Molecular Mechanisms of Mechanosensitive Channel Gating

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DESCRIPTION (provided by applicant): The study of the bacterial mechanosensitive MscL channel has biomedical significance for several reasons. First, the channel serves a vital function in maintaining osmotic homeostasis of microbes; when the channel misfunctions it can lead to the death of the microbial cell, and thus may be a viable pharmacological target. Second, as nanotechnology progresses, the potential for biological sensors, especially MscL, to be used in biomedical nano-devices is being realized. Third, MscL is an excellent model for the study of mechanosensory transduction. Because of its tractable nature and the existence of a crystal structure, MscL is a unique tool to investigate structure-function relationships in a mechanosensory channel. We have been exploiting this system to obtain a better understanding of the molecular mechanisms of how a mechanosensitive channel senses and responds to membrane tension; this is the long-term objective of this proposal. While models for structural transitions during gating have been proposed, they are not consistent or complete, and many of the fundamental features are not yet resolved. The experiments within this proposal are designed to ally the solved structure with molecular, biochemical and electrophysiological analyses to determine the functional role that regions of the protein play in sensing and responding to membrane stretch, to define transitions that occur upon gating, and to determine aspects of the open-pore structure. The approaches used include: the generation of chimeras to determine the structural elements associated with functional differences of homologues, utilizing the results from a sulfhydryl-based post-translational scan to determine residues that should be further tested by mutagenesis for whether they enter or exit a lipid environment upon gating, disulfide trapping to define closed, transition and open states of the channel, and attempts to crystallize the channel in these multiple states.