Carbon-Detected NMR for Mapping Binding Sites in Intrinsically Disordered Regions of a Protein

Ref. No. 391-HC


Until recently, it was widely believed that a protein must have a well-defined 3D-structure to support its function and traditional "lock and key" analogy was used to describe the protein-protein interactions. However, many proteins or regions of proteins are inherently flexible. Such anintrinsically disordered protein (IDP) or region of a protein (IDR) does not necessarily have to assume a unique structure to be biologically active. In fact, IDPs have important biological functions and are involved in numerous signal transduction pathways. However, the mechanism of IDR-mediated protein-protein interactions is poorly understood because of a lack of information on their structural preferences and dynamic properties at atomic resolution. Due to their inherent dynamics, IDRs are not amenable to structure determination by techniques such as x-ray crystallography and conventional nuclear magnetic resonance (NMR), which require a uniquely folded conformation. These regions are often either removed from expression constructs or proteolytically cleaved prior to generation of crystals, and their binding properties and regulatory roles remain poorly understood.Thus, there is a need for improved techniques for probing the highly flexible regions of proteins and their roles in mediating protein-protein interactions.

Summary of the Invention

Researchers at Fox Chase Cancer Center developed methods for identifying the amino acids involved in mediating interactions between an intrinsically disordered region of a polypeptide and a macromolecule. In brief, a 13C-detected 2D NMR spectrum of a uniformly 13C/15N-labeled sample yields unique, well resolved, 13CO-15N cross peaks for most residues of an IDP. By observing the spectral changes (generally a loss in cross peak intensity) upon addition of an unlabeled binding partner (e.g., a protein or nucleic acid), it is possible to identify individual residues of the IDP involved in binding. This CON footprinting technique enables the characterization of the sequence motifs in an IDP involved in recognizing binding partners with single-residue resolution.

Relevant Publication

Cheng H., Schwell V., Curtis B.R ., Fazlieva R., Roder H., & Campbell K.S. Conformational Changes in the Cytoplasmic Region of KIR3DL1 upon Interaction with SHP-2. Structure 2019; Apr 2;27(4):639-650.


  • In contrast to conventional biochemical and molecular biology methods, the CON footprinting technique enables identification of binding sites with single-residue resolution.
  • The quality of NMR spectra of IDRs/IDPs is often poor and uninterpretable due to spectral overlap and line broadening. Compared to conventional (proton-detected) NMR methods, NMR experiments involving direct detection of carbon (13C) signals are less sensitive, but result in improved spectral resolution due to the more favorable relaxation properties and larger spectral dispersion of 13C compared to protons.
  • Standard instrumentation for macromolecular NMR, including a cryogenic probe-head optimized for proton detection, is sufficient for obtaining high-quality spectra of a flexible macromolecule, such as an IDP.
  • The method allows precise definition of binding motifs on IDPs even for weakly interacting systems with dissociation constants in excess of 1 mM.


  • The technique introduced here promises to become an important tool for characterizing protein-protein interactions that involve intrinsically disordered protein regions, including studies of cell signaling, macromolecular interaction networks, and many other biological systems.
  • To map epitopes for antibodies recognizing IDRs or IDPs (with relatively low affinity) forbiotechnology and biomedical research.

Patent Status: Published Patent Application No.US20180080942A1

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Inna Khartchenko, MS, MBA
Director, Technology Transfer and New Ventures
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