Improved 3D gd-HCACO and gd-(H)CACO-TOCSY experiments for isotopically enriched proteins dissolved in H2O.
Pulsed field gradients were incorporated into the HCACO experiment for acquiring spectra on isotopically enriched protein samples dissolved in H2O. Excellent water suppression and spectral quality were achieved using the modified pulse sequence (gd-HCACO), as demonstrated for a 13C-/15N-labeled sample of the SH2 domain from the hematopoietic cellular kinase dissolved in 90% H2O/10% D2O. Strong correlations for all residues were observed in the gd-HCACO spectrum, even for residues having alpha-protons resonating exactly at the H2O frequency. The HCACO-TOCSY experiment was modified to correlate intraresidue 13C alpha (rather than 1H alpha), carbonyl (13C'), and aliphatic side-chain protons [(H)CACO-TOCSY]. Pulsed field gradients were also incorporated into the (H)CACO-TOCSY experiment for water suppression.
Zhang
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Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805, USA.
Gmeiner
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WH
D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015394:Molecular Structure; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011518:Proto-Oncogene Proteins / Q000737:chemistry; D051572:Proto-Oncogene Proteins c-hck; D014867:Water; D018909:src Homology Domains
D011506:Proteins; D011518:Proto-Oncogene Proteins; D014867:Water; D011505:Protein-Tyrosine Kinases; D051572:Proto-Oncogene Proteins c-hck