OPTICAL STUDIES OF PROTEIN STRUCTURE AND FUNCTION

总的目标是开发新的荧光技术，并使用它们 与其他实验方法一起来阐明结构 在未来五年内，我们将重点关注选定蛋白质的动态， (1)触发效应器功能的分子机制 免疫球蛋白，（2）节段柔性的功能意义， 新型藻胆蛋白荧光探针的研制 在高灵敏度荧光分析中。 构象转变 将通过荧光研究检测由抗原结合诱导的 在每个免疫球蛋白结构域的特定位点上附着的探针 和铰链区。 反应性半胱氨酸残基将通过 表面附近丝氨酸和丙氨酸残基的位点特异性诱变。 荧光探针将附着于这些半胱氨酸。 的影响 结合单价和多价抗原将通过 的发射光谱，量子产率，和激发态寿命的 荧光探针。 特异性标记位点对之间的距离 将使用荧光能量转移作为分光光度计来测定 尺子。 使用长寿命铽的扩散增强能量转移 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。 描述了磁畴的旋转运动 通过纳秒荧光偏振光谱。 铰链区和 CH1结构域中的相邻残基将被位点特异性的 诱变以深入了解片段柔性的控制， 其功能意义。 这些研究应该揭示， 补体结合是由Fc单元的聚集触发的， 通过结构域的移动或通过传播的效应位点的暴露， 激活效应位点的构象变化。 的影响 抗原对膜结合免疫球蛋白构象和动力学的影响 也将通过荧光法研究重组膜中的 技术来深入了解跨膜信号。 新 藻胆蛋白荧光探针，发射远红光和近红光， 红外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一个 将制备可裂解键作为酶联底物 免疫测定。 一种优化的用于检测标记颗粒的微荧光计 用藻荧光素（例如，微生物）将被构建。

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.