The Method for Determination of Membrane Protein Structures without Purification

描述(申请人提供)：分子生物学在这十年中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显示，多达30%的总蛋白确实是膜蛋白。然而，尽管到目前为止，PDB中已经沉积了多达17,780个三维结构，但这些结构中只有0.56%是膜蛋白。令人惊讶地缺乏膜蛋白结构研究的原因有很多。首先，膜蛋白是高度疏水的，这使得将它们纯化到结构研究所需的同质性变得极其困难。第二，膜蛋白的表达普遍很差。有没有办法绕过这些问题？如果一个人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，那么膜蛋白的结构研究就可以在不进行纯化的情况下进行。这无疑将给膜蛋白的结构生物学带来革命性的变化，并将对我们理解膜蛋白的结构和功能做出重大贡献。在这项提案中，我将尝试建立生产活细胞中感兴趣的单一膜蛋白所需的技术，而不生产任何其他细胞蛋白。这项被称为“SPP系统”的技术将使我们能够在不进行纯化的情况下确定膜蛋白的核磁共振结构，并探索活细胞中的蛋白质动力学。与公众健康相关：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。 与公众健康相关：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。膜蛋白在活细胞中扮演着各种重要的角色，因此与人类健康直接相关，膜蛋白的三维结构的确定是这十年分子生物学的主要挑战之一。从细菌到人类的基因组分析显示，多达30%的总蛋白确实是膜蛋白。然而，尽管已经确定了多达17,780个三维结构，但这些结构中只有0.56%是膜蛋白。膜蛋白缺乏结构研究的主要原因是膜蛋白的纯化极其困难，得率极低。为了绕过这些问题，我将尝试建立生产活细胞中感兴趣的单一膜蛋白所需的技术，而不生产任何其他细胞蛋白。这项被称为“SPP系统”的技术将使我们能够在不进行纯化的情况下确定膜蛋白的结构，并探索活细胞中的蛋白质动力学。这无疑将使膜蛋白的结构生物学发生革命性的变化，并将对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.