NMR Spectroscopy & Protein Structure Determination

Instructor: Laura Foist

Laura has a Masters of Science in Food Science and Human Nutrition and has taught college Science.

Nuclear magnetic radiation (NMR) spectroscopy is a great method for determining the structures of compounds. It is particularly useful in determining the 3D structure of proteins.

Uses of NMR Spectroscopy

How do we know the 3D structure of the proteins that make up your hair and heart, or the eggs that you eat? The structure we see when we look at an image of the 3D structure of a protein has been determined using several methods. One of these methods uses nuclear magnetic resonance (NMR) spectroscopy.

NMR spectroscopy uses the energy levels from atoms that have been made magnetic, and measures these levels to determine the atoms that are linked together and close to each other. An NMR spectra, with peaks indicating interactions between atoms, is produced from this data. Using an NMR spectra, or the output from the NMR, the protein structure can be determined.

NMR spectroscopy is limited in that it can only determine the structure of small proteins, up to about 50 kDa. Since the average molecular weight of an amino acid is 110 daltons, this means that NMR works for proteins of up to about 450 amino acids. This means that NMR spectroscopy is useful for proteins like the insulin protein (around 51 amino acids), but the hemoglobin protein will be a little too large (around 600 amino acids).

How NMR Spectroscopy Works

In H-NMR, each hydrogen is magnetized by making them the H1 isotope. In C-NMR, each carbon is magnetized by making them the C13 isotope. The magnets in the NMR machine can then feel the vibrations on these isotopes. Based on what the hydrogen or carbon is attached to, it will vibrate at different frequencies.

These frequencies can be measured, and a spectra with the results is printed out.

NMR Spectra

Once the frequencies have been measured, we are given an output that looks like this:

NMR spectra

The top spectra shows the NMR for C13 and the bottom spectra shows the NMR for H1. The horizontal axis shows how much of a shift was seen, while the vertical axis shows the intensity. A short vertical line shows that there isn't much in that intensity range, while a tall one shows a high intensity (or a large amount).

Specific functional groups have been determined for each spectra line. Important functional groups for proteins include amides, which show up in the range of 5.5-8.5 on the H-NMR spectra. On the C-NMR chart, in general, carbon-carbon single bonds are found in the 0-50 ppm range, carbon-oxygen single bonds are found in the 50-100 ppm range, carbon-carbon double bonds are in the 100-150 range, carbon-oxygen double bonds are in the 150-200 range, and carbon-nitrogen bonds are in the 30-65 range.

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