New Approach for Local Structure Analysis of the Tyrosine Domain in Proteins by Using a Site-Specific and Polarity-Sensitive Fluorescent Probe
New Approach for Local Structure Analysis of the Tyrosine Domain in Proteins by Using a Site-Specific and Polarity-Sensitive Fluorescent Probe
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使用位点特异性和极性敏感荧光探针分析蛋白质酪氨酸结构域局部结构的新方法
DOI：
10.1002/cbic.200900003
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发表时间：
2009-05-04
期刊：
影响因子：
3.2
通讯作者：
Ma, Huimin
中科院分区：
文献类型：
--
作者：
Chen, Suming;Li, Xiaohua;Ma, Huimin
据报道，设计和合成了一种新型长波长极性敏感荧光探针6-[9-(二乙氨基)-5-oxo-5H-苯并[α]吩恶嗪-2-基氧基]己-2-烯基乙酸酯，用于选择性修饰酪氨酸残基，目的是提供蛋白质中酪氨酸结构域的局部信息。该探针包含极性敏感的尼罗红荧光团和活性 pi-烯丙基，可以形成 pi-烯丙基钯复合物并选择性地与酪氨酸残基反应。该探针具有以下特点：1）具有>550nm的长波长发射，可以避免常见生物基质的短波长荧光的干扰； 2）最大发射波长仅对极性敏感，对pH或温度不敏感；这样可以准确确定局部极性； 3)它是中性、不带电的分子，不会干扰标记蛋白的整体电荷。利用该探针，首次检测到天然和酸变性和热变性牛铜/锌超氧化物歧化酶中 Tyr108 结构域的极性和构象变化。研究发现，在pH 4和9之间酸变性时，Tyr108结构域的极性几乎没有改变。然而，热变性导致Tyr108结构域变得更加疏水，并伴随着蛋白质的不可逆聚集。此外，探针结合实验表明，热变性后蛋白质表面变得更加疏水；这可以归因于更疏水的聚集体的形成。该策略可能为研究酸或热变性条件下蛋白质中酪氨酸结构域的局部环境变化提供通用方法。
The design and synthesis of a novel long-wavelength polarity-sensitive fluorescence probe, 6-[9-(diethylamino)-5-oxo-5H-benzo[alpha]phenoxazin-2-yloxy]hex-2-enyl acetate, for the selective modification of tyrosine residues with the goal of providing local information on tyrosine domains in proteins, is reported. This probe comprises a polarity-sensitive Nile red fluorophore and an active pi-allyl group that can form pi-allyipalladium complexes and react selectively with tyrosine residues. The probe has the following features: 1) it has a long-wavelength emission of > 550nm, thanks to which interference from short-wavelength fluorescence from common biological matrixes can be avoided; 2) the maximum emission wavelength is sensitive only to polarity and not to pH or temperature; this allows the accurate determination of local polarity; and 3) it is a neutral, uncharged molecule, and does not disturb,the over-all charge of the labelled protein. With this probe the polarity and conformation changes of the Tyr108 domain in native and in acid- and heat-denatured bovine Cu/Zn superoxide dismutase were detected for the first time. It was found that the polarity of the Tyr108 domain hardly alters on acid denaturation between pH 4 and 9. However, heat denaturation caused the Tyr108 domain to be more hydrophobic, and was accompanied by an irreversible aggregation of the protein. In addition, the probe-binding experiments revealed that the surface of the protein becomes more hydrophobic after thermal denaturation; this can be ascribed to the formation of the more hydrophobic aggregates. This strategy might provide a general approach for studying the local environment changes of tyrosine domains in proteins under acid or heat denaturation conditions.