15N-edited three-dimensional NOESY-HMQC with water flipback: enhancement of weak labile 1H resonances of protein side chains contacting DNA.
Two pulse sequences are described that employ a modified water flipback technique to enhance the signal intensity of weak side chain resonances at the protein-DNA interface of the vnd/NK-2 homeodomain/DNA complex in an 15N-edited three-dimensional NOESY-HMQC spectrum. The pulse sequences presented employ water flipback pulses at the beginning of the NOESY mixing time, optimizing the direct NOE transfer of magnetization 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 1H and 15N evolution times is suppressed. A modified version of the WATERGATE water suppression technique is employed 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 critical 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 of the protein/DNA complex.
Gruschus
J M
JM
Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, 20892, USA.
Ferretti
J A
JA
D001665:Binding Sites; D004247:DNA / Q000032:analysis*; D018398:Homeodomain Proteins / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted; D009682:Magnetic Resonance Spectroscopy; D012680:Sensitivity and Specificity; D014867:Water
D018398:Homeodomain Proteins; D014867:Water; D004247:DNA