The proposal seeks to uncover fundamental mechanisms of transmembrane processes at the level of atomic structure. Specific objectives are to understand the mechanisms of transmembrane signaling in the acetylcholine receptor superfamily of pentameric, ion channel neuroreceptors by structural analysis of components, fragments of the AcCh Receptor alpha chain in complex with neurotoxins to which they bind tightly, the extracellular portion of a subunit, and portions of a whole receptor. These are to provide a three dimensional road map of interactions and will have broad impact on the understanding of fundamental elements in neurochemistry, and structure assisted development of selective psychoactive drugs. The structure of a bacterial, monomeric ion channel-forming protein colicin Ia (Col Ia), with similar conductance properties to neuronal receptors, is being extended in resolution from 3.0 Angstrom units to 2.3 Angstrom units. Mutations will be used to define the impact of charge and polar arrangements in regulating conductance I and selectivity of ion channels. Interpretation of the unique structural features, and their role in determining receptor binding, translocation and channel insertion will be defined by a combination of mutational, channel, forming, bacterial targeting, thermodynamic, and structural analysis. Electron microscopic analysis is to assist in defining the membrane bound channel state. The mechanism of host immunity to this bacteriocidal protein involves high affinity intra-membrane association with a three membrane-crossing immunity protein Imm Ia. The structure of this complex will be sought to uncover basic mechanisms of intramembrane interaction, with impact on the design of modulators of transmembrane receptors. The structure of a channel forming Bacillus thuringiensis toxin (CytA) at 2.3 Angstrom units is a paradigm for interpreting the many existing mutations that alter transmembrane assembly, and channel forming properties, to understand the mechanisms of channel formation, and the membrane bound form. The initial 1.6 Angstrom units structure of apo-Cholesterol esterase, an abundant, enzyme important in regulating transmembrane transport of triglycerides and cholesterol from cholesterol esters is to understand membrane active mechanisms, and to provide a template for structure assisted drug development.
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