Emitting State of 5-Hydroxyindole, 5-Hydroxytryptophan, and 5-Hydroxytryptophan Incorporated in Proteins
Emitting State of 5-Hydroxyindole, 5-Hydroxytryptophan, and 5-Hydroxytryptophan Incorporated in Proteins
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DOI：
10.1021/jp406676j
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发表时间：
2013-09-19
影响因子：
3.3
通讯作者：
Broos, Jaap
中科院分区：
文献类型：
--
作者：
Petrovic, Dejan M.;Hesp, Ben H.;Broos, Jaap
5-羟基-L-色氨酸(5HW)已被生物合成到许多蛋白质中，以便于用荧光光谱对其进行表征。这种色氨酸类似物的一个吸引人的特征是它在310-320 nm处的吸光度，允许它在色氨酸背景中进行特定的激发。在色氨酸或吲哚的5-位引入羟基引起吸收红移是由于L-1(B)跃迁能降低所致。因此认为5HI仅具有(1)lb发射特征。最近，对水中5-羟基吲哚(51-11)的计算表明，L-1(A)是发射状态，蛋白质中的5HW也是如此。为了阐明5HI和5HW中的哪种状态发射，我们在这里给出了这些探针和四个标记的蛋白质在表面暴露位置的激发各向异性谱。我们的数据清楚地表明，在所研究的三种蛋白质中，L-1(B)是5HI、5HW和51-PAT的发射状态。对于一种蛋白质，观察到了混合发射，并且发现衰减动力学强烈地依赖于发射波长。这项工作提供了第一个实验证据，证明IL，可以是蛋白质中加入的这种色氨酸类似物的发射状态。
5-Hydroxy-L-tryptophan (5HW) has been biosynthetically incorporated in many proteins to facilitate their characterization using fluorescence spectroscopy. An attractive feature of this tryptophan analogue is its absorbance at 310-320 nm, allowing its specific excitation in a Trp background. The red-shift in absorbance upon introduction of a hydroxyl group at the 5-position of Trp or indole was found to be due to a lowering of the L-1(b) transition energy. It was therefore believed that 5HI only features (1)Lb emission. Recently, calculations for 5-hydroxyindole (51-11) in water revealed L-1(a), is the emitting state, and the same was predicted for 5HW incorporated in proteins. To clarify which state emits in 5HI and 5HW, we present here excitation anisotropy spectra of these probes and of four proteins labeled with at a surface exposed position. Our data clearly show L-1(b) is the emitting state of 5HI, 5HW, and 51-PAT in three of the proteins investigated. For one protein mixed emission was observed, and the decay kinetics were found strongly dependent on the emission wavelength. This work provides the first experimental evidence that IL, can be the emitting state for this Trp analogue incorporated in a protein.