The 522 residue colicin E1 voltage-gated ion channel is used to study basic aspects of the major structure transition of a toxin-like molecule from the water-soluble to the integral membrane-bound state, the properties associated with the unique intramembrane interaction of the colicin channel with its immunity protein, and its translocation across the cell envelope from outer membrane across the periplasmic space to the cytoplasmic membrane. The channel is highly charged with a pronounced C- terminal hydrophobic domain, and its interaction with membranes is representative of the many facets of membrane interactions of proteins contrasted with those of small peptides. Using newly developed DNA constructs and purification procedures, the studies utilize a 190 residue C-terminal colicin channel polypeptide (P190) over-produced under its own promoter, purified 113 residue hydrophobic immunity protein, and purified 368 residue soluble TolA protein from the cell intermembrane translocation system. The studies will combine a combination of structural, biochemical, biophysical, and molecular genetic approaches. The in vitro transition from H2O to the membrane involves structure intermediates, including a partly unfolded state in solution, and an unfolded state on the surface of the membrane. (I) Structure information will be obtained from (i) an X-ray structure determination of the P190 channel; (ii) NMR structure studies including solid-state analysis of the trans-membrane helix, and secondary structure and folding of the immunity protein in a membrane-mimetic organic solvent mixture; (iii) the identity of residues in contact with the channel aqueous lumen measured by single cysteine substitution and chemical modification by channel-blockers at consecutive residues around helix turns; (iv) the identity of specific lysine residues, Lys 362,395,397,402,403, inferred from structural models to be involved in the docking, will be tested by mutagenesis and quantitative analysis of binding parameters; (v) Cys-Cys crosslinking of specific helices will be used to determine the unfolding steps that must accompany binding to the membrane surface; (vi) the significance of an unusual SDS- resistant dimer of the immunity protein will be tested with imm mutants with impaired activity; (vii) imm 2nd-site revertants to existing imm bypass mutants will define sites of interaction; (viii) the TolA-colicin interaction that confers in vivo translocation-competence will be sought; (ix) 2 dimensional crystallization of the immunity protein and structure solution by EM image analysis will be performed. (II) Quantitative determination of equilibrium and kinetic binding parameters will also be carried out as a function of acidic lipid content, along with the solid- state NMR analysis, to test the inference based on data obtained in the last grant period that the insertion of the hydrophobic domain into the membrane, and the resulting channel activity, requires an optimum surface electrostatic interaction. A weak interaction precludes effective binding. One that is too strong prevents insertion into the bilayer.

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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Shapiro, Bert I
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Purdue University
Schools of Arts and Sciences
West Lafayette
United States
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