Protein Folding from Solution to the Living Cell

智力优势：多年来，对蛋白质动态行为的研究一直集中在细胞环境外(体外)操纵的分离蛋白质。只有少数几个系统中的研究人员进入了更复杂的环境，包括活细胞(在体内)。拟议中的研究将开发快速松弛成像，这是一种观察活细胞内生物分子动力学的技术，允许在体外学习的内容与蛋白质在体内的表现之间建立联系。在拟议的实验中，激光诱导的小温度跃升启动了蛋白质折叠动力学、蛋白质-蛋白质相互作用或细胞内的核酸动力学。一台荧光显微镜将产生的动态成像为时间的函数，在细胞内制作一部“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不同隔室中代谢酶和信号酶的折叠。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调控细胞过程(如基因转录、细胞凋亡和细胞分裂)至关重要。定义蛋白质在细胞中的行为也将导致确定哪些方面的调节是进化的，并已成为蛋白质结构选择过程的一部分。这项研究还得到了几个外展项目的补充，包括招募暑期本科生，与河内科技大学的交流和教学项目，以使他们的教学和研究达到现代标准，以及由PIS实验室的一名生物物理学学生建立的K-12外展项目的继续，该项目现在可以作为生物物理学的网络教程：一个学生在教师导师的指导下，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。该仪器可广泛应用于其他实验室。本项目由MCB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.