Entanglement in the Structural Biology of Living Systems

蛋白质从氨基酸的线性聚合物折叠成三维形状是生命所必需的过程。聚合物可能会变得无可救药地缠绕在一起，并导致功能丧失和从细胞中移除。然而，生物多聚体蛋白质中特定位置的纠缠可以导致稳定性的提高，但具体纠缠发生的过程在分子水平上尚不清楚。这个项目试图解决蛋白质如何在细胞的限制性空间内形成一个深而复杂的三叶结，蛋白质是在那里形成的，以及蛋白质结构中纠缠的整体功能是什么。考虑到将一串珍珠链打结成一种能够催化化学反应的酶的挑战性和复杂性，该小组提议将物理化学和物理学的许多方面结合起来，以阐明这种结在生物学中发挥的独特而关键的作用。自从PI发现了一类缠绕的穿孔套索蛋白以来，这一不断增长的类别现在包括350多个蛋白质家族，跨越了广泛的折叠类型和生物功能。通过计算和物理实验的结合，PI寻求进一步了解蛋白质中的穿线和打结，并扩展它们在生物功能中的各自作用。这些原则仍然是数学、物理和生物学中具有挑战性的课题。因此，随着这项研究的广度，对折叠功能相互作用的持续研究提出了行为相互作用的概念和跨学科策略的强度。在这个项目的过程中，PI的小组将在与结形成相关的各种科学领域对本科生进行培训，并向他们介绍科学探究的兴奋。此外，PI还招募了来自不同领域和(不稳定)的学生加入调查团队，丰富了所有人的学习经验。出乎意料的是，蛋白质可以在其天然折叠中将自己捆绑成结。由于蛋白质从非结构多肽链到天然状态的折叠已经很复杂，打结多肽链的存在立即提出了问题：多肽链是如何交叉形成一个结的，这个结是如何影响功能的？为了解决这些问题，PI将研究深结三叶甲基转移酶(MT)家族。结合他们在这项实验和理论工作中的优势，Pi的团队将进一步努力探索调节结穿线/解线的机制以及自然状态下结的作用，以研究结中的特定区域(被识别为订书钉)如何影响蛋白质的折叠、动态和功能。此外，研究人员将研究在三叶打结蛋白中被确定为解开障碍的特定区域是否也有助于功能调节。在这些基础研究的基础上进行进一步的探索，使他们能够研究打结构象中固有的应变拓扑与功能位点之间的通信之间的相互作用。他们之前的发现导致了一项探索穿线机制并解开打结蛋白质的协议。这一实验优势使研究人员能够在蛋白质理论领域提出基本问题：蛋白质是如何形成线状元件的？打结对功能有什么影响？它如何折叠成一种深度打结的生物活性蛋白质？为了充分了解自然界如何控制蛋白质的拓扑结构，需要广泛的理论、数值和实验进展。该项目的目标是进一步扩大这些原则验证实验，通过包括分子生物学、蛋白质化学、光学光谱学和质谱学在内的“全力以赴”的生化方法来探索该方法的结构和功能范围。这个项目需要数学、生物、化学和物理领域的顶尖研究人员共同努力。最终，了解打结蛋白质的生物物理生物化学可用于设计和设计具有优异机械和热稳定性特性的新型蛋白质。蛋白质中的打结、穿线和打滑打结是打结理论中新的和具有挑战性的话题，必须推导出新的形式，并必须考虑到这些生物对象的物理性质。这个项目的广泛影响包括K-12的指导，以及加州大学圣迭戈分校的本科生和研究生。国际学生联合会参与了K-12推广活动，在当地文法学校(少数民族学生占50%以上)进行科学实验，在特殊教育学校进行实验，帮助中学生参加科学奥林匹克竞赛，在暑期接待高中生作为研究实习生，并在她的实验室接待对中小学教学感兴趣的人作为研究助理。该项目由物理部生命系统物理学资助，分子和细胞生物科学部分子生物物理组提供支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为是值得支持的。

Protein folding from a linear polymer of amino acids into a three-dimensional shape is an essential process for life. The polymer may become hopelessly tangled and lead to loss of function and removal from the cell. However, entanglement in specific locations within the biopolymeric protein can lead to enhancement of stability, but the process by which specific entanglement occurs is not understood at the molecular level. This project seeks to address how a protein can form a deep intricate trefoil knot within the restrictive space of the cell, where proteins are formed, as well as what is the overall function of entanglement within the protein structure. Given the challenge and complexity of knotting a “string of pearls” into an enzyme capable of catalyzing a chemical reaction, the group proposes to combine the many facets of physical chemistry and physics to elucidate the unique yet critical role the knot plays in biology. Since the PI's discovery of a class of entangled pierced lasso proteins, this growing class now includes well over 350 protein families, spanning a huge range of fold types and biological functions. Through a combination of computational and physical experiments, the PI seeks to further the understanding of threading and knotting in proteins and expand upon their respective roles in biological function. These principles continue to be challenging topics in mathematics, physics and biology. Thus the continued investigation into the folding function interplay with the breadth of this study advances the concept of interplay in behavior and the strength of the interdisciplinary strategy. In the course of this project the PI's group will train undergraduate students in a variety of scientific areas related to knot formation and introduce them to the excitement of scientific inquiry. In addition, the PI has recruited students from diverse fields and (dis)abiledness to join the team of investigators, enriching the learning experience for all. Unexpectedly, proteins can tie themselves into knots in their native folds. Since protein folding from an unstructured polypeptide chain into the native-state is already complex, the existence of knotted polypeptide chains immediately raises the questions: how does the chain cross itself to form a knot and how does the knot affect function? To address these questions, the PI will study the deep-knotted trefoil methyltransferase (MT) families. Combining their strengths in this experimental and theoretical efforts, the PI’s group will expand their efforts in probing the mechanisms regulating knot threading/unthreading as well as the role of knots in the native state to ask how specific regions (identified as staples) within knots impact the fold, dynamics, and functions of proteins. Furthermore, the investigators will study whether specific regions identified as barriers to untying in the trefoil knotted proteins also contribute to functional regulation. Further exploration building upon these foundational studies allows them to investigate the interplay between the strained topology inherent in a knotted conformation and communication between functional sites. Their previous discoveries led to a protocol to explore threading mechanisms and untie a knotted protein. This experimental advantage allows the investigators to ask fundamental questions in the protein theory field: How can a protein form a threaded element? How does knotting affect function? How can it fold into a deeply knotted biologically active protein? To fully understand how nature controls the topology of proteins, extensive theoretical, numerical, and experimental progress is required. The goal of this project is to further expand upon these proof-of-principle experiments to explore both the structural and functional scope of the approach through an “all hands on deck” biochemical approach that will include molecular biology, protein chemistry, optical spectroscopy, and mass spectroscopy. This project requires the coordinated effort of top researchers working at the interface of mathematics, biology, chemistry, and physics. Ultimately, understanding the biophysical biochemistry of knotted proteins can be used to design and engineer novel classes of proteins that have superior mechanical and thermal stability properties. Knotting, threading and slip-knotting in proteins are new and challenging topics in knot theory for which new formalisms must be derived and must take into account physical properties of these biological objects.Broader impacts of this project include mentoring of K-12, and undergraduate and graduate students at UCSD. The PI has been involved in K-12 outreach activities by performing scientific experiments at local grammar schools, which are greater than 50% minority students, by performing experiments in special education schools, by helping to train children in middle school for the Science Olympiad, hosting High School students as research interns for the summer, and hosting people interested in a career in teaching in primary and secondary schools as research assistants in her laboratory. This project is funded by the Physics of Living Systems in the Division of Physics with support from the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.