OPTICAL STUDIES OF PROTEIN STRUCTURE AND FUNCTION

蛋白质结构和功能的光学研究

• 批准号: 3272023
• 负责人: LUBERT STRYER
• 金额: $ 19.98万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-01-01 至 1987-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3272023
• 关键词: X ray crystallography; analytical method; cell cycle; cell membrane; chemical reaction; chemical synthesis; conformation; embryo /fetus cell /tissue; fluorescence microscopy; fluorescence polarization; fluorescence spectrometry; fluorescent dye /probe; genetic manipulation; immunoglobulin A; immunoglobulin D; immunoglobulin E; immunoglobulin G; immunoglobulin M; immunoglobulins; ion transport; laser spectrometry; membrane model; monoclonal antibody; mutant; neutron diffraction; photography; saltwater environment; single cell analysis

The goal of the proposed research is to develop novel fluorescence techniques and use them n concert with other experimental approaches to elucidate the structure and dynamics of selected proteins in vitro and in intact cells. (1) We will carry out fluorescence studies of monoclonal mouse immunoglobulins to gain insight into how the binding of antigen leads to effector responses such as the activation of complement and the triggering of lymphocytes. The segmental flexibility of anti-dansyl immunoglobulins M, D, G, E, and A will be measured by nanosecond fluorescence polarization spectroscopy. The F(c) unit will be specifically labeled with fluorescent probes to detect propagated conformational changes. Membrane-bound immunoglobulins in intact cells and reconstituted vesicles will be studied by fluorescence techniques. (2) We will continue our x-ray and neutron crystallographic studies of gramicidin A. The goal now is to solve the structure of this channel at atomic resolution. (3) We will construct a time-resolved fluorescence polarization apparatus with a resolution-time of about 10 psec. The picosecond dynamics of a series of proteins containing a single tryptophan residue will be investigated and related to the function of these molecules. This synch-pumped laser and streak camera instrument will also be used for fluorescence microscopic analyses of single cells. (4) Fluorescence energy transfer and polarization techniques will be used to monitor the assembly and turnover of actin filaments in intact cells during processes such as cell division. The aim is to understand the control of the dynamics of actin filaments and their interactions with membranes. The proposed research will provide fundamental information about protein dynamics in processes such as ion transport, immune recognition, and cell motility and lead to new methods for the analysis of assemblies in intact cells.

这项研究的目标是开发新的荧光 技术，并使用它们与其他实验方法， 阐明体外和体内选定蛋白质的结构和动力学， 完整的细胞 (1)我们将进行单克隆的荧光研究， 小鼠免疫球蛋白，以深入了解抗原的结合如何导致 效应反应，如补体的激活和 触发淋巴细胞。 抗丹磺酰的链段柔性 免疫球蛋白M、D、G、E和A将通过纳秒测量 荧光偏振光谱 F（c）股将具体 用荧光探针标记以检测扩增的构象 变化 完整细胞和复溶细胞中的膜结合免疫球蛋白 将通过荧光技术研究囊泡。 (2)我们将继续 我们对短杆菌肽A的X射线和中子晶体学研究 目标 现在要解决的是原子分辨率下这个通道的结构。 (3)我们 将构建一个时间分辨荧光偏振装置， 分辨率约为10 psec。 一系列的皮秒动力学 将研究含有单个色氨酸残基的蛋白质， 与这些分子的功能有关。 这种同步泵浦激光器和 条纹相机仪器也将用于荧光显微镜 单细胞分析。 (4)荧光能量转移和 极化技术将用于监测组装和周转 完整细胞在细胞分裂等过程中的肌动蛋白丝。 目的是了解肌动蛋白丝的动力学控制， 它们与细胞膜的相互作用。 该研究将提供 关于蛋白质动力学的基本信息，如离子 运输，免疫识别和细胞运动，并导致新的方法 用于分析完整细胞中的组装体。