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， 细胞介导的过程。 基质介导的当前概念 生物矿化假设了生物体之间的特定分子关联， 有机基质组成和发展中的无机矿物相 使得矿物成核和随后晶体生长的控制 和习惯发生的方式，产生有序矿化 例如骨骼、牙本质和牙釉质。 的确定 釉质独特结构图案的大小、形状和位置 蛋白质是推进研究的必要先决条件。 釉质基因产物在遗传性疾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 有望引领新型生物陶瓷材料的生产， 也许是基于釉质形成的原理， 临床牙科学 该实验室最近的研究已经建立了 在小鼠中，有7种釉原蛋白由 选择性剪接从单一的X连锁基因，使我们能够 在原核系统中表达这些蛋白质。 我们推测 这些釉原蛋白是正常形成的关键， 牙釉质的发育 本建议旨在确定第二、第三和第四 釉原蛋白的结构。 我们建议使用重组 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。 总之，我们 提出了四个具体目标：（一）表达，分离和纯化每一个 选择性剪接的鼠牙釉原蛋白和tuftelins。 (ii)到 根据其一级结构表征表达的蛋白质 和翻译后修饰。 (iii)为了确定次级 以及成釉细胞和簇蛋白的三级结构。 (iv)到 表征釉原蛋白和tuftelins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 培养并通过色谱法纯化。 主 表达的蛋白质的结构由氨基酸序列确定， 组成和序列分析以及质谱分析 确认分子量。 纯化的特征蛋白 通过核磁共振进行二级和三级分析 共振（NMR）、圆二色性（CD）和蛋白质结晶- X射线衍射程序。 蛋白质-蛋白质相互作用和聚集 自组装机制的特点是结合了动态 光散射（DLS），高分辨率透射电子显微镜 (TEM)，尺寸排阻色谱法（SEC）和原子力显微镜 （AFM）。 最后，整理这些研究的数据， 釉原蛋白结构的分子模型和推测的特异性 蛋白质-矿物质和蛋白质-蛋白质相互作用所需的 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.