STUDIES OF MACROMOLECULAR STRUCTURE BY C-13 NUCLEAR MAGNETIC-RESONANCE .2. A SPECIFIC LABELING APPROACH TO STUDY OF HISTIDINE RESIDUES IN PROTEINS
STUDIES OF MACROMOLECULAR STRUCTURE BY C-13 NUCLEAR MAGNETIC-RESONANCE .2. A SPECIFIC LABELING APPROACH TO STUDY OF HISTIDINE RESIDUES IN PROTEINS
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DOI：
10.1021/ja00785a050
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发表时间：
1973-01-01
影响因子：
15
通讯作者：
WILSON, DM
中科院分区：
文献类型：
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作者：
BROWNE, DT;KENYON, GL;WILSON, DM
通过将标记的组氨酸体内掺入埃希氏菌蛋白质来制备色氨酸合成酶亚基，该亚基特异性富集 13C，并在四个组氨酸残基的 C2（环）位置用氘标记。标记酶的 13C nmr 光谱被确定为磁场强度、浓度、温度、溶剂和解偶联模式的函数。来自组氨酸 C2 碳在 24 kG 和 pD 7 下的 1SC nmr 信号是未解析的单峰，宽度为 50±5 Hz，纵向弛豫时间 (7)) 为 0.5 秒。 C2 碳旋转重新定向的相关时间 tc 经计算为 2.7 X 10-8 秒，表明组氨酸侧链均高度固定在酶内。对确定横向弛豫时间 (T2) 以及来自单个 C2 碳的信号线宽的机制的定量估计表明，各个共振应约为20赫兹宽。对 C2 碳线宽的主要贡献（约 15 Hz）是由直接键合氘的第二类标量弛豫造成的。有人建议，通过采用强氘去耦射频场，可以使来自氘化大分子的 13C nmr 信号变得更窄。
Tryptophan synthetase a subunit specifically enriched in 13C and labeled with deuterium at the C2 (ring) position of the four histidine residues was prepared by in vivo incorporation of labeled histidine into Esche-richia cotí protein. The 13C nmr spectrum of the labeled enzyme was determined as a function of magnetic field strength, concentration, temperature, solvent, and mode of decoupling. The 1SC nmr signal from the histidine C2 carbons at 24 kG and pD 7 is an unresolved singlet of 50±5 Hz width with a longitudinal relaxation time (7)) of 0.5 sec. The correlation time for rotational reorientation, tc, for the C2 carbons is calculated to be 2.7 X 10-8 sec, indicating that the histidine side chains are all highly immobilized within the enzyme. Quantitative estimates of the mechanisms determining the transverse relaxation times (T2’s) and therefore the line widths of signals from single C2 carbons suggest that the individual resonances should be ca. 20 Hz wide. A major contribution (ca. 15 Hz) to C2 carbon line width is made by scalar relaxation of the second kind with the directly bonded deuterium. It is suggested that 13C nmr signals from deuterated macromolecules can be mademuch narrower by employing a strong deuterium decoupling rf field.