A high sensitivity 3D experiment for measuring Calpha-Halpha residual dipolar coupling constants.
A new sensitivity improved approach is presented to measure the Calpha-Halpha scalar and dipolar coupling constants in 13C/15N-labeled proteins using a HA(CA)CONH scheme. The proposed experiment has significantly higher sensitivity than the previously published (HA)CA(CO)NH sequence, and provides accurate and straightforward measurements of the scalar and residual dipolar coupling constants. The sequence is easy to implement, and has been demonstrated on the C-terminal domain of the human Ku-80 protein (152 amino acid residues). On average, sensitivity is improved by 40% for both isotropic and anisotropic samples. The sensitivity enhancement is more pronounced for structured regions than unstructured regions, with an average of 50-60% enhancement being observed in the well-structured regions of the protein.
Hu
Weidong
W
Division of Immunology, Beckman Institute of the City of Hope, Duarte, CA 91010, USA. whu@coh.org
Zhang
Ziming
Z
Chen
Yuan
Y
D000465:Algorithms; D016880:Anisotropy; D034961:Antigens, Nuclear / Q000737:chemistry*; D002244:Carbon / Q000737:chemistry*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005240:Feasibility Studies; D006801:Humans; D006860:Hydrogen Bonding; D000072200:Ku Autoantigen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted; D013113:Spin Labels
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D013113:Spin Labels; D002244:Carbon; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen