RUI Proposal: Learning the Rules that Govern the Folding and Stability of Coiled Coils

RUI 提案：学习控制线圈折叠和稳定性的规则

• 批准号: 0211754
• 负责人: Robert Fairman
• 金额: $ 41.78万
• 依托单位: Haverford College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0211754&HistoricalAwards=false
• 关键词: RUI; Proposal; Learning; Rules; Govern

The objective of this project is to explore the relationship between sequence and structure for very long coiled coils, such as found in myosin, using both protein folding and design approaches. Three-pronged approach will be used to accomplish the goal. First, synthetic genes that encode copolymers of 14-amino acid blocks will be constructed, whose sequences are based on de novo, minimalist-design principles. These genes will be cloned into expression vectors to make dimeric coiled coils that range from 70 residues to greater than 1,000 residues per helix. Specifically, this system will be used to test the role of intermediates in the assembly of long coiled coils, such as monomeric helix formation and nucleation of specific helix pairing interactions to dictate proper phasing of helices. After expressing and purifying these designed proteins, their structures will be characterized using circular dichroism, analytical ultracentrifugation, and single molecule techniques such as atomic force microscopy and laser tweezing. Second, to complement these design studies, a myosin coiled-coil rod domain will be used as a model system for folding studies involving segment swapping between designed and natural sequences. In addition, the myosin coiled coil will be used to help develop biophysical protocols for studying designed coiled coils, using the instruments described above. Finally, these synthetic peptide blocks will be used to make long copolymers for the study of other coiled coil topologies and higher order assembly to form fibrils. Long copolymers are generated by forming staggered helical structures that act as templates for their own head-to-tail self-assembly. Peptides will be synthesized and purified in the laboratory and then characterized using the same biophysical techniques described above.The overall goal of this research is to understand the basic relationship between protein sequence and structure. It is still not possible to predict protein structure and function from first principles, mainly because it is still not understood how proteins balance the major chemical forces in attaining their three dimensional shape. Two approaches have been applied to study this problem, defining the fields of protein folding and design. The two questions are the inverse of one another: scientists in the field of protein folding ask, "Can we predict the structure of a protein given its amino acid sequence?" and those who work on protein design ask, "Can we predict what sequence of a protein will result in a target structure?" Both of these strategies will be used in the study of coiled coils. These structural motifs, predicted to be in 1/3 of all proteins, involve the interaction between two or more alpha-helices. The modular design of the experiments will allow students to make significant achievements over the course of a summer experience and an academic year working towards a senior thesis project. This cohesive program in design, along with a strong modular component, should provide a rewarding experience for students interested generally in interdisciplinary sciences, including elements of biochemistry and biophysics.

这个项目的目标是利用蛋白质折叠和设计方法来探索超长螺旋卷曲之间的序列和结构之间的关系，例如在肌球蛋白中发现的。为实现这一目标，将采取三管齐下的方法。首先，将构建编码14个氨基酸区块的共聚物的合成基因，其序列基于从头开始的极简主义设计原则。这些基因将被克隆到表达载体中，以形成从每螺旋70个残基到超过1000个残基的二聚体螺旋。具体地说，该系统将用于测试中间体在长螺旋线圈组装中的作用，例如单体螺旋的形成和特定螺旋配对相互作用的成核，以指示螺旋的正确定相。在表达和纯化这些设计的蛋白质后，将使用圆二色谱、分析超速离心法以及原子力显微镜和激光镊子等单分子技术来表征它们的结构。其次，为了补充这些设计研究，肌球蛋白卷曲杆结构域将被用作折叠研究的模型系统，涉及设计序列和自然序列之间的片段交换。此外，肌球蛋白卷曲线圈将利用上述仪器，帮助制定研究设计的卷曲线圈的生物物理方案。最后，这些合成的多肽段将被用来制造长共聚物，用于研究其他盘绕线圈的拓扑结构和高阶组装以形成纤维。长共聚物是通过形成交错的螺旋结构而产生的，这些螺旋结构充当它们自己头尾自组装的模板。多肽将在实验室中合成和纯化，然后使用上述相同的生物物理技术进行表征。本研究的总体目标是了解蛋白质序列和结构之间的基本关系。根据第一性原理预测蛋白质的结构和功能仍然是不可能的，主要是因为人们仍然不知道蛋白质如何平衡主要的化学力来获得其三维形状。有两种方法被用来研究这个问题，它们定义了蛋白质折叠和设计领域。这两个问题是相反的：蛋白质折叠领域的科学家提出的问题是，我们能根据蛋白质的氨基酸序列预测其结构吗？那些从事蛋白质设计工作的人问道：“我们能预测出蛋白质的哪个序列会形成一个靶标结构吗？”这两种策略都将用于线圈的研究。这些结构基序预计存在于所有蛋白质的三分之一中，涉及两个或更多个α-螺旋之间的相互作用。实验的模块化设计将允许学生在暑期体验和一学年为高级论文项目而努力的过程中取得显著成就。这一具有凝聚力的设计课程，加上强大的模块组件，应该会为对跨学科科学感兴趣的学生提供有益的体验，包括生物化学和生物物理学的元素。