N-15-EDITED 3-DIMENSIONAL NOESY-HMQC WITH WATER FLIPBACK - ENHANCEMENT OF WEAK LABILE H-1 RESONANCES OF PROTEIN SIDE-CHAINS CONTACTING DNA

Two pulse sequences are described that employ a modified water flipbac k technique to enhance the signal intensity of weak side chain resonan ces at the protein-DNA interface of the vnd/NK-2 homeodomain/DNA compl ex in an N-15-edited three-dimensional NOESY-HMQC spectrum. The pulse sequences presented employ water flipback pulses at the beginning of t he NOESY mixing time, optimizing the direct NOE transfer of magnetizat ion from the water to the protein by maximizing the z-component of the water magnetization, In one of the pulse sequences, radiation damping during the the indirect H-1 and N-15 evolution times is suppressed. A modified version of the WATERGATE water suppression technique is empl oyed during the HMQC portion of the experiment. The signal enhancement is demonstrated for the resonances of the side chain amide of Asn51, an invariant homeodomain residue whose contact with the DNA is critica l for binding. An ancillary advantage of the experiment is the ability to observe NOE transfer of magnetization from water. The information present in the water resonance plane of the three-dimensional spectrum is illustrated in a comparison with the corresponding HMQC spectrum o f the protein/DNA complex. (C) 1998 Academic Press.