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 i s 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.
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