The long-term objective of the research program is to understand how the structure and function of membrane-spanning channels is determined by their amino acid sequence and the properties of the host bilayer. The proposed experiments will examine these problems using the linear gramicidins as a model system. This system offers a unique combination of advantages that, at the present time, sets it apart from all other ion channels (and most, if not all, soluble peptides/proteins): the channel structure is known at atomic resolution, which serves as a guide for the experimental design and data analysis; it is possible to obtain quite detailed structural information about the channels using functional (single-channel) measurements; the channels are permeable only to monovalent cations, which means that single-channel measurements provide direct information about the channels catalytic ability; the channel-forming molecules are made/modified using peptide chemical methods, which allows the convenient introduction of non-genetic amino acids; and gramicidin channels are suitable to be used as molecular force transducers to quantify the energetics of channel/membrane interactions, which provides for a novel way to examine membrane/protein interactions. The proposed experiments address the following questions; what are the molecular determinants of channel folding and membrane insertion; what are the energetics of peptide interactions in a membrane; how does a structural destabilization introduce voltage-dependent gating in an ion channel; how is channel structure and function modulated by the chemical composition and physico-chemical properties of the host bilayer; and what are the molecular and structural determinants of the channels' permeability characteristics? The questions will be examined using a combination of single-channel experiments and molecular modeling: the amplitude of single-channel current transitions will be used to characterize the channels' catalytic efficiency; the formation of heterodimer (hybrid) channels, and their stability and appearance rate relateive to the homodimeric channels, constitutes a new method to elucidate structural questions pertaining to channel folding which is more powerful than many conventional spectroscopic methods. The experimental results will e used to guide conformational energy and molecular dynamics calculations, which in turn will be used to interpret the experimental results and guide the design of new experiments.
The aims are to understand how the primary amino acid sequence and the anisotropic membrane environment interact in determining channel structure and function; how a """"""""stress"""""""" introduced by sequence modifications affect the structure and dynamics, specifically how relatively modest sequence alterations can introduce voltage control of channel function; how alterations in the host bilayer alter channel function-whether alterations in a bilayer's mechanical properties alter the equilibrium distribution between different channel state acid sequence.
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