Protein & Peptide BioNMR Structure Services
SARomics Biostructures is a leading CRO provider of custom protein NMR structural biology services (BioNMR), offering protein and peptide structure determination, ligand-binding analysis, fragment-based screening, and biosimilar HOS comparability analysis.
Leading Structural Biology NMR Services
We provide comprehensive contract NMR structural biology outsourcing services as part of our integrated structural biology platform. Our offerings are leveraged by our company team’s extensive expertise and access to advanced high-field NMR spectroscopy instrumentation. Below is a list of the custom protein NMR service packages provided by our company, each linked to a more detailed description of the experiments:
- Contract protein and peptide NMR structure determination 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.
- Epitope 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 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 Structure Determination Services
Protein and peptide BioNMR is a powerful alternative to X-ray crystallography for determining and analyzing three-dimensional protein structures, especially for peptides, where crystallization is often impossible. Unlike X-ray crystallography, protein NMR spectroscopy analysis only requires the peptide or protein to 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 structure determination 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 determine whether it is possible to determine the structure of a peptide or protein.
Custom Protein-Ligand/Protein-Protein Complex NMR Structure Determination Services
The protein must be assigned to analyze the structure of protein complexes using BioNMR. 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. Unlike X-ray crystallography, even complexes with binders having affinities in the µM to mM range can be analyzed, making it suitable for peptide-protein and protein-protein complexes.
Fragment-Based NMR Screening Services
Our NMR services include custom NMR fragment-based screening for library screening and to validate hits identified at our proprietary weak-affinity chromatography (WAC™) fragment screening 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.
Epitope Mapping and Dissociation Constant Determinations for Compounds
The binding epitope of a compound can be thoroughly analyzed using BioNMR through 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.
Higher Order Structure (HOS) Comparability Analysis Services of Biosimilars, Comparability Studies for Formulation Optimizations
NMR spectroscopy can be utilized to analyze the higher-order structure (HOS) of biosimilars and to compare their structure 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 analyze 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 can be found in the presentation.