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首页> 外文期刊>Biochemistry >Dynamic fluorescence spectroscopy on single tryptophan mutants of EII(mtl) in detergent micelles. Effects of substrate binding and phosphorylation on the fluorescence and anisotropy decay.
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Dynamic fluorescence spectroscopy on single tryptophan mutants of EII(mtl) in detergent micelles. Effects of substrate binding and phosphorylation on the fluorescence and anisotropy decay.

机译：动态荧光光谱对洗涤剂胶束中EII（mtl）的单个色氨酸突变体。底物结合和磷酸化对荧光和各向异性衰减的影响。

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摘要

The effects of substrate and substrate analogue binding and phosphorylation on the conformational dynamics of the mannitol permease of Escherichia coli were investigated, using time-resolved fluorescence spectroscopy on mutants containing five single tryptophans situated in the membrane-embedded C domain of the enzyme [Swaving Dijkstra et al. (1996) Biochemistry 35, 6628-6634]. Since no fluorescent impurities are present in these mutants, the changes in fluorescence and anisotropy could be related with changes in the tryptophan microenvironment. Tryptophans at positions 30 and 42 showed changes in fluorescence intensity decay upon binding mannitol, which were reflected in the changes in lifetime distribution patterns. The disappearance of the shortest-lived decay component in these mutants, as well as in the mutant with a single tryptophan at position 109, indicates a change in the local environment such that quenching via neighboring side chains or solvent is reduced. Phosphorylation at histidine 554 and cysteine 384, located in the cytoplasmatic A and B domains of EII(mtl), respectively, induced an increase in the average fluorescence lifetimes of all of the tryptophans. The effect was most pronounced for tryptophans 30 and 109 which show large increases in the average fluorescence lifetime mainly due to loss of short-lived decay components. A correlation time distribution of the individual tryptophans deduced from an analysis of the anisotropy decay showed that they differed in their rotational mobility with tryptophan 30 showing the least local flexibility. Phosphorylation resulted in immobilization of W109 which, together with changes in the average fluorescence lifetime, is evidence for a conformational coupling between the phosphorylated B domain and the C domain. The influence of mannitol binding on the rotational behavior of the tryptophans is limited; it induces more internal flexibility at all tryptophan positions. A rotational correlation time of 30 ns was resolved for tryptophan 30, which probably represents a rotational mode of the micelle-embedded C-domain of EII(mtl) or a portion thereof. Upon phosphorylation, this rotational correlation time increases to 50 ns, probably reflecting a changed spatial orientation of W30 with respect to the C domain. Although kinetic experiments have shown that none of the tryptophans is essential for the catalytic activity of EII(mtl), it is significant that the residues most sensitive to mannitol binding, W30 and W42, are both located in the first membrane-spanning alpha-helix, a portion of which is highly conserved among mannitol-specific EII's of different bacteria.
机译：使用时间分辨荧光光谱技术，研究了底物和底物类似物的结合和磷酸化对大肠杆菌甘露醇通透酶构象动力学的影响，方法是对位于膜的C嵌入结构域中的五个单个色氨酸的突变体进行时间分辨荧光光谱分析[Swaving Dijkstra等。 （1996）Biochemistry 35，6628-6634]。由于这些突变体中没有荧光杂质，因此荧光和各向异性的变化可能与色氨酸微环境的变化有关。结合甘露醇后，第30和42位的色氨酸显示荧光强度衰减的变化，这反映在寿命分布模式的变化中。这些突变体中最短寿命的衰变成分的消失，以及在109位具有单个色氨酸的突变体中消失，表明局部环境发生了变化，从而减少了通过相邻侧链或溶剂的猝灭。分别位于EII（mtl）胞质A和B域中的组氨酸554和半胱氨酸384的磷酸化作用诱导了所有色氨酸的平均荧光寿命的增加。对于色氨酸30和109的影响最为明显，色氨酸30和109的平均荧光寿命显着增加，这主要是由于短暂衰变成分的损失所致。通过对各向异性衰减的分析得出的各个色氨酸的相关时间分布表明，它们的旋转迁移率有所不同，其中色氨酸30的局部柔性最小。磷酸化导致W109的固定化，以及平均荧光寿命的变化，证明了磷酸化B结构域和C结构域之间构象偶联。甘露醇结合对色氨酸旋转行为的影响是有限的。它在所有色氨酸位置上都具有更大的内部柔韧性。色氨酸30解析了30 ns的旋转相关时间，色氨酸30可能代表了EII（mtl）或其一部分的胶束嵌入C结构域的旋转模式。磷酸化后，该旋转相关时间增加到50 ns，可能反映了W30相对于C结构域的空间取向发生了变化。尽管动力学实验表明，色氨酸都不是EII（mtl）催化活性所必需的，但重要的是，对甘露醇结合最敏感的残基W30和W42都位于第一个跨膜的α-螺旋中，其中一部分在不同细菌的甘露醇特异性EII中高度保守。

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