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

该项目的目的是利用蛋白质折叠和设计方法，探索超长卷曲线圈（如肌球蛋白）的序列和结构之间的关系。为实现这一目标，将采取三管齐下的办法。首先，将构建编码14个氨基酸块共聚物的合成基因，其序列基于从头开始，极简设计原则。这些基因将被克隆到表达载体中，形成二聚体螺旋，每个螺旋的残基从70个到超过1000个不等。具体来说，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，以指示螺旋的适当相位。在表达和纯化这些设计的蛋白质后，它们的结构将使用圆二色性，分析超离心以及原子力显微镜和激光镊子等单分子技术进行表征。其次，为了补充这些设计研究，肌凝蛋白卷曲-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，使用上述仪器，肌球蛋白线圈将用于帮助制定研究设计线圈的生物物理方案。最后，这些合成的肽段将用于制作长共聚物，用于研究其他卷曲线圈的拓扑结构和高阶组装以形成原纤维。长共聚物是通过形成交错的螺旋结构而产生的，这些螺旋结构作为从头到尾自组装的模板。多肽将在实验室合成和纯化，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与结构之间的基本关系。从第一性原理来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了解蛋白质是如何平衡主要的化学力来获得它们的三维形状的。有两种方法被应用于研究这个问题，定义了蛋白质折叠和设计领域。这两个问题是相反的：研究蛋白质折叠的科学家会问：“我们能不能根据氨基酸序列预测蛋白质的结构？”而研究蛋白质设计的科学家会问：“我们能不能预测蛋白质的哪个序列会产生目标结构？”这两种策略都将用于卷绕线圈的研究。这些结构基序，预计存在于三分之一的蛋白质中，涉及两个或多个α -螺旋之间的相互作用。实验的模块化设计将使学生在暑期经历和一学年的毕业论文项目中取得重大成就。这个有凝聚力的设计项目，以及强大的模块化组件，应该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。

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.