Revealing the Interaction Mechanism of Amelogenin with Hydroxyapaptite

揭示牙釉蛋白与羟基磷灰石的相互作用机制

• 批准号: 9113535
• 负责人: Wendy J Shaw
• 金额: $ 51.92万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9113535
• 关键词: Address; Adsorption; Advanced Development; Amelogenesis Imperfecta; Amino Acid Substitution; Animal Model; Atomic Force Microscopy; Automobile Driving; Binding; Data; Databases; Dental; Dental Enamel; Development; Enamel Formation; Engineering; Foundations; Goals; Growth; Health; Histidine; Human body; Hydroxyapatites; In Situ; Investigation; Kinetics; Label; Left; Length; Mechanical Stress; Methods; Minerals; Molecular; Molecular Structure; Mus; Mutation; NMR Spectroscopy; Nanosphere; Physical Chemistry; Physiological; Play; Property; Protein Family; Protein Isoforms; Proteins; RNA Splicing; Recombinants; Research; Research Personnel; Role; Scheme; Series; Site; Steel; Structure; Surface; Techniques; Therapeutic; Tissues; Uncertainty; Variant; Work; ameloblastin; amelogenin; biomineralization; design; enamelin; insight; interfacial; leucine-rich amelogenin peptide; monomer; mutant; protein protein interaction; protein structure; solid state nuclear magnetic resonance

 DESCRIPTION (provided by applicant): The overall goal of this research is to elucidate the interfacial mechanisms of the biomineralization proteins driving the formation of enamel. Enamel is the most highly ordered biomineralization crystal and is uniquely designed to handle abrasions and mechanical stress. Enamelins, ameloblastins and amelogenins are proteins present during enamel formation and all have been suggested to play a critical role in enamel development. Amelogenins, a family of proteins consisting of a full-length isoform, splice variants and cleavage products, consists of at least 90% of the protein present during enamel growth. The full-length isoform is necessary for proper enamel formation and as such, it is the primary focus of the proposed studies. Very little is understood at a mechanistic level about how amelogenin controls crystal growth. Protein structure is thought to play a key role in the function of amelogenin and it is known that amelogenin forms into a self-assembled quaternary structure called nanospheres which are thought to be tied to the elongated growth of enamel crystals during development. However, insight into the secondary and tertiary structure of amelogenin in nanospheres or bound to hydroxyapatite (HAP) has eluded researchers. No single technique will fully characterize the protein- protein and protein-crystal interactions controlling enamel formation mechanisms, however, recent advancements in several experimental techniques present a unique opportunity to begin addressing some of these critical questions. Building on our previous work, these studies will utilize a suite of techniques including advanced, multi-dimensional solution and solid state NMR, and in situ atomic force microscopy, along with other physical chemistry methods to study critical outstanding questions in the molecular mechanism of enamel formation. Using solution and solid state NMR, the secondary and tertiary structure and the orientation of full-length amelogenin and two naturally occurring mutants will be determined in the nanosphere and bound to HAP, the most biologically relevant forms. The application of these techniques to allow the investigation of proteins >60 residues represents a major advancement for amelogenin specifically and biomineralization proteins in general. To establish which residues which are important in binding to HAP, a series of amelogenin proteins with site-specific amino acid substitutions (Probes) will be made. Crystal growth and binding properties will be characterized using constant composition kinetics and adsorption isotherms. The secondary, tertiary and quaternary structure of Probes with modified crystal growth and interaction properties will be determined on HAP and in the nanosphere. Correlating the structure and orientation results for the Probes compared to native amelogenin will provide crucial insight into the interfacial mechanisms used by amelogenin for exquisite control of enamel. These molecular level insights will allow the implementation of bioinspired designs for therapeutic solutions to deficient enamel. More generally, these studies will provide basic insight into protein/crystal interactions dominating the formation of all biominerals.

 描述（由应用程序提供）：这项研究的总体目标是阐明驱动搪瓷形成的生物矿化蛋白的界面机制。搪瓷是最有序的生物矿化晶体，其独特而设计用于处理擦伤和机械应力。牙釉质，氨基蛋白酶和氨基蛋白是搪瓷形成过程中存在的蛋白质，所有这些都被建议在牙釉质发育中起关键作用。 Amelogenin是一个由全长同工型，剪接变体和裂解产物组成的蛋白质家族，由牙釉质生长过程中至少90％的蛋白质组成。全长同工型对于适当的搪瓷形成是必需的，因此，它是拟议研究的主要重点。关于氨基氨酸如何控制晶体生长的机械水平，几乎没有理解。蛋白质结构被认为在功能中起关键作用 氨基蛋白酶的蛋白质蛋白众所周知，氨基蛋白蛋白形成了一种称为纳米球的自组装季结构，被认为与发育过程中搪瓷晶体的细长生长有关。然而，对纳米球蛋白或与羟基磷灰石（HAP）结合的氨基蛋白蛋白的二级和三级结构的洞察力已避免了研究人员。没有任何一种技术能够充分表征控制牙釉质形成机制的蛋白质蛋白质和蛋白质 - 晶体相互作用，但是，几种实验技术的最新进步为开始解决其中一些关键问题提供了一个独特的机会。在我们先前的工作的基础上，这些研究将利用一系列技术，包括先进的，多维溶液和固态NMR，以及原位原子力显微镜，以及其他物理化学方法，以研究搪瓷形成分子机制的关键杰出问题。使用溶液和固态NMR，将在纳米球中确定二级和三级结构以及全长氨基蛋白以及两个天然突变体的方向，并与HAP（最相关的形式）确定。这些技术在允许蛋白质投资> 60结果的应用中的应用代表了蛋白质蛋白的主要进步和一般的生物矿化蛋白。为了确定哪些在与HAP结合的结果中，将制定一系列具有位点特异性氨基酸取代（探针）的氨基蛋白蛋白。晶体生长和结合特性将使用恒定组成动力学和增加等温线进行表征。具有改性晶体生长和相互作用特性的探针的次级，第三和第四纪结构将在HAP和纳米球上确定。将探针的结构和定向结果与天然氨基蛋白相比，将提供对氨基蛋白用于独家控制搪瓷的界面机制的关键见解。这些分子水平的见解将允许实施用于治疗溶液的生物启发设计，从而使牙釉质不足。更普遍地，这些研究将提供基本的见解 进入蛋白质/晶体相互作用，主导所有生物矿物的形成。