INTRINSICALLY DISORDERED PROTEINS IN BIOMINERALIZATION

描述(由申请人提供)：这项提案将探索蛋白质与其靶标相互作用的新结构原理，并将调查牙齿生物矿化领域中“固有无序”蛋白质(IDPs)的流行情况。这项研究的长期目标是促进对细胞外基质蛋白在牙釉质和牙本质中的作用的了解，重点是分析关键蛋白和多肽的折叠和二级结构的无序程度。我们认为细胞外基质蛋白中的无序结构域在生物矿化中起着重要作用。我们假设，牙釉质和牙本质中的程序性蛋白分解活动通过调节蛋白质序列中非结构区域的数量和无序程度来调节蛋白质-矿物、蛋白质-蛋白质和蛋白质-细胞之间的相互作用。这种相互作用将影响化学和细胞事件，如细胞信号、大分子自组装、晶体成核和生长以及蛋白质去除。目标1.应用计算和生物物理方法分析牙釉质和牙本质细胞外基质的关键蛋白及其所选择的蛋白降解片段的二级结构，评价其“无序”程度和性质。目的II：利用圆二色谱(CD)和荧光滴定技术测定成釉蛋白和成釉蛋白关键片段之间的构象变化和相互作用强度，以及DPP与其大分子靶标的相互作用。利用核磁共振波谱监测釉原蛋白序列中发生构象转变的关键氨基酸残基，这些氨基酸残基是结合到配对/靶标上的结果。目的：利用光谱技术(CD和ATR-FTIR)监测釉原蛋白、釉蛋白和成釉蛋白的二级结构变化和构象转变，以及DPP与钙和磷灰石晶体表面的相互作用。总结：牙釉质和牙本质的生物矿化是一系列蛋白质-蛋白质、蛋白质-矿物和蛋白质细胞相互作用协调的结果。我们的目标是系统地剖析主要釉质细胞外基质蛋白以及牙本质涎磷蛋白(DPP)与IDPs相关的功能结构域的序列和二级结构，并提供由于蛋白质-蛋白质、蛋白质-矿物和蛋白质-细胞表面相互作用而导致的二级结构变化的信息。了解内源性紊乱在牙釉质和牙本质细胞外基质蛋白中的作用，将为仿生材料的制备和开发奠定基础，届时合成肽将用于控制晶体的成核和生长过程。识别细胞信号中未折叠的功能结构域将对组织再生领域产生重大影响。因此，我们的研究结果将有可能改进口腔、牙齿和颅面组织的修复和再生治疗。 公共卫生相关性：这项提案将探索蛋白质与其靶标相互作用的新结构原理，并将调查牙齿生物矿化领域中“内在无序”蛋白质(IDPs)的流行情况。这些结果将为仿生材料的制备和开发奠定基础，届时合成肽将可以用于控制晶体的成核和生长过程。识别细胞信号中未折叠的功能结构域将对组织再生领域产生重大影响。因此，我们的研究结果将有可能改进口腔、牙齿和颅面组织的修复和再生治疗。

DESCRIPTION (provided by applicant): This proposal will explore novel structural principles by which proteins interact with their targets and will investigates the prevalence of "intrinsically disordered" proteins (IDPs) in the field of tooth biomineralization. The long term objective of the proposed research is to advance understanding of the role of extracellular matrix protein fragmentation in enamel and dentin, with a focus on the analysis of folding and the degree of disorder in the secondary structures of key proteins and polypeptides. We propose that disordered domains in the extracellular matrix proteins play substantial roles in biomineralization. We hypothesize that the programmed proteolytic activities in enamel and dentin regulate protein-mineral, protein-protein and protein- cell interactions through regulation of the number of unstructured regions and the degree of disorder in the protein sequence. Such interactions will affect chemical and cellular events such as cell signaling, macromolecular self-assembly, crystal nucleation and growth, and protein removal. Three specific aims are proposed: Aim I. To apply computational and biophysical strategies to analyze the secondary structures and evaluate the degree and nature of "disorder" in key proteins of the extracellular matrix of enamel and dentin as well as their selected proteolytic fragments. Aim II: To use circular dichroism (CD) and fluorescence titrations to determine the conformational changes and strength of interactions between key fragments of amelogenin and ameloblastin, as well as DPP with their macromolecular targets. To use NMR spectroscopy to monitor key amino acid residues that undergoes conformational transitions in amelogenin sequence as the result of binding to partners/targets. Aim III: To use spectroscopical techniques (CD and ATR-FTIR) to monitor secondary structural changes and conformational transitions of selected amelogenin, enamelin, and ameloblastin proteolytic fragments, as well as DPP following their interactions with calcium and with apatite crystal surfaces. In Summary: Enamel and dentin biomineralization is the result of orchestration among a series of protein-protein, protein-mineral and protein cell interactions. Our goal is to systematically dissect the sequence and secondary structures of functional domains in major enamel extracellular matrix proteins as well as the C-terminal portion of dentin sialophosphoprotein (DPP) with regard to IDPs, and to provide information on secondary structural alteration as the result of protein-protein, protein-mineral and protein-cell surface interactions. Understanding the role of "intrinsic disorder" in proteins of extracellular matrix of enamel and dentin will prepare the ground for the fabrication and development of biomimetic materials when synthetic peptides can be used to control the processes of crystal nucleation and growth. Identification of unfolded functional domains in cell signaling will have a great impact in the field of tissue regeneration. The outcomes of our study will therefore have the potential to improve treatments for repair and regeneration of oral, dental and craniofacial tissues. PUBLIC HEALTH RELEVANCE: This proposal will explore novel structural principles by which proteins interact with their targets and will investigates the prevalence of "intrinsically disordered" proteins (IDPs) in the field of tooth biomineralization. The outcomes will prepare the ground for the fabrication and development of biomimetic materials when synthetic peptides can be used to control the processes of crystal nucleation and growth. Identification of unfolded functional domains in cell signaling will have a great impact in the field of tissue regeneration. The outcomes of our study will therefore have the potential to improve treatments for repair and regeneration of oral, dental and craniofacial tissues.