Investigation of an unnatural amino acid for use as a resonance Raman probe: Detection limits, solvent and temperature dependence of the νC≡N band of 4-cyanophenylalanine.
Investigation of an unnatural amino acid for use as a resonance Raman probe: Detection limits, solvent and temperature dependence of the νC≡N band of 4-cyanophenylalanine.
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DOI：
10.1002/jrs.2067
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发表时间：
2008-11
影响因子：
2.5
通讯作者：
Spiro, Thomas G.
中科院分区：
文献类型：
--
作者：
Weeks, Colin L.;Polishchuk, Alexei;Getahun, Zelleka;DeGrado, William F.;Spiro, Thomas G.
将非天然氨基酸掺入蛋白质中作为光谱探针可用于研究蛋白质的结构和功能。一种这样的探针是 4-氰基苯丙氨酸 (PheCN)，其腈基团具有伸缩模式，该伸缩模式发生在不包含蛋白质常见成分的任何模式的振动光谱区域，并且波数对其环境的极性敏感。在这项工作中，我们评估了紫外共振拉曼光谱在监测掺入蛋白质的 PheCN νCeqN 带对蛋白质环境的敏感性方面的潜力。 PheCN 拉曼激发曲线的测量表明，使用 UV 激发获得了拉曼信号的显着共振增强，并且在 229 和 244 nm 激发波长下获得了最佳信噪比。蛋白质中 PheCN 的检测限约为 10 μM，比红外研究中使用的浓度低约一百倍，这增加了 PheCN 作为振动探针的潜在应用。 PheCN νCeqN 带的波数强烈依赖于其环境的极性，当溶剂从 H2O 变为 THF 时，波数减少了 8 cm−1。对于经过修饰以含有 PheCN 的肽 mastoparan X，脂质体的存在导致了类似但较小的 νCeqN 减少。 PheCN 共振拉曼光谱的选择性和灵敏度意味着它可以成为蛋白质分子内和分子间相互作用的有用探针，并为其利用时间分辨共振拉曼光谱研究蛋白质动力学打开了大门。
The incorporation of unnatural amino acids into proteins that act as spectroscopic probes can be used to study protein structure and function. One such probe is 4-cyanophenylalanine (PheCN), the nitrile group of which has a stretching mode that occurs in a region of the vibrational spectrum that does not contain any modes from the usual components of proteins and the wavenumber is sensitive to the polarity of its environment. In this work we evaluate the potential of UV resonance Raman spectroscopy for monitoring the sensitivity of the νC≡N band of PheCN incorporated into proteins to the protein environment. Measurement of the Raman excitation profile of PheCN showed that considerable resonance enhancement of the Raman signal was obtained using UV excitation and the best signal-to-noise ratios were obtained with excitation wavelengths of 229 and 244 nm. The detection limit for PheCN in proteins was ~10 μM, approximately a hundred-fold lower than the concentrations used in IR studies, which increases the potential applications of PheCN as a vibrational probe. The wavenumber of the PheCN νC≡N band was strongly dependent on the polarity of its environment, when the solvent was changed from H2O to THF it decreased by 8 cm−1. The presence of liposomes caused a similar though smaller decrease in νC≡N for a peptide, mastoparan X, modified to contain PheCN. The selectivity and sensitivity of resonance Raman spectroscopy of PheCN mean that it can be a useful probe of intra- and intermolecular interactions in proteins and opens the door to its application in the study of protein dynamics using time-resolved resonance Raman spectroscopy.
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