Das K, Ashby K D, Smirnov A V, Reinach F C, Petrich J W, Farah C S
Department of Chemistry, Iowa State University, Ames 50011-3111, USA.
Photochem Photobiol. 1999 Nov;70(5):719-30.
Tropomyosin mutants containing either tryptophan (122W), 5-hydroxytryptophan (5OH122W) or 7-azatryptophan (7N122W) have been expressed in Escherichia coli and their fluorescence properties studied. The fluorescent amino acids were located at position 122 of the tropomyosin primary sequence, corresponding to a solvent-exposed position c of the coiled-coil heptapeptide repeat. The emission spectrum of the probe in each mutant is blue-shifted slightly with respect to that of the probe in water. The fluorescence anisotropy decays are single exponential, with a time constant of 2-3 ns while the fluorescence lifetimes of the probes incorporated into the proteins, in water, are nonexponential. Because tryptophan in water has an intrinsic nonexponential fluorescence decay, it is not surprising that the fluorescence decay of 122W is well described by a triple exponential. The fluorescence decays in water of the nonnatural amino acids 5-hydroxytryptophan and 7-azatryptophan (when emission is collected from the entire band) are single exponential. Incorporation into tropomyosin induces triple-exponential fluorescence decay in 5-hydroxytryptophan and double-exponential fluorescence decay in 7-azatryptophan. The range of lifetimes observed for 5-hydroxyindole and 5-hydroxytryptophan at high pH and in the nonaqueous solvents were used as a base with which to interpret the lifetimes observed for the 5OH122W and indicate that the chromophore exists in several solvent environments in both its protonated and unprotonated forms in 5OH122W.
含有色氨酸（122W）、5-羟色氨酸（5OH122W）或7-氮杂色氨酸（7N122W）的原肌球蛋白突变体已在大肠杆菌中表达，并对其荧光特性进行了研究。荧光氨基酸位于原肌球蛋白一级序列的第122位，对应于卷曲螺旋七肽重复序列中一个暴露于溶剂的c位置。每个突变体中探针的发射光谱相对于水中探针的发射光谱略有蓝移。荧光各向异性衰减为单指数形式，时间常数为2 - 3纳秒，而掺入蛋白质中的探针在水中的荧光寿命是非指数形式的。由于水中的色氨酸具有固有的非指数荧光衰减，因此122W的荧光衰减能用三指数很好地描述也就不足为奇了。非天然氨基酸5-羟色氨酸和7-氮杂色氨酸在水中的荧光衰减（当从整个波段收集发射时）是单指数的。掺入原肌球蛋白会诱导5-羟色氨酸产生三指数荧光衰减，7-氮杂色氨酸产生双指数荧光衰减。在高pH值和非水溶剂中观察到的5-羟吲哚和5-羟色氨酸的寿命范围被用作解释5OH122W观察到的寿命的基础，并表明发色团在5OH122W中以其质子化和非质子化形式存在于几种溶剂环境中。
