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

蛋白质是生命所必需的。为了让人类行走、倾听或触摸，蛋白质必须在折叠成正确的三维结构后产生并对力做出反应。然而，传统的生化技术并不能测量蛋白质对力的反应，这阻碍了机械生物学这一新兴领域的发展。在细胞内部，力可以引起蛋白质结构的细微变化，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上的后果。为了对单个蛋白质施加力并测量这些微小的信号，该项目将使用新型原子力显微镜（AFM）悬臂，这是一种微米级的、像跳板一样的力传感器。该项目将有助于阐明蛋白质对力的反应，这是生物学中一个关键但研究不足的信号机制。更一般地说，用这些新型悬臂研究蛋白质折叠过程将提供对蛋白质折叠方式的洞察，这是一个持续的挑战，尽管五十年的努力。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生和大学生提供优秀的跨学科培训。由于统计学对解释实验至关重要，但许多年轻研究人员对统计学知之甚少，因此该项目还将产生新的培训工具，重点关注统计学和使用生物实例进行数据分析。这些互动教育模拟将与科罗拉多大学博尔德分校PhET项目一起开发，从而利用他们在此类模拟和全球分布方面的专业知识。两个科学目标是(i)表征两种广泛研究的机械敏感蛋白的结构转变，titin的I27结构域和focal adhesion kinase，因为之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转变；（ii）以1微秒的分辨率测量模型双态蛋白的折叠转变时间。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形蛋白质在蛋白质沿着其过渡路径移动的短暂时间内的构象动力学。该项目将通过应用最先进的力精度、稳定性和时间分辨率的组合，通过聚焦离子束修改AFM悬臂梁来克服这些障碍。该项目由分子和细胞生物科学部的分子生物物理集群和物理部的生命系统物理项目共同资助。

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.