OPTICAL STUDIES OF PROTEIN STRUCTURE AND FUNCTION

蛋白质结构和功能的光学研究

• 批准号: 3484510
• 负责人: LUBERT STRYER
• 金额: $ 22.02万
• 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 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3484510
• 关键词: X ray crystallography; actins; analytical method; antibody receptor; cell cycle; cell membrane; chemical reaction; chemical synthesis; complement receptor; conformation; deuterium; 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; laboratory mouse; laser spectrometry; membrane model; monoclonal antibody; mutant; neutron diffraction; photography; protein engineering; protein structure; receptor; saltwater environment; sea urchins; single cell analysis

The overall aims are to develop novel fluorescence techniques and use them in concert with other experimental approaches to elucidate the structure and dynamics of selected proteins in the next five years, we will focus on (1) the molecular mechanism of triggering of effector functions of immunoglobulins, (2) the functional significance of segmental flexibility, and (3) the development of new phycobiliprotein fluorescent probes for use in high-sensitivity fluorescence assays. Conformational transitions induced by the binding of antigen will be detected by fluorescence studies of probes attached to specific sites in each of the immunoglobulin domains and the hinge region. Reactive cysteine residues will be introduced by site-specific mutagenesis of serine and alanine residues near the surface. Fluorescent probes will be attached to these cysteines. The effects of binding univalent and multivalent antigens will be monitored by changes in the emission spectrum, quantum yield, and excited-state lifetime of the fluorescent probes. Distances between pairs of specifically labeled sites will be determined using fluorescence energy transfer as a spectroscopic ruler. Diffusion-enhanced energy transfer using long-lived terbium chelates will provide information concerning the depth and accessibility of labeled cysteine residues. Rotation motions of domains will be delineated by nanosecond fluorescence polarization spectroscopy. The hinge region and adjacent residues in the CH1 domain will be changed by site-specific mutagenesis to gain insight into the control of segmental flexibility and its functional significance. These studies should reveal whether complement fixation is triggered by the clustering of Fc units, the unmasking of effector sites by movements of domains, or by propagated conformational changes that activate the effector site. The effect of antigen on the conformation and dynamics of membrane-bound immunoglobulin in reconstituted membranes will also be investigated by fluorescence techniques to gain insight into transmembrane signaling. New phycobiliprotein fluorescent probes that emit in the far red and near infrared will be synthesized for use in multiparameter fluorescence-activated cell sorting and high-sensitivity fluorescence immunoassays. Phycobiliproteins with energy acceptors joined to them by a cleavable bond will be prepared as substrates for enzyme-linked immunoassays. A microfluorimeter optimized for detecting particles labeled with phycofluors (e.g., microorganisms) will be constructed.

总体目标是开发新型荧光技术并使用它们 与其他实验方法相结合来阐明结构 以及未来五年选定蛋白质的动态，我们将重点关注 (1)效应功能触发的分子机制 免疫球蛋白，(2) 节段灵活性的功能意义， (3)新型藻胆蛋白荧光探针的开发使用 在高灵敏度荧光测定中。 构象转变 抗原结合诱导的荧光研究可检测到 连接到每个免疫球蛋白结构域中特定位点的探针 和铰链区。 反应性半胱氨酸残基将通过以下方式引入 表面附近丝氨酸和丙氨酸残基的定点诱变。 荧光探针将附着在这些半胱氨酸上。 的影响 结合单价和多价抗原将通过变化来监测 发射光谱、量子产率和激发态寿命 荧光探针。 专门标记的位点对之间的距离 将使用荧光能量转移作为光谱来确定 统治者。 使用长寿命铽进行扩散增强能量传输 螯合物将提供有关深度和可及性的信息 标记的半胱氨酸残基。 将描述域的旋转运动 通过纳秒荧光偏振光谱法。 铰链区和 CH1 结构域中的相邻残基将因位点特异性而改变 诱变以深入了解片段灵活性的控制和 其功能意义。 这些研究应该揭示是否 补体固定是由 Fc 单元的聚集触发的， 通过域的移动或传播来揭露效应位点 激活效应位点的构象变化。 的效果 抗原对膜结合免疫球蛋白构象和动力学的影响 重组膜中的成分也将通过荧光进行研究 深入了解跨膜信号传导的技术。 新的 藻胆蛋白荧光探针发射远红光和近红光 红外线将被合成用于多参数 荧光激活细胞分选和高灵敏度荧光 免疫测定。 藻胆蛋白与能量受体通过 将制备可裂解的键作为酶联的底物 免疫测定。 用于检测标记颗粒而优化的微荧光计 与藻氟菌（例如微生物）一起构建。