铁蛋白壳状结构的完全去对称化及利用小分子促使壳状结构形成的研究

Protein-protein interactions are essential to most biological functions causing many of these interactions to be of medicinal interest. Furthermore, the manipulation of protein-protein interactions is becoming an increasingly important priority of the pharmaceutical industry. Therefore understanding the fundamentals of protein-protein interactions and how they control protein oligomer self-assembly could shed light on rudimentary biological processes.. Ferritins are composed of twenty-four identical or homologous subunits that assemble into a large spherical cage with octahedral symmetry. Ferritins and other cage proteins have been the focus of much recent attention as part of drug delivery studies and as platforms of nanostructured materials. Due to the modern interest in their application and the fact that they are composed of monomers folded into a four-helix bundle motif, ferritins could act as an important model system for developing the fundamentals of how to expand the dimensionality of rational protein engineering into the control of self-assembly through the manipulation of protein-protein interactions.. This proposal focuses on establishing the fundamentals of the protein-protein interactions that govern the assembly of the ferritin nano-cage proteins. These proteins form oligomers that are highly symmetric and have a hollow central cavity. Aim 1 sets out to understand how the charged amino acids (R30,D56,E60,K33) at the C2 interface are important for the protein-protein interactions which govern self-assembly in bacterioferritin. They are designed rationally to mutate to the opposite charged amino acids to desymmetrize the octahedral nanocage structure. Aim 2 focuses on the two amino acids N23 and Y114, located on the C2 and C3 interface respectively, that have been discovered to be hot spots on the protein self-assembly. We set out to push the mutations（N23A and Y114A）in reverse directions that utilize small-molecule as the chemical inducers of oligomerization （CIO） to control cage formation by switching the dimer to 24-mer. . Through this endeavour we hope to gain a more thorough understanding of the protein-protein interactions and self-assembly in this model system with the intention of applying what we learn here to the assembly of nano-quaternary structure in general. Moreover, the Chemical Inducers of Oligomerization (CIO) could have eventual applications in controlling delivery, materials formation, and in understanding cellular iron homeostasis.

铁蛋白不仅在药物载体以及纳米结构材料设计等方面已成为研究热点，而且其由24个亚基组成的独特的八面体空间结构也成为研究蛋白质间相互作用的重要模型之一。本项目一方面通过设计将铁蛋白高度对称性结构进行完全去对称化，另一方面从外界引入小分子使已经被破坏的空间结构重组为壳状结构。其中，对已发现的处于C2对称表面的关键氨基酸热点通过计算将其突变成各自相应的反电荷氨基酸以达到获得亚基单体的目的，并研究其对蛋白质结构、热力学性质以及对蛋白自组装的影响，为掌握蛋白质之间相互作用，探讨自组装机理以及为后期蛋白质杂化和应用方面提供理论和实验依据；另外，对前期已经发现的关键氨基酸热点且在溶液中以亚基二聚体形式存在的分别处于C2和C3对称表面的两个突变蛋白，探讨引入小分子作为化学诱导聚合剂促使其重组为24聚体空间结构，为将铁蛋白结构发展成化学诱导聚合系统，最终在控制药物释放、创造新材料等应用方面打下坚实的基础。

铁蛋白是由24个亚基组成的独特的八面体空间结构，是研究蛋白质间相互作用的重要模型。本项目一方面对处于C2对称表面的关键氨基酸热点进行突变以获得亚基单体，并研究突变对蛋白质结构、热力学性质以及蛋白自组装的影响。另一方面，对前期已经发现的处于C2和C3对称表面的氨基酸热点进行研究，探讨引入有机小分子作为化学诱导聚合剂促使其2聚体重组为24聚体空间结构。在所选择的氨基酸热点D56R, E60H, R30D 及D56R/E60H中，除了突变蛋白E60H以24聚体和2聚体混合物形式存在之外，其它三种均只形成2聚体，并没有得到亚基单体，圆二色谱对热力学性质的分析结果与计算软件预测一致。研究证明了处在C2对称的带电荷氨基酸突变会影响铁蛋白的结构及自组装。尝试利用苯、甲苯、苯胺、吲哚等有机小分子促使处在C2对称表面关键突变蛋白N23A由2聚体转为24聚体。非变性凝胶电泳结果显示，小分子的引入未使聚合态发生明显变化。圆二色谱对热力学性质进行分析表明，蛋白质的热力学性质不受小分子苯的影响。将处在大肠杆菌铁蛋白C2对称面上与血红素结合的关键氨基酸残基Met52突变为丙氨酸后，其热力学稳定性明显降低，但可通过外界加入氯化血红素使其稳定性恢复到野生型水平。突变体M52A可通过小分子诱导控制温度稳定性，为后期合成功能性蛋白质纳米材料奠定研究基础。