Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants.
Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants.
复制标题
使用单个色氨酸突变体解析 lac 阻遏物的两个色氨酸残基的荧光衰减。
DOI：
10.1016/s0006-3495(90)82383-3
复制
发表时间：
1990
影响因子：
3.4
通讯作者：
Matthews,KS
中科院分区：
文献类型：
--
作者：
Royer,CA;Gardner,JA;Beechem,JM;Brochon,JC;Matthews,KS
我们已经研究了时间分辨内在色氨酸荧光的乳糖阻遏物（对称四聚体，每个单体含有两个色氨酸残基）和两个单色氨酸突变体阻遏物通过定点诱变，乳糖W201 Y和乳糖W220 Y。这些突变阻遏蛋白分别在201和220位用酪氨酸取代色氨酸，在W201Y突变体的220位和W220Y突变体的201位每个单体亚基留下单个色氨酸残基。发现从野生型数据的分析中恢复的两个衰减速率不对应于从突变蛋白的衰减分析中恢复的速率。突变阻遏物中的这些残基中的每一个显示至少两种衰减速率。然而，从所有三种蛋白质的多波长数据的全球分析，产生的结果与野生型乳糖阻遏物的荧光衰减相一致，简单地对应于从突变蛋白质的衰减的加权线性组合。拮抗配体，诱导剂和操纵基因DNA的连接的效果，是相似的所有三种蛋白质。诱导物糖的结合导致长寿命物种的淬灭，而操作员的结合降低了短组分的寿命。对这三种蛋白质的色氨酸内源荧光的时间分辨各向异性的研究表明，荧光的去偏振是由于大分子的快速运动和整体翻滚造成的。来自三种蛋白质的频域数据集的同时全局分析的结果揭示了大分子的各向异性旋转，与已知的阻遏物四聚体的细长形状一致。此外，色氨酸220的激发态偶极似乎与阻遏物的长轴对齐。
We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor.
登录
查看更多内容
紫胶阻遏蛋白：分子形状、亚基结构以及基于微晶结构研究的操作者相互作用模型。
DOI：
--
发表时间：
1974
影响因子：
11.1
作者：
T. Steitz;T. Richmond;D. Wise;D. Engelman
通讯作者：
D. Engelman
DOI：
--
发表时间：
1971
期刊：
影响因子：
--
作者：
A. Lamola;W. Ware
通讯作者：
W. Ware
影响因子：
2.9
作者：
C. Royer;P. Tauc;G. Hervé;J. Brochon
通讯作者：
C. Royer;P. Tauc;G. Hervé;J. Brochon
影响因子：
29.4
作者：
Zhang F;Wan Y;Zuo T;Yeoh YK;Liu Q;Zhang L;Zhan H;Lu W;Xu W;Lui GCY;Li AYL;Cheung CP;Wong CK;Chan PKS;Chan FKL;Ng SC
通讯作者：
Ng SC
通过单个色氨酸残基 (Trp-59) 的时间分辨荧光研究马心脱辅细胞色素 c 在水溶液中的纳秒动力学。
影响因子：
2.9
作者：
M. Vincent;J. Brochon;F. Mérola;W. Jordi;J. Gallay
通讯作者：
J. Gallay