Substitution of the sole tryptophan of the cupredoxin, amicyanin, with 5-hydroxytryptophan alters fluorescence properties and energy transfer to the type 1 copper site.
Substitution of the sole tryptophan of the cupredoxin, amicyanin, with 5-hydroxytryptophan alters fluorescence properties and energy transfer to the type 1 copper site.
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DOI：
10.1016/j.jinorgbio.2022.111895
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发表时间：
2022-09
影响因子：
3.9
通讯作者：
Davidson, Victor L.
中科院分区：
文献类型：
--
作者：
Pastore, Anthony J.;Ficaretta, Elise;Chatterjee, Abhishek;Davidson, Victor L.
关键词：
阿米青蛋白是一种具有单个色氨酸残基的 1 型铜蛋白。通过遗传密码扩展，色氨酸被选择性地替换为非天然氨基酸 5-羟基色氨酸 (5-HTP)。 5-HTP 取代的阿米蓝蛋白在 300-320 nm 处表现出吸光度，这是 5-HTP 的特征，而在天然阿米蓝蛋白中未见。 5-HTP 取代的阿米蓝蛋白的荧光发射最大值从天然阿米蓝蛋白的 318 nm 红移至 331 nm，以及未折叠蛋白中的 348 nm。 5-HTP取代的阿米蓝蛋白突变体的荧光量子产率远低于天然阿米蓝蛋白。内在荧光的差异可以通过色氨酸与 5-HTP 的激发态以及蛋白质内环境的差异来解释。用 5-HTP 取代色氨酸不会影响 10 Å 之外的铜的可见光吸光度和氧化还原电位。在阿米青蛋白和其他铜氧还蛋白中，观察到结合的铜对固有荧光的无法解释的猝灭。然而，5-HTP取代的氨基花青蛋白的荧光不会被铜猝灭。结果表明，天然阿霉素的猝灭机制是福斯特（Förster），或荧光共振能量转移（FRET）。这种情况不会发生在 5-HTP 取代的阿霉素中，因为荧光量子产率显着降低，并且荧光发射最大值的红移减少与铜的近紫外吸光度的重叠。 5-HTP 相对于阿米青蛋白中色氨酸的独特荧光特性的表征为使用 5-HTP 以及与过渡金属的长距离相互作用对蛋白质微环境进行光谱分析提供了基础。
Amicyanin is a type 1 copper protein with a single tryptophan residue. Using genetic code expansion, the tryptophan was selectively replaced with the unnatural amino acid, 5-hydroxytryptophan (5-HTP). The 5-HTP substituted amicyanin exhibited absorbance at 300-320 nm, characteristic of 5-HTP and not seen in native amicyanin. The fluorescence emission maximum in 5-HTP substituted amicyanin is redshifted from 318 nm in native amicyanin to 331 nm and to 348 nm in the unfolded protein. The fluorescence quantum yield of 5-HTP substituted amicyanin mutant was much less than that of native amicyanin. Differences in intrinsic fluorescence are explained by differences in the excited states of tryptophan versus 5-HTP and the intraprotein environment. The substitution of tryptophan with 5-HTP did not affect the visible absorbance and redox potential of the copper, which is 10 Å away. In amicyanin and other cupredoxins, an unexplained quenching of the intrinsic fluorescence by the bound copper is observed. However, the fluorescence of 5-HTP substituted amicyanin is not quenched by the copper. It is shown that the mechanism of quenching in native amicyanin is Förster, or fluorescence, resonance energy transfer (FRET). This does not occur in 5-HTP substituted amicyanin because the fluorescence quantum yield is significantly lower and the red-shift of fluorescence emission maximum decreases overlap with the near UV absorbance of copper. Characterization of the distinct fluorescence properties of 5-HTP relative to tryptophan in amicyanin provides a basis for spectroscopic interrogation of the protein microenvironment using 5-HTP, and long-distance interactions with transition metals.
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影响因子：
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作者：
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通讯作者：
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DOI：
10.3390/molecules23071662
发表时间：
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期刊：
Molecules (Basel, Switzerland)
影响因子：
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通讯作者：
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DOI：
10.1039/c0mt00061b
发表时间：
2011-03
期刊：
Metallomics : integrated biometal science
影响因子：
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通讯作者：
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