The overall 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 a family of prototypical channel-formers, the linear gramicidins. This system offers several advantages: there is a considerable amount of structural information to guide the experimental design; the channel-forming molecules are modified using peptide chemical methods, which allows the convenient introduction of non-genetic amino acids; and it is possible to obtain structural information about channels formed by sequence-modified gramicidins by making use of the inherent resolution of single-channel measurements. The experiments will address the following questions: what are the determinants of channel folding and membrane insertion; what are the energetics of peptide interactions in a membrane; specifically, what are the determinants of the voltage-dependent gating seen in asymmetrical gramicidin channels; what are the determinants of the channels' permeability characteristics; and how is channel function modulated by the chemical composition and physico-chemical properties of the host bilayer? These questions will be examined in single-channel experiments. The amplitude of the current transitions will be used to characterize the channels' catalytic efficiency. the formation of heterodimer (hybrid) channels and their stability and appearance rate relative to the homodimeric channels will be used to elucidate the structural questions.
The aim i s to understand how a """"""""simple"""""""" channel functions: how the anisotropic membrane environment and the primary amino acid sequence interact in determining the channel structure; how a """"""""stress"""""""" introduced by sequence modifications affect the structure; specifically, how relatively modest sequence alterations can introduce voltage control into the channels; how the rate of ion movement is determined by the channel structure and the amino acid sequence; and how alterations in the host bilayer alter channel function, in particular whether alterations in a bilayer's mechanical properties can alter the equilibrium distribution among different channel states.
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