Heteronuclear single quantum coherence spectroscopy

After a time delay (t1), the magnetization is transferred back to the proton via a retro-INEPT step and the signal is then recorded.

Each residue of the protein, with the exception of proline, has an amide proton attached to a nitrogen in the peptide bond.

Normally the N-terminal residue (which has an NH3+ group attached) is not readily observable due to exchange with solvent.

The sidechain amine peaks from tryptophan are usually shifted downfield and appear near the bottom left corner.

If there is a large cluster of severely overlapped peaks around the middle of the spectrum, that would indicate the presence of significant unstructured elements in the protein.

The assignment of the spectrum is essential for a meaningful interpretation of more advanced NMR experiments such as structure determination and relaxation analysis.

The time-consuming process of structure determination is usually not undertaken until a good HSQC spectrum can be obtained.

The constant time (CT) version of 1H—13C HSQC is normally used as it circumvents the issue of splitting of signal due to homonuclear 13C—13C J couplings which reduces spectral resolution.

1 H– 15 N HSQC polarization scheme for a protein/amino acid.
1 H– 15 N HSQC spectrum of a fragment of an isotopically labeled protein NleG3-2. Each peak in the spectrum represents a bonded N-H pair, with its two coordinates corresponding to the chemical shifts of each of the H and N atoms. [ 2 ]