A PULSED-FIELD GRADIENT ISOTOPE-FILTERED 3D C-13 HMQC-NOESY EXPERIMENT FOR EXTRACTING INTERMOLECULAR NOE CONTACTS IN MOLECULAR-COMPLEXES

A pulsed field gradient three-dimensional isotope-filtered C-13 HMQC-NOESY experiment has been developed to characterize intermolecular contacts in a 37 kDa macromolecular ternary complex consisting of uniformly C-13 labeled trp-repressor, its natural abundance co-repressor, L-tryptophan, and natural abundance operator DNA. The pulse scheme makes use of pulsed field gradients for the removal of artifacts and dephasing of unwanted magnetization during isotope filtering, and employs a strategy to minimize the time that magnetization resides in the transverse plane. The experiment provides solely intermolecular NOE contacts between protons of the labeled protein and protons of the unlabeled species, and has proven to be especially useful in eliminating ambiguities between intra- and intermolecular NOEs in the isotope-edited 3D C-13 HMQC-NOESY spectrum of the complex.