Some major problems of general interest from studies on the pore-forming colicins are: the nature of (i) the large soluble yields membrane-bound structural transition undergone by colicins, toxins, and other membrane-active proteins; (ii) the surface-bound state that potentiates helix insertion; (iii) structure changes associated with voltage-gated channel formation. 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. To initiate studies on the structure transition from closed- to open-channel state, 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. 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. Cross-linking of the polypeptide molecule (dimerization) was detected in parallel experiments with channel domain bound to liposomes. Based on studies of the Antonenko lab on the gramicidin cation-selective channel, it is proposed to investigate the mechanism of sensitized photoinactivation of the colicin E1 channel, and associated structure changes. The Trp-dependence of photoinactivation allows the use of single-Trp mutants to infer the helices and specific side chain involvement in channel formation. New approaches that will be developed in the course of this study will extend the application of Trp- and Cys-scanning mutagenesis. Preliminary experiments show also that colicin E1 membrane-binding and channel formation is affected by the lipid interfacial dipole potential. Both single-and multi- channel measurements will be used to investigate the mechanism photoinactivation and the role of the membrane dipole potential. Colicin photoinactivation will serve as an important model for study of photodamage of membrane proteins and photodynamic therapy, widely used in cancer treatment.
| Zakharov, Stanislav D; Hulleman, John D; Dutseva, Elena A et al. (2007) Helical alpha-synuclein forms highly conductive ion channels. Biochemistry 46:14369-79 |
