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.
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