NMR Spectroscopy Services: How it Works

The following points are guidelines for the NMR spectroscopy experiments. Many applications of NMR work around these conditions. For details of the services, please visit the services page, or enquire for advice on your project.

Protein concentration in sample

In NMR spectroscopy, concentration is proportional to the signal, so the higher the concentration, the better the data acquired. For a peptide sample, 2-5 mM concentrations are usually possible, while the limit is lower for larger proteins. However, a high concentration is not always optimal for systems that form dimers or larger-size oligomers.

Requirements for labeling

Typically for NMR spectroscopy experiments, the protein needs to be labeled. Although, the amount of labeling required depends on the peptide/protein size and concentration. No labeling is strictly necessary for peptides and proteins consisting of up to 40 residues since 15N, and 13C natural abundance spectra can be recorded with modern high-field spectrometers equipped with cryo-probes. However, labeling is beneficial, especially if the concentration is low, and 15N-isotope labeling is usually sufficient.
For proteins above 40 residues, 15N and 13C-labelling is essential, mainly due to spectral overlap in 2D-spectra, where 15N and 13C-labelling allows for the acquisition of 3D-NMR spectra and reduces spectral overlap. 15N and 13C-labelling is, in most cases, quickly done by expression in E. coli using minimal media, where especially the cost of the 15N-isotope labeling is low. For proteins ranging from 20 kDa to 30 kDa and higher, 2H isotope labeling will increase the signal-to-noise ratio and is often required for successful assignment and structure determination.

How large the protein can be for NMR spectroscopy?

For high-resolution tertiary protein structure determination by NMR spectroscopy, the molecular weight of the protein is typically less than 30 kDa. Larger proteins (up to 100 kDa in favorable cases) can be studied in projects aiming to identify ligand binding sites or, e.g., characterize a construct.

Required total amount of protein for the experiment

As a rule of thumb, the typical sample for protein NMR spectroscopy should have a concentration of 0.5-1.0 mM in 500 µl buffer solution, corresponding to 5-10 mg protein for a 20 kDa protein.
Peptide samples for NMR spectroscopy are preferably run at higher concentrations, where 1-5 mM correspond to 1.5-7.5 mg peptide.

Requirements for stability

For structure determinations, the proteins must be stable at room temperature for at least a week. Usually, this is achievable using standard protein purification methods followed by optimization of buffer conditions.
For structure determination of peptides, the peptide needs to be stable in solution for around 2 to 4 days.

The presence of non-natural amino acids

Structure determination of peptides with non-natural amino acids can also be performed with NMR spectroscopy. However, some considerations must be made about peak assignment and parametrization of the amino acids for structure determination.

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