Solid-state NMR studies on the supersecondary structures in fibrous proteins

测定了原肌球蛋白的~&lt；13&gt；C CP/MAS核磁共振波谱，以便通过氨基酸残基的~&lt；13&gt；C核磁共振化学位移及其迁移率来阐明其高级结构。原肌球蛋白的高级结构为盘绕状结构，含有两个不同的位置，分别为内部和外部位置。出现在15ppm-17ppm的13C信号可以很容易地归属于Ala C^；β&gt；碳，并且可以看出该信号由两个峰组成。15.8ppm的峰值比16.7ppm的T_1值长。这表明前一种碳比后一种碳具有更强的流动性。因此，15.8ppm和16.7ppm的两个峰分别归属于盘管结构外部和内部位置的Ala C^&lt；β&gt；碳。羊毛角蛋白在还原二硫键和用碘乙酸保护生成的硫醇基团后可分为三个主要组分，形成S-羧甲基角氨酸(SCMK)。从羊毛和SCMK的~&lt；13&gt；C CP/MAS核磁共振谱表明，由于这些样品中Ala C^&lt；β&gt；碳的^&lt；13&gt；C化学位移与原肌球蛋白中位于盘绕结构内部位置的Ala残基的化学位移值在实验误差上一致，所以存在盘绕结构。利用构象依赖的~&lt；13&gt；C CP/MAS核磁共振化学位移进行构象分析，估算了SCMK经拉伸、加热或蒸汽处理后的构象转变。通过拉伸和蒸汽处理，出现了β-薄片形式。对于加热的SCMK，可以说出现了无规卷曲的形式。然而，尽管出现了随机卷曲形式，但加热后的螺旋结构在一定程度上保持了可察觉的程度，这表明加热只破坏了SCMK中盘绕螺旋结构的堆积，但保留了二级结构。

^<13>C CP/MAS NMR spectra of tropomyosin were measured in order to elucidate the higher order structure through the ^<13>C NMR chemical shifts of the amino acid residues and their mobility. The higher order structure of tropomyosin is a coiled-coil structure, and contains two different sites which are characterized as the internal and external sites. The ^<13>C signal which appear at 15-17 ppm can be easily assigned to the Ala C^<beta> carbons, and it can be seen that this signal was consisted of two peaks. The peak at 15.8 ppm have a longer T_1 value than those at 16.7 ppm. This is indicated that the former carbons are more mobile than the latter carbons. Therefore, the two peaks at 15.8 and 16.7 ppm are assigned to the Ala C^<beta> carbons in the extermal and internal sites of the coiled-coil structure, respectively. Wool keratin can be divided into three main fractions after reducing disulfide bonds and protection of the resulting thiol groups with iodoacetic acid to form S-carboxymethyl kerateine (SCMK). From the ^<13>C CP/MAS NMR spectra of wool and SCMK,it was suggested that the coiled-coil structure exists because the ^<13>C chemical shift values of Ala C^<beta> carbons in these samples coincide in experimental error with that of the Ala residue located in the internal site of the coiled-coil structure in tropomyosin. The conformational analysis using the conformation-dependent ^<13>C CP/MAS NMR chemical shifts were applied to estimate the conformational transition of SCMK by stretching, heating, or steam-treating. The beta-sheet form appears by stretching and steam-treating. For the heated SCMK,it can be said that the random coil form appears. However, the fact that the a-helix form remains to an appreciable extent in the heated one although the random coil form appears, suggests that only the packing of the coiled-coil structure in the SCMK is disrupted by heating, but secondary structure being retained.