CAREER: Mechanical unfolding and refolding of multidomain proteins as a new signaling mechanism, studied using a novel single molecule approach

这个项目将提高我们对蛋白质在体内如何在外力作用下发挥作用的理解。蛋白质是组织和器官的主要组成部分，为了发挥其功能，它们通过一种称为折叠的过程获得了特定的三维结构。调节蛋白质功能的一个关键机制可能是利用它们的展开/再折叠转变来响应它们的生物力学环境。目前对这一过程所知甚少，因为它的研究需要新颖的实验方法来重现单个蛋白质所经历的力。这个CAREER项目旨在超越蛋白质折叠作为一种手段的既定观点，以获得一种特征的三维结构，并表明蛋白质在外力作用下在体内有规律地使用结构域展开和再折叠。了解这些过程将对理解组织的正常功能产生广泛的影响。蛋白质折叠作为一种功能机制，是生物学与化学、工程学和物理学相结合的多学科课题。教育和推广活动，结合拟议的研究目标，包括：建立一个新的科学和技术展览，这将解释蛋白质的展开和再折叠如何在分子水平上工作；改善和扩大K-12学生的生物物理学实习计划；开设生物物理学及相关学科的研究生课程；并将计算机编程作为本科生的新“第二语言”。PI将让学生参与几个科学领域的外联和教育活动，同时鼓励妇女和代表性不足的少数民族的参与。该项目将研究蛋白质在受力下的结构、功能、展开和再折叠动力学之间的关系，以及这一过程如何迅速改变组织的刚度，执行有用的机械功，有效地储存和释放能量，并充当招募和释放配体的开关。PI将利用一种基于磁镊子和共价附着的新型单分子方法。使用这种方法，单个蛋白质分子可以暴露在类似于体内经历的强制协议中，长达15天，并且存在各种缓冲液。该项目旨在建立蛋白质在作用力下展开和再折叠的规则，这些规则决定了自然界中几个重要系统的行为。此外，该项目将为生物医学研究人员提供蛋白质折叠与功能之间关系的新视角，这可能会促进新的治疗干预措施的发展。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project will improve our understanding of how proteins function in vivo under force. Proteins are a major component of tissues and organs and, to perform their function, they acquire a specific 3-dimentional structure via a process known as folding. A key mechanism regulating the functioning of proteins may be the use of their unfolding/refolding transitions in response to their biomechanical environment. Little is currently known about this process, as its investigation requires novel experimental approaches that can reproduce the force experienced by individual proteins. This CAREER project aims to look beyond the established view of protein folding as a mean to acquire a characteristic 3-dimentional structure and show that proteins operating under force use domain unfolding and refolding in vivo regularly. Understanding these processes will have widespread consequences in comprehending the normal functioning of tissues. Protein folding, as a mechanism of function, is a multifaceted topic, wherein biology meets chemistry, engineering and physics. Educational and outreach activities, integrated with the proposed research goals, include: establishing a new science and technology exhibit, which will explain how protein unfolding and refolding works at molecular level; improving and expanding a biophysics internship program for K-12 students; introducing new graduate-level courses in biophysics and related disciplines; and implementing computer programing as a new "second language" for undergraduate students. The PI will engage students in outreach and education activities from several areas of science, while encouraging participation of women and underrepresented minorities.This project will investigate the relation between the structure, function and unfolding and refolding dynamics of proteins under force and how this process can swiftly change the stiffness of a tissue, perform useful mechanical work, efficiently store and release energy, and act as an on/off switch to recruit and release ligands. The PI will take advantage of a novel single molecule approach based on magnetic tweezers and covalent attachment. Using this approach, single protein molecules can be exposed to force protocols resembling those experienced in vivo, for up to 15 days, and in the presence of various buffers. This project aims to establish the rules of how protein unfolding and refolding under force determine the behavior of several important systems in nature. Furthermore, this project will provide biomedical researchers with a new perspective on the relation between protein folding and function, which will likely spur development of new therapeutic interventions.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.