A DNA origami platform for measuring membrane protein interactions

项目总结/摘要 细胞膜上充满了蛋白质，这些蛋白质的相互作用对无数细胞的生长至关重要。 功能，从代谢到信号传导，包括参与过程的许多功能， 神经退化众所周知，膜蛋白质很难处理和研究。对于许多 膜蛋白，它们是否单独存在（作为单体），成对存在（作为二聚体）， 或更大的集合（更高的低聚物）。一种确定多个 蛋白质复合物中的亚基在其天然脂质膜环境中将分解 目前存在的一些争议，并最终会对人类健康产生很大的影响。一 解决这个问题的方法是在单分子水平上研究蛋白质相互作用， 可能需要昂贵且复杂的仪器。另一种方法是使用 相对便宜的，化学自组装的“分子手”来编程相互作用 精确控制蛋白质的数量和比例（它们的“化学计量”）之间的关系。以 第二种方法，我们建议开发一个研究蛋白质-蛋白质的通用平台 脂质膜中的相互作用，DNA折纸环模板脂质体。这个平台将 允许精确控制和测量单个脂质内的蛋白质-蛋白质相互作用 双层，并克服了现有方法的局限性，用于区分单体， 二聚体。将构建一个充满圆盘状脂质体膜的DNA折纸环 具有用于感兴趣的单个蛋白质的附着点。沿着以纳米精度间隔 环的边缘，这些附着点将用于定义蛋白质的数量和类型 可以进入细胞膜的亚基。蛋白质程序性释放进入细胞膜 将通过引入DNA信号来实现，该信号会破坏细胞之间的DNA接头 蛋白质和DNA环的边缘。在我们的第一个目标，我们将原型和故障排除 通过研究荧光标记的DNA测试分子在许多环境中的相互作用， 对照实验在第二个目标中，我们将用蛋白质取代DNA测试分子， 一个已知的相互作用，并验证该平台可用于测量蛋白质-蛋白质 交互.最后，我们将集中精力解决一个长期存在的问题， SNARE复合蛋白Synaptobrevin 2的二聚化，这是一个重要的参与者， 释放神经递质所需的膜融合过程。

Project Summary/Abstract Cell membranes are populated with proteins whose interactions are important for myriad cellular functions, from metabolism to signaling, including many functions implicated in processes such as neurodegeneration. Membrane proteins are notoriously difficult to handle and study. For many membrane proteins it is unknown whether they exist singly (as monomers), in pairs (as dimers), or larger collections (higher oligomers). A method to determine the number and arrangement of subunits in a protein complex within its native lipid membrane environment would resolve a number existing controversies, and would eventually have a large impact human health. One approach to this problem would be to study protein interactions at the single molecule level, which can require expensive and complex instrumentation. Another approach would be to employ a relatively inexpensive, chemically self-assembled “molecular hand” to program the interactions between precisely controlled numbers and ratios of proteins (their “stoichiometry”). Taking the second approach, we propose to develop a general platform for studying protein-protein interactions in lipid membranes, the DNA origami ring-templated liposome. This platform will allow exquisite control and measurement of protein-protein interactions within a single lipid bilayer, and overcome the limitations of existing methods for differentiating monomers from dimers. A DNA origami ring, filled with a disc-shaped liposomal membrane, will be constructed with attachment points for individual proteins of interest. Spaced with nanometer-precision along the edge of the ring, these attachment points will be used to define the number and type of protein subunits that can enter the membrane. Programmed release of the proteins into the membrane will be achieved through the introduction of DNA signals, that break DNA linkers between the proteins and the edge of the DNA ring. In our first aim, we will prototype and troubleshoot the platform by studying the interactions of fluorescently labelled DNA test molecules in a number of control experiments. In a second aim, we will replace the DNA test molecules with proteins having a known interaction, and verify that the platform can be used to measure protein-protein interactions. In a final aim, we will focus on resolving a long-standing question regarding the dimerization of a SNARE complex protein Synaptobrevin 2, which is an important participant in the membrane fusion process required for the release of neurotransmitters.