The Method for Determination of Membrane Protein Structures without Purification

描述（由申请人提供）：在这十年中，分子生物学的主要挑战之一是确定膜蛋白的三维结构。从细菌到人类的基因组分析表明，总蛋白质中有多达30%是膜蛋白。然而，尽管迄今为止已经在PDB中沉积了多达17，780个三维结构，但这些结构中只有0.56%是膜蛋白。膜蛋白结构研究的惊人缺乏有许多原因。首先，膜蛋白是高度疏水性的，这使得将它们纯化到结构研究所需的均一性非常困难。其次，膜蛋白的表达通常很差。有没有办法解决这些问题？如果在活细胞中只产生一种感兴趣的膜蛋白而不产生任何其他细胞蛋白，则可以在不纯化的情况下进行膜蛋白的结构研究。毫无疑问，这将彻底改变膜蛋白的结构生物学，并将为我们理解膜蛋白的结构和功能做出重大贡献。在这个提议中，我将尝试建立在活细胞中产生感兴趣的单一膜蛋白而不产生任何其他细胞蛋白所需的技术。这项技术，被称为“SPP系统”，将使我们能够确定膜蛋白的NMR结构，而无需纯化，并探索活细胞中的蛋白质动力学。与公共卫生的相关性：这项研究将开发的技术将在膜蛋白的结构生物学方面取得重大突破，膜蛋白在人类健康和人类疾病中发挥着许多关键作用。 与公共卫生的相关性：这项研究将开发的技术将在膜蛋白的结构生物学方面取得重大突破，膜蛋白在人类健康和人类疾病中发挥着许多关键作用。 在这十年中，分子生物学的主要挑战之一是确定膜蛋白的三维结构，这些膜蛋白在活细胞中发挥各种重要作用，因此直接与人类健康相关。从细菌到人类的基因组分析表明，总蛋白质中有多达30%是膜蛋白。然而，尽管事实上已经确定了多达17，780个三维结构，但这些结构中只有0.56%是膜蛋白。对膜蛋白结构研究的这种令人惊讶的缺乏的主要原因是由于膜蛋白纯化的极端困难和它们极低的产率。为了解决这些问题，我将尝试建立在活细胞中产生感兴趣的单一膜蛋白而不产生任何其他细胞蛋白所需的技术。这项技术，被称为“SPP系统”，将使我们能够确定膜蛋白的结构，而无需纯化，并探索活细胞中的蛋白质动力学。毫无疑问，这将彻底改变膜蛋白的结构生物学，并将为我们理解膜蛋白在活细胞中的功能做出重大贡献。

DESCRIPTION (provided by applicant): One of the major challenges in molecular biology during this decade is the determination of three-dimensional structures of membrane proteins. The analysis of genomes from bacteria to humans reveals that as much as 30% of the total proteins are, indeed, membrane proteins. However, in spite of the fact that as much as 17,780 three- dimensional structures have been deposited in the PDB to date, only 0.56% of these structures are of membrane proteins. There are a number of reasons for this surprising lack of structural studies of membrane proteins. First, membrane proteins are highly hydrophobic, which makes it extremely difficult to purify them to the homogeneity required for structural studies. Second, the expression of membrane proteins is generally very poor. Is there a way to circumvent these problems? If one can produce only a single membrane protein of interest without producing any other cellular proteins in living cells, the structural study of membrane proteins can be carried out without purification. This will, no doubt, revolutionize the structural biology of membrane proteins and will make a major contribution to our understanding of the structure and function of membrane proteins. In this proposal, I will attempt to establish the technology needed to produce a single membrane protein of interest in living cells without producing any other cellular proteins. This technology, termed the "SPP system", will then enable us to determine the NMR structures of membrane proteins without purification and to explore protein dynamics in living cells. Relevance to public health: The technology that will be developed in this study will make a major breakthrough in the structure biology of membrane proteins, which play numerous crucial roles in human health and human diseases. Relevance to public health: The technology that will be developed in this study will make a major breakthrough in the structure biology of membrane proteins, which play numerous crucial roles in human health and human diseases. One of the major challenges in molecular biology during this decade is the determination of three-dimensional structures of membrane proteins, which play various essential roles in living cells and thereby are directly related with human health. The analysis of genomes from bacteria to humans reveals that as much as 30% of the total proteins are, indeed, membrane proteins. However, in spite of the fact that as much as 17,780 three- dimensional structures have been determined, only 0.56% of these structures are of membrane proteins. The major reason for this surprising lack of structural studies on membrane proteins is due to the extreme difficulty of membrane protein purification and their extremely poor yields. To circumvent these problems, I will attempt to establish the technology needed to produce a single membrane protein of interest in living cells without producing any other cellular proteins. This technology, termed the "SPP system", will then enable us to determine the structures of membrane proteins without purification and to explore protein dynamics in the living cells. This will, no doubt, revolutionize the structural biology of membrane proteins and will make a major contribution to our understanding of how membrane proteins function in living cells.