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

这个项目的目的是探索序列和结构之间的关系非常长的卷曲螺旋，如肌球蛋白中发现的，使用蛋白质折叠和设计方法。 我们会采取三管齐下的方法，以达致这个目标。 首先，将构建编码14个氨基酸嵌段的共聚物的合成基因，其序列基于从头开始的最小设计原则。 这些基因将被克隆到表达载体中，以形成每螺旋70个残基至大于1，000个残基的二聚卷曲螺旋。 具体而言，该系统将用于测试中间体在长卷曲螺旋组装中的作用，例如单体螺旋形成和特定螺旋配对相互作用的成核，以决定螺旋的适当定相。 在表达和纯化这些设计的蛋白质后，将使用圆二色性、分析超离心和单分子技术（如原子力显微镜和激光镊子）来表征它们的结构。 其次，为了补充这些设计研究，肌球蛋白卷曲螺旋杆域将被用作折叠研究的模型系统，涉及设计和天然序列之间的片段交换。 此外，肌球蛋白卷曲螺旋将用于帮助开发生物物理协议，研究设计的卷曲螺旋，使用上述仪器。 最后，这些合成肽嵌段将用于制备长共聚物，用于研究其他卷曲螺旋拓扑结构和更高阶组装以形成原纤维。 长链共聚物是通过形成交错的螺旋结构来产生的，这些螺旋结构充当它们自己的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和结构之间的基本关系。 从第一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它们的三维形状。 两种方法已被应用于研究这个问题，定义蛋白质折叠和设计领域。 这两个问题是彼此相反的：蛋白质折叠领域的科学家们问：“我们能根据蛋白质的氨基酸序列预测它的结构吗？那些从事蛋白质设计的人会问：“我们能预测蛋白质的什么序列会导致目标结构吗？”" 这两种策略都将用于螺旋弹簧圈的研究。 这些结构基序，预计在1/3的蛋白质中，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使学生在夏季体验和一学年的课程中取得重大成就，致力于高级论文项目。 这个有凝聚力的设计方案，沿着一个强大的模块化组件，应该提供一个有益的经验，为学生感兴趣的跨学科科学，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.