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

测定<13>了原肌球蛋白的~ 1C CP/MAS NMR谱，通过<13>氨基酸残基的~ 1C NMR化学位移及其迁移率，阐明了原肌球蛋白的高级结构。原肌球蛋白的高级结构是卷曲螺旋结构，并包含两个不同的位点，其特征在于内部和外部位点。在<13>15-17 ppm处出现的14 C信号可以容易地归属于Ala C14<beta>碳，并且可以看出该信号由两个峰组成。15.8ppm处的峰比16.7ppm处的峰具有更长的T_1值。这表明前一种碳比后一种碳更具有移动的性。因此，在15.8和16.7ppm处的两个峰分别归属于<beta>卷曲螺旋结构的外部和内部位点中的Ala C12碳。羊毛角蛋白经二硫键还原和巯基碘乙酸保护形成S-羧甲基角蛋白（SCMK）后，可分为三个主要组分。从<13>羊毛和SCMK的~ 1C CP/MAS NMR谱中可以看出，卷曲螺旋结构的存在是因为这些样品中的<13>Ala C^碳的~ 1C化学位移值<beta>与原肌球蛋白中位于卷曲螺旋结构内部位置的Ala残基的~ 1C化学位移值在实验误差范围内一致。利用构象依赖的~ 1C <13>CP/MAS NMR化学位移进行构象分析，以估计SCMK在拉伸、加热或水蒸汽处理下的构象转变。β-折叠形式通过拉伸和蒸汽处理出现。对于加热的SCMK，可以说出现了无规卷曲形式。然而，尽管出现无规卷曲形式，但α-螺旋形式在加热的α-螺旋中保持相当程度的事实表明，加热仅破坏了SCMK中卷曲螺旋结构的包装，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.