OPTICAL STUDIES OF PROTEIN STRUCTURE AND FUNCTION

总体目标是开发新的荧光技术并使用它们 与其他实验方法相结合来阐明结构 和动态的选定蛋白质在未来五年，我们将重点放在 (1)触发效应器功能的分子机制 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研制 在高灵敏度的荧光分析中。构象转变 将通过荧光研究来检测由抗原结合所诱导的 连接到每个免疫球蛋白结构域中的特定位置的探针 以及铰链区域。活性半胱氨酸残留物将通过 表面附近丝氨酸和丙氨酸残基的定点突变。 荧光探针将被连接到这些半胱氨酸上。的影响 结合单价和多价抗原将通过以下变化进行监测 分子的发射光谱、量子产额和激发态寿命 荧光探头。特定标记的位置对之间的距离 将使用荧光能量转移作为光谱来确定 尺子。长寿命Tb的扩散增强能量传递 螯合物将提供有关深度和可获得性的信息 标记的半胱氨酸残基。区域的旋转运动将被描绘出来 通过纳秒荧光偏振光谱。铰链区域和 CH1结构域中的相邻残基将因位点而异 突变以了解节段灵活性和节段灵活性的控制 它的功能意义。这些研究应该揭示出 补体结合由Fc单元的聚集触发， 通过域的移动或通过传播来揭开效应器位置的掩蔽 激活效应器部位的构象变化。的影响 膜结合免疫球蛋白构象和动力学的抗原 重组膜中的荧光也将被用来研究 深入了解跨膜信号传递的技术。新的 远红光和近红光发射的藻胆蛋白荧光探针 红外线将被合成用于多参数 荧光激活细胞分选与高灵敏度荧光 免疫分析。藻胆蛋白与能量受体通过一种 将制备可裂解的键作为酶连接的底物 免疫分析。一种用于检测标记的颗粒物的微型荧光仪 利用藻类(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.