珍珠層相關蛋白之結構鑑定及礦化研究

Abstract

生物成礦指的是生物體製造礦化組織的過程，例如：骨骼、牙齒、棘刺、殼……等。在自然界所有的生物礦物中，碳酸鈣是含量最豐富且被最廣泛研究的一種生物礦物，它具有不同的晶相（方解石、球文石、霰石）。碳酸鈣生物礦物細緻的結構、良好的機械強度以及晶相的調控被認為與生物體中的蛋白、多醣類等大分子有關。其中一種生物礦化相關蛋白的例子便是AP24，此蛋白先前從紅鮑的珍珠層中與另一種蛋白AP7一起被純化出來。先前研究指出，這兩種蛋白可能以二聚體的方式在生物礦化過程中執行其功能性。然而，由於AP24在生理pH值下容易沉澱的特性，人們對全長的AP24的結構及功能並不是了解地非常透徹。為了能進一步了解這個蛋白，本研究首先在AP24較不易沉澱的酸性條件下鑑定其結構。我們使用CD及NMR檢視在添加陽離子、改變pH、與AP7共存的不同條件之下AP24的結構變化。我們發現靜電作用力在AP24分子間交互作用的過程中十分重要。當pH及陽離子濃度增加時，AP24可能由原先單體狀態的隨基螺旋，轉變為具有二級結構的穩定寡聚物。NMR的結果也指出AP7並未對單體狀態的AP24的結構造成明顯的影響。碳酸鈣礦化實驗中，無論是單獨添加AP24或是一起添加AP24及AP7，都未能造成碳酸鈣晶相的選擇性。綜合本研究的實驗結果，我們認為AP24與AP7在碳酸鈣成核的階段並非扮演調控晶相的重要角色。這兩種蛋白或許在礦化過程中具有其他功能，如：對碳酸鈣的形貌調控。其他生物體中的有機基質或許在碳酸鈣晶相調控的過程中也是不可或缺的。至於在蛋白的沉澱中，AP7與AP24是否存在交互作用，則需要透過未來進一步的鑑定（如：固態核磁共振）才能回答這個問題。

Biomineralization refers to the process by which living organisms produce mineralized structures such as their bones, teeth, spines and shells. Among all the biominerals produced by organisms in nature, calcium carbonate is the most abundant and most extensively studied mineral, which exists as different polymorphs (calcite, vaterite, aragonite). The delicate structure, fracture toughness and polymorph selection of calcium carbonate biomineral are attributed to macromolecules such as proteins and polysaccharides in organisms. One of these biomineral proteins, AP24, was copurified with another protein, AP7, from the aragonitic nacreous layer of Haliotis rufescens in previous research. It has been suggested that AP24 and AP7 might function as a dimer in biomineralization process. However, little is known about the structure and function of the full-length AP24 due to its aggregation propensity in physiological conditions. To provide additional insights, we characterized the structure of AP24 under acidic pH, where the aggregation problem was mitigated. Using CD and NMR, the structural change of AP24 in response to different factors such as cation, pH, and AP7 was examined. We find that electrostatic force is a key factor for AP24 intermolecular interaction. When concentration of cations and/or pH increased, AP24 could form stable oligomer that possessed different secondary structure compared with its monomeric counterpart. NMR results also revealed that AP7 did not significantly perturb the conformation of monomeric AP24. In vitro mineralization assays indicated neither AP24 nor the synergy of AP24 and AP7 led to phase selection of calcium carbonate. The results suggest AP24 and AP7 are not the major factors in tuning the nucleation stage of calcium carbonate. They might exhibit other functions such as morphology modulation of minerals. The other organic matrix in nacreous layer could also be involved in the polymorphic selection of calcium carbonate biomineral. Further studies are required to clarify whether there exists interaction between AP24 and AP7 in the aggregation form.