The goal of the proposed research is to develop novel fluorescence techniques and use them in concert with other experimental approaches to elucidate the structure and dynamics of selected model and biological membrane systems. We plan to carry out the following studies: (1) We will continue our x-ray crystallographic studies of the transmembrane channel formed by gramicidin A, a peptide antibiotic, and of its complexes with alkali cations. Heavy atom derivatives will be synthesized to solve these structures at high resolution. (2) Our x-ray crystallographic and fluorescence studies have shown that gramicidin A undergoes a major conformational change on binding alkali cations. The dynamics of these structures changes in membranes will be investigated by optical and conductance techniques. Gramicidin analogs will be synthesized with the aim of making the channel voltage-sensitive. (3) A new fluorescence energy transfer approach using long lifetime donors such as chelates of terbium will be developed and applied to obtain information about lateral diffusion, lateral proximity and transverse proximity in membranes. (4) The synchrotron radiation source at SLAC will be used for subnanosecond fluorescence studies of rotational motions of aromatic residues in relation to the conformational flexibility and folding of proteins. The segmental flexibility and antigen-triggered conformational changes in immunoglobulins will also be investigated by this technique. (5) New fluorescent labeling reagents for membrane systems will be designed and synthesized. Emphasis will be given to preparing activatable and cleavable fluorescent groups, and to attaching probes to specific sugars of membrane glycoproteins.
NIH ExPorter
NIH ExPorter