UV resonance Raman determination of protein acid denaturation: Selective unfolding of helical segments of horse myoglobin
UV resonance Raman determination of protein acid denaturation: Selective unfolding of helical segments of horse myoglobin
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DOI：
10.1021/bi971161r
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发表时间：
1998-03-03
期刊：
影响因子：
2.9
通讯作者：
Asher, SA
中科院分区：
文献类型：
--
作者：
Chi, ZH;Asher, SA
我们使用紫外共振拉曼光谱研究了马心水肌红蛋白 (Mb) 在 pH 7.5 和 1.5 之间的酸变性。在 206.5 nm 处激发的拉曼光谱以酰胺振动为主，使用新方法对其进行分析以定量确定 Mb 二级结构。相比之下，229 nm 拉曼光谱以 Tyr 和 Trp 拉曼谱带为主，分析这些拉曼谱带可检查 Tyr 和 Trp 环境的变化，例如暴露于水、氢键，以及 Trp 环及其与蛋白质骨架的连接之间的二面角的任何变化。我们通过将酰胺、Tyr 和 Trp 拉曼光谱信息与血红素吸收光谱信息相结合，独特地确定了哪些 Mb α 螺旋熔化。我们计算出，Mb α-螺旋组成从中性 pH 值时的大约 80% 减少到 pH 3.5 以下的大约 19%。 Trp 拉曼截面在低 pH 值下急剧减小，表明它们完全暴露于水；该结果表明 A 螺旋熔化。 Tyr 拉曼谱带与 pH 无关，这表明 Tyr 残基周围的 G 和 H 螺旋不会熔化。血红素吸收酸变性的显着变化表明血红素袋的重大变化和血红素结合的变化。这些结果表明 A、B、C、D、E 和 F 螺旋以一致的方式熔化，而反向平行的 G 和 H 螺旋仅部分熔化。
We have used UV resonance Raman spectroscopy to study the acid denaturation of horse heart aquometmyoglobin (Mb) between pH 7.5 and 1.5. Raman spectra excited at 206.5 nm are dominated by amide vibrations, which are analyzed by using a new methodology to quantitatively determine the Mb secondary structure. In contrast, the 229-nm Raman spectra are dominated by the Tyr and Trp Raman bands, which are analyzed to examine changes in Tyr and Trp environments, such as exposure to water, hydrogen bonding, and, for Trp, any alterations of the dihedral angle between the Trp ring and its linkage to the protein backbone. We uniquely determined which Mb alpha-helices melt by combining the amide, Tyr, and Trp Raman spectral information with heme absorption spectral information. We calculate that the Mb alpha-helical composition decreases from similar to 80% at neutral pH to similar to 19% below pH 3.5. The Trp Raman cross sections dramatically decrease at low pH to values which indicate that they are fully exposed to water; this result indicates that the A helix melts. The Tyr Raman bands are pH independent, which indicates that the G and H helices around the Tyr residues do not melt. The dramatic heme absorption acid denaturation changes indicate major alterations of the heme pocket and changes in heme binding. These results indicate that the A, B, C, D, E, and F helices melt in a concerted fashion, while the antiparallel G and H helices only partially melt.