Investigation of the structural determinants of the intrinsic fluorescence emission of the trp repressor using single tryptophan mutants.
Investigation of the structural determinants of the intrinsic fluorescence emission of the trp repressor using single tryptophan mutants.
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DOI：
10.1016/s0006-3495(92)81658-2
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发表时间：
1992-09
影响因子：
3.4
通讯作者：
C. Royer
中科院分区：
文献类型：
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作者：
C. Royer
野生型色氨酸阻遏物（TR）的荧光衰减特性的特点是进行了多发射波长的频率响应曲线的研究。衰减最好是在0.5 ns附近的单指数衰减和寿命分布集中在3-4 ns。通过比较回收的衰变相关的光谱和寿命值与阻遏物的结构，从分析中回收的两个衰变组分的两个色氨酸残基，W19和W 99，蛋白质的暂定分配。这些分配包括将短的红色发射分量与W 99的发射联系起来，以及将大部分较长的蓝色发射寿命分布与W19的发射联系起来。接下来，使用阻遏物的单个色氨酸突变体（其中每个色氨酸残基中的一个被苯丙氨酸取代）来确认初步归属，因为0.5 ns组分显然是由于色氨酸99的发射，而导致恢复分布的大部分衰变来自色氨酸19。然而，数据表明，由于野生型发射中的大量组分（至少五个）以及五个寿命组分中的三个在值上非常接近的事实，野生型蛋白质的衰变是不完全可解析的。野生型衰变的荧光衰变被很好地描述为在每个突变体中发现的组分的组合。然而，尽管15个数据集（5个来自野生型和每个突变体）的线性组合分析产生了先前对于两个突变体回收的组分的良好拟合，但野生型中这些组分的振幅没有以预期的比率回收。由于野生型蛋白中蓝移发射的优势，最有可能的是野生型与突变体相比的细微结构差异，而不是从色氨酸19到99的能量转移，是导致线性组合假设失败的原因。
The fluorescence decay properties of wild-type trp repressor (TR) have been characterized by carrying out a multi-emission wavelength study of the frequency response profiles. The decay is best analyzed in terms of a single exponential decay near 0.5 ns and a distribution of lifetimes centered near 3–4 ns. By comparing the recovered decay associated spectra and lifetime values with the structure of the repressor, tentative assignments of the two decay components recovered from the analysis to the two tryptophan residues, W19 and W99, of the protein have been made. These assignments consist of linking the short, red emitting component to emission from W99 and most of the longer bluer emitting lifetime distribution to emission from W19. Next, single tryptophan mutants of the repressor in which one of each of the tryptophan residues was substituted by phenylalanine were used to confirm the preliminary assignments, inasmuch as the 0.5-ns component is clearly due to emission from tryptophan 99, and much of the decay responsible for the recovered distribution emanates from tryptophan 19. The data demonstrate, however, that the decay of the wild-type protein is not completely resolvable due both to the large number of components in the wild-type emission (at least five) as well as to the fact that three of the five lifetime components are very close in value. The fluorescence decay of the wild-type decay is well described as a combination of the components found in each of the mutants. However, whereas the linear combination analysis of the 15 data sets (5 from the wild-type and each mutant) yields a good fit for the components recovered previously for the two mutants, the amplitudes of these components in the wild-type are not recovered in the expected ratios. Because of the dominance of the blue shifted emission in the wild-type protein, it is most likely that subtle structural differences in the wild-type as compared with the mutants, rather than energy transfer from tryptophan 19 to 99, are responsible for this failure of the linear combination hypothesis.