Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor.
Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor.
复制标题
对色氨酸阻遏物温度敏感突变体动力学的远程影响。
DOI：
10.1006/jmbi.1998.2311
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Carey,J
中科院分区：
文献类型：
--
作者：
Jin,L;Fukayama,JW;Pelczer,I;Carey,J
突变体色氨酸阻遏蛋白（TrpR）含有取代苯丙氨酸亮氨酸75已被分离的温度敏感性突变的遗传筛选。二维（2D）1H NMR光谱表明纯化的突变体和野生型蛋白质的总体非常相似的折叠。圆二色谱偏振光谱法表明相对于野生型蛋白质增加的螺旋含量，以及突变蛋白质的尿素变性中点略高，尽管热稳定性没有差异。荧光光谱表明突变蛋白中的一个或两个色氨酸残基的更深埋的环境。从突变体和野生型蛋白质的NMR光谱定量两个色氨酸残基的解析吲哚环质子的质子-氘交换出速率，发现在野生型蛋白质中快约50%。突变蛋白结合辅阻遏物l-色氨酸（l-Trp）的能力比野生型蛋白弱约10倍，但在l-Trp过量时，其DNA结合亲和力仅弱2 - 5倍。两者合计的结果意味着，尽管其保守的化学性质和表面位置在螺旋-转角-螺旋DNA识别基序的螺旋1的C末端，这种突变的变化赋予长期的动态蛋白质的二级和三级结构的影响，而基本上不改变其折叠，并与相对较小的影响蛋白质的功能。
A mutant tryptophan repressor (TrpR) protein containing the substitution of phenylalanine for leucine 75 has been isolated following a genetic screen for temperature-sensitive mutations. Two-dimensional (2D)1H NMR spectra indicate an overall very similar fold for the purified mutant and wild-type proteins. Circular dichroism spectropolarimetry indicates an increased helix content relative to the wild-type protein, and a slightly higher urea denaturation midpoint for the mutant protein, although there is no difference in thermal stability. Fluorescence spectra indicate a more buried environment for one or both tryptophan residues in the mutant protein. The rate of proton-deuterium exchange-out for the resolved indole ring protons of the two tryptophan residues was quantified from NMR spectra of mutant and wild-type proteins and found to be approximately 50 % faster in the wild-type protein. The mutant protein binds the corepressor l-tryptophan (l-Trp) approximately ten times more weakly than does the wild-type protein, but in l-Trp excess its DNA-binding affinity is only two to fivefold weaker. Taken together the results imply that, despite its conservative chemical character and surface location at the C terminus of helix one in the helix-turn-helix DNA recognition motif, this mutational change confers long-range effects on the dynamics of the protein’s secondary and tertiary structure without substantially altering its fold, and with relatively minor effects on protein function.