Modular Design of Voltage-Gated Channel Proteins

描述（由申请人提供）：最终目标是了解序列结构决定论的基本原理，这是当代生物学中尚未解决的主要问题。当前的目标是通过蛋白质设计来解决这个问题，并从其组成电压传感器（VSM）和孔（PM）模块产生电压门控通道，在脂质双层中重构后检索其功能，并通过溶液和固态核磁共振波谱的结合来确定其在膜中的三维结构。电压门控通道的选择是基于它们作为细胞兴奋性的关键控制元件的重要性，电压传感的机制尚不完全清楚，原子分辨率的结构尚不可用。获得了S. lividans KcsA和M. thermoautotrophicum MthK的PM结构。VSM的结构仍然难以捉摸，但它是赋予电压门控通道跨膜电压耦合通道打开能力的独特元件。这是需要做的事情，也是拟议的工作打算实现的目标。修订后的应用程序的具体目标集中在提供含有原核和设计通道蛋白的VSM和PM模块的VSM和完整通道的结构。功能是通过脂质双层中纯化蛋白的重构和哺乳动物细胞中基因产物的表达来建立的。蛋白质结构由氘化脂胶束中同位素标记的蛋白质的多维核磁共振光谱和定向磷脂双层中的固态核磁共振确定。成功设计的最终测试是通过组装VSM和PM并确定其结构来重现整个蛋白质的生物学功能。这些进展可能有助于了解疾病的机制，并为药物设计提供结构蓝图。

DESCRIPTION (provided by applicant): The ultimate goal is to understand the fundamental principles underlying the sequence-structure determinism, a major unsolved issue in contemporary biology. The immediate objective is to approach this question by protein design and produce a voltage-gated channel from its constituents voltage sensor (VSM) and pore (PM) modules, to retrieve its function after reconstitution in lipid bilayers, and to determine its three-dimensional structure in membranes by a combination of solution and solid-state NMR spectroscopy. The choice of voltage-gated channels is based on their importance as key control elements of cellular excitability, the mechanism underlying voltage-sensing is not fully understood, and a structure at atomic resolution is not available. A structure for the PM of S. lividans KcsA and of M. thermoautotrophicum MthK is available. The structure of the VSM remains elusive, yet this is the unique element that endows voltage-gated channels with the ability to couple a transmembrane voltage to channel opening. This is what needs to be done and what the proposed work intends to achieve. The specific aims for the revised application are focused on providing structures of the VSM and the full channel containing both VSM and PM modules of prokaryotic and designed channel proteins. Function is established by reconstitution of purified proteins in lipid bilayers and by expression of gene products in mammalian cells. Protein structure is determined by multidimensional NMR spectroscopy of isotopically labeled proteins in deuterated lipid micelles and by solid-state NMR in oriented phospholipid bilayers. The ultimate test of a successful design is recapitulation of biological function of the whole protein by assembling it from VSM and PM and determining its structure. These advances may contribute valuable insights to understand mechanisms of disease and provide structural blueprints for drug design.