Solid State NMR Structure/Function Studies of Amelogenin

釉原蛋白的固态核磁共振结构/功能研究

• 批准号: 7984641
• 负责人: Wendy J Shaw
• 金额: $ 38.8万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7984641
• 关键词: Address; Affect; Alternative Splicing; Amelogenesis Imperfecta; Amino Acids; Atomic Force Microscopy; Automobile Driving; Binding; Defect; Dental Enamel; Development; Enamel Formation; Environment; Funding; Goals; Growth; Habits; Hydroxyapatites; In Vitro; Ionic Strengths; Kinetics; Knockout Mice; Knowledge; Leucine; Liquid substance; Mechanical Stress; Membrane Proteins; Minerals; Modification; Molecular; Molecular Biology Techniques; Molecular Models; Molecular Structure; Mutate; Mutation; Nanosphere; Play; Point Mutation; Process; Proteins; Quartz; Regulation; Research; Research Design; Research Personnel; Role; Series; Solutions; Staging; Steel; Structure; Surface; Techniques; Therapeutic; Time; Tissues; Variant; Work; amelogenin; biomineralization; bone; design; dimer; extracellular; in vivo; insight; interfacial; leucine-rich amelogenin peptide; loss of function; molecular modeling; monomer; mutant; programs; protein function; protein protein interaction; protein structure; public health relevance; self assembly; 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, 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.

描述（由申请人提供）：本研究的总体目标是阐明驱动牙釉质形成的生物矿化蛋白的界面机制。牙釉质是最有序的生物矿化晶体，经过独特设计，可应对磨损和机械应力。牙釉质、tuftelins、成釉素和釉原蛋白是牙釉质形成过程中存在的蛋白质，所有这些都被认为在牙釉质发育中发挥着关键作用。牙釉质蛋白由牙釉质生长过程中存在的 90% 的蛋白质组成，对于牙釉质的正常形成是必需的，因此，它是拟议研究的主要焦点。在机制层面上，人们对牙釉蛋白如何控制晶体生长知之甚少。众所周知，釉原蛋白形成独特的自组装纳米球，这被认为与发育过程中牙釉质晶体的拉长生长有关。然而，纳米球的组织结构尚未明确，并且蛋白质-羟基磷灰石界面在分子水平上尚不明确。蛋白质结构被认为在牙釉蛋白作为可能的晶体成核剂和生长调节剂的功能中发挥着关键作用，但研究人员一直未能深入了解牙釉蛋白的二级和三级结构。没有任何一种技术能够完全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，然而，几种实验技术的最新进展为开始解决其中一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，特别是关注突变导致的功能丧失。在我们之前的工作基础上，这些研究将利用一系列技术，包括溶液和固态核磁共振、原子力显微镜 (AFM)、石英晶体微天平 (QCM)、恒定成分动力学 (CCK) 和分子建模来研究关键的突出问题牙釉质形成的分子机制。使用 NMR，可以确定天然突变体的二级结构和方向，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态核磁共振来确定，揭示纳米球自组装中涉及的精确残基。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相似条件下生长和成核的差异相关联，将为了解釉原蛋白用于精确控制牙釉质基质的界面机制提供重要的见解。这些见解对于设计针对牙釉质缺陷的治疗方案是必要的。更一般地说，这些研究将为主导所有生物矿物质形成的蛋白质/晶体相互作用提供基本见解。 公共健康相关性：牙釉质是体内矿化程度最高的组织，产生的羟基磷灰石晶体的强度接近钢。釉原蛋白是一种对于这种高度组织化的物质的形成至关重要的蛋白质，但它如何控制牙釉质的形成在分子水平上尚不清楚。我们建议结合现有的最先进技术，阐明蛋白质-蛋白质和蛋白质-羟基磷灰石相互作用机制，这是设计持久疗法之前所必需的见解。