Venters R A, Huang C C, Farmer B T, Trolard R, Spicer L D, Fierke C A
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
J Biomol NMR. 1995 Jun;5(4):339-44. doi: 10.1007/BF00182275.
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.
蛋白质人碳酸酐酶II（HCA II）已用2H、13C和15N进行了同位素标记，用于高分辨率核磁共振归属研究和脉冲序列开发。为了提高几个关键的1H/13C/15N三共振相关实验的灵敏度，已将2H掺入HCA II中，以降低13C和1HN的T2弛豫速率。通过在含有D2O、[1,2-13C2, 99%]乙酸钠和[15N, 99%]氯化铵的限定培养基中培养大肠杆菌，获得了基本上完全掺入脂肪族2H的蛋白质的核磁共振量。完全的脂肪族氘富集对于13C和15N主链核磁共振归属研究是最佳的，因为13C和1HN的T2弛豫时间以及因此的灵敏度被最大化。此外，完全的脂肪族氘代通过消除部分氘代的CHnDm基团观察到的差异2H同位素位移，提高了分辨率和灵敏度。
