Signal enhancement using 45 degrees water flipback for 3D 15N-edited ROESY and NOESY HMQC and HSQC.
A novel implementation of the water flipback technique employing a 45 degrees flip-angle water-selective pulse is presented. The use of this water flipback technique is shown to significantly enhance signal in 3D 15N-edited ROESY in a 20 kDa complex of the vnd/NK-2 homeodomain bound to DNA. The enhancement is seen relative to the same experiment using weak water presaturation during the recovery delay. This enhancement is observed for the signals from both labile and nonlabile protons. ROESY and NOESY pulse sequences with 45 degrees water flipback are presented using both HMQC and HSQC for the 15N dimension. The 45 degrees flipback pulse is followed by a gradient, a water selective 180 degrees pulse, and another gradient to remove quadrature images and crosspeak phase distortion near the water frequency. Radiation damping of the water magnetization during the t1 and t2 evolution periods is suppressed using gradients. Water resonance planes from NOESY-HMQC and NOESY-HSQC spectra show that the HMQC version of the pulse sequences can provide stronger signal for very fast exchanging protons. The HSQC versions of the ROESY and NOESY pulse sequences are designed for the quantitative determination of protein-water crossrelaxation rates, with no water-selective pulses during the mixing time and with phase cycling and other measures for reducing axial artifacts in the water signal.
Gruschus
J M
JM
Laboratory of Biophysical Chemistry, NHLBI, Bethesda, Maryland 20892-0380, USA.
Ferretti
J A
JA
D016477:Artifacts; D004247:DNA / Q000737:chemistry*; D029721:Drosophila Proteins; D018398:Homeodomain Proteins / Q000737:chemistry*; D006801:Humans; D006859:Hydrogen; D007089:Image Enhancement / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008280:Magnetics; D009587:Nitrogen Isotopes; D011522:Protons; D014157:Transcription Factors; D014867:Water / Q000737:chemistry*
D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D009587:Nitrogen Isotopes; D011522:Protons; D014157:Transcription Factors; C094797:vnd protein, Drosophila; D014867:Water; D006859:Hydrogen; D004247:DNA