Signal enhancement using 45 degrees water flipback for 3D N-15-edited ROESY and NOESY HMQC and HSQC

A novel implementation of the water flipback technique employing a 45 degre es flip-angle water-selective pulse is presented. The use of this water fli pback technique is shown to significantly enhance signal in 3D N-15-edited ROESY in a 20 kDa complex of the vnd/NK-2 homeodomain bound to DNA, The enh ancement is seen relative to the same experiment using weak water presatura tion during the recovery delay. This enhancement is observed for the signal s from both labile and nonlabile protons. ROESY and NOESY pulse sequences w ith 45 degrees water flipback are presented using both HMQC and HSQC for th e N-15 dimension. The 45 degrees flipback pulse is followed by a gradient, a water selective 180 degrees pulse, and another gradient to remove quadrat ure images and crosspeak phase distortion near the water frequency. Radiati on damping of the water magnetization during the t(1) and t(2) evolution pe riods is suppressed using gradients, Water resonance planes from NOESY-HMQC and NOESY-HSQC spectra show that the HMQC version of the pulse sequences c an provide stronger signal for very fast exchanging protons, The HSQC versi ons of the ROESY and NOESY pulse sequences are designed for the quantitativ e determination of protein-water crossrelaxation rates, with no water-selec tive pulses during the mixing time and with phase cycling and other measure s for reducing axial artifacts in the water signal.