【作者】 苏境坦；

【导师】 张荣庆；

【作者基本信息】 清华大学 ， 海洋生物学， 2013， 博士

【摘要】 无定形碳酸钙（amorphous calcium carbonate，ACC）是碳酸钙生物矿物矿化的前体。ACC的调控机制是近几年生物矿化研究的前沿与热点。围绕着这个热点，本论文研究了无定形碳酸钙结合蛋白（amorphous calciumcarbonate-binding protein，ACCBP）对ACC形成与转化的调控机制。ACCBP是我们实验室首次分离并鉴定的合浦珠母贝（Pinctada fucata）间液蛋白质，它在体外可以诱导ACC的形成。结合生物化学和生物信息学手段，我们深入研究了ACCBP诱导ACC形成的结构基础。分子排阻色谱、化学交联反应以及负染电镜表明，ACCBP是一个由两个五聚体叠加形成的十聚体；氨基酸序列分析、荧光淬灭实验和体外碳酸钙结晶实验表明，ACCBP具有两个钙离子结合位点（DYDSD和DGD），前一个位点对ACCBP诱导ACC的功能至关重要，后一个位点影响ACCBP诱导ACC的效率；由于ACCBP的同源蛋白是以五聚体形式存在，且ACCBP是一个由五聚体叠加形成的十聚体，以同源性为32%的α-7烟碱受体蛋白（α-7nicotinic receptor）的五聚体晶体结构为模板，进行同源蛋白质结构建模，得到ACCBP的三级结构。结果表明，ACCBP的钙离子结合位点不能直接作为诱导ACC的模板，可能通过其五次轴对称性来保持钙离子的非晶态，促使ACC的形成。体外碳酸钙结晶实验和ACC转化实验表明，ACCBP可以在低镁溶液中诱导ACC转化为文石；负染电镜表明，镁离子不能通过改变ACCBP的寡聚体状态使其形成传统的文石晶核模板；SEM和激光粒度分析表明ACCBP对ACC的形貌、尺寸基本没有影响；Micro-Raman和FTIR光谱分析表明ACCBP对ACC的微观原子排布基本没有影响；ICP分析表明ACCBP对ACC的体相Mg/Ca比例基本没有影响；XPS则表明ACCBP可以显著的提高ACC的表面Mg/Ca比例。结合镁离子对晶型的影响，可以推测，ACCBP通过将镁离子富集到ACC表面，从而调控ACC转化过程中的晶型选择。荧光淬灭实验表明，ACCBP具有比较强的钙离子和镁离子结合能力，这是ACCBP能够将镁离子富集到ACC表面的基础。本论文工作充实了ACC前体的矿化理论，促进了对贝壳珍珠层和珍珠形成机制的了解，对基于生物矿物的材料设计有启示意义。更多还原

【Abstract】 Amorphous calcium carbonate (ACC) plays an important role in biomineralizationprocess for its function as a precursor for calcium carbonate biominerals. Themechanisms for ACC regulation is one of the most interesting questions in the field ofbiomineralization. In this thesis, we studied the function of amorphous calciumcarbonate-binding (ACCBP) during ACC formation and transformation, and themechanisms for it.Biochemical experiments coupled with bioinformatics approaches were usded toexplore the mechanisms of ACC formation controlled by ACCBP. Size-exclusionchromatography, chemical cross-linking experiments and negative staining electronmicroscopy reveal that ACCBP is a decamer composed of two adjacent pentamers.Sequence analyses and fluorescence quenching results indicate that ACCBP containstwo Ca2+-binding sites. The results of in vitro crystallization experiments suggest thatone Ca2+-binding site is critical for ACC formation and the other site affects the ACCinduction efficiency. As homologous proteins of ACCBP are all pentamers and ACCBPitself is a decamer comprosed of two pentamers, a homology modeling was conductedwith the structure of a pentermeric protein, α-7nicotinic receptor. Homology modelingdemonstrates that the Ca2+-binding sites of pentameric ACCBP are arranged in a five-fold symmetry, which is the structural basis for ACC formation. To the best of ourknowledge, this is the first report on the structural basis for protein-induced ACCformation and it will significantly improve our understanding of the amorphousprecursor pathway.Another key question in the amorphous precursor pathway is the polymorphdetermination which is delicately controlled by biomacromolecules, especially proteins.However, the mechanism of protein-mediated polymorph determination is still unclear.In this thesis, in vitro crystallization experiments and ACC transformation experimentsshow that ACCBP induces aragonite via ACC precursor in solution with low Mg/Ca atlow temperature. SEM images show that the ACC precursor in the ACC transformationexperiment is nanograins. XPS, ICP, Raman, and FTIR analyses on the ACC nanograins show that ACCBP increases Mg/Ca molar ratio on the surface of ACC, butdoes not regulate the bulk Mg/Ca ratio or the short-range order structure. It is proposedthat the polymorph switching is controlled by increasing the surficial Mg/Ca of transientACC nanograins, and it may be the mechanism for polymorph selection in vivo. Theseresults would contribute to the understanding of biomineralization, particularly howprotein cooperate with Mg2+on the transformation of amorphous calcium carbonate.In summary, the functions of ACCBP in the biomineralization process werestudied and the mechanisms for ACC formation and polymorph determination in theamorphous precursor pathway were discussed in this thesis.更多还原