Comprehensive secondary‐structure analysis of disulfide variants of lysozyme by synchrotron‐radiation vacuum‐ultraviolet circular dichroism
Comprehensive secondary‐structure analysis of disulfide variants of lysozyme by synchrotron‐radiation vacuum‐ultraviolet circular dichroism
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DOI：
10.1002/prot.22430
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发表时间：
2009-10
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影响因子：
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通讯作者：
K. Matsuo;Hidenori Watanabe;S. Tate;H. Tachibana;K. Gekko
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文献类型：
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作者：
K. Matsuo;Hidenori Watanabe;S. Tate;H. Tachibana;K. Gekko
阐明特定二硫键的作用（Cys 6-Cys 127、Cys 30-Cys 115、Cys 64-Cys 80和Cys 76-Cys 94）对鸡溶菌酶二级结构的影响，真空紫外圆二色性（VUVCD）在pH 2.9和25°C下，使用荧光分光光度计测量13种二硫键缺陷变体的光谱，其中Cys残基被Ala或Ser残基取代，低至170 nm。同步辐射VUVCD分光光度计每个变体都表现出相当数量的残留二级结构的VUVCD光谱特征，这取决于删除的二硫键的位置和数量。使用31个参考蛋白质的VUVCD光谱和PDB数据，使用SELCON 3程序估计α螺旋、β链、转角和无序结构的含量。还根据VUVCD数据估计了α螺旋和β链片段的数量。一般而言，随着二硫桥数量的增加以及它们在一级结构中形成的距离更大，二级结构通过熵力更有效地稳定。三个二硫键变体的结构与野生型相似，但其他变体表现出减少的α螺旋，在两个二硫键变体周围的有序和无序结构之间存在边界。通过将VUVCD数据与神经网络方法相结合，预测所有变体的二级结构序列。这些结果揭示了每个二硫键在二级结构形成中的特征作用。Proteins 2009.© 2009 Wiley利斯公司
To elucidate the effects of specific disulfide bridges (Cys6‐Cys127, Cys30‐Cys115, Cys64‐Cys80, and Cys76‐Cys94) on the secondary structure of hen lysozyme, the vacuum‐ultraviolet circular dichroism (VUVCD) spectra of 13 species of disulfide‐deficient variants in which Cys residues were replaced with Ala or Ser residues were measured down to 170 nm at pH 2.9 and 25°C using a synchrotron‐radiation VUVCD spectrophotometer. Each variant exhibited a VUVCD spectrum characteristic of a considerable amount of residual secondary structures depending on the positions and numbers of deleted disulfide bridges. The contents of α‐helices, β‐strands, turns, and unordered structures were estimated with the SELCON3 program using the VUVCD spectra and PDB data of 31 reference proteins. The numbers of α‐helix and β‐strand segments were also estimated from the VUVCD data. In general, the secondary structures were more effectively stabilized through entropic forces as the number of disulfide bridges increased and as they were formed over larger distances in the primary structure. The structures of three‐disulfide variants were similar to that of the wild type, but other variants exhibited diminished α‐helices with a border between the ordered and disordered structures around the two‐disulfide variants. The sequences of the secondary structures were predicted for all the variants by combining VUVCD data with a neural‐network method. These results revealed the characteristic role of each disulfide bridge in the formation of secondary structures. Proteins 2009. © 2009 Wiley‐Liss, Inc.