Protein Folding from Solution to the Living Cell

智力价值：多年来，对蛋白质动态行为的研究一直集中在细胞环境（体外）之外操纵的分离蛋白质上。只有少数系统中的研究人员进入了更复杂的环境，包括活细胞（体内）。拟议的研究将开发快速弛豫成像，这是一种观察活细胞内生物分子动力学的技术，可以将体外学到的知识与蛋白质在体内的行为联系起来。在拟议的实验中，一个小的激光诱导的温度跳跃启动蛋白质折叠动力学，蛋白质-蛋白质相互作用，或细胞内的核酸动力学。 荧光显微镜将产生的动力学成像为时间的函数，在细胞内制作“折叠电影”。 该实验将用于研究细胞的热休克反应，蛋白质α-突触核蛋白与细胞膜的相互作用，以及不同细胞类型的各个隔室中代谢和信号酶的折叠。更广泛的影响：由于蛋白质动力学对蛋白质功能至关重要，因此了解细胞如何调节其组成生物分子的稳定性和动力学对于了解细胞功能和细胞过程（如基因转录，细胞凋亡和细胞分裂）的调节至关重要。定义细胞中的蛋白质行为也将导致确定哪些方面的调节是进化的，并且是蛋白质结构选择过程的一部分。这项研究是由几个推广计划，包括夏季本科研究生的招聘，与科学的河内大学的交流和教学计划，使他们的教学和研究达到现代标准，并继续K-12推广计划，这是由一个生物物理学学生在PI实验室成立，现在是一个基于网络的生物物理学教程：一名学生在教员导师的指导下与初中和高中教师合作，帮助他们使用生物物理可视化工具创建教育材料。更广泛的影响还包括为拟议的实验开发仪器。本项目由MCB的细胞系统和分子生物物理学联合资助。

Intellectual merit:For many years research on the dynamic behavior of proteins has focused on isolated proteins manipulated outside of the cellular environment (in vitro). There are only a few systems in which investigators have moved into more complex environments, including the living cell (in vivo). The proposed studies will develop Fast Relaxation Imaging, a technique that looks at biomolecular dynamics inside living cells, allowing connections to be made between what has been learned in vitro to how proteins behave in vivo. In the proposed experiments a small laser-induced temperature jump initiates protein folding kinetics, protein-protein interactions, or nucleic acid dynamics inside cells. A fluorescence microscope images the resulting dynamics as a function of time, making a "folding movie" inside the cell. The experiment will be applied to study the heat shock response of cells, interactions of the protein alpha-synuclein with cell membranes, and folding of metabolic and signal enzymes in various compartments of different cell types. Broader Impacts:Because protein dynamics are critical to protein function, understanding how cells modulate the stability and kinetics of their constituent biomolecules is critical to understanding cell function and the regulation of cellular processes such as gene transcription, apoptosis and cell division. Defining protein behavior in the cell will also lead to determining what aspects of modulation are evolved and have been part of the selection process on protein structure. The research is complemented by several outreach programs, including recruitment of summer undergraduate research students, an exchange and teaching program with Hanoi University of Science to bring their teaching and research up to modern standards, and continuation of a K-12 outreach program that was set up by a biophysics student in the PIs lab, and is now available as a web-based tutorial in biophysics: a student, with guidance of a faculty mentor, collaborates with middle- and high school teachers to help them create educational materials employing biophysical visualization tools. The broader impacts also include development of the instrumentation for the proposed experiments. This instrumentation could be become widely used in other laboratories.This project is jointly funded by Cellular Systems and Molecular Biophysics in MCB.