CAREER: Conformations of Beta-Sheet Folding Intermediates: The Discrimination Between Productive Folding and Aggregation

这个CAREER项目的目标是开发b片形成和稳定的通用模型。 几种全b片层蛋白是不寻常的热稳定性，许多功能在恶劣的细胞外环境中，异常高的稳定性是必需的活动。 这些蛋白质中的许多具有简单的结构拓扑（例如，平行b-螺旋），这与它们的热稳定性一起使它们成为设计新功能的“分子支架”的有吸引力的主题。 然而，这些蛋白质有一个致命的弱点：虽然天然蛋白质可能是热稳定的，但折叠中间体通常是非常不耐热的，并且易于聚集。 使这些蛋白质适应替代功能的先决条件是了解它们如何运作。 然而，关于b片层形成的细节，特别是平行b片层形成的细节知之甚少。 该项目将使用生物化学和生物物理学方法，如荧光光谱，蛋白酶灵敏度和分子半径的变化，以检查两个全b-折叠蛋白质折叠中间体的构象非常稳定，但难以形成天然状态。 这项工作将揭示b-折叠中间体的特征，即（1）促进正确折叠到天然结构，（2）抑制非途径聚集反应。 这些研究的结果将有广泛的应用，正确折叠的b-折叠蛋白质的生物技术目的的设计。该项目的教育目标侧重于介绍学生在各个层次的现代科学研究和发展的跨学科方法。 具体目标包括更新本科课程，纳入蛋白质生物物理学新的高年级本科选修课，以及一年级学生新的普通化学课程，重点是现代科学发现的跨学科方法。 目前征聘女性和少数民族研究人员的工作将继续进行并扩大。

The objective of this CAREER project is to develop general models for b-sheet formation and stabilization. Several all-b-sheet proteins are unusually thermostable, and many function in harsh extracellular environments where unusually high stability is required for activity. Many of these proteins have simple structural topologies (for example, the parallel b-helix), which, together with their thermostability, make them attractive subjects for use as 'molecular scaffolds' for designing new functionalities. However, these proteins have an Achilles' heel: while the native protein may be thermostable, the folding intermediates are often quite thermolabile, and prone to aggregation. A prerequisite for adapting these proteins for alternative functions is to understand how they operate. Yet little is known about the details of b-sheet formation, particularly parallel b-sheet formation. This project will use biochemical and biophysical approaches such as fluorescence spectroscopy, protease sensitivity, and changes in molecular radius to examine the conformations of folding intermediates for two all-b-sheet proteins with very stable yet difficult-to-form native states. This work will uncover the features of b-sheet folding intermediates that (1) promote correct folding to the native structure, and (2) suppress the off-pathway aggregation reaction. The results of these studies will have broad application to the design of correctly folded b-sheet proteins for biotechnological purposes.The education goals of this project focus on introducing students at all levels to the interdisciplinary approaches of modern scientific research and development. The specific objectives include updating the undergraduate curriculum to incorporate a new upper-level undergraduate elective in protein biophysics, and a new general chemistry course for first year students, focused on the interdisciplinary approaches of modern scientific discovery. Current efforts to recruit female and minority researchers will be continued and expanded.