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 C -13,N-15-labeled protein. The method uses an adiabatic C-13 inversion pulse optimized to an empirically observed relationship between (1)J(C)H and car bon chemical shift to selectively invert the protein protons (attached to C -13). Two NOESY data sets are recorded where the intermolecular and intramo lecular NOESY cross peaks have either equal or opposite signs, respectively . Addition and subtraction yield two NOESY spectra which contain either NOE s within the labeled protein (or unlabeled ligand) or along the binding int erface. The method is demonstrated with an application to the B-12-binding subunit of Glutamate Mutase from Clostridium tetanomorphum complexed with t he B-12-nucleotide loop moiety of the natural cofactor adenosylcobalamin (C oenzyme B-12).