INTRINSICALLY DISORDERED PROTEINS IN BIOMINERALIZATION

描述（由申请人提供）：本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“内在无序”蛋白质（IDPs）在牙齿生物矿化领域的流行。本研究的长期目标是进一步了解细胞外基质蛋白在牙釉质和牙本质中的作用，重点是分析关键蛋白和多肽的折叠和二级结构的紊乱程度。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化蛋白水解活性通过调节非结构化区域的数量和蛋白质序列的紊乱程度来调节蛋白质-矿物质、蛋白质-蛋白质和蛋白质-细胞的相互作用。这种相互作用将影响化学和细胞事件，如细胞信号传导、大分子自组装、晶体成核和生长以及蛋白质去除。本研究提出了三个具体目标：目的1 .应用计算和生物物理策略分析牙釉质和牙本质细胞外基质关键蛋白的二级结构，评估其“紊乱”的程度和性质，以及它们所选择的蛋白水解片段。目的二：利用圆二色性（CD）和荧光滴定法测定成釉素和成釉素关键片段之间的构象变化和相互作用强度，以及DPP与其大分子靶标之间的相互作用。利用核磁共振波谱监测淀粉原蛋白序列中由于与伴侣/靶标结合而发生构象转变的关键氨基酸残基。目的三：利用光谱学技术（CD和ATR-FTIR）监测选定的变釉素、釉素和成釉素蛋白水解片段以及DPP与钙和磷灰石晶体表面相互作用后的二级结构变化和构象转变。总结：牙釉质和牙本质生物矿化是一系列蛋白质-蛋白质、蛋白质-矿物质和蛋白质细胞相互作用的结果。我们的目标是系统地解剖主要牙釉质细胞外基质蛋白以及牙本质唾液磷蛋白（DPP） c端部分与IDPs相关的功能域的序列和二级结构，并提供蛋白质-蛋白质、蛋白质-矿物质和蛋白质-细胞表面相互作用导致的二级结构改变的信息。了解牙釉质和牙本质细胞外基质蛋白质“内在紊乱”的作用，将为仿生材料的制备和发展奠定基础，合成多肽可用于控制晶体成核和生长过程。识别细胞信号传导中未折叠的功能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善口腔、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.