Modular Design of Voltage-Gated Channel Proteins

描述(由申请人提供)：最终目标是理解序列结构决定论的基本原理，序列结构决定论是当代生物学中一个尚未解决的主要问题。目前的目标是通过蛋白质设计来解决这一问题，并利用其组成的电压传感器(VSM)和孔道(PM)模块制造电压门控通道，在脂质双层中重建后恢复其功能，并通过溶液和固体核磁共振光谱相结合的方法确定其在膜上的三维结构。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，电压敏感的潜在机制还没有完全了解，原子分辨率的结构也是不可用的。可获得变铅青链霉菌KCSA和热自养分支杆菌MthK的质膜结构。VSM的结构仍然难以捉摸，但这是赋予电压门控通道将跨膜电压耦合到通道开口的能力的独特元件。这就是需要做的事情，也是拟议的工作要实现的目标。修订后的应用的具体目标集中在提供VSM的结构和包含原核生物和设计的通道蛋白的VSM和PM模块的全通道。功能是通过在脂双层中重组纯化的蛋白质和通过在哺乳动物细胞中表达基因产物来确定的。蛋白质结构的确定是通过多维核磁共振谱的同位素标记的蛋白质在氚化脂胶束和固体核磁共振在定向磷脂双层。成功设计的最终检验是通过将VSM和PM组装并确定其结构来重现整个蛋白质的生物学功能。这些进展可能为理解疾病机制提供有价值的见解，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.