OPTICAL STUDIES OF PROTEIN STRUCTURE AND FUNCTION

这项拟议研究的目标是开发新的荧光 技术，并将它们与其他实验方法结合使用 阐明选定的蛋白质在体外和体内的结构和动力学 完好无损的细胞。(1)开展单抗的荧光研究 小鼠免疫球蛋白了解抗原结合是如何导致 效应器的反应，如补体的激活和 触发淋巴细胞。抗胆囊素的节段性灵活性 免疫球蛋白M、D、G、E和A将以纳秒为单位进行测量 荧光偏振光谱。F(C)单位将特别 用荧光探针标记检测传播的构象 改变。完整细胞和重组细胞中的膜结合免疫球蛋白 囊泡将用荧光技术进行研究。(2)我们会继续 我们对革兰菌素A的X射线和中子结晶学研究 现在是在原子分辨率下解决这个通道的结构。(3)我们 将建造一台时间分辨的荧光偏振装置， 解决-时间约为10PSEC。一系列星云的皮秒动力学 含有单一色氨酸残基的蛋白质将被研究并 与这些分子的功能有关。这种同步泵浦的激光器和 条纹照相仪器也将用于荧光显微镜 单细胞分析。(4)荧光能量转移和 将使用偏振技术来监控组装和周转 在细胞分裂等过程中，完整细胞中肌动蛋白细丝的数量。 目的是为了了解肌动蛋白细丝和肌动蛋白动力学的控制 它们与膜的相互作用。拟议的研究将提供 离子等过程中蛋白质动力学的基本信息 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件。

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.