STRUCTURAL BIOLOGY OF THE ENAMEL PROTEINS

生物矿化组织是生物陶瓷-生物聚合物复合材料，由 细胞介导的过程。当前的矩阵中介概念 生物矿化作用假设特定的分子联系 有机基质组成与发展中的无机矿物相 这样矿物成核和随后对晶体生长的控制 和习性以一种产生有序矿化的方式发生 骨、牙本质和牙釉质等结构。这一决定决定了 牙釉质独特结构图案的大小、形状和位置 蛋白质是进一步研究的必要前提。 釉质基因产物在遗传病和正常人中的功能作用 釉质生物矿化。此外，蛋白质工程的进展可能 有望导致新的生物陶瓷材料的生产， 可能是基于牙釉质形成的原理，并在 临床牙科。这个实验室最近的研究已经证实 在小鼠体内，七种釉原蛋白由 来自单个X连锁基因的选择性剪接，并使我们能够 在原核系统中表达其中的一些蛋白质。我们假设 这些釉原蛋白对正常的形成和 牙釉质的发展。 这项建议旨在确定第二、第三和第四纪 釉原蛋白的结构。我们建议使用重组人 小鼠釉原蛋白，在原核和真核系统中表达， 来表征和分析这些蛋白质结构。总而言之，我们 提出四个具体目标：(I)表达、分离和纯化每个 交替剪接的小鼠釉原蛋白和端粒蛋白。(Ii)至 根据一级结构对表达的蛋白质进行特征分析 和翻译后修饰。(三)确定次要 釉原蛋白和端粒蛋白的三级结构。(Iv)至 表征釉原蛋白和端粒蛋白的四级结构； 聚集体的形成和相互作用。 从细菌和/或酵母细胞中分离重组蛋白 培养并通过层析程序进行纯化。初级阶段 表达蛋白的结构是由氨基酸确定的 成分和序列分析与质谱学相结合 确认分子量。纯化的特征蛋白 通过核磁进行二次和三次分析 核磁共振、圆二色谱和蛋白质结晶-- X射线衍射法。蛋白质-蛋白质相互作用和聚集体 自组装机构的特征是动态的组合 光散射(DLS)、高分辨率透射电子显微镜 (TM)、尺寸排除层析(SEC)和原子力显微镜 (AFM)。最后，对从这些研究中得出的数据进行整理，以提供 釉原蛋白结构和推测的特异性的分子模型 蛋白质-矿物质和蛋白质-蛋白质相互作用 牙釉质生物矿化的调控。

Biomineralized tissues are bioceramic-biopolymer composites produced by cell-mediated processes. Current concepts of matrix-mediated biomineralization postulate specific molecular associations between the organic matrix constitutes and the developing inorganic mineral phase such that mineral nucleation and the subsequent control of crystal growth and habit occur in a manner which generates ordered mineralized structures such as bone, dentine and enamel. The determination of the size, shape and location of unique structural motifs of the enamel proteins is an essential prerequisite to advance studies of the functional role of enamel gene products in genetic diseases and in normal enamel biomineralization. Moreover, advances in protein engineering may be expected to lead to the production of new bioceramic materials, perhaps based on principles of enamel formation, with applications in clinical dentistry. Recent studies from this laboratory have established that in the mouse, seven amelogenin proteins are expressed by alternative-splicing from the single X-linked gene and have enabled us to express some of these proteins in a prokaryotic system. We postulate that these amelogenin proteins ar pivotal to the normal formation and development of dental enamel. This Proposal seeks to determine the secondary, tertiary and quaternary structures of the amelogenin proteins. We propose to employ recombinant mouse amelogenins, expressed in both prokaryotic and eukaryotic systems, to characterize and analyze these protein structures. In summary, we propose four Specific Aims: (i) To express, isolate and purify each of the alternatively-spliced murine amelogenins and tuftelins. (ii) To characterize the expressed proteins in terms of their primary structures and post-translational modifications. (iii) To determine the secondary and tertiary structures of amelogenis and tuftelins. (iv) To characterize the quaternary structures of amelogenins and tuftelins; aggregate formation and interactions. Recombinant proteins are isolated from bacterial and/or yeast cell cultures and purified by chromatographic procedures. The primary structures of the expressed proteins are established by amino acid composition and sequence analyses together with mass spectrographic confirmation of molecular weights. The purified characterized proteins are subjected to secondary and tertiary analyses through nuclear magnetic resonance (NMR), circular dichroism (CD) and protein crystallization - X-ray diffraction procedures. Protein-protein interactions and aggregate self-assembly mechanisms are characterized by a combination of dynamic light scattering (DLS), high-resolution transmission electron microscopy (TEM), size-exclusion chromatography (SEC) and atomic force microscopy (AFM). Finally, data derived from these studies are collated to provide molecular models for amelogenin structures and for the putative specific protein-mineral and protein-protein interactions required for the regulation of dental enamel biomineralization.