SARomics Biostructures offers a broad range of custom protein NMR spectroscopy services for protein three-dimensional structure determination and compound library screening. Among our services are the following:
The first step in studying a peptide or protein using NMR consists of assigning the available peaks to get an atomic resolution of the NMR spectra. This can be used for various purposes such as primary structure determination and secondary structure estimations for peptides, assignment of 2D 15N NMR spectra for epitope mapping, fragment screening and Kd determinations. The assignment also provides information on whether it is possible to determine the structure of a peptide or protein. Experimental considerations for these types of experiments are detailed in our technology section.
Peptide/protein NMR is an alternative to protein X-ray crystallography for the determination of a protein structure. Especially for peptides, where crystallization often is very hard. In contrast to X-ray crystallography, NMR only requires the peptide/protein to be stable in solution in order to determine the structure. Generally, only proteins up to 30 kDa in size can be determined using NMR. Structure determinations are typically done using iterative restrained molecular dynamic simulations using structural restraints generated from 2D and 3D NMR experiments. Experimental considerations for these types of experiments are detailed in our technology section.
Protein complexes can be determined using NMR if assignment of the protein is available. The complex can be determined from scratch if the protein structure is unknown, otherwise the complex can be determined using available assignments and structures (X-ray or NMR) in combination with filtered NOESY experiments or transfer NOE experiments. It is required that the protein can be 15N and 13C-isotope-labelled. In contrast to X-ray crystallography, even complexes where the binders have affinities in the µM-mM regime can be determined, which can be extra suitable for peptide-protein and protein-protein complexes.
We regularly run NMR screening and hit validation of drug-like compounds and fragments, either in combination with fragment screens using our proprietary weak-affinity chromatography (WAC™) platform or as a stand alone screening method. Depending on the target protein, we have the possibility to either run 1D ligand-detected experiments or 2D spectra.
For 1D ligand-detected experiments, we have the possibility to either run 1H or 19F ligand-detected experiments. Competition experiments with a known binder allows for identification of binding sites. For 2D experiments, generally the protein needs to be labeled with 15N (for large protein 2H-labelling as well), although recent advances in NMR technology makes it possible to run fragment screening on non-isotope labelled samples by looking at the 13C NMR spectra for the methyl region. If assignment is available, the binding epitope of various fragments can be readily identified with atomic resolution.
The binding epitope of a compound can be readily identified using NMR using 1D or 2D experiments, where the choice of experiment is dependent on whether isotope-labelled protein and peak assignments are available. For 1D experiments, ligand-detected NMR experiments in combination with competition using a known binder can identify the binding site of a compound. For 2D experiments, the binding site of a compound can be identified with atomic resolution if assignment is available. However, even without assignment, it is possible to identify the binding site if a known binder is available. The dissociation constant (Kd) can in a similar way be determined using titration experiments using 1D or 2D experiments. For 2D experiments, no assignment is needed for Kd determinations, however it is beneficial if one requires atomic resolution.
NMR spectroscopy can be used for higher order structure (HOS) studies of biosimilars and for comparison of a biosimilar's structure to its originator antibody. For this purpose, 2D 13C NMR spectra of the methyl region of the biomolecule are acquired, where the biosimilar and originator spectra can be compared. No labelling is required and molecules as large as full antibodies can be studied using this approach. In addition, for formulation optimization and batch comparison, the biomolecule can be studied in the actual formulation buffer. Depending on the biomolecule, around 1-2 mg protein can be sufficient to perform HOS studies.
For NMR studies, generally the protein needs to be labelled with 15N and/or 13C in order to assign the 2D NMR spectra with atomic resolution. This is readily done by expression in E. coli using inhouse protocols, where especially 15N-isotope labelling is no more expensive than a standard E. coli expression. In addition, only 15N-isotope labelling is usually sufficient for running 2D NMR fragment screening projects and can as well be used for verification of the folding state of protein constructs. Depending on the project, additional isotope-labelling might be required, especially for larger proteins where deuteration with 2H often gives better NMR spectra.
We have access to state-of-the-art NMR spectrometers. For practical considerations and for the details of the NMR experiments please see our technology page.
Karczewski J, Krasucki S P, Asare-Okai P N, Diehl C, Friedman A, Brown C M, Maezato Y, and Streatfield S J. (2020).
Isolation, Characterization and Structure Elucidation of a Novel Lantibiotic From Paenibacillus sp.
Front. Microbiol. 24. https://doi.org/10.3389/fmicb.2020.598789
Sanchez-Fernandez A, Diehl C, Houston J E, Leung A E, Tellam J P, Rogers S E, Prevost S, Ulvenlund S, Sjögren H, and Wahlgren M. (2020).
An integrative toolbox to unlock the structure and dynamics of protein–surfactant complexes.
Nanoscale Adv 2, 4011-4023. https://doi.org/10.1039/D0NA00194E