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15N-labeled protein was expressed from plasmid pET/BCII in Escherichia coli BL21(DE3). Cells were grown at 30 °C in M9 minimal medium with 10g of glucose and 1 g of 15NH4Cl as the only nitrogen source. Expression was induced by adding 0.5 mM IPTG at an OD600 of 1.00. After 16 h the cells were harvested by centrifugation and then broken by sonication; Bell was purified as described.14 The cadmium enzyme is made by adding gradually, at room-temperature, 2 equiv (1.6 mM) of 112CdCl2 or 113CdCl2 (95.83%, Cambridge Isotope Laboratories MA) to 1 equiv of BCII apo-en/yme (0.8 mM) in 10 mM MES-Na. 100 mM NaCl, pH 6.4, 10% D2O. The apo-enzyme was prepared as described.15
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Paul-Soto, R.; Bauer, R.; Frere, J. M.; Galleni, M.; MeyerKlaucke, W.; Nolting, H.; Rossolini, G. M.; deSeny, D.; HernandezValladares, M.; Zeppezauer, M.; Adolph, H. W. J. Biol. Chem. 1999, 274, 13242-13249.
The 1H-15N HMQC experiment with pulsed field gradients was a modification of that described by Davis et al.17 To detect broad signals in the nitrogen dimension, the original pulse sequence was shortened by removing a 15N 180° refocusing pulse. A 1H 90° selective (Gaussian) water (lip-back pulse18 just before the first 1H pulse greatly improved the water suppression as well as the intensities of the imidazole one-bond NH cross-peaks. The pulse sequence used is: g1-wfbq1-90q1(H)-Δ-90 q2(N)-t1/2-180x(H)-t1/2-g 2-180x(N)-g3-τ1-90 x(N)-τ2-g4-acq with phase cycling: q1 = 2(x,-x); q2 = 2(x)2(-x); receiver = (x,-x,-x, x). The carrier frequencies are centered at 4.8 (water) and 160 ppm for 1H and 15N, respectively. The water flip-back (wfb) is a 90° water-selective pulse of 2.8 ms with a Gaussian profile. Delays are: Δ = 17 ms; τ1 = τ180(H) + t10 (where τ180(H) is the duration of the 180° (H) pulse and t10 the initial incremental delay); τ2 = Δ-τg2-τg3-τg4-τ 180-(N)-τ1 (where τg are the duration of the gradient pulses). The duration and strengths of the gradients were: g1: 1 ms, 9 G/cm; g2: 0.5 ms, 27 G/cm; g3: 0.5 ms, -27 G/cm; g4: 0.5 ms, 5.6 G/cm. For each value of t1, N- and P-type coherences were obtained by recording two data sets, the sign of the gradient g4 being inverted for the second data set. The phase q2 was incremented by 180° along with the phase of the receiver for each t1 increment. The spectral widths were 15 kHz (1H) and 10 kHz (15N), and the relaxation delay between scans was Is. The data consisted of 150 complex points in the 15N dimension and IK complex points in the 1H dimension, with 64 scans for each FID.
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