Revealing the Interaction Mechanism of Amelogenin with Hydroxyapaptite

 描述(申请人提供)：这项研究的总体目标是阐明生物矿化蛋白驱动牙釉质形成的界面机制。牙釉质是最有序的生物矿化晶体，专为处理磨损和机械应力而设计。釉质蛋白、成釉蛋白和成釉蛋白是釉质形成过程中存在的蛋白质，它们都被认为在釉质发育中起着关键作用。釉原蛋白是由全长异构体、剪接变异体和切割产物组成的蛋白质家族，由釉质生长过程中存在的至少90%的蛋白质组成。全长异构体是牙釉质正常形成所必需的，因此，它是拟议研究的主要重点。在机理水平上，人们对釉原蛋白如何控制晶体生长知之甚少。蛋白质结构被认为在功能中起着关键作用 已知，釉原蛋白形成一种自组装的四元结构，称为纳米球，被认为在发育过程中与釉质晶体的拉长生长有关。然而，研究人员一直未能深入了解釉原蛋白在纳米球或结合到羟基磷灰石(HAP)中的二级和三级结构。没有一种单一的技术可以完全描述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，然而，最近几项实验技术的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。在我们以前工作的基础上，这些研究将利用一系列技术，包括先进的多维溶液和固体核磁共振，以及原位原子力显微镜，以及其他物理化学方法来研究牙釉质形成分子机制中的关键悬而未决的问题。利用溶液和固体核磁共振，全长釉原蛋白和两个自然产生的突变体的二级和三级结构和取向将在纳米球中确定并与生物相关性最强的形式HAP结合。这些技术的应用使得对蛋白质&GT；60残基的研究成为专门针对釉原蛋白和生物矿化蛋白的重大进步。为了确定哪些残基是与HAP结合的重要残基，将制作一系列具有位点特异性氨基酸替换的釉原蛋白(探针)。晶体生长和结合性能将使用恒定组成动力学和吸附等温线来表征。具有改进的晶体生长和相互作用性质的探针的二级、三级和四级结构将在HAP和纳米球中确定。将探针的结构和取向结果与天然釉原蛋白进行比较，将有助于深入了解釉原蛋白用于精细控制牙釉质的界面机制。这些分子水平的洞察将允许实施生物灵感设计，用于治疗牙釉质缺陷的解决方案。更广泛地说，这些研究将提供基本的见解 蛋白质/晶体相互作用主导了所有生物矿物的形成。

 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.