Teeter M M, Whitlow M
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167.
Proteins. 1988;4(4):262-73. doi: 10.1002/prot.340040405.
Methods that analyze protein circular dichroism (CD) spectra for fractions of secondary structure are evaluated for the plant protein crambin, which has a known high-resolution crystal structure. In addition, a two-step secondary structure prediction scheme is presented and used for the toxins homologous to crambin, shown by others to have secondary structures similar to crambin. The test of CD spectral analysis methods with the protein crambin employed two computer programs and several CD basis sets. Crambin's crystal structure, known to 0.945A resolution (Hendrickson, W.A., Teeter, M.M. Nature 290:107-113, 1981), allows accurate evaluation of results. Analysis with the protein spectra basis sets (Provencher, S.W., Glöckner, J. Biochemistry 20:33-37, 1981) as modified (Manavalan, P., Johnson, W.C., Jr. Anal. Biochem. 167:76-85, 1987) agreed most closely with crambin's crystal structure. This method was then applied to the CD spectra of the membrane-active toxins homologous to crambin (alpha 1- and beta-purothionin, phoratoxin A and B, and viscotoxin A3 and B). The new program SEQ (pronounced "seek") was developed to assign the secondary structure along the protein chain in a hierarchical fashion and applied to the plant toxins. The method constrained the secondary structure fractions to those from CD analysis and combined standard statistical methods with amphipathic helix location. Both CD-arrived secondary structure percentages and sequence assignment indicate that the viscotoxins are structurally most similar to crambin. Purothionin's secondary structure was predicted to be fundamentally similar to crambin's with a difference at the start of the first helix. This assignment agreed with Raman and NMR analyses of purothionin and lends validity to the method presented here. Differences from the NMR in the CD secondary structure fraction analysis for phoratoxin suggest interference in the CD from tryptophan residues.
针对具有已知高分辨率晶体结构的植物蛋白克拉宾，对分析二级结构片段的蛋白质圆二色性（CD）光谱的方法进行了评估。此外，还提出了一种两步二级结构预测方案，并将其用于与克拉宾同源的毒素，其他人已证明这些毒素具有与克拉宾相似的二级结构。用蛋白质克拉宾对CD光谱分析方法进行测试时使用了两个计算机程序和几个CD基组。克拉宾的晶体结构分辨率为0.945埃（亨德里克森，W.A.，蒂特，M.M.《自然》290:107 - 113，1981），这使得能够准确评估结果。使用经修改的（马纳瓦兰，P.，约翰逊，W.C.，Jr.《分析生物化学》167:76 - 85，1987）蛋白质光谱基组（普罗文彻，S.W.，格洛克纳，J.《生物化学》20:33 - 37，1981）进行分析，与克拉宾的晶体结构最为吻合。然后将该方法应用于与克拉宾同源的膜活性毒素（α1 - 和β - 硫堇、毒莠蛋白A和B以及蓖麻毒蛋白A3和B）的CD光谱。开发了新程序SEQ（发音为“seek”）以分层方式沿着蛋白质链确定二级结构，并将其应用于植物毒素。该方法将二级结构片段限制为来自CD分析的片段，并将标准统计方法与两亲性螺旋定位相结合。CD得出的二级结构百分比和序列确定都表明，蓖麻毒蛋白在结构上与克拉宾最相似。硫堇的二级结构预计与克拉宾基本相似，但在第一个螺旋开始处有所不同。这一确定与硫堇的拉曼和核磁共振分析结果一致，从而验证了本文提出的方法。在毒莠蛋白的CD二级结构片段分析中与核磁共振结果的差异表明色氨酸残基对CD有干扰。
