Protein NMR Spectroscopy Services: How it Works
NMR spectroscopy (BioNMR) has become an indispensable method in structural biology. It has a long history, with Nobel Prizes awarded to Felix Bloch and Edward Purcell, 1952, Richard Ernst, 1991 & Kurt Wüthrich, 2002). It plays an essential role in protein structure analysis and, generally, in structural biology.
At SARomics Biostructures, we offer custom peptide and protein NMR spectroscopy structure determination, peptide NMR & protein NMR assignment, protein-ligand & protein-protein complex structure determination, epitope mapping, and dissociation constant (Kd) determinations for compounds, higher order structure (HOS) analysis of biosimilars and comparison with originator antibody, fragment library screening using 1D or 2D NMR. We can also offer expression and purification of 2H, 15N, and 13C-labelled proteins in E. coli for NMR spectroscopy studies and structure determination.
Here we provide guidelines for 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.
At SARomics Biostructures, we offer custom peptide and protein NMR spectroscopy structure determination, peptide NMR & protein NMR assignment, protein-ligand & protein-protein complex structure determination, epitope mapping, and dissociation constant (Kd) determinations for compounds, higher order structure (HOS) analysis of biosimilars and comparison with originator antibody, fragment library screening using 1D or 2D NMR. We can also offer expression and purification of 2H, 15N, and 13C-labelled proteins in E. coli for NMR spectroscopy studies and structure determination.
Here we provide guidelines for 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.
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.
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.
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.
Links to related services
X-ray crystallography
Protein NMR spectroscopy
Antibody and antibody-antigen complex structures
Weak-affinity chromatography
Integrated drug discovery
in silico lead discovery