STRUCTURAL BIOLOGY OF THE ENAMEL PROTEINS

牙釉质蛋白质的结构生物学

• 批准号: 6104691
• 负责人: ALAN G FINCHAM
• 金额: $ 38.86万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-15 至 2001-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6104691
• 关键词: X ray crystallography; amelogenin; animal tissue; atomic force microscopy; chromatography; circular dichroism; dental development; endopeptidases; intermolecular interaction; molecular assembly /self assembly; nuclear magnetic resonance spectroscopy; posttranslational modifications; protein purification; protein sequence; protein structure function; recombinant proteins; tooth enamel; transmission electron microscopy

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.

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。 基质介导的当前概念 生物矿化假设了特定的分子关联 有机基质构成和发展中的无机矿物相 以便矿物成核和随后的晶体生长控制 和习惯以产生有序矿化的方式发生 骨骼、牙本质和牙釉质等结构。 的确定 牙釉质独特结构图案的大小、形状和位置 蛋白质是推进蛋白质研究的重要前提 牙釉质基因产物在遗传疾病和正常人中的功能作用 牙釉质生物矿化。 此外，蛋白质工程的进步可能 预计将导致新型生物陶瓷材料的生产， 或许基于牙釉质形成原理，并应用于 临床牙科。 该实验室最近的研究表明 在小鼠中，七种釉原蛋白由 来自单个 X 连锁基因的选择性剪接使我们能够 在原核系统中表达其中一些蛋白质。 我们假设 这些釉原蛋白对于正常形成至关重要 牙釉质的发育。 本提案旨在确定二级、三级和四级 釉原蛋白的结构。 我们建议采用重组 小鼠釉原蛋白，在原核和真核系统中表达， 表征和分析这些蛋白质结构。 综上所述，我们 提出四个具体目标： (i) 表达、分离和纯化每一个 选择性剪接的鼠牙釉蛋白和 tuftelins。 (二) 至 根据表达的蛋白质的一级结构表征其特征 和翻译后修饰。 (iii) 确定次要 以及 amelogenis 和 tuftelins 的三级结构。 (四) 至 表征牙釉蛋白和 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 培养物并通过色谱程序纯化。 初级 表达蛋白质的结构由氨基酸确定 成分和序列分析以及质谱分析 分子量的确认。 纯化的特征蛋白 通过核磁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动态的组合 光散射 (DLS)、高分辨率透射电子显微镜 (TEM)、尺寸排阻色谱 (SEC) 和原子力显微镜 （原子力显微镜）。 最后，将从这些研究中获得的数据进行整理以提供 牙釉蛋白结构和假定的特定分子模型 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节。