Structure determination of proteins in 2H2O solution aided by a deuterium-decoupled 3D HCA(N)CO experiment.
We developed an NMR pulse sequence, 3D HCA(N)CO, to correlate the chemical shifts of protein backbone (1)Halpha and (13)Calpha to those of (13)C' in the preceding residue. By applying (2)H decoupling, the experiment was accomplished with high sensitivity comparable to that of HCA(CO)N. When combined with HCACO, HCAN and HCA(CO)N, the HCA(N)CO sequence allows the sequential assignment using backbone (13)C' and amide (15)N chemical shifts without resort to backbone amide protons. This assignment strategy was demonstrated for (13)C/(15)N-labeled GB1 dissolved in (2)H(2)O. The quality of the GB1 structure determined in (2)H(2)O was similar to that determined in H(2)O in spite of significantly smaller number of NOE correlations. Thus this strategy enables the determination of protein structures in (2)H(2)O or H(2)O at high pH values.
Ogura
Kenji
K
Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Kita-ku, Sapporo, Japan.
Kumeta
Hiroyuki
H
Inagaki
Fuyuhiko
F
D002247:Carbon Isotopes / Q000737:chemistry; D017666:Deuterium Oxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
D002247:Carbon Isotopes; D011506:Proteins; D017666:Deuterium Oxide