The structure of the colicin El channel domain is studied by (i) spectroscopic (FTIR and CD) determination of the secondary structure of the channel peptide incorporated into artificial membranes; (ii) site-directed mutagenesis of the prominent hydrophobic sequence near the COOH-terminus to determine the limits of the membrane-spanning domain, the residues that interact with the polar channel interior, and the regions of local hydrophobicity. Mutagenesis will also be used to test two other highly amphipathic nineteen residue segments that are proposed to be membrane spanning alpha=helices, and to generate Cys-containing mutants that can be labeled with heavy atoms for the phase analysis of well-diffracting crystals ([2.5 Alpha resolution_ of tryptic and thermolytic channel peptides. Initial experiments on preparation of heavy atom derivatives will use the one cysteine in the channel peptides, Cys-505, that has been previously modified without loss of function. (iii) The minimum fully active peptide will be determined, as will the monomeric or multimeric state of the functional channel. Studies on channel formation and the associated translational dynamics would focus on: (iv) tryptophan residues in site-directed mutants containing a single tryptophan; (v) the using directed mutagenesis to test residues implicated in binding to the membrane surface, using directed mutagenesis to test residues implicated from the structure of the soluble colicin Alpha peptide; (vi) the dynamics and the time course of voltage-dependent insertion into the membrane bilayer, as well as pH-dependent reversible insertion and extrusion. The segments of the channel peptide that insert into the bilayer in the presence of the segments and individual amino acids by lipophilic and phospholipid photoaffinity probes. Initial studies have identified one such segment. (vii) The colicin E3-btuB and El colicin and receptor binding domains defined, the role of receptor examined in the unfolding of the imported colicin, and the mechanism of receptor into liposomes. The structure of the colicin channel and the mechanism and dynamics of its import bear on the problems of protein import and secretion, translocation of toxin molecules, eukaryotic voltage-dependent and ligand-gated channels, and receptor function.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biophysical Chemistry Study Section (BBCB)
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Purdue University
Schools of Arts and Sciences
West Lafayette
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