Simultaneous measurement of intra- and intermolecular NOEs in differentially labeled protein-ligand complexes*

A new NOE strategy is presented that allows the simultaneous observation of intermolecular and intramolecular NOEs between an unlabeled ligand and a ¹³C,¹⁵N-labeled protein. The method uses an adiabatic ¹³C inversion pulse optimized to an empirically observed relationship between ¹ J _CH and carbon chemical shift to selectively invert the protein protons (attached to ¹³C). Two NOESY data sets are recorded where the intermolecular and intramolecular NOESY cross peaks have either equal or opposite signs, respectively. Addition and subtraction yield two NOESY spectra which contain either NOEs within the labeled protein (or unlabeled ligand) or along the binding interface. The method is demonstrated with an application to the B₁₂-binding subunit of Glutamate Mutase from Clostridium tetanomorphum complexed with the B₁₂-nucleotide loop moiety of the natural cofactor adenosylcobalamin (Coenzyme B₁₂).