Voltage-Gated Insertion of Colicin into Planar Bilayers

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DESCRIPTION (provided by applicant) Studies on membrane import and channel formation of the pore-forming colicins concern: the nature of (i) the large soluble --> membrane-bound structural transition undergone by colicins, toxins, and other membrane-active proteins, (ii) the surface-bound state that potentates helix insertion, (iii) structure changes associated with voltage-gated channel formation, and (iv) the pathway of protein insertion into the membrane, and (v) the mechanism by which the MW = 65,000 colicins are translocated across the E. coli outer membrane. The structure of the colicin E1 channel domain, solved at atomic resolution, allows structure-based mutagenesis strategies to test models for structural transitions upon membrane-binding and channel formation. Single-Trp and -Cys mutants were used in fluorescence quenching and fluorescence resonance energy transfer to define the colicin channel bound in the membrane interfacial layer as an extended, flexible, two-dimensional helical net. Planar lipid bilayer experiments have been carried out in collaboration with the lab of Y. N. Antonenko (Moscow, Russia) to observe the kinetics of colicin channel formation and related properties. There have been 4 collaborative projects: (1) Colicin channel activity was photoinactivated in the presence of sensitizing dyes, and this effect depended on the presence of Trp495 in helix 9 of channel domain. Colicin photoinactivation will serve as an important model for study of photodamage of membrane proteins and photodynamic therapy, widely used in cancer treatment. (2) Colicin import and channel formation was found to be very sensitive to membrane anionic lipid content and to be "tuned" at a surface potential of -60 +/- 5 mV. (3) The channel activities of purified outer membrane receptors proteins were found to be occluded by exogenous colicin. (4) Preliminary experiments indicate that colicin El membrane-binding and channel formation is affected by the lipid interfacial dipole potential. It is proposed to simultaneously measure channel current and fluorescence with horizontal planar bilayers and to analyze the kinetics and pathway of voltage-gated colicin insertion into, and channel formation in, the membrane.