Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution.
Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution.
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DOI：
10.1073/pnas.140161997
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发表时间：
2000-07
影响因子：
11.1
通讯作者：
R. A. Silva;J. Kubelka;P. Bouř;S. Decatur;T. Keiderling
中科院分区：
文献类型：
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作者：
R. A. Silva;J. Kubelka;P. Bouř;S. Decatur;T. Keiderling
了解蛋白质折叠的详细机制需要动态的、位点特异性的立体化学信息。振动光谱的短时间响应允许评价随着肽展开而快速平衡的群体分布。与同位素标记相关的光谱位移沿着与振动圆二色性（VCD）的局部立体化学灵敏度允许确定展开的片段序列。对于一系列富含丙氨酸的肽，在水溶液中形成α-螺旋，我们使用同位素标记和VCD证明，α-螺旋非合作解绕从末端随着温度的升高。对于这些封闭的肽，C-末端在5 ℃下磨损。从头算水平的理论模拟的IR和VCD带的形状被用来分析光谱，并确认的构象标记的组件。与标记残基相关的VCD信号通过与分子的未标记部分偶联而被放大。因此，小标记的片段是可检测的，并在本报告中的位点特异性肽VCD构象分析中的中等大肽的立体化学定义。
Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form alpha-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the alpha-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5 degrees C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis.