Protein NMR Spectroscopy: Practical Aspects & Frequently Asked Questions
The practical aspects of protein and peptide NMR spectroscopy are crucial to consider when preparing the experiment. SARomics Biostructures’ leading NMR structural biology services include custom peptide and protein NMR assignment and analysis, protein-ligand and protein-protein complex structure elucidation, epitope mapping, compound library screening, and dissociation constant (Kd) determinations. We also provide higher-order structure (HOS) analysis and comparability assessment of biosimilars. Furthermore, we offer expression and purification of 2H, 15N, and 13C-labeled proteins in E. coli for NMR spectroscopy studies.
Here we discuss some frequently asked questions about NMR experiments, along with 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 NMR services page or contact us to inquire and get expert advice on your project.
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 oligomers.
Labeling requirements
Proteins typically require labeling in NMR spectroscopy experiments. The amount of labeling required depends on the peptide or protein’s size and concentration. Labeling is not 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 when the concentration is low, and 15N-isotope labeling is typically sufficient.
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 typically performed 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 molecular weights below 30 kDa. Larger proteins, up to 100 kDa in some cases, can be studied in projects focused on identifying ligand-binding sites or characterizing a specific protein construct.
How much protein is needed for the experiment?
When preparing for protein NMR spectroscopy, it is recommended to use a sample at 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. Stabilization can generally be accomplished by using standard protein purification methods and adjusting buffer conditions as needed. Our general protein sample preparation and shipping guidelines for crystallography can also be helpful for NMR experiments.
When determining peptide structure, 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 (e.g., those found in macrocyclic peptides). However, peak assignment and amino acid parametrization are essential considerations for structure determination.