2 i, j
α-helix 2.8 Å 4.2 Å
β-sheet (parallel) 4.3 Å 4.8 Å
β-sheet (antiparallel) 4.2 Å 3.3 Å

You have several options of how to identify which are your neighbouring strips. Probably the easist option initially is to use the Match Peaks function. An alternative would be to select the assignment panel for each NOE peak (hold the mouse over the peak and press a) and then make a note of each of the spin systems that is an option for that NOE. Then you can line their strips up using the procedure outlined here and look at the matches.