Upon binding of a ligand to a protein, the chemical shifts of signals for both protein and ligand will be affected. This effect, in the protein, is greatest for the residues located at the binding site and as protons are the most sensitive nuclei in NMR experiments, it is sometimes possible to detect binding of a ligand to a protein by observation of changes in the chemical shift of well-resolved protein protons. Unfortunately, it is usually extremely difficult to unambiguously assign the chemical shift changes of protein proton signals due to severe overlap, making it impossible to determine the amino acid residues involved in binding.
One solution to this problem is to label the target protein with the stable isotopes 15N and/or 13C. Binding of a ligand changes the resonance signals at the binding site, and these changes can be measured using 15N/1H and/or 13C/1H correlation spectra. The 15N/1H or 13C/1H heteronuclear single quantum correlation (HSQC) spectrum of the labelled protein is acquired as a reference. Upon addition of a binding ligand followed by acquisition of a second HSQC spectrum, changes will be observed between the two spectra, particularly at the protein residues at the binding site. To simply detect binding, it is only necessary to observe changes between the two spectra. However, to determine the residues involved in binding it is necessary to assign the 15N or 13C HSQC signals to the amino acids in the protein.
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