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Folding and Mechanical Response of Single Proteins Probed at High Spatio-Temporal Resolution

在高时空分辨率下探测单个蛋白质的折叠和机械响应

基本信息

批准号：
1716033
负责人：
Thomas Perkins
金额：
$ 68.4万
依托单位：
University of Colorado at Boulder
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1716033&HistoricalAwards=false
关键词：
Folding Mechanical Response Single Proteins

项目摘要

Proteins are essential to life. For humans to walk, hear, or touch, proteins must generate and respond to forces after folding into their correct three-dimensional structure. Yet, traditional biochemical techniques do not measure a protein's response to force, hindering progress in the emerging field of mechanobiology. Inside a cell, force can induce subtle change in a protein's structure, fully unfold a protein, or disrupt the interaction between two proteins. These effects all have biological consequence. To exert a force on individual proteins and measure these tiny signals, this project will use novel atomic force microscope (AFM) cantilevers, which are micron-scale, diving-board like force sensors. This project will help elucidate the response of proteins to force, a critical but understudied signaling mechanism in biology. More generally, studying the process of protein folding with these novel cantilevers will provide insight into how proteins fold, an ongoing challenge despite five decades of effort. By using the tools of physics and nanoscience to solve exciting problems in biology, this project will provide excellent interdisciplinary training for high school and colleges students just starting their research career. As statistics are vital to interpreting experiments but are poorly understood by many young researchers, this project will also generate novel training tools focused on statistics and data analysis using biological examples. These interactive education simulations will be developed in conjunction with the CU-Boulder PhET program and thereby leverage their expertise in such simulations and world-wide distribution.The twin scientific goals are (i) to characterize structural transitions in two widely studied mechano-sensitive proteins, titin's I27 domain and the focal adhesion kinase, since previous studies have either yielded conflicting results or failed to resolve a predicted mechanically induced transition; and (ii) measure a model two-state protein's folding transition time with 1-microsecond resolution. The key technical hurdle is that traditional AFM studies lack the spatial precision to resolve subtle changes in protein structure and lack the temporal precision to characterize the conformational dynamics of a traditional globular protein during the brief time the protein moves along its transition path. This project will overcome these obstacles by applying a state-of-the-art combination of force precision, stability, and time resolution enabled by focused-ion beam modification of AFM cantilevers. This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems Program in the Division of Physics.

蛋白质是生命所必需的。对于人类来说，要走路、听或触摸，蛋白质必须在折叠成正确的三维结构后产生并响应力。然而，传统的生物化学技术不能测量蛋白质对力的反应，这阻碍了新兴的机械生物学领域的进展。在细胞内，力可以引起蛋白质结构的微妙变化，完全展开蛋白质，或破坏两种蛋白质之间的相互作用。这些影响都有生物学后果。为了对单个蛋白质施加力并测量这些微小信号，该项目将使用新型原子力显微镜（AFM）杠杆，这是微米级的，潜水板状的力传感器。该项目将有助于阐明蛋白质对力的反应，这是生物学中一种关键但未充分研究的信号机制。更一般地说，研究蛋白质折叠的过程与这些新的杠杆将提供深入了解蛋白质如何折叠，一个持续的挑战，尽管50年的努力。通过使用物理和纳米科学的工具来解决生物学中令人兴奋的问题，该项目将为刚刚开始研究生涯的高中和大学生提供优秀的跨学科培训。由于统计数据对于解释实验至关重要，但许多年轻研究人员对此知之甚少，该项目还将产生新的培训工具，重点是使用生物学实例进行统计和数据分析。这些交互式教育模拟将与CU-Boulder PhET计划一起开发，从而利用他们在此类模拟和全球分布方面的专业知识。双重科学目标是：（i）表征两种广泛研究的机械敏感蛋白质，肌联蛋白的I27结构域和黏着斑激酶的结构转变，因为先前的研究要么产生了相互矛盾的结果，要么未能解决预测的机械诱导的过渡;和（ii）测量模型的两个状态的蛋白质的折叠过渡时间与1微秒的分辨率。关键的技术障碍是，传统的AFM研究缺乏空间精度来解决蛋白质结构的细微变化，缺乏时间精度来表征传统球状蛋白质在沿着其过渡路径运动的短暂时间内的构象动力学。本项目将通过应用最先进的力精度，稳定性，和时间分辨率，使聚焦离子束修改的原子力显微镜杠杆。该项目由分子和细胞生物科学部的分子生物物理学集群和物理学部的生命系统物理学项目共同资助。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations

DOI：
10.1073/pnas.2015728118
发表时间：
2021-03-23
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Edwards, Devin T.;Leblanc, Marc-Andre;Perkins, Thomas T.
通讯作者：
Perkins, Thomas T.

Type III secretion system effector proteins are mechanically labile

DOI：
10.1073/pnas.2019566118
发表时间：
2021-03-23
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Leblanc, Marc-Andre;Fink, Morgan R.;Sousa, Marcelo C.
通讯作者：
Sousa, Marcelo C.

Quantifying the Native Energetics Stabilizing Bacteriorhodopsin by Single-Molecule Force Spectroscopy

通过单分子力谱定量稳定细菌视紫红质的天然能量