Mapping proximity within proteins using fluorescence spectroscopy. A study of T4 lysozyme showing that tryptophan residues quench bimane fluorescence
Mapping proximity within proteins using fluorescence spectroscopy. A study of T4 lysozyme showing that tryptophan residues quench bimane fluorescence
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DOI：
10.1021/bi011198i
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发表时间：
2002-02-26
期刊：
影响因子：
2.9
通讯作者：
Farrens, DL
中科院分区：
文献类型：
--
作者：
Mansoor, SE;Mchaourab, HS;Farrens, DL
我们提出了一种新的方法来映射蛋白质内的邻近。该方法利用附近的色氨酸残基的荧光标记bimane的淬灭。在T4溶菌酶的研究中，我们发现这种效应似乎是距离依赖性和方向特异性的。具体而言，我们表明，近端色氨酸残基可以降低bimane荧光强度高达500%，并诱导复杂的荧光衰减动力学。用苯丙氨酸取代邻近的Trp残基可以消除这些光谱扰动。使用双甲烷荧光的尖端猝灭用于蛋白质结构研究的优点包括所需蛋白质的量低和标记策略的实质性简化。我们预计这种方法将被证明适用于广泛的高通量蛋白质研究，如蛋白质折叠，蛋白质-蛋白质相互作用的检测，最重要的是，构象变化的动态监测。
We present a novel method for mapping proximity within proteins. The method exploits the quenching of the fluorescent label bimane by nearby Trp residues. In studies of T4 lysozyme we show that this effect appears to be distance dependent and orientation specific. Specifically, we show that a proximal Trp residue can reduce bimane fluorescence intensity by up to 500% and induce complicated fluorescence decay kinetics. Replacing the neighboring Trp residue with phenylalanine removes these spectral perturbations. The advantages of using the Tip quenching of bimane fluorescence for protein structural studies include the low amount of protein required and the substantial simplification of labeling strategies. We anticipate this method will prove suitable for a wide array of high-throughput protein studies such as protein folding, the detection of protein-protein interactions, and, most importantly, the dynamic monitoring of conformational changes.