【作者】 李欣欣；

【导师】 张密林；

【作者基本信息】 哈尔滨工程大学 ， 材料学， 2009， 博士

【摘要】 蛋白质晶体学是一门研究蛋白质结构与功能的边缘学科。随着生物学领域对精确的生物大分子结构需求的日益增加,尤其是利用单晶X射线衍射法得到的三维结构,使得研究生物大分子晶体的生长越来越受到重视。然而,生物大分子的结晶相当困难,从某种意义上讲这是生物大分子结构测定的限制因素。离子液体具有独特的结构和物理化学性质,已经成为目前化学界的热点课题之一,而且在生命科学领域也开始得到应用,尤其是作为生物催化反应的溶剂或共溶剂的研究更是备受关注,近年来,离子液体也开始应用于蛋白质结晶方面。本论文将一种新体系——离子液体-水用于蛋白质的结晶。在该体系中,实现了蛋白质晶体在离子液体中的可控生长,培养了符合X射线衍射条件的蛋白质单晶,对离子液体在蛋白质结晶过程中的作用机理进行了探讨,并剖析了离子液体含量与蛋白质晶体的晶型、形貌和品质间的关系。此外,从动力学角度系统地分析了离子液体在蛋白质结晶过程中对晶体生长速率的控制作用,进而优化结晶条件,改善晶体质量。采用一步法和两步法分别合成了离子液体[BMIM]Cl（1-丁基-3-甲基咪唑氯化物）、[BMIM]BF₄（1-丁基-3-甲基咪唑四氟硼酸盐）和[BMIM]PF₆（1-丁基-3-甲基咪唑六氟磷酸盐）,借助红外光谱分析表征了其化学结构。其次,为了进一步研究离子液体对蛋白质晶体生长过程的影响机制,采用BCA法通过紫外—可见分光光度计测定蛋白质在离子液体—水混合溶液中的溶解度,结果表明,离子液体在体系中起到了类似无机盐类沉淀剂的作用,促进了蛋白质分子从溶液中的析出。通过测定[BMIM]BF₄-乙酸乙酸钠-NaCl离子液体双水相体系的三元体系相图,研究了培养溶液中各组分间的溶解度关系,并将其用于蛋白质晶体的培养,以控制了溶液的局部过饱和度。采用悬滴法和批量法两种结晶方法,在离子液体-水体系中实现了对溶菌酶晶体的可控生长,得到了空间群为P4₃2₁2的四方晶系晶体,并对其结构进行了模型解析工作。考察了沉淀剂种类、溶液过饱和度、离子液体的含量、结晶温度等实验参数的改变对溶菌酶晶型、晶体形貌和品质的影响。研究表明,[BMIM]BF₄更适于溶菌酶晶体的培养,在该体系中,[BMIM]BF₄与NaCl一起作为联合沉淀剂使用,晶体尺寸明显增大,结晶速率得以控制,晶体质量有所改善。另外,随着[BMIM]BF₄含量的增加,晶体形貌逐渐由块状变为棒状,且长径比也随之增大。采用悬滴结晶法,在[BMIM]BF₄-水体系中实现了对索马甜蛋白单晶体的可控生长,得到了空间群为P4₁2₁2的四方晶系晶体,并解析了其初始模型结构。在该体系中,[BMIM]BF₄被单独的作为沉淀剂使用,并且研究了结晶温度、离子液体[BMIM]BF₄的含量等培养条件对索马甜蛋白晶型和品质的影响。离子液体作为一种具有特殊结构和功能的溶剂,在蛋白质结晶的过程中具有与无机盐类沉淀剂相类似的作用。同时形成双水相体系降低溶液的局部过饱和度,发挥了模板剂、联合沉淀剂和添加剂的多重作用,有效的控制晶核的生成数量,提高单晶出现的几率。可以通过调节离子液体的含量来控制蛋白质晶体的生长速率、大小和质量。更多还原

【Abstract】 Protein crystallography is an interdisciplinary subject in the research of the structure and function of the protein. With the increasing demands of precise biomolecular 3-D structures, especially protein structures by X-ray diffraction, people pay more and more attention to the study of protein crystal growth. However, the crystallization of protein is very difficult and become a bottleneck in structural genomics, to some extent.Ionic liquids （ILs） with unique structure and outstanding physical and chemical properties have become the focus in the chemistry field. And it is also used in the field of life science, especially in the biocatalyst reaction as solvent or cosolvent. Recently, ionic liquids are also applied in protein crystallization.In this work, a new system of ionic liquid-water was tested for the protein crystallization. The procedure of crystallization could be controllable conducted in this system. The mechanisms for formations of controlled growth and the relationship among polymorph, morphology, quality and ionic liquid content were investigated. Moreover, the limitation effect of ionic liquid on the rate of crystal growth was disscussed from the view of kinetics.Ionic liquids of [BMIM]Cl, [BMIM]BF₄ and [BMIM]PF₆ were prepared through Direct Method and Two-step Method, respectively. And the chemical structures of the final products were characterized by Infrared spectra. For the further studies on the effects of the ILs on the crystal growth, the solubility of protein was mensurated with ultraviolet-visible light spectrophotometer by the way of BCA method. It was found that ionic liquid had similar effect as precipitants or additions, which was favourable for the single crystals. On the other hand, in order to understand their dissolved relationship among each components in the solution, the equilibrium solubility of a ternary system of [BMIM]BF₄, NaCl and buffer as well as the data of liquid-liquid equilibrium of that system are determined at 25℃and atmospheric pressure. It was used for the protein crystallization, and played a role in the conrol of local supersaturation.Two crystallization methods （hanging drop and sitting drop crystallization） were used for the lysozyme crystallization in the ionic liquids with different buffer/IL proportions. Lysozyme crystal belonged to the tetragonal space group P4₃2₁2 and its 3-D model was analyzed. The influence of growth conditions such as the kind of precipitants, solution supersaturation, ionic liquids content and temperature on polymorph, morphology, quality of crystals was discussed. It showed that [BMIM]BF₄ was more suitable for lysozyme crystallization than other ILs. [BMIM]BF₄ and NaCl were used as union-precipitants in the system of [BMIM]BF₄-water. More lager single crystals with controllable morphologies could be obtained due to the manageable crystal growth velocity. In addition, the morphology of lysozyme crystals were changed from bolck to rod and the L/D ratio increased with the proportion of IL.Futhermore, thaumatin crystal, which was belonged to the tetragonal space group P4₁2₁2, could be obtained with hanging drop crystallization. [BMIM]BF₄ was used as a precipitant, and the effect of ionic liquids content, temperature on polymorph and quality of crystals was investigated.On a whole, ionic liquid was a solvent with special structure and function. More importantly, aqueous two-phase system was appeared with diffusion of water molecules. As a new system, it might play an important role in promoting the growth of single crystals as opposed to polycrystals, in limiting nucleation, and in controlling the supersaturation of solution. The size, quality and growth rate of crystals might be controlled by adjusting content of ionic liquid.更多还原