A pulsed field gradient isotope‐filtered 3D 13C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes

Abstract

A pulsed field gradient three‐dimensional isotope‐filtered ¹³C HMQC‐NOESY experiment has been developed to characterize intermolecular contacts in a 37 kDa macromolecular ternary complex consisting of uniformly ¹³C 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 ¹³C HMQC‐NOESY spectrum of the complex.