Carbon-detected NMR for mapping binding sites in intrinsically disordered regions of a protein
Carbon-detected NMR is well-suited for mapping binding sites in intrinsically disordered regions of a polypeptides, and for mapping of binding motifs in intrinsically disordered regions with single-residue resolution. Provided are methods of carbon-detected NMR for determining the amino acids that mediate the interaction between an intrinsically disordered polypeptide or protein, or an intrinsically disordered region of a polypeptide, and a biomolecule such as another polypeptide or a nucleic acid.
1. A method for determining the amino acids mediating an interaction between an intrinsically disordered region of a first polypeptide and a biomolecule, comprising:
labeling the intrinsically disordered region of the first polypeptide with C and N, thereby producing a labeled first polypeptide;
interacting the labeled first polypeptide with the biomolecule;
recording a nuclear magnetic resonance (NMR) spectrum of the labeled first polypeptide interacted with the biomolecule;
detecting the decrease in intensity of, a loss of 13 CO— 15 N peaks, or both the decrease in intensity and the loss of 13 CO— 15 N peaks in the recorded NMR spectrum relative to a reference NMR spectrum of the first polypeptide, and
from the loss of intensity and/or the loss of 13 CO— 15 N peaks, the amino acids on the first polypeptide that participate in the interaction with the biomolecule are thereby detected and a binding footprint is established for the intrinsically disordered region of the first polypeptide and the biomolecule.
2. The method according to claim 1 , wherein the first polypeptide is a protein of about 200 amino acids or less in length.
3. The method according to claim 1 , wherein the first polypeptide is an intrinsically disordered protein of about 200 amino acids or less in length.
4. The method according to claim 1 , wherein the first polypeptide is phosphorylated.
5. The method according to claim 1 , wherein the first polypeptide is acetylated.
6. The method according to claim 1 , wherein the first polypeptide is methylated.
7. The method according to claim 1 , wherein the first polypeptide comprises a post-translational modification.
8. The method according to claim 1 , wherein the first polypeptide has a solubility in an aqueous medium of at least 0.5 mM.
9. The method according to claim 1 , wherein the biomolecule comprises a second polypeptide that is not intrinsically disordered.
10. The method according to claim 9 , wherein the second polypeptide is a protein.
11. The method according to claim 1 , wherein the second polypeptide comprises an antibody.
12. The method according to claim 1 , wherein the biomolecule comprises a nucleic acid.
13. The method according to claim 12 , wherein the nucleic acid comprises DNA.
14. The method according to claim 12 , wherein the biomolecule comprises a protein-nucleic acid complex.
15. The method according to claim 1 , wherein the step of recording a nuclear magnetic resonance (NMR) spectrum of the labeled first polypeptide interacted with the second polypeptide is carried out using an NMR spectrometer equipped with a cryogenic probe having a high level of carbon sensitivity.