Solid State NMR Structure/Function Studies of Amelogenin

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、tuftelins、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。釉原蛋白由釉质生长过程中存在的90%的蛋白质组成，是适当的釉质形成所必需的，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。已知釉原蛋白形成独特的自组装纳米球，其被认为与发育期间釉质晶体的伸长生长有关。然而，纳米球的组织没有很好地定义，并且蛋白质-羟基磷灰石界面没有在分子水平上被理解。蛋白质结构被认为在釉原蛋白作为一种可能的晶体成核剂和生长调节剂的功能中起着关键作用，但对釉原蛋白的二级和三级结构的了解一直困扰着研究人员。没有一种技术能够完全描述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。将蛋白质-蛋白质和蛋白质-表面相互作用与功能联系起来将是拟议工作的重点，特别是关注由于突变而导致的功能丧失。在我们以前的工作的基础上，这些研究将利用一套技术，包括溶液和固态NMR，原子力显微镜（AFM），石英晶体微量天平（QCM），恒定组成动力学（CCK）和分子建模，以研究釉质形成的分子机制中的关键突出问题。使用NMR，将确定天然存在的突变体的二级结构和取向，并与野生型蛋白质的结构进行比较。还将研究pH、离子强度和蛋白质浓度的影响。AFM将用于确定吸附蛋白质的四级结构。蛋白质-蛋白质相互作用将使用溶液状态NMR来确定，从而揭示参与纳米球自组装的精确残基。为了提供结构和功能之间的相关性，QCM和CCK将用于在结构研究中使用的相同条件下研究成核速率、生长抑制和晶体修饰。将结构和取向结果与在类似条件下生长和成核的差异相关联，将为釉原蛋白用于釉质基质的精细控制的界面机制提供至关重要的见解。这些见解是必要的治疗解决方案，以缺乏牙釉质的设计。更一般地说，这些研究将提供基本的洞察蛋白质/晶体相互作用主导所有生物矿物的形成。 公共卫生相关性：牙釉质是人体内矿化程度最高的组织，产生的羟基磷灰石晶体强度接近钢。釉原蛋白是一种对这种高度组织化材料的形成至关重要的蛋白质，但它如何控制釉质形成还没有在分子水平上被理解。使用现有的最先进的技术相结合，我们建议阐明蛋白质-蛋白质和蛋白质-羟基磷灰石相互作用机制，在设计持久的治疗方法之前，这是必要的。

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, tuftelins, ameloblastins and amelogenins are proteins present during enamel formation and all have been suggested to play a critical role in enamel development. Amelogenin consists of 90% of the protein present during enamel growth, 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. It is known that amelogenin forms into unique self assembled nanospheres which are thought to be tied to the elongated growth of enamel crystals during development. However, the organization of the nanosphere is not well defined, and the protein- hydroxyapatite interface is not understood on a molecular level. Protein structure is thought to play a key role in the function of amelogenin as a possible crystal nucleator and growth regulator, but insight into the secondary and tertiary structure of amelogenin has eluded researchers. No single technique will fully characterize the protein-protein and protein-crystal interactions controlling enamel formation mechansims, however, recent advancements in several experimental techniques present a unique opportunity to begin addressing some of these critical questions. Relating the protein-protein and protein-surface interactions to function will be the emphasis of the proposed work, particularly focusing on the loss of function as a result of mutation. Building on our previous work,these studies will utilize a suite of techniques including solution and solid state NMR, atomic force microscopy (AFM), quartz crystal microbalance (QCM), constant composition kinetics (CCK) and molecular modeling to study critical outstanding questions in the molecular mechanism of enamel formation. Using NMR, the secondary structure and the orientation of naturally occurring mutants will be determined and compared to the structure of the wildtype protein. The affect of pH, ionic strength and protein concentration will also be investigated. AFM will be used to determine the quaternary structure of the adsorbed protein. Protein-protein interactions will be determined using solution state NMR, revealing precise residues involved in nanosphere self-assembly. To provide a correlation between structure and function, QCM and CCK will be used to investigate nucleation rates, growth inhibition and crystal modification under identical conditions used in the structural studies. Correlating the structure and orientation results with differences in growth and nucleation under similar conditions will provide crucial insight into the interfacial mechanisms used by amelogenin for exquisite control of the enamel matrix. These insights are necessary for the design of theraputic solutions to deficient enamel. More generally, these studies will provide basic insight into protein/crystal interactions dominating the formation of all biominerals. PUBLIC HEALTH RELEVANCE: Enamel is the most highly mineralized tissue in the body, and produces hydroxyapatite crystals with a strength approaching that of steel. Amelogenin is a protein that is critical to the formation of this highly organized material, but how it controls enamel formation is not understood on a molecular level. Using a combination of the most advanced techniques available, we propose to elucidate the protein-protein and protein-hydroxyapatite interaction mechanisms, insights that are necessary before long-lasting therapeutics can be designed.