Studies of p53 and Amyloidogenic Proteins

本提案的中心主题是氧化还原调节的蛋白质在表面/溶液界面的相互作用。其中一个子项目涉及p53（一种肿瘤抑制蛋白）的电化学研究以及外源电位与p53/DNA结合程度的相关性。另一个子项目将处理淀粉样蛋白固有的氧化还原反应及其与蛋白质纤维性颤动/聚集过程的关系。这项工作将利用各种分析和表面技术来研究DNA/蛋白质和蛋白质/小分子的相互作用。这些技术包括但不限于伏安法、流动注射石英晶体微天平、表面等离子体共振、扫描隧道显微镜、扫描电化学显微镜和原子力显微镜。特别强调将放在伏安法和表面表征p53蛋白和α -突触核蛋白和β -淀粉样蛋白。我们将探索各种固定方案，以使蛋白质具有最佳的表面结构（即表面覆盖和分子取向），并且没有显著的构象变化。动力学测量也将对这些物种与其生物伙伴的相互作用进行（例如，在各种应用潜力下p53与其共识DNA的结合以及潜在治疗药物与淀粉样蛋白的相互作用）。还将研究各种物质（如氧化还原活性金属离子和有机分子以及电活性金属蛋白）对p53和淀粉样蛋白的伏安行为和结合动力学的影响。这些蛋白质的氧化还原特性将与动力学研究和表面表征的结果相关联。还将尝试鉴定淀粉样蛋白纤维性和聚集性的中间产物。本文提出的研究将有助于阐明这两种重要蛋白质在生理相关条件下与其他物种相互作用的机制，并为有效开发癌症和神经退行性疾病的治疗药物提供有用的信息。

The central theme of this proposal is the redox-modulated protein interactions at the surface/solution interface. One subproject is concerned with the electrochemical studies of p53 (a tumor suppression protein) and the correlation of the externally applied potential to the extent of p53/DNA binding. The other subproject will deal with the redox reactions inherent in amyloidogenic proteins and their relationships with the protein fibrillation/aggregation processes. The proposed work will utilize a variety of analytical and surface techniques to study DNA/protein and protein/small molecule interactions. These techniques include, but are not limited to, voltammetry, flow injection quartz crystal microbalance, surface plasmon resonance, scanning tunneling microscopy, scanning electrochemical microscopy, and atomic force microscopy. Particular emphasis will be placed on the voltammetric and surface characterization of p53 protein and alpha-synuclein and beta-amyloid. Various immobilization schemes will be explored to immobilize proteins with optimal surface configuration (i.e., surface coverage and molecular orientation) and without significant conformational changes. Kinetic measurements will also be performed on the interaction of these species with their biological partners (e.g., binding of p53 to its consensus DNA under various applied potentials and interaction of potential therapeutic drugs with amyloidogenic proteins). The influence of various species (e.g., redox-active metal ions and organic molecules and electroactive metalloproteins) on the voltammetric behavior and binding kinetics of p53 and amyloidogenic proteins will also be investigated. The redox properties of these proteins will be correlated with findings from kinetic studies and surface characterization. Attempts will also be made in the identification of intermediates of amyloidogenic protein fibrillation and aggregation. The studies proposed herein should help elucidate the mechanisms involved in the interactions of the two important types of proteins with other species under physiologically relevant conditions and provide useful information to the effective development of therapeutic drugs for cancer and neurodegenerative diseases.