![]() Each peak represents the 15N- 1H of a unique amino acid along the backbone of the amino acid. In this diagram, each peak corresponds to a cross peak, showing coupling between sets of 1H and 15N nuclei. This is a strong advantage of heteronuclear NMR. Notice the greater clarity of spectra of the HSQC experiment. The next cross peak at (1.6, 20.8) is a methylene group, The final cross peak is (0.9, 10.2) is a methyl group.Īnother example HSCQ spectrum from ubiquitin is shown below. The cross peak at (2.0, 21.8) is the methyl group that has no coupling neighboring hydrogens. In propyl acetate, oxygen is the most electron withdrawing group, so the piece we have at this cross peak is -CH 2-O. If you look at a 13C data table, then you can see where different carbon groups show up. The cross peak at (3.9, 66) is a methylene attached to a carbon that is attached to an electron-withdrawing group. The peak at 77 ppm is residual solvent, in this case CDCl 3. This means that this carbon does not have any hydrogens attached, therefore, this is our carbonyl carbon. The peak at 171 ppm in the 13C has no cross peak. Looking along the x axis, we the 1H NMR spectrum and along the y axis we see the 13C NMR spectrum displayed. The purpose of a HSQC is to determine which protons are coupled to what other specific carbon or heteroatom in the molecule through bonds.Īn HSQC experiment spreads things out into two dimensions just like the homonuclear experiments did in the previous section. Cross-peaks show which proton is attached to which carbon or heteroatom. In an HSQC spectrum, a 13C or heteroatom spectrum is displayed on one axis and a 1H spectrum is displayed on the other axis. The signal from the 1H nuclei is recorded. The pulse sequence for a typical HSQC experiment is detailed below involving 1H and 15N nuclei. This polaraization is then transferred back to the 1H nuclei. In an HSQC experiment, polarization is transferred from a 1H nuclei to a neighboring heteroatom ( 13C or 15N). While the DEPT may give the same information for 13C to 1H correlations, HSQC is more sensitive, therefore, it may be more adventageous to use this type of experiment in complex situations. It is also known as heteronuclear multiple quantum coherence (HMQC). Hetereonuclear Single Quantum Coherence (HSQC) is used to determine the proton to carbon or heteroatom (often nitrogen) single bond correlations. By comparing the HSQC of the free protein with the one bound to the ligand, it is possible to find the changes in the chemical shifts of the peaks, which is most likely to occur in the binding interface.\) The HSQC experiment is also useful for detecting interactions with ligands, such as other proteins or drugs. The assignment of the spectrum is usually the first step in a structure determination, and is essential for a meaningful interpretation of more advanced NMR experiments. ![]() This process can be done in different ways as outlined in the protein NMR article. It is not possible to assign the HSQC spectrum by itself, or in other words to determine which peaks correspond to a particular residue in the protein. The labour-intensive process of structure determination is usually not undertaken until a good HSQC is obtained. It will probably be difficult to solve the structure of the protein if this is not the case. The number of peaks in the spectrum should match the number of residues in the protein (though sidechains with nitrogen-bound protons will add additional peaks). Being a relatively cheap and quick experiment, the HSQC is useful to screen candidates for structure determination by NMR. If the protein is folded, the peaks are usually well dispersed, and most of the individual peaks can be distinguished. The latter can be recorded on much lower concentrations of protein, but requires recombinant expression of the protein.Įach residue of the protein (except proline) has an amide proton attached to a nitrogen in the peptide bond. The HSQC experiment can be performed either using the natural abundance of the 15N isotope, or using isotopically labeled protein. The 15N HSQC experiment is probably the most frequently recorded experiment in protein NMR. Correct Test Weight Handling Guide: 12 Practical Tips HSQC in protein NMR ![]()
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