A conformation-switching peptide probe for rapid, specific, quantitative and sensitive detection of amyloid aggregates

知识价值。该项目的目标是开发一种构象切换肽探针，用于快速，特异，定量和敏感地检测构象不同但相似的分析物。我们的目标分析物是由天然未折叠肽形成的聚集体，&amp；淀粉样蛋白（Aβ）与老年痴呆症有关？阿尔茨海默病（AD），已知形成寡聚体和原纤维组装体。异质A&amp；#946；在聚集过程中形成的聚集体表现出不同的构象，并发挥不同程度的毒性作用。从而准确可靠地检测出不同的A&amp；#946；聚合是朝着更好地理解自然的确切性质迈出的重要一步。早期诊断和治疗药物的聚集和发展。不幸的是，不同的原子之间存在相似的构象。聚合使得特定的检测极其困难。快速、特异、定量和敏感的检测尤为重要，但目前在探测方面还无法实现。寡聚物，被认为是AD的主要毒性物质。在我们之前的研究中，我们开发了一种肽探针PG46，用于快速，特异性和定量检测A&amp；#946；低聚物。PG46是通过整合A&amp；#946；自我识别序列与构象敏感的双砷染料，闪光。PG46被发现特异性结合到A&amp；#946；在这种结合上显示出闪光荧光的增加。快速定量测量A&amp；#946；PG46也可能存在低聚物。A&amp；#946检测灵敏度；对PG46进行了进一步的工程修饰。我们目前的肽探针的灵敏度虽然显著，但仍然不是最佳的。肽探针检测其他A&amp；#946；还迫切需要聚合体来准确分析A&amp；#946；聚合。此外，对我们的肽探针检测机制的理解是有限的。更广泛的机械理解不仅对于改进和优化我们的初始设计原则以开发高效的A&amp；#946；-特异性探针，也是我们的策略的延伸，以创建用于检测由其他天然未折叠蛋白形成的淀粉样蛋白聚集体的分子探针。拟议项目的主要目标是：(1)创建一类新型肽探针，用于高度特异性和敏感性检测不同的a &amp；#946；(2)揭示了A&amp；#946；通过我们的肽探针聚合。更广泛的影响。我们的策略是创建肽探针检测代表了构象切换生物传感器设计的新范式，受到Nature？利用天然未折叠蛋白作为快速和特异性信号传导过程的结构切换生物传感器。一旦成功完成，我们的肽探针将作为生物传感工具(1)推进对A&amp；#946分子基础的理解；生理相关条件下的聚集，(2)在高通量分析中确定针对特定聚集步骤的聚集抑制剂/调节剂，最终(3)建立a &amp；#946；生物样品中的聚集谱和A&amp；#946；聚合。我们的机制研究结果有望为以下方面提供重要的基础：(1)理解由天然未折叠蛋白衍生的构象开关生物传感器；(2)进一步优化A&amp；#946；特异性探针和(3)用于检测涉及20种蛋白质错误折叠疾病的其他淀粉样蛋白聚集体的生物传感器的创建。研究，教育和推广的整合将导致K-12，本科生和研究生以及教师在生物化学，生物物理学和蛋白质工程重叠的多学科领域的教育和专业发展。通过研究项目进行的问题驱动型学习、PI教授的生化工程课程以及通过纽约科学馆开展的推广活动将培养学生。创造性和解决问题的能力。拟议的教育和推广计划与研究计划紧密结合，并将重点放在扩大未被充分代表的少数民族学生参与科学和工程研究上。研究结果和教育资料亦会以不同的格式提供给公众，包括在PI？美国网站。

INTELLECTUAL MERIT. The goal of this project is to develop a conformation-switching peptideprobe for rapid, specific, quantitative and sensitive detection of conformationally distinct yet similar analytes. Our target analytes are aggregates formed by a natively unfolded peptide, β amyloid (Aβ) implicated in Alzheimer?s disease (AD), known to form oligomeric and fibrillar assemblies. The heterogeneous Aβ aggregates formed during aggregation display different conformations and exert a varying extent of toxic effects. Thus, accurate and reliable detection of distinct Aβ aggregates representsa significant step toward a better understanding of the exact nature of Aβ aggregation and the development of early diagnostics as well as therapeutic drugs. Unfortunately, the existence of conformational similarity among distinct Aβ aggregates makes specific detection extremely difficult. Rapid, specific, quantitative and sensitive detection is particularly important yet currently unavailable in probing Aβ oligomeric aggregates, which are believed to be the major toxic agents in AD. In our previous studies, we developed a peptide probe, PG46, for rapid, specific and quantitative detection of Aβ oligomers. PG46 was created by integrating Aβ self-recognition sequences with the conformation-sensitive biarsenical dye, FlAsH. PG46 was found to specifically bind to Aβ oligomers and display an increase in FlAsH fluorescence upon such binding. Rapid, quantitative measurements of Aβ oligomers were also possible with PG46. Sensitivity for detection of Aβ oligomers was further improved by engineering of PG46. The sensitivity of our current peptide probes, though significant, is still not optimal. Peptide probes detecting other Aβ aggregates are also urgently needed for accurate profiling of Aβ aggregation. Moreover, understanding of the detection mechanism of our peptide probes is limited. A broader mechanistic understanding is critical for not only the refinement and optimization of our initial design principle to develop highly effective Aβ-specific probes, but also the extension of our strategy to create molecular probes for detection of amyloid aggregates formed by other natively unfolded proteins. The principal objective of the proposed project is to (1) create a novel class of peptide probes for highly specific and sensitive detection of distinct Aβ aggregates at physiological concentrations and (2) reveal the mechanism of detection of Aβ aggregates by our peptide probes.BROADER IMPACT. Our strategy to create peptide probes for Aβ detection represents a new paradigmfor design of conformation-switching biosensors, motivated by Nature?s use of natively unfolded proteinsas structure-switching biosensors for rapid and specific signaling processes. When successfully completed, our peptide probes will serve as biosensing tools to (1) advance understanding of the molecular basis of Aβ aggregation under physiologically relevant conditions, (2) identify, in a highthroughput assay, aggregation inhibitors/modulators targeting a specific aggregation step and, ultimately (3) establish correlations between Aβ aggregation profiles in biological samples and cellular/clinical manifestations of Aβ aggregation. Outcomes from our mechanistic studies are anticipated to provide an important foundation for (1) understanding of conformation-switching biosensors derived from natively unfolded proteins, (2) additional optimization of Aβ specific probes and (3) the creation of a biosensor for detection of other amyloid protein aggregates, implicated in 20 protein misfolding diseases. The integration of research, education and outreach will result in the educational and professional development of K-12, undergraduate and graduate students as well as teachers in a multidisciplinary area that overlaps biochemistry, biophysics and protein engineering. The problem-driven learning through research projects, the biochemical engineering courses taught by the PI and an outreach effort through New York Hall of Science will foster students? creativity and problem solving ability. The proposed education and outreach plans tightly integrate with the research plan, and will focus on expanding the participation of underrepresented minority students in the study of science and engineering. The research results and educational materials will also be made available to the public in various formats including a video file on the PI?s web site.