Molecular Mechanisms of Mechanosensitive Channel Gating

描述（由申请人提供）：由于几个原因，细菌机械敏感性MscL通道的研究具有生物医学意义。首先，该通道在维持微生物的渗透稳态中起着至关重要的作用；当通道功能失调时，它可能导致微生物细胞死亡，因此可能是一个可行的药理学靶点。其次，随着纳米技术的发展，生物传感器，特别是微型微型传感器在生物医学纳米器件中的应用潜力正在实现。第三，MscL是研究机械感觉传导的一个很好的模型。由于其易于处理的性质和晶体结构的存在，MscL是研究机械感觉通道中结构-功能关系的独特工具。我们一直在利用这个系统来更好地理解机械敏感通道如何感知和响应膜张力的分子机制；这是本提案的长期目标。虽然已经提出了浇注过程中结构转变的模型，但它们并不一致或完整，而且许多基本特征尚未得到解决。本提案中的实验旨在将解决的结构与分子，生化和电生理分析结合起来，以确定蛋白质区域在感知和响应膜拉伸时所起的功能作用，定义门控时发生的转变，并确定开孔结构的各个方面。使用的方法包括：产生嵌合体来确定与同源物功能差异相关的结构元件，利用基于巯基的翻译后扫描结果来确定应该通过诱变进一步测试的残基，以确定它们是否在门化时进入或退出脂质环境，二硫捕获来定义通道的关闭，过渡和开放状态，并尝试在这些多种状态下使通道结晶。

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.