Protein & Peptide NMR Services
SARomics Biostructures is a leading NMR structural biology services provider, offering protein and peptide structure determination, protein-ligand and protein-protein complex structure analysis, and ligand binding site mapping, fragment library screening, and biosimilar higher-order structure comparability analysis.
Leading NMR Structural Biology Services
We provide comprehensive NMR structural biology services, including protein and peptide NMR structure determination, protein-ligand and protein-protein complex characterization, and biosimilar higher-order structure comparability analysis. Our offerings are leveraged by our company team’s extensive expertise and access to advanced high-field NMR spectroscopy instrumentation. Below is a detailed list of the custom protein NMR services packages offered by our company, each linked to a more detailed description of the experiments:
- Protein and peptide NMR structure services (see also our lists of off-the-shelf FastLane™ proteins).
- Peptide NMR & protein NMR assignment, secondary structure assessment.
- Protein-ligand & protein-protein complex structure determination services.
- Fragment-based library screening NMR services using 1D or 2D NMR spectroscopy.
- Binding site mapping and dissociation constant (Kd) determinations for compounds.
- Biosimilars higher order structure (HOS) comparability analysis services, formulation characterization, and optimization, both by NMR spectroscopy and protein X-ray crystallography. See also our blog post on this subject.
- Expression and purification of 2H, 15N, and 13C-labeled protein in E.coli for NMR spectroscopy studies and structure determination.
For details on our leading NMR service packages, please see below.
For practical aspects of NMR spectroscopy experiments, such as sample preparation, labeling requirements, the amount of protein required, its size and stability, and the presence of non-natural amino acids (sometimes seen, e.g., in macrocyclic peptides), please refer to the section on practical aspects of BioNMR. Please also view the article on the different strategies used in structure-based drug design. Feel free to reach out directly to discuss your project.
Protein NMR Services
Protein NMR is a powerful alternative to X-ray crystallography for determining and analyzing 3D protein structures, especially for peptides, where crystallization is often impossible. Unlike X-ray crystallography, protein NMR spectroscopy requires only that the peptide or protein be stable in solution to determine its structure. Typically, protein NMR can determine the structure of proteins up to 30 kDa in size. Our custom protein NMR services typically employ iterative restrained molecular dynamics simulations, utilizing structural restraints derived from 2D and 3D NMR spectroscopy experiments.
NMR Peptide and Protein Assignment and Secondary Structure Analysis
In peptide and protein NMR spectroscopy, the first step is to assign the peaks in the NMR spectrum to the corresponding atoms. This assignment is essential for determining the primary structure and assessing the secondary structure of peptides. It is also used to assign 2D 15N NMR spectra for epitope mapping, fragment screening, and Kd determinations. Additionally, the assignment helps to assess whether the structure of a peptide or protein can be determined.
Custom Protein-Ligand/Protein-Protein Complex NMR Structure Determination Services
The protein must be assigned to analyze the structure of protein complexes using protein NMR. If the structures of the individual components are unknown, it is still possible to determine the structure of the complex. Alternatively, available assignments and structures (from X-ray crystallography or protein NMR spectroscopy) can be combined with filtered NOESY experiments or transfer NOE experiments to analyze the complex. The protein should be labeled with 15N and 13C. Complexes with binders having affinities in the µM to mM range can be analyzed, making it suitable for peptide-protein and protein-protein complexes.
NMR Fragment Screening Services
SARomics Biostructures offers leading NMR fragment screening and hit validation services for hits identified using our proprietary weak-affinity chromatography (WAC™) platform. We can run 1D ligand-detection experiments or 2D spectra depending on the target protein. For 1D ligand detection, we can perform 1H or 19F experiments. We can identify potential binding sites by conducting competition experiments with a known binder. In 2D experiments, the protein must usually be labeled with 15N (or 2H for large proteins). However, recent advances in NMR technology enable us to conduct NMR fragment-based screening on non-isotope-labeled samples by examining the methyl region of the 13C NMR spectrum. If the assignment is available, we can readily identify the binding epitope of various fragments at atomic resolution.
Learn more about our fragment screening services on our Drug Discovery Services page.
Ligand Binding Site Mapping and Dissociation Constant Determinations
A ligand-binding site can be thoroughly analyzed using NMR spectroscopy via one-dimensional (1D) or two-dimensional (2D) experiments. The choice between the two depends on the availability of isotope-labeled protein and peak assignments. For 1D experiments, ligand-detection NMR experiments, combined with competition experiments using a known binder, can effectively identify the binding site of a compound. For 2D experiments, the binding site of a compound can be precisely identified at an atomic resolution if an assignment is available. However, even without an assignment, it is entirely feasible to identify the binding site if a known binder is available. Similarly, the dissociation constant (Kd) can be accurately determined using titration experiments detected by 1D or 2D NMR. No assignment is needed for Kd determinations in 2D experiments, although it is advantageous if atomic resolution is required.
NMR Higher Order Structure (HOS) Analysis of Biosimilars, Comparability Studies for Formulation Optimizations
NMR spectroscopy can be used to analyze the higher-order structure (HOS) of biosimilars and compare it with that of the original antibody. For this purpose, 2D 13C NMR spectra of the methyl region of the biomolecule are obtained and compared with those of the biosimilar and the original. No labeling is necessary, and this method can be applied to large molecules such as whole antibodies. Additionally, the biomolecules can be studied in the formulation buffer for formulation optimization and batch comparison. Depending on the biomolecule, approximately 1-2 mg of protein can be adequate for HOS analysis. You can also read our blog post on why NMR spectroscopy is the best method for studying the comparability of biosimilars.
Expression and Purification of 2H, 15N, and/or 13C-labelled Protein in E.coli
To determine the structures of peptides and proteins using BioNMR, the protein must be labeled with 15N and 13C to enable analysis of 2D NMR spectra at the atomic level. This can be achieved by expressing the protein in E. coli using established protocols, where 15N-labeling is not significantly more expensive than standard E. coli expression. 15N-labeling is generally adequate for conducting 2D NMR fragment screening and confirming the folding status of protein constructs. Depending on the specific project, additional isotope labeling may be necessary, particularly for larger proteins, which often produce clearer NMR spectra when labeled with 2H.
Case Study
Petruk G, Puthia P, Samsudin F, Petrlova J, Olm F, Mittendorfer M, Hyllén S, Edström D, Strömdahl A-C, Diehl C, Ekström S, Walse B, Kjellström S, Bond PJ, Lindstedt S & Schmidtchen A (2023). Nat Commun, 14, 6097. https://doi.org/10.1038/s41467-023-41702-y
PDB entries: 8BWW – Targeting Toll-like receptor-driven systemic inflammation by engineering an innate structural fold into drugs
Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Using a combination of structure- and in silico-based design, nuclear magnetic resonance (NMR) spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, the paper presents the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18.
Discover more examples of projects we’ve contributed to by visiting our publications page.
Additional technical details, information, and case studies on our protein & peptide NMR services are available in the presentation.