Revealing the Interaction Mechanism of Amelogenin with Hydroxyapaptite

 描述（由申请人提供）：本研究的总体目标是阐明生物矿化蛋白驱动釉质形成的界面机制。牙釉质是最高度有序的生物矿化晶体，并且被独特地设计为处理磨损和机械应力。釉蛋白、成釉蛋白和釉原蛋白是存在于釉质形成过程中的蛋白质，并且都被认为在釉质发育中起关键作用。釉原蛋白是由全长同种型、剪接变体和切割产物组成的蛋白质家族，其由存在于釉质生长期间的至少90%的蛋白质组成。全长同种型对于适当的釉质形成是必需的，因此，它是拟议研究的主要焦点。关于釉原蛋白如何控制晶体生长的机制，人们知之甚少。蛋白质结构被认为在功能中起关键作用 并且已知釉原蛋白形成称为纳米球的自组装四级结构，其被认为与发育期间釉质晶体的伸长生长有关。然而，深入了解纳米球或结合到羟基磷灰石（HAP）的釉原蛋白的二级和三级结构的研究人员已经逃避。没有一种技术能够完全表征控制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，最近几种实验技术的进展为开始解决这些关键问题提供了独特的机会。建立在我们以前的工作，这些研究将利用一套技术，包括先进的，多维的解决方案和固态核磁共振，并在原位原子力显微镜，沿着与其他物理化学方法来研究关键的突出问题的分子机制的釉质形成。使用溶液和固态NMR，全长釉原蛋白和两种天然存在的突变体的二级和三级结构和方向将在纳米球中确定并与HAP结合，HAP是最生物相关的形式。这些技术的应用，允许研究蛋白质>60个残基代表了一个重大的进展，釉原蛋白具体和生物矿化蛋白一般。为了确定哪些残基在与HAP结合中是重要的，将制备一系列具有位点特异性氨基酸取代的釉原蛋白（探针）。晶体生长和结合特性将使用恒定的组成动力学和吸附等温线来表征。将在HAP和纳米球中测定具有改性晶体生长和相互作用性质的探针的二级、三级和四级结构。与天然釉原蛋白相比，将探针的结构和取向结果相关联，将为釉原蛋白用于精细控制釉质的界面机制提供重要的见解。这些分子水平的见解将允许实施生物启发的设计，用于治疗有缺陷的牙釉质的解决方案。更一般地说，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.