Protein & Peptide NMR Spectroscopy Services: Practical Aspect
The practical aspects of protein and peptide NMR spectroscopy are most important to observe when preparing the experiment. The services at SARomics Biostructures include custom peptide and protein NMR spectroscopy structure determination, peptide NMR and protein NMR assignment, protein-ligand and protein-protein complex structure determination, epitope mapping, compound library screening, and dissociation constant (Kd) determinations for compounds. We also provide higher-order structure (HOS) analysis and comparability assessment of biosimilars. Furthermore, we guarantee the expression and purification of 2H, 15N, and 13C-labeled proteins in E. coli for NMR spectroscopy studies and structure determination.
Here are some frequently asked questions about NMR experiments and their answers. Our guidelines for protein NMR spectroscopy experiments are tailored for standard NMR applications. For more comprehensive details about our services, we encourage you to visit our services page or inquire for expert advice on your project.
Protein NMR spectroscopy services
Fragment screening by NMR spectroscopy
Biosimilars comparability analysis by NMR
Protein sample concentration
In NMR spectroscopy, the signal is directly proportional to the concentration, meaning higher concentrations yield better data. Concentrations of 2-5 mM are typically achievable when working with peptide samples, but the limit for larger proteins is lower. However, it’s important to note that high concentrations may not be ideal for systems that form dimers or larger-size oligomers.
Labeling requirements
Proteins typically need to be labeled in NMR spectroscopy experiments. The amount of labeling required depends on the size and concentration of the peptide/protein. No labeling is strictly necessary for peptides and proteins with up to 40 residues since modern high-field spectrometers equipped with cryo-probes can record 15N and 13C natural abundance spectra. However, labeling is beneficial, especially if the concentration is low, and 15N-isotope labeling is usually enough.
For proteins with more than 40 residues, 15N and 13C labeling is essential due to spectral overlap in 2D spectra. This labeling allows for the acquisition of 3D-NMR spectra and reduces spectral overlap. 15N and 13C labeling is usually done by expressing the protein in E. coli using minimal media, which keeps the cost 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 necessary for successful assignment and structure determination.
Size in protein NMR spectroscopy
In NMR spectroscopy, high-resolution tertiary protein structure determination typically involves proteins with a molecular weight of less than 30 kDa. Larger proteins, up to 100 kDa in some cases, can be studied in projects focusing on identifying ligand binding sites or characterizing a specific protein construct.
How much protein do we need for the experiment?
When preparing for protein NMR spectroscopy, it is recommended to have a sample with a concentration of 0.5-1.0 mM in a 500 µl buffer solution. This corresponds to 5-10 mg of protein for a 20 kDa protein. It’s better to use higher concentrations for peptide samples, with 1-5 mM corresponding to 1.5-7.5 mg of peptide.
How stable should the protein be?
Protein structures must remain stable at room temperature for at least a week. This can generally be accomplished by employing standard protein purification methods and adjusting the buffer conditions as necessary. Our protein sample preparation guidelines for crystallography can also be used for NMR experiments.
When determining the structure of peptides, the peptides should remain stable in solution for approximately 2 to 4 days.
The presence of non-natural amino acids
NMR spectroscopy can also be used to determine the structure of peptides containing non-natural amino acids. However, peak assignment and parametrization of the amino acids are important considerations for structure determination.