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

职业：使用新型单分子方法研究多域蛋白的机械展开和重折叠作为一种新的信号传导机制

• 批准号: 1846143
• 负责人: Ionel Popa
• 金额: $ 74.17万
• 依托单位: University of Wisconsin-Milwaukee
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1846143&HistoricalAwards=false
• 关键词: CAREER; Mechanical; unfolding; refolding; multidomain

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.

该项目将增进我们对蛋白质在力作用下如何在体内发挥作用的理解。蛋白质是组织和器官的主要组成部分，为了发挥其功能，它们通过折叠过程获得特定的三维结构。调节蛋白质功能的一个关键机制可能是利用其解折叠/重折叠转变来响应其生物力学环境。目前人们对这个过程知之甚少，因为它的研究需要新的实验方法来重现单个蛋白质所经历的力。该职业项目旨在超越蛋白质折叠作为获得特征性三维结构的一种手段的既定观点，并表明在力作用下运行的蛋白质在体内有规律地使用结构域展开和重折叠。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，是一个多方面的主题，其中生物学与化学、工程学和物理学相遇。与拟议的研究目标相结合的教育和外展活动包括：建立一个新的科学技术展览，这将解释蛋白质如何在分子水平上展开和重折叠；改进和扩大 K-12 学生的生物物理学实习计划；引入生物物理学及相关学科的新研究生课程；将计算机编程作为本科生的新“第二语言”。该项目负责人将让学生参与多个科学领域的外展和教育活动，同时鼓励女性和代表性不足的少数群体参与。该项目将研究受力下蛋白质的结构、功能和展开和重折叠动力学之间的关系，以及该过程如何迅速改变组织的硬度、执行有用的机械功、有效地存储和释放能量，以及充当招募和释放配体的开关。 PI 将利用基于磁力镊子和共价连接的新型单分子方法。使用这种方法，单个蛋白质分子可以在各种缓冲液存在下接受类似于体内经历的强制方案长达 15 天。该项目旨在建立蛋白质在受力下的展开和重折叠如何决定自然界中几个重要系统行为的规则。此外，该项目将为生物医学研究人员提供关于蛋白质折叠与功能之间关系的新视角，这可能会刺激新治疗干预措施的开发。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions

• DOI: 10.1016/j.saa.2021.120133
• 发表时间: 2021-07-07
• 期刊: SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
• 影响因子: 4.4
• 作者: Adhikari, Dhruba P.;Biener, Gabriel;Raicu, Valerica
• 通讯作者: Raicu, Valerica

Chemical unfolding of protein domains induces shape change in programmed protein hydrogels

• DOI: 10.1038/s41467-019-13312-0
• 发表时间: 2019-11-29
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Khoury, Luai R.;Popa, Ionel
• 通讯作者: Popa, Ionel

Using Magnets and Flexible 3D-Printed Structures to Illustrate Protein (Un)folding

使用磁铁和灵活的 3D 打印结构来说明蛋白质（解）折叠

• DOI: 10.1021/acs.jchemed.2c00231
• 发表时间: 2022
• 期刊: Journal of Chemical Education
• 影响因子: 3
• 作者: Popa, Ionel;Saitis, Florin
• 通讯作者: Saitis, Florin

Cation-induced shape programming and morphing in protein-based hydrogels

• DOI: 10.1126/sciadv.aba6112
• 发表时间: 2020-04-01
• 期刊: SCIENCE ADVANCES
• 影响因子: 13.6
• 作者: Khoury, Luai R.;Slawinski, Marina;Popa, Ionel
• 通讯作者: Popa, Ionel

Does protein unfolding play a functional role in vivo ?

蛋白质解折叠在体内发挥功能作用吗？

• DOI: 10.1111/febs.15508
• 发表时间: 2020
• 期刊: The FEBS Journal
• 作者: Sharma, Sabita;Subramani, Smrithika;Popa, Ionel
• 通讯作者: Popa, Ionel