【作者】 刘明；

【导师】 朱良均；

【作者基本信息】 浙江大学 ， 特种经济动物饲养， 2006， 硕士

【摘要】 蛋白质是生命科学的重要研究对象，而蛋白质结构研究又是研究的重点与难点。近年来，有关分子构象病，仿生蛋白纤维等方面的研究使得人们充分认识到了研究蛋白构象与功能的关系、构象转变机制的重要性。傅立叶变换红外光谱（FTIR，Fourier transform infrared）技术是研究蛋白质二级结构的强有力手段。上世纪八十年代起，红外光谱法对蛋白质二级结构的研究步入了定量阶段，对蛋白质的红外光谱，应用去卷积、二阶导数和曲线拟合等方法，定量分析了多种蛋白质的二级结构。丝素蛋白是一种容易获得而又相对比较稳定的模式蛋白。本研究以丝素蛋白为研究对象，主要利用傅立叶变换红外光谱（FTIR）技术分析了丝素蛋白的构象特点、构象转变过程以及影响构象转变的因素。通过不同溶解体系对丝素蛋白分子量和结构的影响研究发现，①不同盐溶液的影响：相同条件下，LiBr比CaCl₂对丝素蛋白有更好的溶解性，丝素蛋白降解程度更高。②温度和溶解时间的影响：在盐溶解体系中，98±2℃（水溶液沸腾温度）为丝素蛋白长肽链裂解为短肽链的裂解温度，在温度低于98±2℃时，丝素蛋白分子被水膨润，主要解离为长的丝素肽链。当温度达到98±2℃时，大量的长肽链被分解为短肽链及游离氨基酸。③有机溶剂加入的影响：有机溶剂的加入会加快丝素溶解速率，有助于从丝素肽链上解离氨基酸，但对丝素蛋白分子量影响不大。不同的溶解体系对丝素蛋白分子的二级结构影响不大，但LiBr（98±2℃）、CaCl₂（98±2℃）两种溶解体系，分解丝素蛋白能力强，得到的丝素溶液中多为短肽链，影响了丝素蛋白二级结构的完整性，导致在丝素蛋白溶液成膜过程中较难形成α螺旋结构。研究表明，在60℃条件下，浓度为9.5mol／L的LiBr溶液是制取丝素蛋白进行构象变化研究的最佳溶剂。利用傅立叶变换红外光谱分析了七种状态下丝素蛋白二级结构情况，并用傅立叶变换拉曼光谱进行了验证。通过比较各种状态下丝素蛋白红外光谱二阶导数谱和二级结构定量分析结果，发现丝素蛋白溶液的构象以无规卷曲结构为主，而冷冻干燥的丝素蛋白溶液、丝素烘干膜及丝素风干膜的构象则以α螺旋结构为主，丝素凝胶、冷冻干燥的丝素凝胶及乙醇处理过的丝素风干膜的构象则以β折叠结构为主。同时发现水分在丝素蛋白构象形成和构象转变中起着重要作用，在丝素溶液与丝素凝胶中，由于水分的存在，强极性的水分子促使丝素蛋白分子与水分子之间形成了分子间氢键，这两种状态下的丝素蛋白中含有较多的分子链间β折叠结构；在丝素蛋白溶液形成丝素膜的过程中，由于水分减少，链间氢键随之减少，丝素蛋白链内分子运动的作用形成链内氢键，主要是螺旋结构和链内β结构。随着温度的升高，丝素蛋白中不稳定的螺旋结构向着更稳定的β折叠结构转变，因而温度高的烘干膜中比室温下所成膜含有更多的β折叠结构；丝素蛋白冷冻干燥后，β折叠结构的含量下降。利用乙醇诱导使丝素蛋白溶液发生快速凝胶化。通过研究凝胶化过程中丝素蛋白的形态及红外光谱酰胺Ⅰ带(1600 cm^-1—1670 cm^-1)二阶导数谱变化，发现加入乙醇后，首先肽链间β结构减少，肽链内β结构增加，部分丝素蛋白聚集为白色絮状物。在这些絮状物小核诱导作用下，链间β结构再逐渐增加，丝素蛋白溶液形成凝胶。定量分析的结果表明，丝素蛋白β折叠结构的含量由溶液状态中的17.79％上升到凝胶后的47.20％。热分析的结果显示，凝胶后丝素蛋白的热稳定性增加。探索研究丝素蛋白的构象特点、构象转变过程以及影响构象转变的因素等，对于进一步开发利用丝素蛋白生物材料，解明家蚕成丝机理，拓展蚕丝蛋白用途等具有重要意义，也能为研究其它蛋白的构象转变提供参考。更多还原

【Abstract】 Protein is an important topic in life sciences, and the structure of proteins is one of the most important and difficult aspects. Nowadays, the connection of function and conformation acquired more and more attention with the studies of molecule conformation diseases and man-made protein fibers.FTIR technology is one of the most powerful means in studying the secondary structure of proteins. This method came to a quantitative stage since the 1980s. With the technology of deconvolution, secondary derivative and curve-fitting, a large number of quantitative information of proteins’ secondary structure was obtained.Silk fibroin is an easily obtained and comparatively stable mode protein. Its conformation characteristic, the conformation transition process and the factors affect the process was studied by FTIR.By studying the effects of different solvent systems on the molecular weight and structure of silk fibroin, It’s discovered that: （1）Effect of different salt solutions: LiBr has better dissolving capacity than CaCl₂ in the same condition when dissolving silk fibroin, and the decompostion degree is higher. （2）Effect of temperature and dissolved time: 98±2℃（the boiling temperature of aqueous solutions） is the dissolving temperature when the polypeptides chains were break up to short ones. When it’s below 98±2℃, the silk fibroin was swelled by the solution and mostly dissolved to long polypeptides. When the temperature reached 98±2℃, lots of long polypeptides were decomposed to short polypeptides and single amino acid. （3）Effect of adding organic solvents: Dissolution of silk fibers and decomposition of amino acid from polypeptides will be accelerated with the addition of organic solvents, however, the molecular weight of silk fibroin was unchanged. Different solvent systems have little effect on the secondary structure of silk fibroin. However, the solvent systems: LiBr（98±2℃） and CaCl₂（98±2℃）, which has a better dissolving capacity, decomposed silk fibroin to short polypeptides. These short polypeptides have a worse integrality in secondary structure, which result in difficulty in formation ofα-helix. It’s showed that, 60℃, 9.5mol/L的LiBr solution is the best choice for preparing silk fibroin solution in studying conformation transition.Secondary structure of silk fibroin in different states were studied by FTIR, the result was validated by FTR. Comparing the secondary derivative spectrum and quantitative analyses of fibroin in different states, it’s found that the main conformation in fibroin aqueous solutions was random coils. In the fibroin aqueous solutions（lyophilized）, drying films and air-dry films the main conformation wasα -helix, and it’sβ-sheet in the other states. It’s also found that water plays an important role in the conformation formation and conformation transition. In the solutions and gels, intramolecule hydrogen bonds formed between fibroin molecules and water molecules with the presence of water. So there are much intramoleculeβstructure in these two states; When the water was losing and film gradually formed, the intramolecule hydrogen bonds decreased, the hydrophobic region exposed and interacted to form intermolecule hydrogen bonds, which mainly helix andβstructure. When the temperature rose, the unstable helix changed toβ-sheet, which result in much moreβ-sheet in the drying films than air-dry films. When silk fibroin were lyophilized, content ofβ-sheet was decreased as the water was disappeared.The silk fibroin solution became gel induced by ethanol. Through investigating the change of the shape and the second derivative resolution enhancement spectrum of the amide I (1600 cm^-1 -1670 cm^-1) region, We showed that intramolecularβ-structure in silk fibroin decreased while intermolecularβ-structure increased after the ethanol was added, at the same time white floc appeared. The intramolecularβstructure increased then induced by these nuclears, and the silk fibroin solution became gel formation gradually. The quantitative result showed that the content ofβ-sheet increased from 17.79% in solution to 47.20% in the gel. The thermal stability of silk fibroin enhanced in the process as the DSC result showed.Exploring studying of conformation characteristic, the conformation transition process and the factors that affect the process in silk fibroin has a important significance in exploring the utilization of silk fibroin biomaterials and explaining the mechanism of silk process in Bombyx mori, and providing reference to the studies of other proteins.更多还原