The Evaluation of Isotope Editing and Filtering for Protein—Ligand Interaction Elucidation by Nmr

A series of experiments that aid in the structural characterization of protein—ligand complexes by NMR have been assessed. Methods have been established to identify intermolecular NOEs between labeled proteins and unlabeled peptides and/or drug ligands, while omitting signal from intramolecular NOEs within both labeled and unlabeled constituents. The protein—peptide complex chosen to illustrate the value of such techniques is the C-terminal domain of the cardiac muscle regulatory protein Troponin C (cCTnC_91–161) bound to the N-terminal domain of the inhibitory protein Troponin I (cTnI_35–72). The measurement of intermolecular NOE contacts between cCTnC and cTnI_35–72 was accomplished by the ¹³C-edited/filtered NOESY-HSQC [19] and ¹³C-edited/filtered HMQC-NOESY [9] experiments. The assignment of the bound peptide was facilitated by the ¹³C, ¹⁵N-filtered TOCSY, and ¹³C, ¹⁵N-filtered NOESY experiments [4, 6, 15].