High-level 2H/13C/15N labeling of proteins for NMR studies.
The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with 2H, 13C and 15N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments, 2H has been incorporated into HCA II in order to decrease the rates of 13C and 1HN T2 relaxation. NMR quantities of protein with essentially complete aliphatic 2H incorporation have been obtained by growth of E. coli in defined media containing D2O, [1,2-13C2, 99%] sodium acetate, and [15N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for 13C and 15N backbone NMR assignment studies, since the 13C and 1HN T2 relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential 2H isotopic shift observed for partially deuterated CHnDm moieties.
Venters
R A
RA
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Huang
C C
CC
Farmer
B T
BT
Trolard
R
R
Spicer
L D
LD
Fierke
C A
CA
D002247:Carbon Isotopes; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics; D003903:Deuterium; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D011506:Proteins / Q000737:chemistry*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D003903:Deuterium; D002256:Carbonic Anhydrases