Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy
Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy
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DOI：
10.1021/bm049717v
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发表时间：
2004-11-01
影响因子：
6.2
通讯作者：
Pézolet, M
中科院分区：
文献类型：
--
作者：
Rousseau, ME;Lefèvre, T;Pézolet, M
本文首次利用拉曼显微光谱技术定量测定了家蚕、麻蚕和尼泊尔蛛单丝中β -薄片的取向。结果表明，对于丝等单轴对称体系，可以通过偏振拉曼光谱的强度比确定取向分布函数的序参量和取向分布函数。首先推导了圆柱拉曼张量振动的偏振拉曼微光谱计算方程，然后将其应用于酰胺I波段，该波段主要是由于肽基团的C=O拉伸振动引起的。用甲醇处理的家蚕丝各向同性薄膜测定了酰胺I振动的拉曼张量的形状。家蚕丝蛋白和蓖麻丝蛋白的值分别为-0.36 +/- 0.03和0.19 +/- 0.02，尽管两种家蚕丝蛋白的初级序列存在较大差异。对于Nephila edulis拖丝，分别为-0.32 +/- 0.02和0.13 +/- 0.02。这些结果清楚地表明，羰基高度垂直于纤维轴取向，β -片平行于纤维轴取向，与先前的x射线和核磁共振结果一致。利用信息熵理论，从P、)和（P4）的值计算出最可能的方向分布。对于这三种类型的丝，β -薄片高度平行于纤维轴。蚕丝蛋白和蜘蛛拖丝的β -薄片的取向分布近似于高斯函数，其宽度分别为32度和40度。除了这些结果之外，对不同丝绸样品的拉曼光谱记录和几个波段的极化依赖关系的比较已经允许澄清一些重要的波段分配。
Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the beta-sheets in silk monofilaments from Bombyx mori and Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters and of the orientation distribution function from intensity ratios of polarized Raman spectra. The equations allowing the calculation of and using polarized Raman microspectroscopy for a vibration with a cylindrical Raman tensor were first derived and then applied to the amide I band that is mostly due to the C=O stretching vibration of the peptide groups. The shape of the Raman tensor for the amide I vibration of the beta-sheets was determined from an isotropic film of Bombyx mori silk treated with methanol. For both the Bombyx mori and Samia cynthia ricini fibroin fibers, the values of and obtained are equal to -0.36 +/- 0.03 and 0.19 +/- 0.02, respectively, even though the two types of silkworm fibroins strongly differ in their primary sequences. For the Nephila edulis dragline silk, values of and of -0.32 +/- 0.02 and 0.13 +/- 0.02 were obtained, respectively. These results clearly indicate that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the beta-sheets are oriented parallel to the fiber axis, in agreement with previous X-ray and NMR results. The most probable distribution of orientation was also calculated from the values of P,) and (P4) using the information entropy theory. For the three types of silk, the beta-sheets are highly oriented parallel to the fiber axis. The orientation distributions of the beta-sheets are nearly Gaussian functions with a width of 32degrees and 40degrees for the silkworm fibroins and the spider dragline silk, respectively. In addition to these results, the comparison of the Raman spectra recorded for the different silk samples and the polarization dependence of several bands has allowed to clarify some important band assignments.