Membrane ion-channels are extremely important components of biological membrane systems, controlling many important cell functions. Unfortunately, these systems are poorly understood, especially at the level of molecular structure and function. Part of the difficulty in obtaining this information lies in the fact that many structural techniques applicable to water soluble protein systems can not be used on these large membrane proteins. The work proposed here is aimed at providing molecular information on the structure of a simpler extrinsic ion-channel formed by the macrolide antibiotic amphotericin B. It is anticipated that the molecular features learned from this simpler ion-channel will provide insight into the function of native channels. The development and assessment of techniques used in this project will also provide an experimental and theoretical foundation for work on more complex systems. In addition, amphotericin B is an extremely important medicinal agent that is used clinically to treat systemic fungal infections. Its greater toxicity for fungal cells is believed to result from its preferential association with ergosterol (vs. cholesterol) to form ion-channels. There is clearly a need to develop new analogues of this macrolide as it proves to be quite toxic to mammalian systems. Work in this proposed project will also facilitate the development of new, badly needed pharmaceuticals. Several novel magnetic resonance methods will be utilized in this work. Newly developed two-dimensional nuclear magnetic resonance methods will yield information on structure and a number of powerful electron paramagnetic resonance techniques will facilitate electrostatic measurements of ion-channel activity. Finally, new synthetic derivatives of amphotericin B will be utilized to determine important structural features for the formation of macrolide-sterol ion-channels.
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