Resonance Raman evidence that the thioester-linked 4-hydroxycinnamyl chromophore of photoactive yellow protein is deprotonated.
Resonance Raman evidence that the thioester-linked 4-hydroxycinnamyl chromophore of photoactive yellow protein is deprotonated.
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DOI：
10.1021/bi00039a024
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发表时间：
1995-10
期刊：
影响因子：
2.9
通讯作者：
Munsok Kim;R. Mathies;W. Hoff;K. Hellingwerf
中科院分区：
文献类型：
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作者：
Munsok Kim;R. Mathies;W. Hoff;K. Hellingwerf
利用微旋转样品池，在413.1 nm激发下，获得了光活性黄蛋白(PYP)基态的共振拉曼光谱。比较了蛋白质中硫酯连接的4-羟基肉桂基发色团的共振拉曼光谱与4-羟基肉桂酸模型化合物和对羟基肉桂酸模型化合物在不同pH值下的预共振拉曼光谱。在4-羟基肉桂基苯硫酯的阴离子形式的拉曼光谱中，1568、1542、1500、1434和1166 cm-1处的谱带被证明是生色团去质子化的特征。对PYP中具有非常相似的频率和强度模式的谱带的观察提供了强有力的证据，表明PYP中的生色团在pH为7.4时是以酚类阴离子稳定的，这支持了结晶学研究的结论。此外，PYP拉曼光谱对蛋白质放置在D2O缓冲液中的不敏感性与肉桂基发色团上没有可交换的酚质子是一致的。这些结果为利用共振拉曼光谱阐明这种新型光敏色素光信息转导的分子机制奠定了基础。
Resonance Raman spectra of the ground state of photoactive yellow protein (PYP), a photoactive pigment found in Ectothiorhodospira halophila, have been obtained with excitation at 413.1 nm using a microspinning sample cell. The resonance Raman spectra of the thioester-linked 4-hydroxycinnamyl chromophore in the protein are compared with the preresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and 4-hydroxycinnamic acid model compounds at various pH values. Bands at 1568, 1542, 1500, 1434, and 1166 cm-1 in the Raman spectrum of the anionic form of the 4-hydroxycinnamyl phenyl thioester are shown to be characteristic for the deprotonation of the chromophore. The observation of bands in PYP exhibiting very similar frequency and intensity patterns provides strong evidence that the chromophore in PYP is stabilized as a phenolate anion at pH 7.4, in support of conclusions from crystallographic studies. Furthermore, the insensitivity of the PYP Raman spectrum to placement of the protein in D2O buffer is consistent with the absence of the exchangeable phenolic proton on the cinnamyl chromophore. These results establish the feasibility of elucidating the molecular mechanism of light-to-information transduction by this new photosensory pigment with resonance Raman spectroscopy.