UNS: COLLABORATIVE RESEARCH: Study of Amyloid Protein Oligomerization Using Microchannel Electrophoresis

1511562/1511876 MOSS/Hestekin淀粉样蛋白的聚集参与了广泛的过程，包括许多疾病的发病机制(阿尔茨海默病、2型糖尿病等)。以及与食品生产或水净化等应用相关的生物污垢。更好地了解聚集过程不仅有助于防止不必要的蛋白质聚集，还可以用于设计小规模的制造过程，其中分子能够以有序和可重复的方式组装，例如用于电子产品的纳米线的生产。这个项目的目标是通过研究与疾病和生物污垢有关的蛋白质来发现启动蛋白质聚集的主要机制。为了了解这一机制，蛋白质的关键性质将以受控的方式改变，并将使用分析聚集大小和结构的方法来探索它们对蛋白质聚集的影响。此外，还将研究在抑制分子存在的情况下淀粉样蛋白行为的变化。通过提高我们对蛋白质聚集早期阶段的了解，我们将能够设计出更好的药物来治疗衰弱疾病，减少不必要的生物污垢，并促进工业应用。拟议的工作采用了定制的微通道电泳(ME)系统，该系统利用混合聚合物基质来促进分离跨越广泛分子量范围的单个寡聚体群体。将研究三种模型蛋白：参与阿尔茨海默病发病机制的淀粉样β蛋白，2型糖尿病的致病蛋白淀粉蛋白，以及对生物污垢至关重要的卓别林H蛋白。ME将被用来阐明蛋白质氨基酸序列，包括疏水延伸的长度和蛋白质的灵活性，对寡聚体聚集体进化的影响。此外，ME还将用于研究8种已知的小分子聚集抑制物调节低聚物形成的能力。低聚物大小的确定以及识别疏水结构域和淀粉样结构的荧光染料的结合将与时间相关的研究相结合，以确定低聚物的构象、大小、出现时间和形成的相对数量。定制ME的双光学检测能力将被利用，通过UV和结合的荧光染料同时检测低聚物。了解蛋白质氨基酸序列和小分子抑制剂对淀粉样蛋白齐聚的影响，将为抑制和操纵蛋白质聚集及其在药物开发、药物生产、生物污垢和纳米技术中的应用开辟新的方向。实验工作将与生物医学工程教育相结合。具体地说，拟议的工作将建立一个面向六名本科生的协作本科生研究交流，并将开发一个基于设计的模块，分发给高中和中学教师，让大学预科学生熟悉蛋白质聚集和蛋白质聚集分离的概念。该奖项由CBET分部生物技术和生化工程计划共同资助，由材料研究部生物材料计划和刺激竞争性研究的实验计划(EPSCoR)共同资助。

1511562/1511876 Moss/HestekinThe aggregation of amyloid proteins is involved in a wide range of processes including the pathogenesis of numerous diseases (Alzheimer's disease, type 2 diabetes, etc.) as well as in biofouling associated with applications such as food production or water purification. A better understanding of the aggregation process will not only help to prevent unwanted protein aggregation, but may also be used to design small-scale manufacturing processes where molecules are able to assemble in an ordered and repeatable fashion, such as in the production of nanowires for electronics. The goal of this project is to discover the primary mechanism involved in the initiation of protein aggregation by examining proteins involved in diseases and biofouling. In order to understand this mechanism, key properties of the proteins will be altered in a controlled manner and their effect on protein aggregation will be explored using methods that analyze aggregate size and structure. In addition, changes in amyloid behavior in the presence of inhibitor molecules will be examined. By advancing our understanding of these early stages of protein aggregation, we will be able to design better drugs for treating debilitating diseases, reduce unwanted biofouling, and facilitate industrial applications.The proposed work employs a custom Microchannel Electrophoresis (ME) system that utilizes a blended polymer matrix to facilitate separation of individual oligomer populations that span a wide range of molecular weights. Three model proteins will be studied: amyloid-beta, involved in the pathogenesis of Alzheimer's disease, amylin, a pathogenic protein in type 2 diabetes, and chaplin H, a protein critical for biofouling. ME will be employed to elucidate the effect of protein amino acid sequence, including the length of hydrophobic stretches and protein flexibility, on the evolution of oligomeric aggregates. In addition, ME will be employed to investigate the ability of eight known small molecule inhibitors of aggregation to modulate oligomer formation. Oligomer size determination as well as binding of florescent dyes that recognize hydrophobic domains and amyloid structure will be coupled with time-dependent studies to define the conformation, size, time to appearance, and relative quantity of oligomers formed. The custom ME's dual optical detection capabilities will be exploited to simultaneously detect oligomers via UV and bound fluorescent dyes. The advancement in understanding the influences of protein amino acid sequence and small molecule inhibitors on amyloid protein oligomerization will define new directions for the inhibition and manipulation of protein aggregation with applications in drug development, pharmaceutical production, biofouling, and nanotechnology. The experimental work will be integrated with biomedical engineering education. Specifically, the proposed work will establish a collaborative undergraduate research exchange for six undergraduate students and will develop a design based module to be distribution to high school and middle school teachers that familiarizes precollege students with the concepts of protein aggregation and separation of protein aggregates. This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Biomaterials Program of the Division of Materials Research and by the Experimental Program to Stimulate Competitive Research (EPSCoR).