Protein structure and interactions by combined use of sequential NMR assignments and isotope labeling

The 10 leucyl residues in the DNA-binding domain consisting of the 76 N-terminal residues of Salmonella phage P22 c2 repressor were labeled with15N. By use of a noveli5N(o>2) half-filter technique, simplified two-dimensional nuclear Overhauser enhancement spectra were obtained, which contain exclusively resonance peaks relating to at least one15N-bound hydrogen atom. Observation of nuclear Overhauser effects in these edited subspectra greatly facilitated sequential resonance assignments leading to sequence-specific assignments for all 10 leucines. Further potentialities of the combined use of residue-selective isotope labeling and sequential assignment procedures for studies of protein conformation in solution and for investigations of intermolecular interactions with proteins are discussed.